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Abstract

Oncolytic viruses (OVs) are engineered and/or evolved to propagate selectively in cancerous tissues. They have a dual mechanism of action; direct killing of infected cancer cells crossprimes anticancer immunity to boost the killing of uninfected cancer cells. The goal of the field is to develop OVs that are easily manufactured, efficiently delivered to disseminated sites of cancer growth, undergo rapid intratumoral spread, selectively kill tumor cells, cause no collateral damage and pose no risk of transmission in the population. Here we discuss the many virus engineering strategies that are being pursued to optimize delivery, intratumoral spread and safety of OVs derived from different virus families. With continued progress, OVs have the potential to transform the paradigm of cancer care.

Ov spread

Replication and amplification in the tumor is the major feature that sets OVs apart from other anticancer drugs. No traditional chemotherapy, immunotherapy or small molecule inhibitor can target tumor cells and then amplify at the site of action and spread to other sites of tumor growth. Specificity as well as speed and extent of IT virus replication are the key determinants of therapeutic index (efficacy/toxicity ratio) for an OV therapeutic, and each of these parameters can be modified by virus engineering. Additionally, the kinetics of spread can be impacted by combining the virus with immunomodulatory drugs. The idea of a virus with selective tropism for cancerous tissue has obvious appeal. Even for naturally occurring viruses that have not been tropism modified, a tumor offers a favorable environment to support a productive infection, and this is borne out by case reports of temporary remission or regression of different cancers concurrent with viral infections [4, 93] . There are several reasons why tumors are generally more susceptible than normal tissues to virus attack; poorly developed lymphatics, high nonsuppressible metabolic activity, resistance to apoptosis, poor responsiveness to interferon and intrinsic suppression of immune effector cells. But as we move in the direction of intentionally using virus infections to mediate tumor destruction, it is apparent that a targeted virus with exquisite tumor specificity will be superior to its non targeted counterpart, allowing for the administration of higher tumor destructive doses without toxicity to normal tissues.

viruses as anticancer drugs

Oncolytic viruses (OVs), evolved and engineered for cancer specificity, are gaining momentum as a new drug class in the fight against cancer. Besides, causing the death of virus-infected cancer cells, the spreading intratumoral (IT) infection can also boost the anticancer immune response, leading to immune destruction of uninfected cancer cells. The paradigm of OVs has been reviewed extensively [1] [2] [3] [4] [5] [6] [7] .

Arming Ovs: extending the range

No matter how extensively an OV infection spreads through a tumor, a sizable percentage of the cancer cells will escape infection [61] . Killing of these uninfected (bystander) cancer cells is therefore critical if oncolytic virotherapy is to become a curative, as opposed to just a tumor debulking strategy. Bystander killing can be achieved, both locally at the site of a spreading infection and systemically at uninfected tumor sites, by genetically arming the virus using one of several possible approaches. For example, an OV can be engineered to encode a secreted protein that selectively mediates the destruction of neighboring and/or distant cancer cells. Alternatively, it can be armed with a 'suicide gene' or prodrug convertase whose encoded protein converts a harmless prodrug to a diffusible anticancer drug. In a third approach, the OV can be armed with a 'radioconcentrator gene' so that infected tumor cells are able to concentrate a β-emitting radioisotope whose emitted electrons damage adjacent uninfected tumor cells. Fourth, it can be engineered to fuse infected tumor cells with uninfected neighboring cells. And last, but by no means least, it can be engineered to express one or more genes capable of amplifying immune-mediated killing of uninfected tumor cells. Each of these approaches is discussed below using specific examples to illustrate the concepts.

• Limiting transmissibility

An alternative strategy often used to side step antibody neutralization, at least of the first dose of virus administered, is to use OVs derived from zoonotic animal viruses such as New Castle disease virus (chicken), VSV (cattle), myxomavirus (rabbit), Seneca Valley virus (pig) or mengovirus (e.g., mouse and monkey). Additional regulatory executive summary viruses as anticancer drugs • Viruses naturally possess many properties that favor infection of cancer cells. Enhancing these natural properties and adding new properties through directed evolution and genetic engineering are used to create oncolytic viruses (OVs), which are emerging as a new anticancer drug class.

viruses as anticancer drugs

It is well established in several rodent cancer models that a single dose of an effective OV can completely cure disease [8, 9] . This has been shown for DNA and RNA viruses in diverse tumor models. However, while the single shot cure is an exciting prospect for cancer therapy, to date clinical outcomes have typically fallen short of this, and repeat IT virus administration has proven to be a more reliable approach [6, 10] . But there are a number of anecdotal case reports that give credence to the idea that a single shot OV cure may be achievable in clinical practice [4, 11] suggesting that OVs have the potential to transform the practice of oncology.

engineering viruses: a diversity of platforms

In nature, viruses are continuously evolving and adapting to occupy almost every imaginable biological niche. Viruses infect bacterial, archaea, protist, fungal, plant and animal cells. Their genomes are composed of DNA or RNA, which may be single or double stranded, positive or negative sense, ranging in size from 2 to 300 kb, in complexity from 1 to 300 genes and in capacity for foreign genetic material from a few hundred bases to several kilobases. The particles of viruses from different families also vary enormously in size and structure, ranging from 20 to 1000 nm, from icosahedral to helical symmetry, with or without lipid envelope, integument or matrix and with variable susceptibility to physical disruption. Naturally occurring viruses also offer a vast diversity of virus life cycles, cell entry and replication mechanisms, cell and species tropisms, cycle times, burst sizes, innate immune evasion, apoptosis, antiviral state prevention and immune combat strategies, modes of transmission and pathogenic mechanisms. The diversity of viruses that have been investigated as oncolytic platforms and the different strategies used to improve their efficacy continue to be expanded (Table 1) .

Delivery of Ovs

Mouse models with implanted flank tumors provide a convenient model for IT injection as the tumor is superficial, but in human patients with spontaneous malignancies tumors often develop internally in visceral organs. Melanoma, head and neck cancer, and lower gastrointestinal malignancies may be better suited for IT injections due to anatomical position, although with advanced interventional radiology using computed tomography or MRI image guidance to place the needle many more tumors may be accessible for IT virotherapy. One additional advantage of IT injection over the IV route is that the threshold concentration of input virus required to initiate a spreading infection in the tumor tissue can be more easily achieved.

• Limiting viral neutralization & clearance

For high seroprevalence viruses, one approach to circumvent viral neutralization by preformed antibodies is to engineer or switch the viral coat proteins. Some OVs (e.g., adenovirus) offer a menu of different serotypes, providing a basis for serotype switching between successive doses of the therapy to avoid antibody neutralization, although this does greatly complicate the product development pathway since each serotype is considered to be a distinct pharmaceutical product [82] . For monotypic viruses such as measles, serotype switching is not an option, but antimeasles antibodies can be circumvented by substituting the surface glycoproteins of measles with those of a related but noncross-reactive morbillivirus such as canine distemper virus [83] . Alternatively, the immunodominant epitopes of the measles surface glycoproteins can be modified by mutating key surface residues to eliminate them, or by introducing glycosylation signals so they are shielded by N-linked glycans [84] . It is worth noting that all of these virus engineering strategies have the potential to alter viral receptor usage and hence tumor cell tropism which may limit their utility [83, 85] .

Ov spread

Engineering tumor specificity can also be achieved by detargeting viruses to ablate unwanted tropisms that can cause off target pathology in normal tissues. MircroRNA targeting is the best example of this approach. For example, miRNA targeting was used to control the tropism of an oncolytic coxsackievirus A21 virus which caused rapid tumor regression followed by fatal myositis in murine models of myeloma and melanoma [119] . Insertion of muscle-specific miRNA targets into the viral genome eliminated muscle toxicity but left the antitumor potency of the virus intact. This was shown to be due to miRNA-mediated recognition and rapid destruction of the viral genome in muscle cells. miRNA targeting has since been applied to many OVs from diverse virus families and provides a convenient and economical strategy (using sequence insertions of only ∼100 bases) to control unwanted virus tropisms [25] .

• viral amplification & spread

In the case of free virus particles, which are susceptible to antibody neutralization, the stroma of the tumor also has the potential to limit IT diffusion and block systemic release. Fibroblasts, endothelial cells, immune cells and the ECM make up the tumor stroma, and the exact composition varies widely depending on tumor type [125] . The ECM is a collagenous matrix of protein fibrils, adhesive proteins and proteoglycans that create a web with pore sizes similar or slightly smaller than virions [126] . Destruction of ECM components can facilitate viral spread throughout the tumor and can be achieved using conventional chemoradiotherapy or by delivering matrix-degrading enzymes such as collagenase and hyaluronidase [127] . An alternative approach is to encode a matrix-degrading enzyme or inducer of matrixdegrading enzymes in the viral genome. For example, hyaluronidase and relaxin encoding oncolytic adenoviruses have each been shown to spread more efficiently in experimental tumors [128] [129] [130] .
Another factor that significantly impacts the kinetics of virus spread is the burst size, or the number of progeny viruses released by a productively infected cell, which varies widely between viral families. Picornaviruses, VSV and vaccinia virus can release up to 10,000 progeny from a single infected cell after a delay of only 6-18 h [131] [132] [133] . In addition to innate antiviral immunity, adaptive cell-mediated immune responses are typically required for the complete elimination of a viral infection and act by eliminating infected cells before progeny can be released. Oncolytic virotherapy can therefore be viewed as a race between the spreading virus and the responding immune system. For this reason, faster moving viral infections are often considered capable of inflicting greater damage to an infected tumor before they can be contained by the immune system [134] . However, in defense of the viruses with smaller burst sizes, or which release progeny by budding, they tend to be less review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group rapidly controlled both by the innate and adaptive host immune responses [135] . Therefore, as with the classic race between the hare and the tortoise, it is very difficult to predict whether a fast or slow replicating virus will show superior efficacy in a given preclinical cancer model.

• • Limiting pathogenicity

Understanding the selective pressures that operate within the tumor and the host, as well as the role of viral quasispecies in treatment outcomes is an active area of research. RNA and DNA viruses exist as a population of quasispecies or collections of related viral genomes undergoing variation and selective pressure [174] . Generation of quasispecies occurs when viral genomes are copied during the replication cycle. Viral polymerases typically have an error rate that introduces an average of one or more base mutations per progeny genome [175] . In general, the larger the virus genome, the lower the polymerase error rate. Picornaviruses are among the smallest viruses being developed for oncolytic therapy with positive sense RNA genomes ranging from 7 to 9 kb in length, and their RNA polymerases have a correspondingly high intrinsic error rate. Production of a virus for clinical application is a highly regulated process and involves multiple rounds of replication to achieve enough virus for patients. The viral product is therefore already a swarm of quasispecies at the time it is administered to the patient and mutates further as it undergoes additional rounds of replication in vivo. Studies of the mutation rates of viral polymerases, the generation of quasi species, the evolution of viral populations and the evolution between dominant subspecies within a virus population are therefore of great interest and relevance to the OV field.
Polymerase infidelity is not the sole driver of virus genome diversification and evolution. For many viruses, recombination between homologous viral genomes can occur when two related viruses infect the same cell [178] . This type of recombination is not a feature of negative strand RNA virus evolution because the nascent progeny viral genomes are cotranscriptionally incorporated into a helical ribonucleocapsid structure which prevents recombination. However, for positive sense RNA picornaviruses, and DNA viruses such as adenovirus, HSV and vaccinia, it is important to consider the possibility that the input virus may encounter and recombine with a homologous 'wild-type' virus in a treated patient. While such recombination has never been documented in OV trials, there are well described examples in the field of vaccinology, such as the re-emergence of pathogenic polioviruses through the recombination of vaccine genomes and naturally circulating picornavirus genomes [179] . In view of these risks, it is generally considered inadvisable to arm viruses with therapeutic transgenes that have theoretical potential to increase pathogenicity if transferred to a related naturally circulating virus. Genes encoding immunosuppressive, antiapoptotic or directly cytotoxic proteins are on this list.

• Limiting transmissibility

In light of the inconvenience and undesirability of OV shedding, there is interest in engineering strategies that may selectively interfere with the process. As one example, wild-type measles virus is a highly transmissible airborne virus that uses the nectin-4 receptor to enter into airway epithelial cells from whence its progeny are shed into respiratory secretions [192, 193] . Eliminating the nectin-4 tropism by strategically mutating key surface residues in the hemagglutinin attachment protein results in a virus that still causes measles in nonhuman primates, but which is no longer shed into respiratory secretions or urine [194] . Measles virus RNA (but not infectious virus) was detected in the blood, urine and saliva of myeloma patients up to 3 weeks after IV administration of an oncolytic measles virus, especially at higher dose levels, and it is possible that this shedding might be eliminated by using a nectin-4 blind version of the virus [195] . Virus shedding has also been detected in human clinical trials of HSV, reovirus, vaccinia, reovirus and adenovirus oncolytics, but was less readily detected following IT therapy with oncolytic VSV and polioviruses [167] . However, future science group www.futuremedicine.com it should be noted that while genome sequences have been detected in shed material, infectious virus particles have not been recovered.

viral spread

• Targeting viral spread to tumor cells can be accomplished by transductional targeting (modifying receptor tropism), transcriptional targeting (controlling virus gene expression with tumor-specific promoters), physiologic targeting (disrupting viral immune combat proteins), apoptosis targeting (disrupting viral antiapoptotic proteins) or miRNA targeting.

viruses as anticancer drugs

The key desirable characteristics of any OV are specificity, potency and safety; specificity for the targeted cancer, potency to kill infected cells and cross-prime antitumor immunity, and safety to avoid adverse reactions and pathogenic reversion.

Delivery of Ovs

Viral infections are well understood but for OV applications must be viewed as drugs obeying pharmacological principles. A drug is typically administered to a patient in an extremely controlled way to produce a reliable, consistent and predictable pharmacokinetic profile (absorption, biodistribution, metabolism and excretion) and bioavailability. Natural viral infections do not obey these rules since the inoculum is of variable size; host resistance varies from person to person; and the kinetics of the adaptive immune response differ greatly between individuals. Thus, the outcomes of natural infections with a given pathogenic virus range from asymptomatic seroconversion to full blown disease. The primary virus inoculum must contain sufficient virus particles to overcome initial host defenses at the site of entry. Some viruses enter through mucous membranes in the GI or respiratory tracts, while others enter via direct inoculation into the blood stream following a needle stick or arthropod blood meal [58] . OVs are delivered in the same way as traditional drugs, by introducing a highly concentrated virus inoculum into the body via oral, intravenous (IV), intranasal, transdermal, subcutaneous or intramuscular routes whereupon the dispersion of the inoculated virus, or its progeny, takes it to the targeted cancerous tissues. A major factor distinguishing OVs from traditional drugs is that they self-amplify and spread after delivery so their peak concentration may not be reached until sometime after the treatment is administered. Biological amplification by viral replication is the most important difference between viral therapies and traditional drugs. The concentration of a drug diminishes over time at a very well described rate depending on the clearance and elimination from the body. Through the course of a viral infection the viral load is initially small and then increases and finally decreases rather than just following a specific rate law of elimination. Limited amounts of virus particles replicate at the site of inoculation [59] . Input or progeny viruses then can either drain with the lymphatic fluid to the nearest lymph node, go directly into circulation or spread locally before spreading systemically to eventually arrive at a specific target organ. Pathology is induced by high levels of viral replication in the target organ directly killing infected cells or recruiting the immune system to kill them, at the same time provoking a local inflammatory response.

• • Limiting pathogenicity

Although it will be interesting to determine whether OVs encoding modified polymerases with higher fidelity will exhibit an improved safety profile [176] , it should be noted that OV reversion to a more pathogenic state has not yet been observed clinically. This is also true of many time-honored viral vaccines. For example, in 50 years of widespread measles vaccination using attenuated Edmonston lineage substrains, there has never been a documented reversion back to a wild-type phenotype, even after IV doses as high as 10 11 TCID 50 were administered to immunosuppressed, measles antibody negative myeloma patients in an oncolytic virotherapy study [177] .
Certain viruses with segmented genomes, most notably the orthomyxoviruses (e.g., influenza), are capable of rapid evolution by genome fragment reassortment in multiple infected cells. Reassortment of genome fragments in virus-infected birds and pigs is considered to be a key driver of the antigenic shifts that occur during evolution of new pathogenic influenza virus strains [180] . This important safety concern may explain why oncolytic influenza viruses have not yet been advanced to human clinical trials [181] . Because of the toxicity risks associated with the proliferative and evolutionary capacity of OVs, there is considerable interest in contingency plans to terminate the spread and/or transmission of an OV infection. Reliable antiviral medications are available for TK-expressing herpes viruses, but this is not the case for the majority of viruses being developed as OV platforms [182, 183] . Attention has therefore been directed to the development of safety switches that can be engineered into the OV genome and triggered on demand to terminate in vivo replication.
Limiting the pathogenic potential of OVs can also be accomplished by enhancing the host innate immune response. VSV has been engineered to express IFN-β, which induces an antiviral state and reduces proliferation of cells. Cells capable of responding to interferon signaling will limit viral replication in response to the virally produced interferon, further restricting viral replication to tumors with defective innate responses [188] . The expression of IFN-β from infected tumor cells can also prevent off-target infection of nearby tissue through paracrine signaling. Another way to achieve a similar effect is mutating the VSV matrix protein, which is responsible for limiting cellular production of type I interferons [189] . This, and similar approaches, to inactivate viral genes that combat innate immunity has been extensively utilized in the OV field, across a broad range of viral families. Sensitization of OVs to innate immune responses can limit off-target infections and enhance tumor-specific tropism while increasing the therapeutic index.

• • Sodium iodide symporter (radioconcentrator)

Iodide is a critical component of thyroxine and is concentrated in thyroid follicular cells by the thyroidal sodium iodine symporter (NIS), a cell surface glycoprotein with 13 transmembrane domains [146] . Radioiodine is therefore used routinely in the clinic for thyroid imaging and for ablation of overactive thyroid tissue, including metastatic thyroid cancer. This is facilitated by the ready availability of several iodine radioisotopes, most notably 123 Ifor γ camera and single-photon emission computed tomography imaging, 124- for PET imaging and 131 I -, a β-emitting isotope, for thyroid ablation. Besides radioiodine, NIS can concentrate several related anions, of equal or greater value for single-photon emission computed tomography ( 99m TcO 4 -, pertechnetate), PET (B 18 F 4 -, tetrafluoroborate) and tissue ablation ( 211 At -, astatide, 188 ReO 4 -, perrhenate) [147] . The β emissions of 131 Ihave an average path length of approximately 1.8 mm in tissue and can therefore inflict significant damage on cells adjacent to an 131 I --loaded NIS expressing cell. For 188 ReO 4 the β emission path length is longer such that NIS negative tumor cells are even more likely to be damaged in the β particle crossfire when this radioisotope is used. Unsurprisingly, therefore, the NIS gene has been engineered into several OVs whose IT spread has been elegantly mapped and monitored in tumor-bearing mice by serial radiotracer imaging, and whose potency has been substantially boosted by appropriately timed administration of 131 I - [148] . At least two NIS-expressing OVs, a prostate-targeted oncolytic adenovirus and a CD46-targeted measles virus, have been advanced to human clinical trials with positive imaging data reported [11, 149] .

viruses as anticancer drugs

Based on these clinical advances, development of OVs is rapidly accelerating. The purpose of this review is to discuss the virus engineering approaches and OV performance optimization strategies that are being pursued in the field, and to point out some of the challenges that remain. We believe this perspective will be particularly valuable to virologists entering the field. We apologize to the numerous investigators whose work has not been acknowledged; we are well aware of these omissions but due to space constraints our approach has been choosing illustrative examples that demonstrate the key principles. The review is divided into five main sections: (i) an introduction to viruses and virus engineering, (ii) delivery, (iii) spread, (iv) arming and (v) safety. All five topics are highly interdependent.

engineering viruses: a diversity of platforms

Aside from their differences, viruses share fundamental similarities including their dependence on the host cell to provide a suitable environment for genome amplification, gene expression and progeny virus production and their adaptation to a specific set of host cell conditions and factors such that propagation is precluded in cells that fail to provide the necessary environment. These characteristics allow viruses to be targeted to cancer cells as a self-replicating antineoplastic therapy.

Delivery of Ovs

OV therapy begins with the administration of the virus to the patient just as with any other drug. Several approaches can be taken, each with to optimize oncolytic virotherapy indicated that maximizing delivery, so more tumor cells are infected, has a profound impact on the degree to which the virus must propagate from each infected cell for curative therapy [60] . The higher the number and the more random the dispersion of infectious centers established in a tumor after delivery of virus, the more effective the virus will be, although these parameters must be optimized for each virus and model [61] . IV and IT administrations of OVs are common delivery methods that introduce large amounts of virus into one compartment in a very short time. This rapid delivery of a massive virus inoculum does not parallel what typically occurs in a natural infection and potentially allows the IT spread of the OV infection to outpace host defenses. Successful delivery in the context of an OV infection requires that a critical viral concentration is achieved within the tumor to allow for sufficient oncolysis and systemic spread of the virus to all sites of disease in a safe and reproducible pattern.
• • iv delivery IV delivery of an OV seems advantageous in the setting of metastatic disease, allowing the virus to potentially access all sites of disease via the circulation soon after infusion. In a clinical setting, systemic delivery may be preferred since it is more broadly applicable than IT inoculation, regardless of tumor location and total tumor burden. Yet, attempts at systemic delivery have shown limited success; the administered virus is immediately diluted in the circulating blood volume (∼5 l), so extremely high doses are needed to achieve meaningful circulating titers. A clinical trial using an oncolytic pox virus, JX-594, demonstrated that virus could only be detected in tumors of patients that received at least 10 9 p.f.u. per IV dose without any virus being detected in tumors of the lower dose cohorts of patients [62] . Because of this important dilution effect, doses required for IV therapy may have to be up to 1000-fold higher than for IT administration, which results in significant manufacturing challenges as well as a unique set of toxicities [2] . Administration of high titers of virus intravenously can lead to hepatotoxicity, thrombocytopenia and lymphopenia [33, 63] .
Following the path of an OV from the IV line to the tumor will quickly illustrate some of the challenges for IV delivery. First, the virus is rapidly diluted in the circulation, where its infectivity is neutralized by serum proteins including antibodies and complement, and a large proportion is sequestered by reticuloendothelial phagocytes in the spleen and liver. Any virus particles that reach the tumor vasculature without first being neutralized must then extravasate through fenestrations, and pores between the vascular endothelial cells of the tumor capillaries, and once in the interstitial space of the tumor, must negotiate the extracellular matrix (ECM) to reach and infect the resident cancer cell [64] .
Tumor vasculature can both aid and limit viral access. Tumors typically have an unorganized growth pattern, producing vasculature that is tumultuous and insufficient resulting in heterogeneous perfusion throughout the parenchyma [65] . Decreased perfusion limits the formation of infectious centers evenly throughout a tumor. Manipulating physiological parameters such as blood pressure and systemic vascular resistance can preferentially increase tumor perfusion and promote better delivery of viral vectors [66] . Once in a tumor vessel, the virus must move from the intravascular compartment into the interstitium by crossing the vascular endothelium to continue its journey through the interstitial space until finally reaching a permissive tumor cell.
At least in theory, viruses can cross the endothelial lining of tumor neovessels by diffusion, active infection, or by trafficking inside of or on the surface of marrow-derived cells that are capable of diapedesis. Tumors generally have poor lymphatic drainage and can have leaky blood vessels with a wide range of pore sizes which helps to explain the phenomenon of enhanced permeability and retention of nanoparticles such as OVs in the tumor parenchyma [67] . In some tumors, the pores and fenestrations in tumor capillaries may be large enough to permit passive diffusion of viruses (depending on their size) but in many tumors review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group the endothelium is sufficiently organized that this type of extravasation is simply not possible.
Another approach to help OVs' extravasate is by temporally creating pores throughout the tumor microvasculature to enhance transport of the virus into tumor interstitium. This can be done through application of focused ultrasound in the presence of a contrast agent or a polymer. The microbubbles or polymers are injected intravenously and can freely circulate without harming tissues, but when they are exposed to ultrasound waves they can oscillate and induce pore formation or cavitation in the surrounding tissue. The ultrasound can be focused on a tumor just after the infusion of the virus to increase viral extravasation. This has been shown to enhance vaccinia virus delivery to tumor xenografts and can increase viral transgene expression by more than a 1000-fold in the tumor [71, 72] . Many strategies are being developed to enhance OV IV delivery.
• • iT delivery IT administration directly delivers a high concentration of the OV into the parenchyma of the injected tumor but may not result in spread of the virus to distant sites of metastasis. T-Vec is administered by IT injection to accessible skin tumors of patients with metastatic malignant melanoma. The injection is repeated every 2 weeks until lesions have resolved or failed to respond [12, 73] . Interestingly, viremia was not documented in patients, suggesting that the regression of noninjected lesions was likely immune-mediated and not due to direct oncolysis [74] .
Immediately following IT delivery, small levels of virus can be detected in the blood from leakage through injured tumor vasculature [75] , but that small amount of virus is quickly cleared from the system. Also, virus that has been injected into a tumor is frequently extruded immediately from the injection track once the syringe is removed, especially in smaller higher pressure tumors. However, systemic viral spread can occur following IT administration if the initially exposed tumor cells are capable of amplifying the input virus and releasing progeny into the bloodstream. Such secondary viremia may not peak until several days after IT virus delivery and, with currently used OVs, is often not detected. Secondary viremia, when it occurs, more closely mimics the natural propagation of a viral infection from the site of inoculation to more distant target organs.

• Other routes of delivery

Intraperitoneal virus administration is often pursued in virotherapy studies aiming to impact ovarian cancer and other disseminated intraperitoneal malignancies, while intrapleural administration is pursued for mesothelioma therapy. Both of these approaches are similar to IT administration in that the virus comes into direct contact with tumor cells in the injected cavity, although there is a greater risk that input virus will be immediately neutralized by antibodies and other proteins in ascites or pleural fluid. Intravesical instillation of virus is the favored route of administration for treatment of early-stage bladder cancer, requiring only that future science group www.futuremedicine.com the input virus be stable in urine. Also, immediately after brain cancer surgery, in an attempt to control residual disease, virus is often instilled directly into the resection cavity.

• Limiting viral neutralization & clearance

Perhaps the most significant barrier to widespread OV delivery is rapid neutralization and clearance of circulating virus particles. Antibodies and complement proteins can coat the virus blocking its ability to interact with its cellular receptor and accelerate Fc receptor-mediated clearance by splenic macrophages and hepatic Kupffer cells.
Mengovirus, VSV and Newcastle disease virus are examples of animal pathogens being developed as oncolytic agents. Their appeal as oncolytic platforms is due to the coupling of their ability to selectively propagate in human tumors with their low seroprevalence in the human population [77] . Unlike viruses such as measles which have almost exclusively human cell tropism, these zoonotic viruses are capable of infecting both rodent and human tumor cells allowing preclinical testing in more informative immunocompetent mouse cancer models.
Virus-infected cell carriers can also be used to transport viruses via the bloodstream to sites of tumor growth. In part, this is possible because there is an 'eclipse period' after the cell carriers have been infected during which they do not display viral proteins on their surface and are therefore not bound by virus neutralizing antibody, but are still able to extravasate from tumor neovessels and release infectious progeny into the tumor parenchyma. Several cell carrier/OV combinations have been used including mesenchymal stem cells, dendritic cells, T cells and endothelial progenitor cells [89] [90] [91] [92] . Some carrier cells are believed to home more efficiently to tumors responding to chemotactic signals arising from hypoxia or IT inflammation.

Ov spread

Virus tropism is determined by many factors, most of which, if understood sufficiently, can be manipulated to enhance tumor specificity. The receptor tropisms of naturally occurring viruses are rarely of interest for tumor targeting, but this is not true for tissue-culture-adapted vaccine lineage viruses, some of which have evolved in the laboratory to use receptors more abundantly expressed on cancer cells. Examples include the CD46 receptor tropism of vaccine lineage measles virus and the heparan sulfate tropism of laboratory-adapted Sindbis virus [94, 95] . Where greater specificity is desired, it may be possible to engineer new receptor tropisms by modifying the structure of a viral attachment protein, for example, by displaying polypeptide ligands at the extreme C-terminus of the measles H glycoprotein [96, 97] . However, for many viruses, displaying a polypeptide ligand on the surface does no more than redirect attachment and does not confer a new receptor tropism [98] [99] [100] . This remains an area of active investigation.
In addition to their dependence on specific entry receptors, the surface glycoproteins of many enveloped viruses (and sometimes other critical viral proteins) must be proteolytically activated before they are competent to mediate virus entry [101] . By engineering the protease target sequences in these viral glycoproteins it is possible to generate viruses whose propagation is now dependent on exposure to a specific protease with high IT abundance, such as urokinase, matrix metalloproteinase 1 or cathepsin D [102] [103] [104] .
Beyond the step of cell entry, viruses are exquisite sensors of intracellular processes and can therefore be adapted or engineered in several ways for intracellular targeting of cancer cells. This is best understood by considering some fundamental aspects of the interplay between a virus and an infected cell. The incoming virus aims to usurp the cellular synthetic machinery for generation of progeny viruses. The cell resists this takeover bid by rapidly detecting virus invasion, then triggering a signaling cascade that leads to establishment of an antiviral state and release of interferon which induces an anti viral state in adjacent cells [105, 106] . The antiviral state is very complex but suppression of protein translation is a key component. Apoptosis is also triggered by the virus detection machinery so that the infected cell dies before it is able to manufacture virus progeny [107] .
For a virus to be 'successful' it must combat these host cell responses, avoiding detection, as long as possible, for suppressing the establishment of an antiviral state and preventing apoptosis. Virtually all naturally occurring viruses therefore encode proteins that inhibit apop tosis and the antiviral state [108] . Removing these accessory functions from the viral genome leads to virus attenuation in normal tissues, but to a much lesser degree in cancerous tissues. This is because cancer cells are intrinsically resistant to apoptosis and to the establishment of an antiviral state, making them highly susceptible to attenuated viruses that are no longer able to control those processes. This has provided a mechanistic basis for physiologic targeting of several viruses; VSV by mutating the matrix gene whose encoded protein blocks the interferon response [109] ; HSV by mutating both copies of the γ-34.5 gene which interferes with interferonmediated shutoff of host protein synthesis and enhances neurovirulence [110, 111] ; adenovirus by mutating the E1B protein, one of whose actions is to inhibit the apoptotic activity of p53 [112] .
Viruses can be further engineered to exclusively replicate in tumor cells by combining a virus' needs with physiologic peculiarities intrinsic in tumorigenesis. For example, a virally encoded thymidine kinase (TK) is required for HSV and vaccinia virus infection to increase the supply of deoxynucleotide triphosphates required for synthesis of progeny virus genomes [113, 114] . Elimination of the TK future science group www.futuremedicine.com gene from the viral genome restricts viral replication to cancer cells where there is an upregulation of human TK [115, 116] . Another example of a virus that exploits the high replication rate of tumor cells is Toca-511, a replication competent C-type retrovirus encoding the drug activating enzyme cytosine deaminase (CD) [117] . Since the integration of C-type retroviruses is S-phase dependent [118] , this virus is selectively amplified in rapidly proliferating tumor tissue.

• viral amplification & spread

After an appropriately targeted virus has infected a tumor cell, it is the extent of its subsequent propagation that becomes the key driver of potency. Data-driven mathematical models of systemic oncolytic virotherapy indicate that tumor eradication is dependent on two major parameters: the initial density and distribution of infectious foci in the tumor; and the ultimate size of the infectious centers arising from each individual infected cell (i.e., virus spread) [120] . Viral spread may occur by various mechanisms. Local spread may occur by intercellular fusion, by direct transfer of virus from infected to adjacent cells, or by release and local migration of progeny virions through the interstitial space. Systemic spread as free virus particles or as virusinfected migratory cells occurs via lymphatic channels or via the bloodstream.
Direct cell-to-cell transfer of viruses has the advantage of stealth as the virus cannot be neutralized by antiviral antibodies in the interstitial fluid [121] . Nonfusogenic viruses can be armed with fusogenic membrane glycoproteins (FMGs) to enable stealthy spread through intercellular fusion leading to the formation of large, nonviable multinucleated syncytia which may also serve as excellent antigen presenting cells for amplification of the antitumor immune response [122] . By way of example, VSV encoding measles fusogenic glycoproteins and HSVs encoding the fusogenic gibbon ape leukemia virus glycoproteins have shown superior efficacy when compared with their corresponding parental viruses [123, 124] .

• • Secreted toxins

As a general rule, if a virus is to be armed with a gene encoding a secreted toxin, that toxin should be targeted so that it can kill only cancer cells. In the absence of such targeting, there would be little prospect of avoiding off-target toxicities. Immunotoxins are bifunctional proteins in which plant or bacterial toxins (typically ribosomal inhibitors) are fused to a single chain antibody or other polypeptide domain to target endocytosis in cancer cells [136] . In theory, such molecules that have been extensively investigated and advanced to human clinical trials particularly for the treatment of B-cell and T-cell malignancies [137] , could be expressed from an engineered OV genome.

• • Fusogenic membrane glycoproteins

Fusion of the lipid envelope of an incoming virus with the limiting membrane of its target cell is a necessary step in the life cycle of an enveloped virus, to deliver the encapsidated viral genome into the target cell cytoplasm. This virus-to-cell fusion reaction is mediated by FMGs embedded in the envelope of the virus, and may occur at the cell surface at neutral pH, or in the endo-somal compartment at acidic pH [150] . Neutral pH fusion is triggered by receptor attachment. Cells expressing certain virally encoded, neutral pH-active FMGs on their surface may fuse with neighboring receptor positive cells (cell-to-cell fusion), giving rise to multinucleated syncytia, the hallmark cytopathic signature of a fusogenic virus (e.g., measles) [151] . But more often the FMG is activated only after its incorporation into a budding virus particle, so cannot cause cell-tocell fusion, and must be modified, for example, by cytoplasmic tail truncation, to render it constitutively fusogenic. Either way, FMG-driven fusion of OV-infected cells with uninfected neighboring cells leads to increased bystander killing because multinucleated syncytia are nonviable as well as being highly immunogenic [152] . Based on these observations, nonfusogenic OVs have been armed with FMGs thereby conferring superior oncolytic potency [123, 153] . In one example, an oncolytic herpes virus was rendered highly fusogenic when engineered to encode a cytoplasmically truncated gibbon ape leukemia virus envelope glycoprotein [154] [155] [156] while in another study, an oncolytic VSV was rendered highly fusogenic by replacing its surface glycoprotein (G) with the hemagglutinin and fusion glycoproteins of measles virus.

• • Arming Ovs to amplify antitumor immunity

Each step of the above process can be impacted by virus engineering. Not only can the kinetics and mode of cell killing be manipulated (see the previous section on virus spread), but also the OVs can be armed with a wide variety of immunoregulatory genes whose products will be secreted into the interstitial fluid space. Thus, OVs have been engineered to express the following: high levels of type I interferons to better drive the early innate/inflammatory response; GM-CSF to stimulate the phagocytic activity and lymph node trafficking of professional antigen presenting cells; chemokines to enhance the IT recruitment of immune and inflammatory effector cells, especially cytotoxic T cells; cytokines to drive the activation and proliferation of tumor-resident cytotoxic T cells; bispecific T-cell engagers to enable tumor cell killing by T cells not recognizing tumor antigens; checkpoint inhibitor antibodies to block the protective PD-L1/B7.1 shield that protects tumor cells from T-cell attack; cytokines to increase MHC-peptide neoantigen expression on uninfected tumor cells; or cloned tumor antigens to further amplify tumor-specific immunity [165] .
While each of these approaches has proven beneficial in selected preclinical animal models where immune destruction of the tumor is the dominant OV effect, they are equally likely to decrease potency by speeding virus elimination in situations where the spread of the virus is dominant.

Safety

Engineering viruses for increased stealth, greater specificity, faster spread and enhanced potency is not without risk. As OVs gain new properties, checks and balances must be applied to assure that they do not readily evolve into pathogens, let alone transmissible pathogens that could pose a risk not only to immediate contacts of the treated patient, but also to the greater population.

• • Limiting pathogenicity

Viral cytotoxicity is the basis of tumor cell death necessary for oncolytic activity. Off-target infection and killing of normal cells by poorly targeted OVs or by OVs that have evolved new tropisms in a treated patient can cause unwanted normal tissue pathology. OVs are therefore delicately balanced between retaining enough virulence to substantially decrease tumor burden versus being sufficiently targeted (or attenuated) to not cause a new disease in the patient. Cancer-specific targeting is the most critical safety feature, but viruses evolve and viral populations are dynamic [172] . Evolution is a constant accompaniment of a spreading virus infection, whether or not the virus is oncolytic; in vivo progeny of the therapeutic OV differs on average by a single -point mutation per genome from the input virus [173] . Hence, as the input virus is amplified, it generates a swarm of quasispecies viruses, each one a slightly imperfect replica of the input virus. This swarm of progeny viruses is subjected to selective pressure as it encounters new biological niches in the treated cancer patient. Thus, if it so happens that a member of the swarm is capable of infecting a normal host tissue, the virus may have gained a new foothold from which to further evolve. Gaining new cell tropisms or losing restriction factors is therefore a significant theoretical concern in oncolytic infections, but has not yet been documented in human trials, nor in preclinical models. However, given the importance of this particular scenario, a great deal of attention is paid to the problem not just by investigators, but also by regulatory agencies.

• Limiting transmissibility

For obvious reasons OV transmission from a treated patient to a caregiver, family member, coworker, pet or other species is highly undesir-able. Infectious virus particles may be present in the blood of a treated patient, and may be shed into the environment in urine, feces, saliva and other bodily secretions. Contact with the body fluids of an OV-treated patient therefore may have the potential to spread an infection to new hosts [190] .
Because the risks of OV transmission are typically unknown when first-in-human Phase I trials are initiated, it is usual to implement standard infection containment measures throughout these studies and to carefully monitor body fluids for the appearance and disappearance of viral genomes and infectious virus progeny [191] . For certain viruses, containment may be considered unnecessary, particularly when there is already widespread population immunity to the OV in question (e.g., measles) or when there has been extensive human experience of exposure to a related virus in the form of a live viral vaccine (e.g., vaccinia). Contingency plans may also be required calling for quarantine of OV-treated patients if treatment toxicity is associated with a longer period of shedding. In reality, to date there has been no instance in which transmission of an OV from a patient to a caregiver or other contact has been demonstrated, and there are no examples of long-term virus persistence or shedding in a treated patient.
In an OV-treated patient pre-existing antiviral immunity can be a formidable barrier to efficacy, particularly if the virus is administered intravenously. However, pre-existing antiviral immunity in caregivers and patient contacts provides reassuring protection against virus transmission. Conversely, when this particular efficacy barrier is circumvented by using OVs engineered for antibody evasion or selected for low seroprevalence, the risk of epidemic spread in the human population, unchecked by pre-existing herd immunity, looms larger.
• The diversity of virus families and engineering techniques allows for the creation of OVs with a wide range of properties that can be tailored for each type of cancer.

Delivery of Ovs

• Intravenous delivery allows a virus to reach distant sites of metastasis via the circulation, but extravasation into the tumor parenchyma is inefficient.
• Intratumoral injection can concentrate virus at a site of tumor growth, but regression of distant tumors requires that the virus spread systemically or induce a systemic antitumor immune response.
• Neutralizing antibodies, hepatosplenic sequestration of the virus by macrophages and dilution of the virus in blood or tissue may limit the effectiveness of treatment.

Safety

• Ideally, OVs should be nontransmissible. scrutiny is generally required for these viruses (e.g., from the US Department of Agriculture) to address the additional risks of environmental release and epidemic spread in domestic animals, particularly as they relate to agricultural livestock.

Conclusion & future perspective

Viruses are at last being harnessed for the benefit of cancer patients. The OV field has moved well beyond proof of principle in human studies, and virus engineering will be the key to its continued advancement in the coming years. Virtually every component of every naturally occurring or laboratory-adapted virus can be engineered and/or evolved to enhance its suitability for cancer therapy and we are currently witnessing unstoppable creative activity in this area. Safety is obviously of paramount importance in this relatively new field, and is therefore closely regulated from the design stage to clinical implementation. Considering the current trajectory of OV research, there can be little doubt that viruses are on their way to becoming one of the foundational modalities of future cancer treatment regimens.

viruses as anticancer drugs

In light of recent clinical progress, interest in the approach is burgeoning. One critical milestone was the 2015 marketing approval granted in Europe and the USA for talimogene laherparepvec (T-Vec, Imlygic™), an engineered HSV encoding GM-CSF. This virus, administered intratumorally every 2 weeks for malignant melanoma, led to complete resolution in 47% of injected tumors and boosted systemic antitumor immunity leading to resolution of 9% of distant un infected visceral tumors [12] . Subsequent clinical studies have shown that responses are more frequent and more durable when T-Vec (and other OVs such as coxsackievirus A21) is combined with immune checkpoint For reprint orders, please contact: reprints@futuremedicine.com review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group inhibitor antibody therapy [13] . Another significant milestone was the demonstration that a systemically administered oncolytic measles virus can target and destroy disseminated cancer in a human subject [11] .

engineering viruses: a diversity of platforms

As might be expected, proponents of a given OV are typically able to advance strong arguments to support their choice of platform, emphasizing unique features such as replication kinetics, genome plasticity, targetability, seroprevalence and stability that may lead to superior oncolysis. However, it is too early to determine which unique viral characteristics will be the critical drivers of clinical success for a given cancer type. Reverse genetics systems are available for virtually all virus families, and the rules of engagement for new virus creation are well established. In general, the most effective strategy is to combine rational design with evolution, allowing each engineered virus to mutate and fully adapt to its intended target cells after it has been rescued. Biosafety oversight is in place at all academic centers responsibly engaging in virus engineering activities, and it is now a relatively straightforward matter to generate and test new virus configurations using what now amounts to the world's best lego set. Viral gene and noncoding sequences can be modified in a variety of ways to add or eliminate functions and nonviral genes or noncoding regulatory elements, whether synthetic or naturally occurring, can be added into viral genomes to confer additional desirable properties. The overarching engineering goal for the oncolytic virotherapy field is to generate viruses that can be efficiently delivered to disseminated tumors in the body where they will spread and selectively kill both infected and uninfected tumor cells, without causing collateral damage and posing no risk of transmission to the population.

Delivery of Ovs

Clearly, the ability to infect the activated endothelial cells of tumor neovessels would be an attractive targeting property for OVs to enhance entry into the tumor parenchyma by releasing viral progeny on the ablumenal side of the blood vessel. Intravascular coagulation in the capillary could also be provoked by virusinfected endothelial cells reacting with clotting and inflammatory factors. Virus engineering strategies have been pursued to achieve this goal, for example, by displaying polypeptide ligands echistatin and urokinase plasminogen activator on the surface of measles virus to target integrin αVβ3 and UPaR endothelial cell surface receptors, respectively [68, 69] . Also, at least one OV (vesicular stomatitis virus VSV) has been shown naturally capable of infecting neovessel endothelium in implanted mouse tumors, but the study did highlight the potential toxicity of the approach, namely intravascular coagulation requiring heparin therapy for its prevention [70] .

• Limiting viral neutralization & clearance

Antibody neutralization has been shown to reduce the efficacy of systemically administered OVs such as measles, VSV and vaccinia in preclinical models [76] [77] [78] . When previous exposure has occurred, preformed antibody reduces the effectiveness of the initial treatment. However, the situation is typically far more problematic for second and subsequent doses, even for low seroprevalence viruses, because the first dose induces a powerful primary antiviral antibody response or boosts the pre-existing response [79, 80] . These troublesome responses can be constrained by coadministering immunosuppressive drugs such as cyclophosphamide. Cyclophosphamide administered concurrently with an OV has been shown to suppress or delay the development of humoral and cytotoxic antiviral T-cell responses [81] .
Another strategy for avoiding neutralization is to block the reticuloendothelial system with polyinosinic acid or with clodronate-loaded liposomes which poison or deplete splenic macrophages and Kupffer cells. This approach has been shown to slow the clearance of circulating virus particles that have been coated with antibodies or complement [86] [87] [88] .

• • Prodrug convertases

CD converts 5-flurocytosine (5-FC), an inert small molecule that is administered intravenously, to 5-flurouracil (5-FU), an antimetabolite that irreversibly inhibits thymidylate synthase and is an approved chemotherapeutic agent for a variety of cancers (anal, breast, colorectal, esophageal, stomach, pancreatic and skin) [29] . The argument supporting the CD/5-FC system is that local production of 5-FU in the OV-infected tumor will create a 5-FU concentration gradient that will expose tumor cells to a higher concentration of the drug compared with distant tissues, thereby ameliorating toxicity and enhancing the therapeutic index of the drug. However, 5-FU is freely diffusible so local production of the drug in a CD-positive tumor exposed to high concentrations of 5-FC can lead to systemic toxicity [140] . The dose of 5-FC must therefore be adjusted accordingly. CD has been incorporated into several OVs including adenoviruses, paramyxoviruses and poxviruses, but the one that has advanced ahead of all others in clinical testing is a C-type retrovirus, Toca-511, which is currently being evaluated in a Phase III clinical trial for the treatment of patients with malignant glioma [141] [142] [143] . Besides the TK and CD prodrug convertase systems, there are reports of OVs engineered to express the cyclophosphamide-activating protein CYP2B1, future science group www.futuremedicine.com the CPT11-activating secreted human intestinal carboxylesterase (shiCE) and the fludarabine phosphate activating purine nucleotide phosphorylase [144, 145] .

Safety

So far OVs have an exceptional safety record in the clinic with few serious adverse events and minimal mortality reported from human trials [166, 167] . However, since efficacy has also been limited, it remains possible that toxicity profiles may appear less favorable as doses are increased and as newer OVs or virus-drug combinations are advanced to clinical testing. Currently, there are over 40 active clinical trials ongoing using OVs alone or in combination with other therapies [168] . As the number of patients participating in trials grows so will the insight into rare adverse effects. The most common adverse effects reported in recent OV trials are fever and flu-like symptoms [169] . However, the potential for severe adverse reactions was much more clearly demonstrated in the earliest virotherapy trials undertaken in the 1950s and 1960s when deaths and severe adverse reactions were not infrequently recorded due to presumed OV replication in normal tissues, notably the brain, especially in immunocompromised cancer patients [4, 170, 171] .

• • Limiting pathogenicity

Two so-called suicide genes, TK and inducible caspase 9, are of interest in this regard. Both of these genes have been used as a safety switches to eliminate genetically modified T cells that were causing graft versus host disease in human clinical trials [184] [185] [186] . Viruses engineered to encode TK have also been controlled with ganciclovir therapy [187] , but this cannot be considered a reliable safety switch because of the ever-present risk of the emergence of viral quasispecies with TK inactivating mutations. The development of a universal and highly reliable safety switch remains one of the significant research challenges facing the field of oncolytic virotherapy.

• • Prodrug convertases

Unlike secreted toxins, prodrug convertases do not pose a risk of increasing OV virulence because their toxic potential is manifest only in the presence of an exogenously added prodrug. For this reason, the approach has been extensively studied. Perhaps the most well-known convertase-prodrug combination is HSV TK, used with ganciclovir. TK converts ganciclovir to ganciclovir monophosphate which is further processed intracellularly to ganciclovir triphosphate, a DNA synthesis chain terminator that kills dividing cells as they enter S phase [138] . A major weakness of the TK-ganciclovir system, aside from its inability to kill nondividing tumor cells, is that it has very limited bystander killing potential. This is because ganciclovir monophosphate is not released from the cell in which it is generated, so does not impact uninfected tumor cells unless they are connected to the infected cell via gap junctions through which it can pass [139] . Encoding connexin, a gap junction protein, in the OV genome can enhance the bystander killing effect of TK, but attention is shifting to other convertases, most notably CD.

Arming viruses with transgenes

• Secreted toxins, prodrug convertases and immunostimulatory proteins have been incorporated into OVs to increase treatment efficacy.
26 section matches

INTRODUCTION

From the 1940s to 2007, 73% of the 155 small molecules approved as anticancer drugs were of natural origin, either directly or derived (Newman and Cragg, 2007) . As opposed to this, few natural products (NPs) had been approved for use in antiviral therapy, although they were the inspiration for antiviral nucleoside analogs (Chung and Cutler, 1992) .

NVP018 Is an Inhibitor of HCV Replication In Vitro

Until recently the standard-of-care treatment for chronic HCV infection has been a combination of interferon and ribavirin for 6-12 months. Use of recently approved drugs with high barrier to resistance, such as the nucleotide polymerase inhibitor (NPI) sofosbuvir, leads to high levels of viral clearance (AASLD 2011, abstract; Sulkowski et al., 2012) . Cyclophilin inhibition is one of the few mechanisms of action of HCV inhibition that consistently shows a high barrier to resistance, and in phase 2 trials some patients were clear of the virus after being treated for 24 weeks with 1000 mg/day ALV monotherapy (Pawlotsky et al., 2012) . Alisporivir, a nonimmunosuppressive semisynthetic derivative of CsA, is a potent cyclophilin inhibitor which was in phase 3 trials for the treatment of HCV infection. NVP018 could provide a suitable alternative backbone for use in an interferon-free combination. Comparison of the inhibitory potential of NVP018 with the semisynthetic sangamide, BC544, and ALV against HCV replicons revealed that NVP018 was much more potent in all assays, and was similarly active against all genotypes tested (see Table 3 ). Analysis of the effect of increasing levels of either human serum or fetal calf serum (FCS) also confirmed that there was no effect on antiviral potency with addition of up to 40% serum (see Table S4 ).
To compare the barrier to resistance of NVP018 in HCV, Huh7-Con1 cells were serially passaged for 7 weeks in the presence of 300 mg/ml G418 and increasing concentrations of NVP018 or ALV. Colonies were amplified, and cellular RNA extracted and analyzed by quantitative RT-PCR. For all ALV-resistant replicons, only the previously described and clinically relevant D320E mutation was consistently found in all clones analyzed. In the case of NVP018-resistant replicons, all clones analyzed contained the same five mutations: L27I, R318W, D320E, S370P, and C446S. When NVP018 was tested against separate replicons containing individual mutations from this list, only the D320E mutation was shown to confer more than 2-fold resistance alone. The combination of all five mutations only led to $4-fold resistance compared with the wild-type.

NVP018 Is an Inhibitor of HBV Replication In Vitro

There have been previous reports that cyclophilin inhibitors such as CsA have an inhibitory effect on HBV replication (Xia et al., 2005) . We therefore tested NVP018 in two in vitro models to look for inhibitory activity. The first involved transfecting Huh7 cells with HBV DNA (8 mg) in the presence of 2 mM NVP018, ALV, or DMSO. After days 0, 1, 2, and 3, amounts of de novo HBV proteins HBsAg and HBeAg present in cell extracts or supernatants were quantified by ELISA, in triplicate. Data shown in Table 3 revealed that NVP018 and ALV potently inhibited HBV replication at this concentration. An additional assay was run using HepG2 cells stably expressing replicating HBV. These were treated with DMSO, NVP018, or ALV at three concentrations (0.1, 1, and 10 mM). The effect on HBV replication was monitored by treating HepG2 cells with test article at the relevant

NVP018 Is a Potent Inhibitor of HIV-1 Replication In Vivo

To enable comparison with data from previous studies with approved agents in the model, NVP018 or ALV was dosed orally bidaily for 14 days, starting 1 day before inoculation with the NL4.3 virus, at 50 mg/kg/day or 100 mg/kg/day. Administration of NVP018 gave >6 log 10 reductions in HIV-1 RNA, and was more potent than ALV at protecting thymocytes and in reducing HIV-1 RNA and p24 levels at all concentrations tested (see Figure 3 ). No overt signs of toxicity were seen in any of the mice at any dose level. The potency of NVP018 compares well with data previously published by Stoddart et al. (2007) , as the >6 log 10 HIV-1 RNA log drop seen following NVP018 dosing was more pronounced than for approved therapies such as efavirenz, atazanavir, emtricitabine, and nevirapine, which showed 1-4 log 10 viral RNA reductions after 21 days' bidaily dosing at 100 mg/kg.

DISCUSSION

Second-generation cyclophilin inhibitors, such as NVP018, have the potential for therapeutic application in a number of areas, in particular chronic viral infection. This is due to their potency, high barrier to resistance, activity on multiple steps of the viral life cycle, and oral bioavailability. It is also worth noting the recent publications suggesting that these compounds might also have additional impact on interferon regulatory factors, which have long been implicated in the escape of certain viruses, such as HCV and HIV, from the immune system (Obata et al., 2005; Bobardt et al., 2013) . While cyclophilin inhibitors do not exhibit inhibition of all viruses, they have been shown to have activity against a wide variety of both RNA viruses (e.g. coronaviruses, HIV, and HCV) and DNA viruses (human CMV, HBV, varicella zoster virus). It is therefore tempting to speculate that cyclophilin inhibitors may be a step in the direction of agents with broad antiviral activity (Katze et al., 2002) .

HCV Replicon Antiviral Assays

The replicon cells (subgenomic replicons of genotype 1a [H77], 1b [Huh7], and 2a [JFH-1]) were grown in DMEM, 10% fetal bovine serum (FBS), 1% penicillinstreptomycin (pen-strep), 1% glutamine, 1% nonessential amino acids, and 250 mg/ml G418 in a 5% CO 2 incubator at 37 C. All cell culture reagents were purchased from Mediatech (Herndon, VA). The replicon cells were trypsinized and seeded at 5 3 10 3 cells per well in 96-well plates with the above media without G418. On the following day, the culture medium was replaced with DMEM containing compounds serially diluted in the presence of 5% FBS. The cells containing the HCV replicon were seeded into 96-well plates and test articles were serially diluted with DMEM plus 5% FBS. The diluted compound was then applied to appropriate wells in the plate. After 72 hr of incubation at 37 C, the cells were processed. The intracellular RNA from each well was extracted with an RNeasy 96 kit (Qiagen). The level of HCV RNA was determined by a real-time RT-PCR assay using TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, CA) and an ABI Prism 7900 sequence detection system (Applied Biosystems). The cytotoxic effects were measured with TaqMan Ribosomal RNA Control Reagents (Applied Biosystems) as an indication of cell numbers. The amount of the HCV RNA and rRNA were then used to derive applicable EC 50 values (concentration that would inhibit the replicon replication by 50%).
In Vitro Assays for Assessment of HBV Antiviral Activity HepG2 cells stably replicating robustly HBV were treated with DMSO, tenofovir, BC544, NVP018, and ALV at three concentrations (0.1, 1, and 10 mM). Effect on HBV replication was monitored by treating HepG2 cells with test article at the relevant concentration for 3 days, removing the supernatant, retreating, and analyzing after an additional 72 hr by HBsAg ELISA, HBeAg ELISA, and HBV DNA in triplicate in cell extracts. These data were used to generate test/control values, which were plotted against the test article concentration and used to calculate IC 50 values. Huh7 cells were transfected with HBV DNA (8 mg) in the presence of DMSO, tenofovir, BC544, NVP018, and ALV at 2 mM. After day 3, amounts of de novo HBsAg protein present in cell extracts or supernatants were quantified by ELISA in triplicate. This was used to calculate a test/control value.

SCID-hu Thy/Liv Mouse Model

Stocks of NL4-3 (X4 virus) were prepared by transfection of 293T cells and collection of supernatants on day 2. Supernatants were filtered, aliquoted, and frozen at À80 C until use. Amounts of virus were quantified by p24 ELISA and infectivity of viral stocks verified using CD4+ HeLa-b-galactosidase reporter cells.
Implants were dispersed through nylon mesh into single-cell suspensions and assessed for p24 by ELISA (p24 [ELISA]: intracellular Gag pg/10 6 cells), for viral RNA (bDNA assay: copies/10 6 cells), and for depletion of thymocyte subsets by flow cytometry (% of CD3, CD4, CD8) as described previously (Stoddart et al., 2007; Rabin et al., 1996; Stoddart et al., 2000) . Specifically, implant cells were stained with phycoerythrin cyanine dye CY7-conjugated anti-CD4 (BD Biosciences), phycoerythrin cyanine CY5.5-conjugated anti-CD8 (Caltag), allophycocyanin cyanine CY7-conjugated anti-CD3 (eBiosciences), and phycoerythrin-conjugated anti-W6/32 (DakoCytomation). Cells were fixed and permeabilized with 1.2% paraformaldehyde and 0.5% Tween 20, stained with fluorescein isothiocyanate-conjugated anti-p24 (Beckman Coulter), and analyzed on an LSR II (BD Biosciences). After collecting 100,000 total cell events, percentages of marker-positive (CD4+, CD8+, and CD4+CD8+) thymocytes in the implant samples were determined by first gating on a live lymphoid cell population identified by forward-and sidescatter characteristics and then by CD3 expression. Total RNA was extracted from frozen thymocyte pellets using Trizol LS (Invitrogen) and resuspended in nuclease-free water. The capsid region of the HIV-1 gag was amplified by RT-PCR using 10 ml of purified RNA and AmpliTaq Gold (Applied Biosystems) according to the manufacturer's instructions.

Solubility analysis

Solubility was measured by diluting test compounds in DMSO (10 mM) into PBS at pH 7.4 to a target concentration of 100 µM with a final DMSO concentration of 1%. Sample tubes were gently shaken for 4 h at room temperature, centrifuged and supernatants diluted into PBS. Diluted samples were mixed with the same volume (1:1) of methanol, then the same volume (1:1) of acetonitrile containing internal standard for LC-MS/MS analysis (see general methods).

INTRODUCTION

Treatment of chronic viral diseases by direct inhibition of viral targets frequently leads to rapid development of virally encoded resistance. Therapies targeted to host proteins involved in the viral life cycle offer an opportunity to both raise the barrier to generation of resistance and generate antivirals able to treat a broad range of viral diseases. However, this has been hindered in the past by the relative rarity of targets essential to the virus but nonessential to the host, and the inherent complexity of discovering and developing drugs that effectively target these proteins.
Cyclophilins are a class of peptidyl-prolyl isomerases, proteins that catalyze the cis-trans isomerization of the peptide bond preceding prolyl residues. Knockout studies in several species, including mice and human cells, confirm that they have limited or no effect on cellular growth and survival (Luvisetto et al., 2008; Elrod et al., 2010; Dolinski et al., 1997; Colgan et al., 2000) . However, cyclophilins recruited from host cells have been shown to have essential roles in many viral life cycles. Initially, cyclophilin A was shown to be incorporated into HIV-1 virions (Thali et al., 1994; Franke et al., 1994) involved in viral replication, its expression level in patients being related to the speed of progression to AIDS . Isomerase activity of cyclophilin A, and potentially others such as cyclophilin B and cyclophilin 40, have been shown to be required for hepatitis C virus (HCV) replication (Chatterji et al., 2009; Yang et al., 2008; Kaul et al., 2009) . Other viruses in which cyclophilin involvement has been implicated in their life cycle or cyclophilin inhibitors have shown inhibitory activity include vaccinia virus (Damaso and Moussatché , 1998) , West Nile virus, Dengue virus, yellow fever virus (Qing et al., 2009 ), hepatitis B virus (HBV) (Ptak et al., 2008) , human papilloma virus (Bienkowska-Haba et al., 2009) , cytomegalovirus (CMV) (Kawasaki et al., 2007) , SARS coronavirus (Pfefferle et al., 2011) , Japanese encephalitis virus (Kambara et al., 2011) , and influenza A (Liu et al., 2012) .

HCV Replicon Antiviral Assays

In Vitro Assay for Assessment of HIV Antiviral Activity Antiviral efficacy against HIV may be tested as follows. Blood-derived CD4+ T lymphocytes and macrophages were isolated as described previously (Bobardt et al., 2008) . In brief, human peripheral blood mononuclear cells (PBMCs) were purified from fresh blood by banding on Ficoll-Hypaque (30 min, 800 g, 25 C). Primary human CD4+ T cells were purified from PBMCs by positive selection with anti-CD4 Dynabeads and subsequent release using Detachabead. Cells were cultured in RPMI medium 1640 (Invitrogen) supplemented with 10% FCS, MEM amino acids, L-glutamine, MEM vitamins, sodium pyruvate, and penicillin plus streptomycin, and were subsequently activated with bacterial superantigen staphylococcal enterotoxin B (SEB; 100 ng/ml) and mitomycin C-killed PBMCs from another donor (10:1 PBMC:CD4 cell ratio). Three days after stimulation, cells were split 1:2 in medium containing interleukin-2 (IL-2) (200 units/ml final concentration). Cultures were then split 1:2 every 2 days in IL-2 medium and infected with HIV at 7 days after stimulation. For generating primary human macrophages, monocytes were purified from human PBMCs by negative selection and activated and cultured at a cell concentration of 10 6 /ml in DMEM, supplemented with 10% FCS, MEM amino acids, L-glutamine, MEM vitamins, sodium pyruvate, and penicillin (100 units/ml), streptomycin (100 mg/ml), and 50 ng/ml recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF), and maintained at 37 C in a humidified atmosphere supplemented with 5% CO 2 . To obtain monocytederived macrophages, cells were allowed to adhere to plastic and cultured for 6 days to allow differentiation. CD4+ HeLa cells, Jurkat cells, activated CD4+ peripheral blood T lymphocytes, and macrophages (500,000 cells/100 ml) were incubated with pNL4.3-GFP (X4 virus) or pNL4.3-BaL-GFP (R5 virus) (100 ng of p24) in the presence of increasing concentrations of test article; 48 hr later, infection was scored by analyzing the percentage of GFP-positive cells by FACS and the EC 50 calculated.

SCID-hu Thy/Liv Mouse Model

Human fetal thymus and liver were obtained from Advanced Bioscience Resources in accordance with federal, state, and local regulations. Coimplantation of thymus and liver fragments under the kidney capsule to create SCIDhu Thy/Liv mice and inoculation of the Thy/Liv implants with HIV-1 (1000 TCID50 HIV-1 per Thy/Liv implant by direct injection) were carried out as described (Rabin et al., 1996; Namikawa et al., 1990 ). Male C.B-17 SCID (model #CB17SC-M, homozygous, C.B-Igh-1b/IcrTac-Prkdcscid) mice were obtained at 6-8 weeks of age from Taconic, and cohorts of 50-60 SCID-hu Thy/Liv mice were implanted with tissues from a single donor. Implants were inoculated 18 weeks after implantation with 50 ml of stock virus or complete DMEM (control infection) by direct injection. Animal protocols were approved by the TSRI Institutional Animal Care and Use Committee. Groups of six mice each were treated with NVP018 or ALV at 50 or 100 mg/kg/day by twice-daily oral gavage until implant collection 2 weeks after inoculation.

In Brief

Hansson et al. describe the generation and preclinical analysis of a bacterial natural product with activity as a hosttargeted antiviral drug. This was generated by a combination of biosynthetic engineering and semisynthetic chemistry.

INTRODUCTION

The tools available for optimizing NPs are ever increasing. Specifically, bioengineering has been proposed as a way to reinvigorate NP drug discovery (Hutchinson, 1994; Wilkinson and Micklefield, 2007) . In the past, semisynthetic approaches have been the most frequently used route to improve the drug-like properties of an NP hit. However, the available semisynthetic options are predetermined by the array of functional groups on the NP. In contrast, bioengineering options are predetermined by the biosynthetic pathway. The potential changes are thus orthogonal to those available to semisynthesis.
We now describe the use of combined bioengineering and semisynthetic approaches to optimize the drug-like properties of sanglifehrin A, an NP cyclophilin inhibitor, to generate NVP018 (formerly BC556). Preclinical analysis reveals NVP018 to be a molecule displaying in vitro inhibition of HBV and HCV and potent in vitro and oral in vivo inhibition of HIV-1.

DISCUSSION

We also anticipate that in the era of rapid genome sequencing and gene synthesis, bioengineering applications will continue to become more amenable and offer a more flexible route to improve NPs with potent inhibition of cellular targets, but with less than optimum drug-like properties. We hope that in the future, bioengineering and semisynthesis will be used more frequently in combination to optimize NPs with an aim to select candidates for clinical development. antiviral drug. This was achieved by a combination of biosynthetic engineering and semisynthetic chemistrybringing together the advantages of each method to more efficiently optimize a bacterially produced, genetically encoded natural product. The preclinical analysis reveals potent activity against HCV, HIV, and HBV and a significantly improved profile compared with both the parent natural product and the cyclosporin A (CsA) class of cyclophilin inhibitors, of which alisporivir was in phase 3 for treatment of chronic HCV infection. Semisynthesis of NVP018 from BC457 NVP018 was synthesized in three steps from BC457 using the same protocol as for compound BC544. In brief, BC457 was treated via modified Sharpless dihydroxylation conditions to form a diol, which was subsequently cleaved to generate an allylic aldehyde by the action of sodium periodate. Finally, the resultant allylic aldehyde was coupled with a suitable amide under Horner-Wadsworth-Emmons coupling conditions.

INTRODUCTION

Whole-genome sequencing is now straightforward and easily affordable, and for NP classes such as the modular polyketide synthases (PKSs), knowledge of the DNA sequence encoding the biosynthetic gene cluster enables rapid understanding of gene product function. When combined with improved techniques for DNA transfer and the rapid targeted alteration of biosynthetic genes, this provides a powerful platform for focused drug discovery efforts with the aim of improving drug-like properties and pharmacokinetics and reducing offtarget effects. These bioengineering techniques are readily combined with semisynthesis to identify molecules with further improved properties. In particular, inactivation of precursor pathways can allow mutasynthesis, the process of feeding a synthetic analog of the precursor which is then incorporated, biosynthetically, into the final molecule (Gregory et al., 2005; Kennedy, 2008) . This enables a combinatorial element in bioengineering.

RESULTS

Bioengineering and Mutasynthesis of Streptomyces sp. A92-308110 Previous semisynthetic derivatization to replace the spirolactam fragment of sanglifehrin A led to generation of the sangamides, molecules with improved solubility, potency, and selectivity (Moss et al., 2012) . This approach moved the immunosuppressive NPs from tool compounds appropriate for chemical genetics to being compounds that could reasonably be pursued as therapeutic agents. We then used bioengineering to make further alterations to the macrocyclic portion of the molecule, changes that could not have otherwise been done without complex total synthesis. The major aim of this work was to further improve the drug-like properties of the final molecule by specific manipulation of the parts of the molecule involved in cyclophilin binding.

Synthesis and Selection of NVP018

A screen was then carried out by feeding a selection of appropriate meta-L-tyrosine analogs to BIOT-4585 to determine its ability to generate sanglifehrin analogs (see Figure 1 ). Fermentations with those analogs successfully accepted as substrates were scaled up to allow isolation of new sanglifehrins. The antiviral activity and ADME (absorption, distribution, metabolism, excretion) properties of each analog were analyzed, and the best of the analogs were subjected to semisynthesis to yield the corresponding sangamides as described previously (Moss et al., 2012) . The resulting sangamides were again analyzed for antiviral activity and ADME properties, and from this NVP018 was chosen as a potential drug candidate (data not shown). NVP018 is derived from a sanglifehrin resulting from incorporation of 5-fluoro-meta-L-tyrosine. Semisynthesis replaced the spirolactam-containing portion of the sanglifehrin with a tertiary hydroxamic amide moiety (Table S1 ). Of the sangamides tested, NVP018 had the best combination of properties (Table 1) , including more potent inhibition of CypA PPIase activity and inhibition of HCV replicons.

NVP018 Has Reduced Off-Target Inhibition of Drug Transporters

One of the issues with cyclosporin-based cyclophilin inhibitors is off-target inhibition of transporter proteins. Alisporivir, for example, inhibits multiple transporter proteins, including OATP1B1, OATP1B3, and MRP2, which are involved in bilirubin transport. It is this inhibition which is now thought to be the mechanism for the dose-limiting hyperbilirubinemia (Avila et al., 2012) . These compounds also have drug-drug interaction concerns through inhibition of xenobiotic transporters such as Pgp and BSEP (Kapturczak and Kaplan, 2004; Wring et al., 2010) . Comparison of the inhibition of these transporters using vesicular transport or uptake transporter inhibition assays, and in vitro inhibition of the xenobiotic transporters Pgp and BSEP revealed that while alisporivir (ALV) and cyclosporin A (CsA) potently inhibited many of them, BC544 (Figure 1 , previously stated as compound 3o in Moss et al., 2012) and NVP018 showed minimal or no inhibition (see Table 2 ). To test the effect of NVP018 on bilirubin levels, we then dosed three mice with up to 250 mg/kg/day NVP018 for 7 days. In all cases, almost no increase was seen in either bilirubin or liver enzymes when measured at the end of the study (see Table S2 ).

DISCUSSION

The cyclophilin inhibitor NVP018 was generated using a combination of semisynthetic and bioengineering methods. These are frequently used independently, but in the past have been less commonly used together (Kennedy, 2008; Eichner et al., 2012) . This enabled a combinatorial approach to NP drug discovery and led to the selection of NVP018, a compound with more potent cyclophilin inhibition, antiviral potency versus HBV, HCV, and HIV-1, and reduced off-target transporter inhibition compared with the parent compound, sanglifehrin A. A fluorine was introduced into the compound by mutasynthesis, a method that has been successful in introducing this substitution pattern in a less invasive way than semisynthetic chemistry (Weist et al., 2002; Hojati et al., 2002; Goss et al., 2010; Knobloch et al., 2012; Almabruk et al., 2013) .

PPIase Inhibition Analysis

The inhibition of the PPIase activity of CypA was used to compare the inhibitory potential of sanglifehrin derivatives as an indication of their binding affinity to CypA. The PPIase activity of recombinant CypA, produced by thrombin cleavage of GST-CypA, is determined by following the rate of hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by chymotrypsin. Chymotrypsin only hydrolyzes the trans form of the peptide, and hydrolysis of the cis form, the concentration of which is maximized by using a stock dissolved in trifluoroethanol containing 470 mM LiCl, is limited by the rate of cis-trans isomerization. CypA was equilibrated for 1 hr at 5 C with selected sanglifehrin derivatives using a drug concentration range from 0.1 to 20 nM. The reaction was started by addition of the peptide, and the change in absorbance was monitored spectrophotometrically at 10 data points per second. The blank rates of hydrolysis (in the absence of CypA) were subtracted from the rates in the presence of CypA. The initial rates of the enzymatic reaction were analyzed by first-order regression analysis of the time course of the change in absorbance. All sanglifehrin derivatives exhibited anti-PPIase activity that HCV Antiviral Assay Huh 5-2 Anti-HCV assay in Huh 5-2 cells was performed by seeding 6.5 3 10 3 cells per well in a tissue-culture-treated white 96-well view plate (Packard, Canberra, Canada) in Dulbecco's modified essential medium (DMEM) supplemented with 250 mg/ml G418. Following incubation for 24 hr at 37 C (5% CO 2 ), medium was removed and 3-fold serial dilutions in complete DMEM (without G418) of the test compounds were added in a total volume of 100 ml. After 4 days of incubation at 37 C, cell culture medium was removed and luciferase activity was determined using the luciferase assay system (Promega, Leiden, the Netherlands); the luciferase signal was measured using a Safire2 (Tecan, Switzerland). Relative luminescence units were con-verted to percentage of untreated controls. The 50% effective concentration (EC 50 ) was defined as the concentration of compound that reduced the luciferase signal by 50%.

In vitro assessment of inhibition of Pgp transporters using MDCK cells

To assess the inhibition of the P-glycoprotein (Pgp/MDR1) transporter, an in vitro ATPase assay from Cyprotex was used. MDR1-MDCK cells obtained from the NIH (Rockville, MD, USA) were used. Following culture, the monolayers were prepared by rinsing both basolateral and apical surfaces twice with buffer at pH 7.4 and 37 °C. Cells were then incubated with pH 7.4 buffer in both apical and basolateral compartments for 40 min at 37 °C and 5% CO2 with a relative humidity of 95 % to stabilize physiological parameters. For the apical to basolateral study (A-B), buffer at pH 7.4 was removed from the apical compartment and replaced with loperamide dosing solutions before being placed in the 'companion' plates. The solutions were prepared by diluting loperamide in DMSO with buffer to give a final loperamide concentration of 5 μM (final DMSO concentration adjusted to 1 %). The fluorescent integrity marker Lucifer yellow was also included in the dosing solution. The experiment was performed in the presence and absence of the test compound (applied to both the apical and basolateral compartments). For basolateral to apical (B-A) study, the P-glycoprotein substrate, loperamide (final concentration = 5 μM) was placed in the basolateral compartment. The experiment was performed in the presence and absence of the test compound (applied to the apical and basolateral compartments). Incubations were carried out in an atmosphere of 5% CO2 with a relative humidity of 95% at 37 °C for 60 min. After the incubation period, the companion plate was removed and apical and basolateral samples diluted for analysis by LC-MS/MS. A single determination of each test compound concentration was performed. On each plate, a positive control inhibitor was also screened. The test compound was assessed at 0.1, 0.3, 1, 3, 10, 30 and 50 μM. The integrity of the monolayers throughout the experiment was checked by monitoring Lucifer yellow permeation using fluorimetric analysis. After analysis, an IC50 was calculated (i.e., inhibitor concentration (test drug) achieving half maximal inhibition effect).
25 section matches

Abstract

Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent, high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed non-ribosomal peptide/polyketide origin in order to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of HBV, HCV and HIV-1 replication, shows minimal inhibition of major drug transporters and has a high barrier to generation of both HCV and HIV-1 resistance.

Introduction

From the 1940s to 2007, 73% of the 155 small molecules approved as anticancer drugs were of natural origin, either directly or derived 1 . As opposed to this, few natural products had been approved for use in antiviral therapy, although they were the inspiration for antiviral nucleoside analogues 2 .

NVP018 is an inhibitor of HCV replication in vitro

Until recently the standard of care treatment for chronic HCV infection has been a combination of interferon and ribavirin for 6-12 months. Use of recently approved high barrier to resistance drugs, such as the nucleotide polymerase inhibitor (NPI) sofosbuvir leads to high levels of viral clearance 37, 38 . Cyclophilin inhibition is one of the few mechanisms of action of HCV inhibition which consistently shows a high barrier to resistance, and in Phase 2 trials, some patients were clear of the virus after being treated for 24 weeks with 1000 mg/day alisporivir monotherapy 39 . Alisporivir, a nonimmunosuppressive semisynthetic derivative of cyclosporin A, is a potent cyclophilin inhibitor in Phase III trials for the treatment of HCV infection. NVP018 could provide a suitable alternative backbone for use in an interferon-free combination. Comparison of the inhibitory potential of NVP018 with the semisynthetic sangamide, BC544 and alisporivir against HCV replicons revealed that NVP018 was much more potent in all assays, and was similarly active against all genotypes tested (see table 3 ). Analysis of the effect of increasing levels of either human serum or FCS also confirmed that there was no effect on antiviral potency with addition of up to 40% serum (see supplementary information table S4).
To compare the barrier to resistance of NVP018 in HCV, Huh7-Con1 cells were serially passaged for 7 weeks in the presence of 300 µg/mL G418 and increasing concentrations of NVP018 or alisporivir. Colonies were amplified, cellular RNA extracted and analyzed by RT-qPCR. For all alisporivir-resistant replicons, only the previously described and clinically relevant D320E mutation was consistently found in all clones analyzed. In the case of NVP018 resistant replicons, all clones analyzed contained the same five mutations: L27I, R318W, D320E, S370P and C446S. When NVP018 was tested against separate replicons containing individual mutations from this list, only the D320E mutation was shown to confer more than 2-fold resistance alone. The combination of all five mutations only led to ~4-fold resistance compared to the wild-type.

NVP018 is an inhibitor of HBV replication in vitro

There have been previous reports that cyclophilin inhibitors such as cyclosporin A have an inhibitory effect on HBV replication 40 . We therefore tested NVP018 in two in vitro models to look for inhibitory activity. The first involved transfecting Huh7 cells with HBV DNA (8 µg) in the presence of 2 µM NVP018, alisporivir or DMSO. After days 0, 1, 2 and 3, amounts of de novo HBV proteins HBsAg and HBeAg present in cell extracts or supernatants were quantified by ELISA, in triplicate. Data is shown in Table 3 and revealed that NVP018 and alisporivir potently inhibited HBV replication at this concentration. An additional assay was run using HepG2 cells stably expressing replicating HBV. These were treated with DMSO, NVP018 or alisporivir at three concentrations (0.1, 1 and 10 µM). The effect on HBV replication was monitored by treating HepG2 cells with test article at the relevant concentration for 3 days, removing the supernatant, retreating and analyzing after an additional 48 and 72 h by HBsAg ELISA, HBeAg ELISA and for HBV DNA, in triplicate, in both cell extracts and supernatants. This assay confirmed a dose dependent effect of NVP018 against HBV replication at the final time point.

NVP018 is a potent inhibitor of HIV-1 replication in vivo

To enable comparison with data from previous studies with approved agents in the model, NVP018 or alisporivir were dosed orally bi-daily for 14 days, starting one day before inoculation with the NL4.3 virus, at 50 mg/kg/day or 100 mg/kg/day. Administration of NVP018 gave >6 log 10 reductions in HIV-1 RNA, and was more potent at all concentrations tested than alisporivir at protecting thymocytes, and in reducing HIV-1 RNA and p24 levels (see figure 3) . No overt signs of toxicity were seen in any of the mice at any dose level. The potency of NVP018 compares well with data previously published by Stoddart et al. as the >6 log 10 HIV-1 RNA log drop seen following NVP018 dosing was more pronounced than for approved therapies such as efavirenz, atazanavir, emtricitabine and nevirapine, which showed 1-4 log 10 viral RNA reductions after 21 days bi-daily dosing at 100 mg/kg.

Discussion

Second generation cyclophilin inhibitors, such as NVP018, have the potential for therapeutic application in a number of areas, in particular chronic viral infection. This is due to their potency, high barrier to resistance, activity on multiple steps of the viral life-cycle and oral bioavailability. It is also worth noting the recent publications which suggest that these compounds might also have additional impact on Interferon Regulatory Factors, which have long been implicated in the escape of certain viruses, such as HCV and HIV, from the immune system 49, 50 . Whilst cyclophilin inhibitors do not exhibit inhibition of all viruses, they have been shown to have activity against a wide variety of both RNA viruses (eg Coronaviruses, HIV and HCV) and DNA viruses (HCMV, HBV, VZV). It is therefore tempting to speculate that cyclophilin inhibitors may be a step in the direction of agents with broad antiviral activity 51 .

HCV Replicon antiviral assays

The replicon cells (subgenomic replicons of genotype 1a (H77), 1b (Huh7) and 2a (JFH-1)) were grown in DMEM, 10 % fetal bovine serum (FBS), 1 % penicillin-streptomycin (penstrep), 1 % glutamine, 1 % non-essential amino acids, 250 µg/ml G418 in a 5% CO 2 incubator at 37 °C. All cell culture reagents were purchased from Mediatech (Herndon, VA). The replicon cells were trypsinized and seeded at 5 × 10 3 cells per well in 96-well plates with the above media without G418. On the following day, the culture medium was replaced with DMEM containing compounds serially diluted in the presence of 5 % FBS. The cells containing the HCV replicon were seeded into 96-well plates and test articles were serially diluted with DMEM plus 5 % FBS. The diluted compound was then applied to appropriate wells in the plate. After 72 hours incubation at 37 °C, the cells were processed. The intracellular RNA from each well was extracted with an RNeasy 96 kit (Qiagen). The level of HCV RNA was determined by a reverse transcriptase-real time PCR assay using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, CA) and an ABI Prism 7900 sequence detection system (Applied Biosystems). The cytotoxic effects were measured with TaqMan® Ribosomal RNA Control Reagents (Applied Biosystems) as an indication of cell numbers. The amount of the HCV RNA and ribosomal RNA were then used to derive applicable EC 50 values (concentration that would inhibit the replicon replication by 50%).

In vitro assays for assessment of HBV antiviral activity

HepG2 cells stably replicating robustly HBV were treated with DMSO, tenofovir, BC544, NVP018 and ALV at 3 concentrations (0.1, 1 and 10 µM). Effect on HBV replication was monitored by treating HepG2 cells with test article at the relevant concentration for 3 days, removing the supernatant, retreating and analyzing after an additional 72 hours by HBsAg ELISA, HBeAg ELISA and HBV DNA in triplicates in cell extracts. These data were used to generate test/control values which were plotted against the test article concentration and used to calculate IC 50 values.
Huh7 cells were transfected with HBV DNA (8 µg) in the presence of DMSO, tenofovir, BC544, NVP018 and ALV (alisporivir) at 2 µM. After day 3, amounts of de novo HBsAg protein present in cell extracts or supernatants was quantified by ELISA in triplicate. This was used to calculate a test/control value.

SCID-hu Thy/Liv mouse model

Stocks of NL4-3 (X4 virus) were prepared by transfection of 293T cells and collection of supernatants on day 2. Supernatants were filtered, aliquoted and frozen at −80 °C until use. Amounts of virus were quantified by p24 ELISA and infectivity of viral stocks verified using CD4+ HeLa-betagalactosidase reporter cells.
Implants were dispersed through nylon mesh into single-cell suspensions and assessed for p24 by ELISA (p24 (ELISA): intracellular Gag pg/10 6 cells), for viral RNA (bDNA assay: copies/10 6 cells), and for depletion of thymocyte subsets by flow cytometry (% of CD3, CD4, CD8) as described previously 29, 53, 55 . Specifically, implant cells were stained with phycoerythrin cyanine dye CY7-conjugated anti-CD4 (BD Biosciences), phycoerythrin cyanine CY5.5-conjugated anti-CD8 (Caltag), allophycocyanin cyanine CY7-conjugated anti-CD3 (eBiosciences), and phycoerythrinconjugated anti-W6/32 (DakoCytomation). Cells were fixed and permeabilized with 1.2 % paraformaldehyde and 0.5 % Tween 20, stained with fluorescein isothiocyanate-conjugated anti-p24 (Beckman Coulter), and analyzed on an LSR II (BD Biosciences). After collecting 100,000 total cell events, percentages of marker-positive (CD4+, CD8+, and CD4+CD8+) thymocytes in the implant samples were determined by first gating on a live lymphoid cell population identified by forward-and side-scatter characteristics and then by CD3 expression. Total RNA was extracted from frozen thymocyte pellets using Trizol LS (Invitrogen) and resuspended in nuclease-free water. The capsid region of the HIV-1 gag was amplified by RT-PCR using 10 µL of purified RNA and AmpliTaq Gold (Applied Biosystems) according to the manufacturer's instructions.

Highlights

• Preclinical analysis revealing potent antiviral activity. Overview of (bio)synthetic medicinal chemistry route to generate BC544 and NVP018 Mean blood concentration-time profiles of NVP018 following a single po dose of 5 mg.kg −1 to CD-1 mice, SD rats or beagle dogs Comparison of implant P24 levels, T-lymphocytes and HIV-1 RNA in the SCID-hu Thy/Liv mouse model, following bi-daily doses of either 50 or 100 mg.kg −1 alisporivir (ALV) or NVP018 for two weeks. *=p≤0.05, compared with untreated mice

Introduction

Treatment of chronic viral diseases by direct inhibition of viral targets frequently leads to rapid development of virally-encoded resistance. Therapies targeted to host proteins involved in the viral life-cycle offer an opportunity to both raise the barrier to generation of resistance and generate antivirals able to treat a broad range of viral diseases. However, this has been hindered in the past by the relative rarity of targets essential to the virus, but nonessential to the host, and the inherent complexity of discovering and developing drugs that effectively target these proteins.

SCID-hu Thy/Liv mouse model

Human fetal thymus and liver were obtained from Advanced Bioscience Resources in accordance with federal, state, and local regulations. Co-implantation of thymus and liver fragments under the kidney capsule to create SCID-hu Thy/Liv mice and inoculation of the Thy/Liv implants with HIV-1 (1000 TCID50 HIV-1 per Thy/Liv implant by direct injection) were carried out as described 53, 54 . Male C.B-17 SCID (model #CB17SC-M, homozygous, C.B-Igh-1b/IcrTac-Prkdcscid) mice were obtained at 6-8 weeks of age from Taconic, and cohorts of 50-60 SCID-hu Thy/Liv mice were implanted with tissues from a single donor. Implants were inoculated 18 weeks after implantation with 50 µL of stock virus or complete DMEM (control infection) by direct injection. Animal protocols were approved by the TSRI Institutional Animal Care and Use Committee. Groups of 6 mice each were treated with NVP018 or alisporivir (ALV) at 50 or 100 mg/kg/day by twice-daily oral gavage until implant collection 2 weeks after inoculation.

Introduction

The tools available for optimizing NPs are ever increasing. Specifically, bioengineering has been proposed as a way to reinvigorate natural products drug discovery 6, 7 . In the past, semisynthetic approaches have been the most frequently used route to improve the drug-like properties of a natural product hit. However, the available semi synthetic options are predetermined by the array of functional groups on the NP. In contrast, bioengineering options are pre-determined by the biosynthetic pathway. The potential changes are thus orthogonal to those available to semisynthesis.

Significance

Moss et al., describes the generation and preclinical analysis of a bacterial natural product with activity as a host targeted antiviral drug. This was generated by a combination of biosynthetic engineering and semisynthetic chemistry -bringing together the advantages of each method to more efficiently optimise a bacterially produced, genetically encoded natural product. The preclinical analysis reveals potent activity against HCV, HIV and HBV and a significantly improved profile as compared to both the parent natural product and the cyclosporine A (CsA) class of cyclophilin inhibitors, of which alisporivir is in phase III for treatment of chronic HCV infection.

Introduction

Whole genome sequencing is now straightforward and easily affordable, and for NP classes such as the modular polyketide synthases (PKSs), knowledge of the DNA sequence encoding the biosynthetic gene cluster enables rapid understanding of gene product function. When combined with improved techniques for DNA transfer and the rapid targeted alteration of biosynthetic genes this provide a powerful platform for focused drug discovery efforts with the aim of improving drug-like properties, pharmacokinetics and reducing offtarget effects. These bioengineering techniques are readily combined with semisynthesis to identify molecules with further improved properties. In particular, inactivation of precursor pathways can allow mutasynthesis, the process of feeding a synthetic analogue of the precursor which is then incorporated, biosynthetically, into the final molecule 8, 9 . This enables a combinatorial element to bioengineering.
Cyclophilins are a class of peptidyl-prolyl isomerases, proteins which catalyse the cistrans isomerization of the peptide bond preceding prolyl residues. Knockout studies in several species, including mice and human cells, confirm that they have limited or no effect on cellular growth and survival [10] [11] [12] [13] . However, cyclophilins recruited from host cells have been shown to have essential roles in many viral life-cycles. Initially, cyclophilin A was shown to be incorporated into HIV-1 virions 14, 15 , involved in viral replication, and its expression level in patients related to the speed of progression to AIDS 16 . Cyclophilin A isomerase activity, and potentially other cyclophilins such as B and cyclophilin 40, have been shown to be required for HCV replication [17] [18] [19] We now describe the use of combined bioengineering and semisynthetic approaches to optimize the drug-like properties of sanglifehrin A, a NP cyclophilin inhibitor, to generate NVP018 (formerly BC556). Preclinical analysis reveals NVP018 to be a molecule displaying in vitro inhibition of HBV and HCV and potent in vitro and oral in vivo inhibition of HIV-1.

Bioengineering and mutasynthesis of Streptomyces sp. A92-308110

Previous semisynthetic derivatization to replace the spirolactam fragment of sanglifehrin A led to generation of the sangamides, molecules with improved solubility, potency and selectivity 28 . This approach moved the immunosuppressive NPs from tool compounds appropriate for chemical genetics to being compounds that could reasonably be pursued as therapeutic agents. We then used bioengineering to make further alterations to the macrocyclic portion of the molecule, changes which could not have otherwise been done without complex total synthesis. The major aim of this work was to further improve the drug-like properties of the final molecule by specific manipulation of the parts of the molecule involved in cyclophilin binding.

Synthesis and selection of NVP018

A screen was then carried out by feeding a selection of appropriate meta-L-tyrosine analogues to BIOT-4585 in order to determine its ability to generate sanglifehrin analogues (see figure 1 ). Fermentations with those analogues successfully accepted as substrates were scaled up to allow isolation of new sanglifehrins. The antiviral activity and ADME properties of each analogue were analysed, and the best of the analogues were subjected to semisynthesis to yield the corresponding sangamides as described previously 28 . The resulting sangamides were again analysed for antiviral activity and ADME properties, and from this NVP018 was chosen as a potential drug candidate (data not shown). NVP018 is derived from a sanglifehrin resulting from incorporation of 5-fluoro-meta-L-tyrosine. Semisynthesis replaced the spirolactam containing portion of the sanglifehrin with a tertiary hydroxamic amide moiety (table S1). Of the sangamides tested, NVP018 had the best combination of properties (table 1), including more potent inhibition of CypA PPIase activity and inhibition of HCV replicons.

NVP018 has reduced off target inhibition of drug transporters

One of the issues with cyclosporin-based cyclophilin inhibitors is off-target inhibition of transporter proteins. Alisporivir, for example, inhibits multiple transporter proteins, including OATP1B1, OATP1B3 and MRP2, which are involved in bilirubin transport. It is this inhibition which is now thought to be the mechanism for the dose limiting hyperbilirubinaemia 34 . These compounds also have drug-drug interaction concerns through inhibition of xenobiotic transporters such as Pgp and BSEP 35, 36 . Comparison of the inhibition of these transporters using vesicular transport or uptake transporter inhibition assays, and in vitro inhibition of the xenobiotic transporters, Pgp and BSEP revealed that whilst alisporivir and CsA potently inhibited many of them, BC544 (figure 1, previously stated as compound 3o in Moss et al., 2011) and NVP018 showed minimal or no inhibition (see table 2 ). To test the effect of NVP018 on bilirubin levels, we then dosed three mice with up to 250 mg/kg/day NVP018 for 7 days. In all cases, almost no increase was seen in either bilirubin or liver enzymes when measured at the end of the study (see supplementary information, table S2).

Discussion

The cyclophilin inhibitor NVP018 was generated using a combination of semisynthetic and bioengineering methods. These are frequently used independently, but in the past have been less commonly used together 42, 43 . This enabled a combinatorial approach to natural product drug discovery and led to the selection of NVP018, a compound with more potent cyclophilin inhibition, antiviral potency versus HBV, HCV and HIV-1, and reduced offtarget transporter inhibition as compared to the parent compound, sanglifehrin A. A fluorine was introduced into the compound by mutasynthesis, a method which has been successful in introducing this substitution pattern in a less invasive way than semisynthetic chemistry 44, 45, 46, 47, 48 .
We also anticipate that in the era of rapid genome sequencing and gene synthesis, bioengineering applications will continue to become more amenable and offer a more flexible route to improve natural products with potent inhibition of cellular targets, but with less than optimum drug-like properties. We hope that in the future, bioengineering and semisynthesis will be used more frequently in combination to optimize natural products with an aim to select candidates for clinical development.

PPIase Inhibition Analysis

The inhibition of the PPIase activity of CypA was used to compare the inhibitory potential of sanglifehrin derivatives as an indication of their binding affinity to CypA. The PPIase activity of recombinant CypA, produced by thrombin cleavage of GST-CypA, is determined by following the rate of hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by chymotrypsin. Chymotrypsin only hydrolyzes the trans form of the peptide, and hydrolysis of the cis form, the concentration of which is maximized by using a stock dissolved in trifluoroethanol containing 470 mM LiCl, is limited by the rate of cis-trans isomerization. CypA was equilibrated for 1 hour at 5 °C with selected sanglifehrin derivatives using a drug concentration range from 0.1 to 20 nM. The reaction was started by addition of the peptide, and the change in absorbance was monitored spectrophotometrically at 10 data points per sec. The blank rates of hydrolysis (in the absence of CypA) were subtracted from the rates in the presence of CypA. The initial rates of the enzymatic reaction were analyzed by firstorder regression analysis of the time course of the change in absorbance. All sanglifehrin derivatives exhibited anti-PPIase activity that correlated well with their capacities to prevent CypA-NS5A interactions analyzed by ELISA or pulldown.
13 section matches

Abstract

The n-butanol fraction (BF) obtained from the crude extract of the marine sponge Petromica citrina, the halistanol-enriched fraction (TSH fraction), and the isolated compounds halistanol sulfate (1) and halistanol sulfate C (2), were evaluated for their inhibitory effects on the replication of the Herpes Simplex Virus type 1 (HSV-1, KOS
Mar. Drugs 2013, 11 4177 strain) by the viral plaque number reduction assay. The TSH fraction was the most effective against HSV-1 replication (SI = 15.33), whereas compounds 1 (SI = 2.46) and 2 (SI = 1.95) were less active. The most active fraction and these compounds were also assayed to determine the viral multiplication step(s) upon which they act as well as their potential synergistic effects. The anti-HSV-1 activity detected was mediated by the inhibition of virus attachment and by the penetration into Vero cells, the virucidal effect on virus particles, and by the impairment in levels of ICP27 and gD proteins of HSV-1. In summary, these results suggest that the anti-HSV-1 activity of TSH fraction detected is possibly related to the synergic effects of compounds 1 and 2.

Introduction

Pharmaceutical interest in marine organisms has provided thousands of new and novel compounds that have shown important biological properties, such as anticancer, antiviral, antiprotozoal, and antibacterial activities [2, [5] [6] [7] [8] . In this context, marine sponges have been a prolific source of diverse secondary metabolites with complex and unique structures [2, [9] [10] [11] [12] [13] . Some of them were used as lead compounds to obtain new drugs that are currently used in clinics, such as acyclovir, vidarabine, cytarabine, eribulin mesylate, and others, that are now in clinical stages of evaluation such hemiasterlin [14] [15] [16] . In addition, several highly active compounds from marine sponges have been reported as new biologically active structures [17] [18] [19] [20] [21] [22] [23] [24] .

General Experimental Procedures

General 1D and 2D NMR experiments were performed on a Bruker Avance 2 (500 MHz) instrument at 500 MHz for 1 H and 125 MHz for 13 C. All spectra were recorded in CD 3 OD using the signals of residual non-deuterated solvent as internal reference. Mass spectrometric analyses were performed using a Bruker micrOTOF-Q II mass spectrometer (Bruker ® Daltonics, Billerica, MA, USA), equipped with ESI. Multi-point mass calibration was carried out using a mixture of sodium formate from m/z 50 to 900. Data acquisition and processing were carried out using the Bruker Compass Data Analysis version 4.0 software supplied with the instrument. All the analytical solutions (0.5 mg/mL) were prepared using methanol LCMS grade. Compounds were infused into the source using a KDS 100 syringe pump (

Introduction

The drug of choice for the prophylaxis and treatment of Herpex Simplex Virus (HSV) infections is acyclovir (ACV), which selectively inhibits HSV DNA replication with low host-cell toxicity. However, the intensive use of antiviral drugs has led to the emergence of resistant viruses [1] [2] [3] . Recently, De Clercq [4] described the evolution of antiviral agents against some viral infections, including HSV, confirming that the search for new antiviral agents is still relevant.

Antiviral Activity

Pretreatment of Vero cells with TSH fraction and compounds 1 and 2 for three hours before viral infection showed that these samples did not affect viral infectivity suggesting that they did not exert protective effects against the HSV-1 infection process (data not shown). The direct virus inactivating activity of the tested samples, in the absence of cells, was also evaluated. It was also observed that the TSH fraction and compounds 1 and 2 were able to reduce HSV-1 infectivity at concentrations 8×, 12× and 6× lower than their IC values ( Table 2 ). This is in accordance with previous studies that have reported the virucidal activity of halistanol sulfates F and H against HIV replication [34, 38] . In order to determine whether these samples were able to interfere with early events of HSV infection, their effects on HSV-1 attachment and penetration were investigated separately. All the tested samples inhibited virus attachment and penetration, as shown in Table 2 . Therefore, the inactivation of HSV-1 could be related to virions binding to heparan sulfate receptors, inhibiting these two early stages of viral replication. Other natural sulfated molecules, such as sulfated polysaccharides, were also active against HIV, HSV-1, and HSV-2 replication [43] [44] [45] [46] , inhibiting these same early events of viral replication.

Antiviral Activity Assays

Pretreatment: This assay was performed as described by Bettega et al. [57] . Briefly, Vero cell monolayers were pretreated with different concentrations of samples for 3 h at 37 °C prior virus infection. After washing, cells were infected with 100 PFU of HSV-1 for 1 h at 37 °C. The infected cells were washed, overlaid with MEM containing 1.5% CMC, incubated for 72 h, and treated as described earlier for the viral plaque number reduction assay.

Conclusions

In summary, these results suggest that the TSH fraction and compounds 1 and 2 present antiherpes activity through the reduction of viral infectivity, inhibition of virus entry into the cells, and by the impairment of levels of ICP27 and gD proteins of HSV-1.

Bioguided Fractionation of the n-Butanol Fraction of P. citrina

In addition, there are many other reports about different members of the halistanol series that have shown important pharmacological properties. One of the first reported members of this series was ibisterol sulfate, isolated from Topsentia sp., which showed anti-HIV activity [41] . Other examples of halistanol-type compounds with antiviral activity are weinbersterol disulfates A and B isolated from the sponge Petrosia weinbergi which exhibited activity against leukemia virus (FeLV), mouse influenza virus (PR8), and mouse coronavirus (A59) replication [42] . As compounds with sulfated groups are described to have antiviral properties [34, 38, [43] [44] [45] [46] , and due to the anti-herpetic activity shown by the BF fraction, we decided to verify the anti-HSV-1 activity of compounds 1 and 2 and the TSH fraction and to elucidate their mode of action.

Antiviral Activity Assays

Viral plaque number reduction assay: To evaluate the anti-herpes activity, a plaque reduction assay was performed following the general procedures described by Silva et al. [55] . Vero cell monolayers were infected with approximately 100 PFU of the virus for 1 h at 37 °C, then overlaid with MEM containing 1.5% carboxymethylcellulose (CMC; Sigma-Aldrich ® , St. Louis, MO, USA) either in the presence or absence of different concentrations of the samples. After 72 h of incubation at 37 °C, cells were fixed and stained with naphtol blue-black (Sigma-Aldrich ® , St. Louis, MO, USA ), and the plaques were counted. The IC 50 of each sample was calculated as the concentration that reduced the number of viral plaques in 50%, when compared to the untreated controls. ACV was used as a positive control. The selectivity index (SI = CC 50 /IC 50 ) was calculated for each sample tested.
Adsorption and penetration assays: These assays followed the procedures described by Silva et al. [55] , with minor modifications. Briefly, for the adsorption assay, confluent Vero cells, pre-chilled at 4 °C for 1 h, were infected with 100 PFU of HSV-1 and treated with different concentrations of samples, then incubated at 4 °C for 2 h. The unabsorbed viruses were removed by washing with cold PBS, the cells were covered with overlay medium, the temperature was raised to 37 °C, and treated as described earlier for viral plaque number reduction assay. Dextran sulfate (Sigma) was used as a positive control. For the penetration assay, 100 PFU of HSV-1 was adsorbed for 2 h at 4 °C on confluent Vero cells, after that incubated at 37 °C for 5 min to maximize virus penetration. The cells were then treated with different concentrations of samples. After 1 h at 37 °C, unpenetrated viruses were inactivated with warm citrate-buffer (pH 3.0) for 1 min. The cells were washed with PBS and treated as described above for the viral plaque number reduction assay.
Western blotting analysis: Procedures were performed as described by Bertol et al. [58] . Briefly, Vero cell monolayers were infected with HSV-1 at MOI 0.2 for 1 h. Next, residual viruses were removed with PBS and the cells were submitted to the different treatments for 18 h. The proteins were then extracted from the cells, separated on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked with 5% non-fat milk in blotting buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween 20] . The membranes were incubated for 90 min with the following primary antibodies: Goat monoclonal antibody against ICP27 protein (1:700 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal antibody against UL42 protein (1:5000 dilution) (Millipore ® , St Charles, MO, USA); mouse monoclonal antibody against gD (1:5000 dilution) (Santa Cruz ® Biotechnology, Santa Cruz, CA, USA); mouse monoclonal antibody against gB (1:5000 dilution) (Millipore ® , St Charles, MO, USA); and rabbit monoclonal antibody against beta-actin (1:5000 dilution) (Millipore ® , St Charles, MO, USA). After washing, the membranes were incubated with the respective secondary antibodies for 1 h. The immunoblots were developed and detected using the Pierce ECL Western Blotting Substrate (Thermo ® Scientific, Rockford, IL, USA), according to the manufacturer's instructions.

Antiviral Activity

Given the fact that TSH fraction, compounds 1 and 2 seemed to act in a different way than ACV, the potential synergistic effects between them were tested at different concentrations ( Table 3 ). The results obtained suggest a strong synergism between TSH fraction, compound 2 and compounds 1 + 2 and ACV, and a moderate synergism when compound 1 was tested with this drug, at the higher concentration (2 × IC 50 ). When compounds 1 and 2 were tested in association, a strong synergism was also detected, at the three tested concentrations. In relation to the other combinations, a slight or a moderate antagonism was detected, exception to the association of ACV and TSH fraction, at the intermediate concentration (1 × IC 50 ), when an additive effect was detected. The observed synergism between these samples and ACV could be explained by the fact that the samples act in different steps of HSV-1 replication than those affected by this anti-herpes drug, which could be considered an interesting result. Therefore, the most important result obtained was when compounds 1 and 2 were tested in association showing that the detected anti-HSV activity could be explained by the strong synergic effects of these major compounds present in the TSH fraction. Other natural compounds with anti-herpes activity, such as sulfated polysaccharides [44, 45] , docosanol [51] , and oxiresveratrol [52] have already been reported to present synergistic effects with ACV, which corroborate our results.
6 section matches

Abstract

To study the influence of a linker rigidity and donor-acceptor properties, the P-CH 2 -O-CHR-fragment in acyclic nucleoside phosphonates (e.g., acyclovir, tenofovir) was replaced by the P-CH 2 -HN-C(O)-residue. The respective phosphonates were synthesized in good yields by coupling the straight chain of x-aminophosphonates and nucleobase-derived acetic acids with EDC. Based on the 1 H and 13 C NMR data, the unrestricted rotation within the methylene and 1,2-ethylidene linkers in phosphonates from series a and b was confirmed. For phosphonates containing 1,3-propylidene (series c) fragments, antiperiplanar disposition of the bulky O,O-diethylphosphonate and substituted amidomethyl groups was established. The synthesized ANPs P-X-HNC(O)-CH 2 B (X = CH 2 , CH 2 CH 2 , CH 2-CH 2 CH 2 , CH 2 OCH 2 CH 2 ) appeared inactive in antiviral assays against a wide variety of DNA and RNA viruses at concentrations up to 100 lM while marginal antiproliferative activity (L1210 cells, IC 50 = 89 ± 16 lM and HeLa cells, IC 50 = 194 ± 19 lM) was noticed for the analog derived from (5-fluorouracyl-1-yl)acetic acid and O,O-diethyl (2-aminoethoxy)methylphosphonate.

Introduction

The search for new compounds endowed with antiviral activity has been underway for decades. Several research groups have been active in this field, both in academia and pharmaceutical industry. Despite these efforts, for many viruses efficient drugs have not been yet discovered. In addition, anticancer drugs available so far exhibit limited applicability. The high mutation rate observed for some viruses makes the issue highly complex. Within medications applied to treat viral infections, acyclic nucleoside phosphonates (ANPs) play an important role [1] [2] [3] . The prototype of the acyclic nucleoside phosphonates (ANPs), (S)-HPMPA (3), was never commercialized but it gave rise to three marketed products [cidofovir (4) , adefovir (1) , and tenofovir (2) ] that are often prescribed by physicians. So far known structural modifications of compounds 1-4 accomplished within a chain connecting nucleobase and phosphonic acid moieties did not lead to discovery of analogs having higher antiviral activity (Fig. 1) .

Antiviral activity

All phosphonates 17-25 were evaluated for inhibitory activity against a wide variety of DNA and RNA viruses, using the following cell-based assays: (e) MDCK cell cultures: influenza A virus (H1N1 and H3N2 subtypes) and influenza B virus. Ganciclovir, cidofovir, acyclovir, brivudine, zalcitabine, zanamivir, alovudine, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight 10,000, DS-10000), mycophenolic acid, Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA) were used as the reference compounds. The antiviral activity was expressed as the EC 50 : the compound concentration required to reduce virus plaque formation (VZV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses). None of the tested compounds showed appreciable antiviral activity toward any of the tested DNA and RNA viruses at concentrations up to 100 lM, nor affected cell morphology of HEL, HeLa, Vero, MDCL, and CrFK cells.

Conclusion

Replacement of the P-CH 2 -O-CHR-fragment in ANPs (e.g., acyclovir, tenofovir) by the P-CH 2 -HN-C(O)residue was introduced to study the influence of a linker rigidity and changes in donor-acceptor properties. To elaborate an appropriate synthetic methodology, a series of the 36 respective phosphonates as O,O-diethyl esters was prepared in good yields by the EDC-induced coupling of the straight chain x-aminophosphonates and nucleobasederived acetic acids. Besides the rigidity of the amide bond based on the 1 H and 13 C NMR data, it was concluded that the unrestricted rotation within the methylene (series a) and 1,2-ethylidene (series b) linkers takes place while for phosphonates containing 1,3-propylidene (series c) fragments antiperiplanar disposition of the bulky O,Odiethylphosphonate and substituted amidomethyl groups was observed. The phosphonates 17a-25d appeared inactive in antiviral assays against a wide variety of DNA and RNA viruses at concentrations up to 100 lM. Marginal antiproliferative activity (L1210, IC 50 = 89 ± 16 lM and HeLa, IC 50 = 194 ± 19 lM) was noticed for the phosphonate 18d derived from (5-fluorouracyl-1-yl)acetic acid and O,O-diethyl (2-aminoethoxy)methylphosphonate. Studies on the analogous phosphonates containing functionalized linkages are currently ongoing in our laboratory and the most active diethyl esters will be transformed into the free ANP and further derivatized to selected prodrug phosphonates [37] .
Experimental 1 H NMR spectra were recorded in CD 3 OD, CDCl 3 , or DMSO-d 6 on the following spectrometers: Varian Gemini 2000BB (200 MHz) and Bruker Avance III (600 MHz) with TMS as internal standard. 13 C NMR spectra were recorder for CD 3 OD, CDCl 3 , or DMSO-d 6 solution on the Bruker Avance III at 151.0 MHz. 31 P NMR spectra were performed on the Varian Gemini 2000BB at 81.0 MHz or on Bruker Avance III at 243.0 MHz. IR spectral data were measured on a Bruker Alpha-T FT-IR spectrometer. Melting points were determined on a Boetius apparatus. Elemental analyses were performed by Microanalytical Laboratory of this Faculty on Perkin Elmer PE 2400 CHNS analyzer and their results were found to be in good agreement (±0.3%) with the calculated values.

Results and discussion

To synthesize the first series of the amides 5, xaminophosphonates 7a-7d containing straight chain linkers were selected (Scheme 2). The x-aminophosphonates 7a-7d were prepared according to the described procedures [12] [13] [14] . Among them, aminomethylphosphonate 7a was considered as the most important since it was later transformed into the analogs 17a-24a in which nitrogen atoms (N1 or N9) in nucleobases and the phosphorus atom are separated by four bonds, thus providing compounds structurally closest to the drugs 1-4.
2 section matches

Background

Cyrtomium fortumei (J.) Smith belongs to the Dryopteridaceae family which comprises approximately 14 genera and 1700 species throughout the world, and is widely spread in tropical and subtropical regions. The Chinese Pharmacopoeia (2005 edition) listed Cyrtomium fortumei (J.) Smith as an official drug, which showed that the plant could be used as anticancer herbs [1] . In the past, people used the rhizomes and extracts from the plant as vermifuges [2] . The plant can be used to cure many diseases, such as influenza, acute and chronic pharyngitis, cancer, and migraine [3] . In addition, the herb was used as antiviral agents to cure severe acute respiratory syndrome, a life-threatening viral respiratory illness believed to be caused by a coronavirus [4] . Furthermore, phoroglucinols and flavonoids are known to display a wide array of pharmacological and biochemical actions, and have been isolated from many species of the Dryopteridaceae family [5] . However, so far the constituents with anticancer activity of the plant remain unclear. Thus it is necessary to identify the potent antitumor compounds from this plant.

General procedures and reagents

The melting points of the products were determined using an XT-4 binocular microscope (Beijing Tech Instrument Co. Ltd., Beijing, China). Infrared spectra were recorded on a Bruker VECTOR22 spectrometer in KBr disks. 1 H-NMR and 13 C-NMR were recorded using a JEOL-ECX500 spectrometer at 22°C, with tetramethylsilane as the internal standard and CDCl 3 , DMSO-d 6 , CD 3 COCD 3 , or CD 3 OD as the solvent. Column chromatography was performed using silica gel (200-300 meshes) (Qingdao Marine Chemistry Co., Qingdao, China) and silica gel H (Qingdao Marine Chemistry Co., Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), HP-20 (Mitsubishi Chemical Corp., Toukyu Met, Japan), YMC RP-18 (YMC Corp., Kyoto, Japan) and MCI-gel CHP 20P (Mitsubishi Chemical Corp., Toukyu Met, Japan). All other chemicals were of analytical reagent grade and used without further purification.