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Abstract

Abstract: Chloroquine diphosphate (CQ) is a hydrophilic drug with low entrapment efficiency in hydrophobic nanoparticles (NP). Herpes simplex virus type 1 (HSV-1) is an enveloped double-stranded DNA virus worldwide known as a common human pathogen. This study aims to develop chloroquine-loaded poly(lactic acid) (PLA) nanoparticles (CQ-NP) to improve the chloroquine anti-HSV-1 efficacy. CQ-NP were successfully prepared using a modified emulsification-solvent evaporation method. Physicochemical properties of the NP were monitored using dynamic light scattering, atomic force microscopy, drug loading efficiency, and drug release studies. Spherical nanoparticles were produced with modal diameter of <300 nm, zeta potential of −20 mv and encapsulation efficiency of 64.1%. In vitro assays of CQ-NP performed in Vero E6 cells, using the MTT-assay, revealed different cytotoxicity levels. Blank nanoparticles (B-NP) were biocompatible. Finally, the antiviral activity tested by the plaque reduction assay revealed greater efficacy for CQ-NP compared to CQ at concentrations equal to or lower than 20 µg mL −1 (p < 0.001). On the other hand, the B-NP had no antiviral activity. The CQ-NP has shown feasible properties and great potential to improve the antiviral activity of drugs.

Introduction

Chloroquine diphosphate (CQ) is an antimalarial drug that has been used as the first-line treatment against Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum infections. In addition to the antimalarial activity, CQ has been used to treat chronic diseases such as rheumatoid arthritis and systemic lupus erythematosus [10] [11] [12] . Recently, this drug has also attracted attention due to its anticancer [13] [14] [15] [16] [17] and antiviral activity [18] [19] [20] [21] [22] [23] [24] . In this context, CQ is a hydrophilic drug with low toxicity that has shown some limitations to reach an effective intracellular concentration. One of the main disadvantages of the conventional malaria treatment is the non-specific targeting to intracellular parasites, which leads to high dose administration, resulting in toxicity [25] .

Preparation of Drug-Loaded Nanoparticles by the Emulsification-Solvent Evaporation Method

In this approach, an adjusted emulsification-solvent evaporation method was used, applying a small-scale liquid-liquid partition to provide the maximum concentration of chloroquine free base (CQ-fb) in the organic phase. All experiments have the CQ-fb amount in organic phase analytically controlled using UV spectrophotometry. An average level of 97.51% of CQ-fb dissolved in the DCM was achieved. NP < 300 nm with negative zeta potential and PdI of about 0.3 were produced using the selected parameters ( Table 2 ). The strategy to induce CQ-fb in an immiscible organic phase structured as droplets dispersed in an alkaline 0.5M NaHCO 3 aqueous solution (pH = 8.4) provided an excellent EE% of 64.1%. This formulation (formulation 6) was then selected for further studies. Note: Data are expressed as mean ± standard deviation (n = 3). 1 PdI, polydispersity index; 2 ZP, zeta potential; 3 EE, encapsulation efficiency; values of drug release rate constant (k); correlation coefficient (R).

Discussion

In this study, the feasibility of biodegradable PLA nanoparticles as a promising approach to improve antiviral activity of Chloroquine diphosphate (CQ) has been investigated. Small biodegradable nanoparticles have the ability to overcome biological barriers, such as the efflux cotransport proteins at cell membrane and until brain-blood barrier [26, 29] . In this context, Vero E6 cells were infected with a common human pathogen, the herpes simplex virus type 1 (HSV-1). The antiviral activity of CQ has been reported in the literature [18] [19] [20] [21] [22] [23] [24] . However, its anti-HSV-1 activity was not yet been well described. Indeed, due to the similar characteristic with previous described virus infections studies using CQ, a promising effect against HSV-1 infection could be hypothesized using this drug. In addition, its encapsulation in the biodegradable and biocompatible NP could solve its non-specific targeting against intracellular pathogens [25] .
The biocompatible and biodegradable poly(lactic acid) (PLA) was chosen for this purpose. However, this polymer is a hydrophobic polyester [29] and produce NP for encapsulating hydrophilic drugs, such as the CQ, consists of an interesting challenge [30, 31] . Thus, two different methods of preparation of nanoparticles well applied for this class of polymer were carefully tested. The small size of the system and the drug encapsulation efficiency (EE) were selected as the main performance parameters. Physicochemical properties of the formulations produced by the nanoprecipitation method revealed that the drug/polymer ratio directly affected both particle size and EE% (Table 1) . As expected, the particle size increased according to the polymer concentration in the organic phase. This effect generally improves the EE% of hydrophobic drugs [32, 33, 47, 48] . In contrast to this fact, Formulations 1, 2, and 3 showed low EE%, which decrease with the polymer increment. Chloroquine Diphosphate (CQ) is a low-weight drug with multiple pKas, and solubility behavior dependent on pH. At this pH = 6.4, this hydrophilic compound is unable to favorable interaction with hydrophobic polyesters such as PLA. The increment of PLA leads to drug displacement from organic phase to aqueous phase, resulting in low entrapment efficiency [30] [31] [32] [33] 49] .
In this study, the feasibility of biodegradable PLA nanoparticles as a promising approach to improve antiviral activity of Chloroquine diphosphate (CQ) has been investigated. Small biodegradable nanoparticles have the ability to overcome biological barriers, such as the efflux cotransport proteins at cell membrane and until brain-blood barrier [26, 29] . In this context, Vero E6 cells were infected with a common human pathogen, the herpes simplex virus type 1 (HSV-1). The antiviral activity of CQ has been reported in the literature [18] [19] [20] [21] [22] [23] [24] . However, its anti-HSV-1 activity was not yet been well described. Indeed, due to the similar characteristic with previous described virus infections studies using CQ, a promising effect against HSV-1 infection could be hypothesized using this drug. In addition, its encapsulation in the biodegradable and biocompatible NP could solve its non-specific targeting against intracellular pathogens [25] .
The biocompatible and biodegradable poly(lactic acid) (PLA) was chosen for this purpose. However, this polymer is a hydrophobic polyester [29] and produce NP for encapsulating hydrophilic drugs, such as the CQ, consists of an interesting challenge [30, 31] . Thus, two different methods of preparation of nanoparticles well applied for this class of polymer were carefully tested. The small size of the system and the drug encapsulation efficiency (EE) were selected as the main performance parameters. Physicochemical properties of the formulations produced by the nanoprecipitation method revealed that the drug/polymer ratio directly affected both particle size and EE% ( Table 1) . As expected, the particle size increased according to the polymer concentration in the organic phase. This effect generally improves the EE% of hydrophobic drugs [32, 33, 47, 48] . In contrast to this fact, Formulations 1, 2, and 3 showed low EE%, which decrease with the polymer increment. Chloroquine Diphosphate (CQ) is a low-weight drug with multiple pKas, and solubility behavior dependent on pH. At this pH = 6.4, this hydrophilic compound is unable to favorable interaction with hydrophobic polyesters such as PLA. The increment of PLA leads to drug displacement from organic phase to aqueous phase, resulting in low entrapment efficiency [30] [31] [32] [33] 49] .
The selected parameters for the emulsification-solvent evaporation method induced larger particles compared to that produced by the nanoprecipitation technique (Table 2) . NP have shown negative zeta potential and narrow particle size (PdI = 0.3) with mean diameter <300 nm. The best results using the nanoprecipitation method were reached using the CQ:PLA ratio of 1:5 w/w and 0.5 M NaHCO 3 aqueous solution (pH = 8.4). These work conditions were repeated for the emulsification-solvent evaporation method, with an excellent average EE% level of about 64.1%. This fact corroborated with the successful partition of CQ-fb to the organic phase and the importance of the alkaline aqueous phase to maintain the drug and polymer in the droplets of the emulsion. The pH of the aqueous phase (0.5 M NaHCO 3 , pH = 8.4) assured a maximum of non-ionized of chloroquine's amine groups (Pka 1 = 8.4, Pka 2 = 10.8), compared to the previous tested aqueous phase (pH = 6.4). At contact with the organic immiscible solvent (DCM), the phase equilibrium between the ionized and the non-ionized species of CQ in the aqueous phase changes constantly, with the partition of non-ionized specie of the drug to the interface, and finally to the organic solvent. The displacement of non-ionized molecules of drug occurs until its exhaustion and transport of the total amount to the organic phase, as suggested, for example, in the Figure 5 . The mass ratio maintained between the two phases could be explained by the principle of mass conservation. [50] . The hypothesis of drug precipitation in the aqueous phase, due to the strongest alkaline pH, should also be considered.
The selected parameters for the emulsification-solvent evaporation method induced larger particles compared to that produced by the nanoprecipitation technique (Table 2) . NP have shown negative zeta potential and narrow particle size (PdI = 0.3) with mean diameter < 300 nm. The best results using the nanoprecipitation method were reached using the CQ:PLA ratio of 1:5 w/w and 0.5 M NaHCO3 aqueous solution (pH = 8.4). These work conditions were repeated for the emulsificationsolvent evaporation method, with an excellent average EE% level of about 64.1%. This fact corroborated with the successful partition of CQ-fb to the organic phase and the importance of the alkaline aqueous phase to maintain the drug and polymer in the droplets of the emulsion. The pH of the aqueous phase (0.5 M NaHCO3, pH = 8.4) assured a maximum of non-ionized of chloroquine's amine groups (Pka1 = 8.4, Pka2 = 10.8), compared to the previous tested aqueous phase (pH = 6.4). At contact with the organic immiscible solvent (DCM), the phase equilibrium between the ionized and the non-ionized species of CQ in the aqueous phase changes constantly, with the partition of nonionized specie of the drug to the interface, and finally to the organic solvent. The displacement of non-ionized molecules of drug occurs until its exhaustion and transport of the total amount to the organic phase, as suggested, for example, in the Figure 5 . The mass ratio maintained between the two phases could be explained by the principle of mass conservation. Figure 5 . Suggested chloroquine partitioning scheme. At the first moment, a theoretical maximum ratio of ionized chloroquine molecules occurs in the polar phase following by a gradually partitioning to the organic phase at the second moment. This transport follows until the exhaustion with the total partition of the drug to the organic apolar phase. Figure 5 . Suggested chloroquine partitioning scheme. At the first moment, a theoretical maximum ratio of ionized chloroquine molecules occurs in the polar phase following by a gradually partitioning to the organic phase at the second moment. This transport follows until the exhaustion with the total partition of the drug to the organic apolar phase. The Formulation 6 was selected for further experiments. The AFM images (Figure 1 ) suggested spherical NP with slightly smooth surface. It was also possible to observe NP in the range of 200 nm, corroborating to the DLS experiments. Hoo et al. (2008) reported some differences among AFM and DLS experiments, mainly due to the differences in sample preparing procedures, which can induce some particle agglomeration [53] .
The Formulation 6 was able to generate the desired slow drug release, corroborating with the drug-loading experiments (Figure 2 ). Considering the emulsification-solvent evaporation method, the EE% level superior to 60% is considered excellent, which induced a slow drug release profile adjusted by the parabolic diffusion mathematical model (r ≥ 0.95) ( Table 2) . This fact suggested a diffusion-controlled transport of CQ from the polymeric matrix of NP [43] . In addition, it is important to note that, on the best of our knowledge, no data in the literature describes the slow chloroquine release from PLA nanoparticles. Previous studies have shown a release of 52.40% of hydroxyl chloroquine sulphate from Eudragit ® RL-100 NP (EE% = 63.14%) at 8 h [54] . The encapsulation of chloroquine diphosphate was described in chitosan NP prepared by ionic gelation technique (EE% = 59.5%), which induced the release of 40% of the drug at 24 h [55] . In the present study, the amount of drug released at 10 h was about 40%, which is much slower than the previously cited studies. In this approach, the residual amount of non-ionized specie of CQ loaded into PLA nanoparticles seems to be strongly interacting with PLA matrix. The parabolic model fitted the experimental data much better (r = 0.95) ( Figure 2E ), which suggested a drug release controlled by diffusion at the first moment. However, the erosion of polymeric matrix of particles should also be considered for release of residual entrapped drug, which could occur in a second moment [44, 48, 49, 51] .
In this context, this work focused on the role of CQ, which is a highlighted lysosomotropic drug among the molecules with proved antiviral activity [18] [19] [20] [21] [22] [23] [24] 63] . The CQ has shown pronounced antiviral action against different viruses [64] . The study reported by Koyama and Uchida (1984) demonstrated that the multiplication of the HSV-1 strain HF in Vero cells was inhibited by ammonium chloride and chloroquine. It was also observed that the maturation of the intracellular virus was prevented immediately after the addition of weak bases in the late stage of infection, indicating that the site of inhibition by weak bases is one step in the maturation process of HSV-1 [34] . This present study showed that Vero E6 cells infected with HSV-1 and treated with CQ and CQ-NP at 30 µg mL −1 have 100% of viral replication inhibition after 48 h ( Figure 4A ). At the concentration range from 2.5 to 20 µg mL −1 , the PLA NP significantly improved the anti-HSV-1 of CQ ( Figure 4A ). The CQ-NP induced an IC 50 considerable lesser than that identified for CQ (Table 3 ). In addition, the CQ-NP (at 10 µg mL −1 and 20 µg mL −1 ) induced similar HSV-1 viral inhibition than that observed for the positive control, acyclovir, at 20 µg mL −1 ( Figure 4B ).

Conclusions

In this study, we found that nanoprecipitation was not the best method to encapsulate the hydrophilic CQ drugs when compared to emulsification-solvent evaporation. However, as shown in our experiments, we tried different strategies and considered important parameters to improve drug loading. Biocompatible PLA nanoparticles improved the anti-HSV-1 activity of chloroquine. The data have demonstrated an efficient nanoencapsulation method, using emulsification-solvent evaporation, able to produce sub-300nm NP, with high EE% of the hydrophilic drug, and able to induce slow CQ release. The experimental results also revealed that CQ-NP induced similar HSV-1 inhibition that observed for the first line of the anti-HSV-1 drug acyclovir. In addition, it could be suggested that PLA NP improved CQ uptake by infected Vero E6 Cell, consisting in a promising approach to solve the drug limitations to overcome biological barriers and targeting affected cells. Further studies are required to corroborate with this hypothesis using animal models or more complex tests to assess possible strategies for functionalizing NP.
11 section matches

Abstract

Emerging viruses such as HIV, dengue, influenza A, SARS coronavirus, Ebola, and other viruses pose a significant threat to human health. Majority of these viruses are responsible for the outbreaks of pathogenic lethal infections. To date, there are no effective therapeutic strategies available for the prophylaxis and treatment of these infections. Chloroquine analogs have been used for decades as the primary and most successful drugs against malaria. Concomitant with the emergence of chloroquine-resistant Plasmodium strains and a subsequent decrease in the use as antimalarial drugs, other applications of the analogs have been investigated. Since the analogs have interesting biochemical properties, these drugs are found to be effective against a wide variety of viral infections. As antiviral action, the analogs have been shown to inhibit acidification of endosome during the events of replication and infection. Moreover, immunomodulatory effects of analogs have been beneficial to patients with severe inflammatory complications of several viral diseases. Interestingly, one of the successful targeting strategies is the inhibition of HIV replication by the analogs in vitro which are being tested in several clinical trials. This review focuses on the potentialities of chloroquine analogs for the treatment of endosomal low pH dependent emerging viral diseases.

Introduction

Chloroquine and its structural analogs such as hydroxychloroquine, pamaquine, plasmoquine, primaquine, mefloquine, or ferroquine (ferrocenic analog of chloroquine) have been used for decades as the primary and most successful drugs against malaria. Concomitant with the emergence of chloroquine-resistant Plasmodium strains and a subsequent decrease in the use as antimalarial drugs, new potential uses of the cheap and available analogs have been investigated. Due to their immunomodulatory effects, the analogs have been used as secondary drugs to treat a variety of chronic autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus etc.), tumors, and nonmalarial infections (Al-Bari 2015) . Recently, several efforts have been made to identify effective, inexpensive, and universally available antiviral agents. In these senses, the analogs have been suggested as such antiviral agents by inhibiting the replications and infections (Geisbert et al. 2003; Savarino et al. 2003 ; Barrow et al., 2013) .

HIV

In vitro chloroquine and its analog hydroxychloroquine are endowed with broad-spectrum anti-HIV-1 and HIV-2 activity at clinically achievable concentrations (0-12.5 lmol/L) (Savarino et al. 2001a) . Chloroquine also inhibits HIV-1 in post-integrational event by affecting newly produced viral envelope glycoproteins. In vitro, chloroquine exerts an additive anti-HIV-1 effect in combination with other anti-retroviral agents (e.g. zidovudine, didanosine and hydroxyurea) without cellular toxicity or apoptosis Savarino et al. 2004 ). Since chloroquine and hydroxychloroquine appear to have a similar site of action (i.e. post-transcriptional inhibition of gp120); these drugs can be useful in combination with other anti-retroviral agents for the treatment for HIV-1 infected individuals in the developing world Savarino et al. 2001b; Naarding et al. 2007 ). As a HIV inhibitor, chloroquine alone inhibits HIV replication and viral particle glycosylation and synergizes the inhibitory effects with protease inhibitors such as indinavir, ritonavir, or saquinavir (Savarino et al. 2004 ). Thus, it is suggested the use of chloroquine analogs in the management of routine HIV disease in vivo (Romanelli et al. 2004; Parris 2006; Naarding et al. 2007 ). HIV-1 transmission and replication on CD4 + T-lymphocytes are reduced in presence of chloroquine, suggesting that the analogs exert anti-HIV-1 activity through a number of mechanisms in vivo including modulations of the gp120 structure (Naarding et al. 2007) . As an inhibitor of route of entry, chloroquine vaginal gel formulation also exerts anti-HIV-1 activity in vitro (Brouwers et al. 2008) . pDC cells recognize microbial products and viruses via TLR7 or TLR9, and produce IFNs. The presence of elevated IFN-a level in HIV infected cells leads to contribute the immune activation. Chloroquine blocks TLR-mediated activation of pDC and MyD88 signaling by decrease in the levels of the downstream signaling molecules IRAK-4 and IRF-7 and by inhibition of IFN-a synthesis (Ewald et al. 2008; Martinson et al. 2014) . Chloroquine also decreases CD8 + T-cell activation induced by HIV-1. These results suggest that chloroquine analogs have a preventive role in HIV pathogenesis by blocking TLR stimulation and IFN-a production pathway (Martinson et al. 2014) . Interestingly, recently in order to find out, screen and evaluate other anti-HIV compounds such as cell-penetrating peptides or polyfunctional styryl thiazolopyrimidines, the analogs can be used as standard drugs for comparison purposes (Fatima et al. 2012; Mizuguchi et al. 2015) .

Dengue and hepatitis C viruses

By increasing endosomal pH, chloroquine inhibits dengue virus type 2 replication (DENV-2)in Vero cells (Farias et al. 2013) , and U937 cells (Farias et al. 2014 ) at a nontoxic dose of 50 lg/mL in vitro. Amodiaquine, one of the 4-aminoquinoline drugs inhibits DENV-2 replication and infectivity with EC 90 value 2.69 lmol/L in the replicon expressing cells (Boonyasuppayakorn et al. 2014) . Chloroquine also inhibits DENV-2-induced membrane TNFrelated apoptosis-inducing ligand (mTRAIL) relocalization and IFN-a production by pDCs in vitro and in vivo (Gandini et al. 2013) . Chloroquine interferes in DENV-2 replication in Aotus monkeys. The serum concentrations of TNFa and IFNc are statistically significant reduced in 2017 | Vol. 5 | Iss. 1 | e00293 Page 4 chloroquine treated groups (Farias et al. 2015) . Several analogs of chloroquine have been attempted to use in several randomized, double-blind studies and the outcomes of the therapies are shown in Tables 1, 2. Chloroquine analogs also reduce HCV entry, replication and infection by interfering endosomal acidification. Treatment of different cells such as JFH-1 or Huh-7 with chloroquine suppresses entry and replication of HCV in a dosedependent manner (Blanchard et al. 2006; Mizui et al. 2009 ). Chloroquine shows more than 50% reduction in infectivity of HCV at 50 lmol/L concentrations in liver cells (Ashfaq et al. 2011) . Furthermore, combination treatment of chloroquine to IFNa enhanced the antiviral effect of IFNa and prevents re-propagation of HCV (Mizui et al. 2009 ). Ferroquine, a ferrocenic analog of chloroquine, potently inhibits HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. In addition, the analog also inhibits HCV RNA replication, and impairs the fusion process. The analog also suppresses HCV cell-to-cell spread between neighboring cells. Combinations of the analog with IFN, or an inhibitor of HCV NS3/4A protease, result in additive to synergistic activity (Vausselin et al. 2013 ). By reducing acidification of endocytic system, chloroquine enhances human CD8 + T cell responses against soluble viral antigens (derived from HCV, hepatitis B virus, or HIV) in vivo (Accapezzato et al. 2005) . A case report suggests that a patient with HCV infection is associated with porphyria cutanea tarda and chloroquine (200 mg twice weekly) results in a gradual regression of the skin lesions including porphyria (Pellicelli et al. 2012) .

Influenza A and Newcastle disease viruses

In vitro, chloroquine is able to inhibit IAV replication at lower plasma concentration than that reached during treatment of acute malaria (Ooi et al. 2006) . Chloroquine increases endosomal pH and impairs IAV release into the cytosol (Fedson 2008) . The inhibitory effect of chloroquine is maximal when the drug has been given at the time of infection and is lost after 2 h postinfection (Di Trani et al. 2007 ). These results suggest that the treatment timing approximately corresponds to that of virus/cell fusion. Moreover, there is a clear correlation between the EC 50 of chloroquine in vitro and the electrostatic potential of HA mediating the virus/cell fusion process. Thus, treatment should be started within time of virus/cell fusion process with exact effective concentration.
Although in vitro results are promising, chloroquine is not effective as preventive therapy in vivo in standard mouse and ferret models of human IAV infection (Vigerust and Mccullers 2007) . Recently, chloroquine is highly effective in treating avian IAV infection in an animal model (Yan et al. 2012) . Chloroquine improves CD8 + T cell responses in mice following a single administration of influenza vaccines (Garulli et al. 2013) . Although the analogs have been reported to be effective against IAV in vitro and used in in-vivo experiments and clinical trial for prevention or treatment of influenza (Paton et al. 2011; Borba et al. 2012) , the effectiveness of analogs as anti-influenza drugs is questioned, and cautions in their uses are recommended (Wu et al. 2015) .
Newcastle disease virus (NDV) is the causative agent of veterinary diseases (birds). NDV enters the cell by direct fusion of the viral envelope with the cellular membrane and by low pH-and receptor-dependent endocytosis (Sanchez-Felipe et al. 2013) . In vitro, optimal NDV infection of the host cells is significantly affected by drugs such as chloroquine that inhibit endosomal acidification (Sanchez-Felipe et al. 2013) . NDV infection induces autophagy and inhibition of autophagy by the analog reduces the viral replication and infection (Meng et al. 2012; Sun et al. 2013) . Importantly, as a pharmacological modulator of autophagy, chloroquine potentiates NDV-mediated oncolysis in mice bearing cisplatin-resistant lung cancer cells (Jiang et al. 2014 ).

Ebola and marburg viruses

Filoviruses (Ebola and Marburg) cause severe hemorrhagic fever in humans and nonhuman primates. The peplomers of Ebola viruses are composed of trimerized heterodimers of glycoproteins 1 and 2, which are heavily glycosylated with both N-linked and O-linked glycans (Geisbert et al. 2015) . The glycoproteins of Ebola peplomers have broad tropism for a variety of host cells due to their ability to bind either specifically or non-specifically to various cell surface molecules and facilitate the pHdependent endosomal entry to the host cells (Yang et al. 1998 (Yang et al. , 2000 Bhattacharyya et al. 2010) . Thus, despite this broad tropism, infection by filoviruses greatly depends on acidic pH (Chandran et al. 2005; Marzi et al. 2012) . Using in vitro cell culture assays, a systematic screening of FDA-approved drugs for inhibitors of biological threat agents such as Ebola and Marburg viruses has been performed and found that chloroquine is the most noteworthy antiviral compound among the identified multiple virus-specific inhibitors. In this report, it has been suggested that chloroquine disrupts viral entry and replication in vitro; protects mice against Ebola virus challenge in vivo (Madrid et al. 2013 ). Long 2015 (Long et al. 2015 confirmed that chloroquine is capable to inhibit viral entry in a pH specific manner and considered it as a priority candidate for treatment of Ebola viruses. Later on, reports suggested chloroquine inhibits the virus replication in vitro but is unable to treat in patient (Bishop 2014) ; mouse, hamster (Falzarano et al., 2015) and guinea pigs (Dowall et al. 2015) .

SARS and MERS viruses

Registered effective prophylactics or postexposure therapeutics for the treatment of coronaviral infections are not currently available. It has been reported that chloroquine has strong antiviral effects on SARS-CoV infection and spread in vitro (Keyaerts et al. 2004; Vincent et al. 2005; De Wilde et al. 2014 ). In addition to the wellknown functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, ACE2. This may negatively affect the virus-receptor binding and abrogate the infection. The IC 50 of chloroquine for inhibition of SARS-CoV in vitro (8.8 AE 1.2 lmol/L) is significantly lower than its cytostatic activity which approximates the plasma chloroquine concentrations reached during treatment of acute malaria. More interestingly, the suppressing effect is observed when the cells are treated with chloroquine either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage (Keyaerts et al. 2004; Vincent et al. 2005) . There are screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture and only four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) have been identified to inhibit the viral replication (50% effective concentrations, EC 50 3-8 lmol/L).

Enigma of clandestine association with failure of chloroquine analogs clinically

Chloroquine analog in combination with other antiviral drugs is considered for effective treatment of the viral diseases in order to avoid the interaction of P-glycoprotein and multidrug-resistance associated proteins of 2017 | Vol. 5 | Iss. 1 | e00293 Page 8 these viruses, which extrude drugs from the cells and other anatomic compartments (Savarino 2011) . It is also noted that the combined drugs must not be interacted with the analogs (Zhou et al. 1995) . 3 The maximum inhibitory effect of chloroquine analogs is observed immediately started and treatment is ineffective when the virus successfully passes through the replication events (Stock 2009; Kaur and Chu 2013) . It is also noted that the efficacy of chloroquine analog is markedly dependent on the acute stage and severity of infections (De Lamballerie et al. 2008) . Thus, the starting time of treatment, doses and dose regimens (therapeutic loading dose and subsequent maintenance dose to achieve steady state blood chloroquine concentration) are important factors for efficacy in these viral infections.

Conclusions and Future Perspective

Since inhibition of acidification of endosomes during replication courses of the emerging viruses by chloroquine analogs have been reported to be endowed with a wide range of viral diseases including HIV, the following consequence can be taken into considerations (1) Chloroquine inhibits viral entry, replication and infection. (2) The analogs exert an inhibitory effect on several opportunistic pathogens including viruses (in AIDS). (3) The analogs exert an inhibitory effect on the synthesis of several pro-inflammatory cytokines that may play a pathogenic role in the progression of viral infection. (4) The drugs have the potential to restrict iron accumulation in various tissues that may play a negative role in viral infection. (5) The analogs have practical advantages, as they are widely distributed, inexpensive and not stigmatizing. (6) The analogs may be of potential benefit in decreasing the rate of mother-to-child transmission of viruses like HIV.
6 section matches

Inhibition of Endosomal Fusion

Fusion of the endosome to lysosome is a critical step in releasing virus from endosomes. Obatoclax is a potential anti-neoplastic and pro-apoptotic synthetic small molecule Bcl-2 inhibitor. Its mesylate salt is reported to reduce the acidity of endolysosomal vesicles in in vitro model. Bcl-2 antagonists are effective only against viruses that require a low pH for fusion and entry, such as ZIKV, WNV, YFV, and others. Despite this limitation, Obatoclax works as a broad-spectrum anti-viral agent (Varghese et al., 2016) . However, in clinical phase I and II trials while treating hematological and myeloid malignancies, Obatoclax did not produce satisfactory results, possibly due to inadequate inhibition of Bcl-2 family proteins. Chloroquine, which is an anti-malarial drug, raises endolysosomal pH and inhibits ZIKV infection in human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres (Delvecchio et al., 2016) . Similarly, SaliPhe, a molecule under pre-clinical study and vATPase inhibitor, was tested as an inhibitor of endocytosis to obstruct ZIKV infection (Adcock et al., 2017) . Griffithsin, a lectin isolated from the red alga Griffithsia sp., is a potent flaviviral entry inhibitor. It can cross-link high-mannose oligosaccharides present on the viral E glycoproteins and has shown wide anti-viral activity against HIV (Alexandre et al., 2011) , HPV (Levendosky et al., 2015) , HSV (Nixon et al., 2013) , HCV (Takebe et al., 2013), and SARS (O'Keefe et al., 2010) . Squalamine, a FDA approved cationic chemical, which act by disturbing electrostatic interactions between the virus and host membranes during fusion and budding (Zasloff et al., 2011) , has been found well tolerated as component of eye drop in clinical studies conducted on human participants. Therefore, such potent drugs can be used as an anti-viral agent against ZIKV too.

Inhibition of Virus Replication

Numerous drugs involved in inhibition of virus replicationhave been portrayed in Figure 2 . (6) The positive-sense genomic ssRNA is translated into a polyprotein, which is cleaved into all structural and non-structural proteins. Replication occurs at the surface of endoplasmic reticulum in cytoplasmic viral factories. A dsRNA genome is synthesized from the genomic ssRNA(+) (7) Virus assembly takes place at the endoplasmic reticulum. (8) At the endoplasmic reticulum, virions bud and are transported to the golgi apparatus. (9) In the golgi, prM protein is cleaved and maturation of the virion takes places. (10) Virions are released by exocytosis. (11) Obatoclax and chloroquineinhibit the acidic environment of endolysosomal vesicles. Squalamine, a cationic chemical, disturbs the electrostatic interaction between virus and host membranes during fusion and budding. (12) Cavinafungin, an alaninal-containing lipopeptide of fungal origin, inhibits ZIKV polyprotein processing and also the cleavage of signal peptide of host proteins. (13) Nanchangmycin, a polyether obtained from Streptomyces nanchangensis; small drug-like molecules, ZINC33683341 and ZINC49605556 block the receptor thus inhibiting the ZIKV entry. (14) TIM1 mediated entry is inhibited by Duramycin-biotin.

Inhibition of the RdRp domain

(2) Methyltransferase domain is responsible for transferring mRNA cap. Sinefungin, an adenosine derivative, isolated from Streptomyces griseoleus, inhibit S-adenosyl-1-methionine (SAM), the natural substrate for methyltransferases and inhibit the methyltransferase activity. (3) Helicase crystal structure reveals a conserved triphosphate pocket and a positively charged tunnel for the accommodation of RNA. The helicase-activation is inhibited in the presence of divalent cation, due to extended conformation adopted by GTPγS in such conditions. (4) Tetrapeptide-Boronic acid is a potent inhibitor of NS2B-NS3 protease. Berberine, Myricetin, Epigallocatechingallate binds with affinity to NS3 protease and also inhibit the ZIKV replication. (5) Small-molecule inhibitor ST-148 inhibits capsid. (6) Ribavirin inhibits host inosine monophosphate dehydrogenase and viral polymerase. (7) Repurposed drugs like Chloroquine, azithromycin, niclosomide are used to treat ZIKV infection. efficacy in animal models, the drug remained unsuccessful in the phase I clinical trial conducted. Adenosine analog NITD008 was found to be effective against flaviviruses including ZIKV in both in vitro and in vivo studies and exhibited reduced viremia in mice (Deng Y.Q. et al., 2016) . Unfortunately, in pre-clinical animal testing, it was found to be too toxic to be suitable for human trials.

Drug Repurposing

Drugs take decades to develop and test for efficacy and safety. Since there is presently no approved vaccine or drug available for ZIKV, the major focus of researchers, therefore, is on attempting drug repurposing. Scientists are evaluating repurposing of several FDA approved drugs against ZIKV infections. In this direction, a few promising drug candidates have been shortlisted by adapting various screening methodologies. For example, chloroquine, a 4-aminoquinoline, readily increases the pH of acidic vesicles (Akpovwa, 2016) and inhibits a conformational change essential for fusion between the virus envelope and endosomal membrane (Smit et al., 2011) . In vitro studies revealed that chloroquine decreases the number of ZIKV-infected neural cells in different cell models and protects cellular death (Delvecchio et al., 2016) . Other anti-malarial drugs such as quinacrine, mefloquine, and GSK369796 also demonstrate anti-ZIKV activity by inhibiting autophagy (Balasubramanian et al., 2016) . During the screening of a library of FDA-approved drugs, both established antivirals like bortezomib and mycophenolic acid and compounds with no previously reported anti-viral activity (e.g., daptomycin) were found to inhibit ZIKV replication in human cervical, placental, neural stem, and primary human amniotic cells (Barrows et al., 2016) . screened a panel of compounds containing FDA-approved drugs, drugs in clinical trials, and pharmacologically active compounds to suppress infection-induced caspase activity. Human neural progenitor cells and glial SNB-19 cells infected with ZIKV were used as models to quantify ZIKV-induced caspase-3 activity. Of these 1 http://bioinfo.imtech.res.in/manojk/zikavr/ compounds, a pro-caspase inhibitor, emricasan, successfully protected both neural cell monolayers and three-dimensional organoid cultures of neural cells by decreasing ZIKV-induced caspase-3. Similarly, screening of 725 FDA-approved chemically diverse compounds in ZIKV-infected Huh7 cells at a 20-µM concentration led to the selection of lovastatin, a drug used to reduce cholesterol; 5-fluorouracil used as a cancer treatment; 6-azauridine, a broad-spectrum antimetabolite; palonosetron, which is used to treat chemotherapy-induced nausea and vomiting; and kitasamycin, a macrolide antibiotic. The selection criteria included a selectivity index, maximum activity, and the EC 50 of compounds (Pascoalino et al., 2016) .

Development of Pregnancy-Safe Drugs

The ability of ZIKV to infect fetuses and cause severe disease requires the development of drugs that function during pregnancy and that are safe for both the pregnant mother and fetus. The drugs must be able to cross the placental barrier to reach the fetus and to cross the blood-brain barrier to reach neural cells, the main targets of ZIKV. Khandia et al. (2017) summarized FDA-approved category B drugs (adequate animal study data shows no risk to fetuses, but controlled studies on pregnant women are unavailable) and category C drugs (animal studies revealed few teratogenic effects on fetuses, but control studies on pregnant women are unavailable; however, the potential benefits of using the drug may outweigh the risks). The list contains several drugs including the FDA category B drugs sofosbuvir (Sacramento et al., 2017) , azithromycin (Retallack et al., 2016) , niclosamide , palonosetron (Pascoalino et al., 2016) , mefloquine (Balasubramanian et al., 2016) , and daptomycin B (Barrows et al., 2016) , category C drugs chloroquine (Delvecchio et al., 2016) , amodiaquine, quinacrine hydrochloride (Balasubramanian et al., 2016) , auranofin, clofazimine, deferasirox, methoxsalen, micafungin, sertraline-HCl, fingolimod, ivermectin, digoxin (Barrows et al., 2016) , and seliciclib , which could be repurposed for treating ZIKV infection.

CONCLUSION AND FUTURE PERSPECTIVES

Encouraging results with repurposed drugs, as shown by the use of chloroquine, a malaria drug, has led to the screening of several other FDA-approved drugs, including niclosamide, emricasan, and daptomycin, palonosetron, kitasamycin, and many more, for ZIKV treatment. Another valuable strategy for the discovery of ZIKV preventives and anti-virals is the use of computational analysis.
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Abstract

Chloroquine is a 4-aminoquinoline previously used in malaria therapy and now becoming an emerging investigational antiviral drug due to its broad spectrum of antiviral activities. To explore whether the low pH-dependency of influenza A viruses might affect the antiviral effects of chloroquine at clinically achievable concentrations, we tested the antiviral effects of this drug on selected human and avian viruses belonging to different subtypes and displaying different pH requirements. Results showed a correlation between the responses to chloroquine and NH 4 Cl, a lysosomotropic agent known to increase the pH of intracellular vesicles. Time-of-addition experiments showed that the inhibitory effect of chloroquine was maximal when the drug had been added at the time of infection and was lost after 2 h post-infection. This timing approximately corresponds to that of virus/cell fusion. Moreover, there was a clear correlation between the EC 50 of chloroquine in vitro and the electrostatic potential of the HA subunit (HA2) mediating the virus/ cell fusion process. Overall, the present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents.

Background

A second look at selected compounds is giving new life to several abandoned therapies and new applications for existing drugs [1] [2] [3] . One such example is provided by chloroquine, being dismissed from antimalarial treatment and finding new applications in the clinical management of autoimmune diseases, tumours and nonmalarial infections [4, 5] . The use of chloroquine in the clinical management of a viral infection was first consid-ered in the 1990s, on the basis of its effects on HIV-1 [6, 7] . The drug is now being tested as an investigational antiretroviral [8] .
Some of us previously analysed the reported effects of chloroquine on replication of several viruses and concluded that the drug should be studied as a broad spectrum antiviral agent against emerging viral infections, being relatively well tolerated, cheap, and immediately available worldwide [9] . As a weak base capable of accumulating within cellular organelles, chloroquine appears to be capable of interfering with pH-dependent steps in the replication of several viruses. Other mechanisms of viral inhibition by chloroquine, such as inhibition of polynucleotidyl transferases have, however, been considered [7] . In 2003-2005, chloroquine was studied as a promising in vitro anti-SARS agent [9] [10] [11] and recently entered clinical trials against chikungunya fever [12] .
The broad-spectrum antiviral effects of chloroquine deserve particular attention in a time in which there are several cases of avian influenza A virus transmission to humans from poultry, and the availability of antiviral drugs is fundamental during preparation and evaluation of effective vaccines. Chloroquine inhibition of both type A and B influenza viruses was first described in the 1980s [13, 14] . The concentrations employed in these studies were however too high to allow a theoretical transposition to in-vivo settings. Anecdotal reports of clinical benefits derived from a related compound, i.e. quinine, date back to the Spanish influenza pandemic of 1918/19. However, it was not until last year that the anti-influenza virus effects of chloroquine at clinically achievable concentrations were studied, in view of a possible application of this drug in the clinical management of influenza [4, 15] . Investigations still have to be done on this topic. For example, the mechanisms of orthomyxovirus inhibition by chloroquine have been uncertain at the clinically achievable concentrations adopted in the most recent studies [4, 15] , as well as the effects of chloroquine on field isolates, including avian strains potentially transmittable to humans.

Results

We first tested the effects of chloroquine on low-pathogenic (LP) A/Ck/It/9097/97 (H5N9) virus, isolated from poultry in Italy. We found that chloroquine dose-dependently inhibited the viral cytopathic effect with a 50% effective concentration (EC 50 ) of 14.38 μM, in cells infected with the H5N9 virus at approx. 10 4 50% tissue culture infecting doses (TCID 50 )/ml (Fig. 1a) . Although this value was rather high, some of the inhibitory concentrations matched the blood concentrations reported in individuals under acute antimalarial treatment (1-15 μM) . The inhib-itory effects were confirmed using quantitative reverse transcritptase real-time PCR (qRRT-PCR) (Fig. 1b) . Ooi et al. (2006) [15] recently reported that chloroquine inhibited human H3N2 and H1N1 viruses with EC 50 values in the range of 0.84 -3.60 μM. To investigate whether the discrepancies with the inhibitory values reported above were due to the type of virus or to the different conditions and methods adopted, we tested the effects of chloroquine on replication of recent human H3N2 and H1N1 viral isolates under conditions similar to those adopted for the H5N9 avian influenza virus. Using the test of inhibition of viral cytopathogenicity, we found that chloroquine inhibited the H3N2 virus (10 4 TCID 50 /ml) with an EC 50 of 1.53 μM (Fig.1c) . Inhibition was confirmed using qRRT-PCR, both under similar conditions and at lower MOIs (Fig.1d) , thus confirming the results of Ooi et al. (the assay adopted by these authors employs a lower MOI than routinely used by our group). Results obtained with H1N1 viruses (10 4 TCID 50 /ml) showed a similar drug susceptibility for the human strain (EC 50 = 1.26 μM), in full agreement with Ooi et al. [15] , but no response to clinically achievable drug concentrations in an avian strain (IC 50 > 20 μM; data not shown). These data suggest a more pronounced inhibitory effect of chloroquine on human H3N2 and H1N1 viruses than on avian H5N9 virus replication.
To further explore this possibility, the isoelectric point was calculated for HAs of all viruses used in the present study, and the electrostatic potential was mapped on the protein surfaces of 3D models obtained by homology Figure 1 Inhibition of H5 and H3 influenza A virus replication by CQ in MDCK cells. Cells were incubated with chloroquine (CQ) after virus inoculation or mock-infection and tested for cell viability and viral RNA copies at 24 h post-infection. A) Viability of cells infected with A/Chicken/Italy/9097/97 (H5N9) and treated with increasing concentrations of CQ as detected by colorimetric test. Assays were performed as described in the text. The dotted line indicates inhibition of uninfected cell viability, the solid line indicates inhibition of infected cell viability. Results are presented as the curves that best fit the data points. B) Results of one representative experiment showing inhibition by CQ of A/Chicken/Italy/9097/97 viral RNA production. Virus infected MDCK cells were incubated for one day in the presence of 0, 5, 10, 20 or 25 μM chloroquine. Cell supernatants were used for viral RNA extraction and subjected to a quantitative real-time RT-PCR (qRRT-PCR) assay. Oseltamivir (OS; 20 nM) was used as a positive control. C and D) as in A and B), respectively, using A/Panama/2007/99-like (H3N2) virus. In D) both results obtained with inocula containing 10 4 and 10 3 TCID 50 /ml are reported. Results in B) and D) are displayed for purely representative reasons to show that there is inhibition of virus production, and cannot be compared with each other or with those in A) and C), due to the high intra-and inter-assay variability of the qRRT PCR assay (see Ref. [29] ). Fig. 2 ). Instead, no correlation was found with the isoelectric point of HA1 (P > 0.05; data not shown). Although the viruses studied here belonged to different subtypes, chloroquine-resistant viruses, independently of the subtype, showed a more marked negative surface potential of HA2 than chloroquine-sensitive viruses (Fig. 2) . Viruses with intermediate drug sensitivity showed intermediate characteristics (Fig. 2 ). We conclude that structural determinants in HA2 are associated with response of influenza A viruses to chloroquine.

Inhibition of H5 and H3 influenza A virus replication by CQ in MDCK cells

If the hypothesis of a pH dependent inhibitory action of chloroquine was correct, the timing of drug inhibition should match that of virus/cell fusion, an early step of virus replication occurring in endosomes and requiring a low pH (approx. pH 5-5.5) in several, but not all, influenza A viruses, as shown by previous studies [17] . As the assays for detection of antiviral effects adopted in the first Correlation between electric characteristics of haemagglutinin subunit 2 (HA2) and response to chloroquine of influenza A viruses Figure 2 Correlation between electric characteristics of haemagglutinin subunit 2 (HA2) and response to chloroquine of influenza A viruses. A) Correlation between EC 50 of chloroquine (CQ) on viral cytopathogenicity (presented as Log values, x axis) and isoelectric point of HA2 (pH value at which the protein is neutral; y axis). The line best fitting the data points is shown. Isoelectric points were calculated based on the protein sequence using the web interface in Ref. [35] . B-G) Theoretical three-dimensional models for HA2 subunits of the viruses adopted in the present study, shown in ranked order of sensitivity to chloroquine (from resistant to clinically achievable concentrations to fully sensitive). part of this study were designed to allow multiple cycles of viral replication, we designed time-of-addition experiments using the chloroquine-sensitive human H3N2 virus. Chloroquine was added during virus adsorption onto cells (i.e. time 0; T 0 ) and/or at 1, 2, 3 and 4 h postinfection (T 1-4 ) Using qRRT-PCR, we found that the inhibitory effect of chloroquine was highest when the drug was added at T 0 (inhibition of viral replication corresponding to 89,36%) and at T 1 (inhibition of replication corresponding to 15,53%), whereas the inhibitory activity was completely lost at T 2 .
If this timing was correct, chloroquine should inhibit influenza A replication by a novel mechanism, and therefore exert additive effects in combination with oseltamivir, inhibiting neuraminidase activity at the late stages of viral replication cycle. To test this hypothesis, human H3N2 virus-infected cells were treated with different chloroquine concentrations in the presence or absence of oseltamivir (10 nM). The virus-infected cells were also incubated with oseltamivir alone, EC 50 = 20 nM. Isobologram analysis showed that the two drugs exerted an additive effect (sum of FICs = 1) (data not shown). This result provides further evidence that chloroquine inhibits viral replication by a mechanism different from that of one major anti-influenza drug.

Discussion

Chloroquine was found to inhibit a number of cellular processes, some of which do not depend on low pH but might anyway interfere with viral replication. For example, the drug was found to inhibit viral nucleotidyl transferases such as HIV-1 integrase [7] . If chloroquine inhibited influenza A RNA-dependent RNA polymerase, the timing of viral inhibition would not be consistent with that observed in the present study, because RNA replication occurs in the nucleus at later stages [18] .
Based on bioinformatic studies, it was recently hypothesized that chloroquine might inhibit UDP-N acetylglucosamine transferase [1] , a limiting enzyme in sialic acid synthesis. This specific issue has not been addressed here. Nonetheless, if the antiviral effect of chloroquine reported here were due to inhibition of UDP-N acetylglucosamine transferase, the drug should likely have antagonized the antiviral effect of the neuraminidase inhibitor oseltamivir (acting on detachment of sialic acid-bound virions from parent cells), rendering oseltamivir inhibition unnecessary. Instead, chloroquine was found in the present study to exert antiviral effects that were additive to those of oseltamivir.
Although a comprehensive study on the variation of fusion pH requirements of influenza A viruses of all HA subtypes and isolates from different hosts is not available, several authors have documented that the threshold pH, at which the HA conformational change and virus-cell fusion occur, is strain-specific [24] [25] [26] . Interestingly, the viruses showing highest chloroquine sensitivity also displayed the highest HA2 isoelectric points. Thus, a relation-ship between isoelectric point and response to pH is apparent. However, a broad study relating the surface electrostatic potential with inactivation by pH would be required to analyse the molecular details of the HA/pH interplay. Analyses of virus production before and after exposure to chloroquine and of the possible changes in HA2 surface potential in viruses rendered resistant to chloroquine after long exposure to the drug will also be necessary.

Conclusion

Although association between variables cannot be considered to be equivalent to causation, the results of the present study strongly suggest that pH critically determines the antiviral activity of chloroquine by regulating virus/host cell interactions. The potential use of this compound as an antiinfluenza drug should take into consideration the possibility that even within the same subtype, different strains may present significantly divergent sensitivities to chloroquine as a consequence of their different pH requirements. Moreover, sensitivity to chloroquine may vary in different cell populations susceptible to influenza A virus infection, depending on different capabilities of endosome acidification. Mutations affecting the electrostatic potential of the the HA2 protein subunit of various isolates of the same virus could also be relevant. All these factors should be carefully evaluated when hypothesising a potential clinical utilisation of chloroquine against influenza A viruses.

Assessment of the effects of two drugs in combination

To measure the anti-influenza effects of chloroquine/oseltamivir drug combinations, cell pellets were resuspended in media containing increasing concentrations of the antimalarial in the presence or absence of oseltamivir. A fractional inhibitory concentration (FIC) was then calculated as the ratio: 50% effective concentration (EC 50 ) of drug A in combination with drug B/EC 50 of drug A alone. The effect was considered to be additive when the sum of FICs was between 0.8 and 1.2, as previously described [8] Time-of-addition assay Monolayers of MDCK cells in 96-well plates were infected with 100 μl of medium containing approximately 10 4 TCID 50 of H3N2 subtype. After 1 hour of adsorption, cell monolayers were washed twice with serum-free MEM and incubated in fresh medium containing TPCK-trypsin and chloroquine at a concentration of 10 μM. Chloroquine was added at the time of infection or at four different time points thereafter. Eight hours post-infection, a time point at which all progeny virus in the supernatants is derived from the first replication cycle, cell supernatants were collected, viral RNA was extracted and the antiviral activity was determined by using the qRRT-PCR described above.
5 section matches

Abstract

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres.

Discussion

Although Zika virus was first identified in Uganda in 1947, from January 2007 to April 2016, ZIKV transmission has been reported in 64 countries and territories [35] . The Zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. Prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce ZIKV transmission and avoid the spread of the disease by travelers. Moreover, a drug that blocks placental transfer of the virus could decrease the Figure 6 . Chloroquine inhibits ZIKV infection in mouse neurospheres. Mouse neurospheres were infected with ZIKV MR766 (2.5 × 10 5 PFU and were treated with chloroquine for 3 days. Neurospheres were analyzed by phase contrast microscopy (A-C), and triple stained for envelope viral protein (green), microtubule-associated protein 2 (Map-2, red), a neuron-specific protein, and DAPI (blue) (D-F).
Here we demonstrated that chloroquine decreases the number of ZIKV-infected cells and protected cells from ZIKV infection as measured by cell viability at non-cytotoxic concentrations (Figures 1, 4 and 5) . The EC50 or concentration of chloroquine that protected 50% of the cells from ZIKV infection assessed by cell viability, was 9.82-14.2 µM depending on the cell model and the CC50 ranged from 94.95 to 134.54 µM ( Table 1) . The values of EC50 obtained for ZIKV MR766 are lower than those obtained for DENV inhibition (~25 µM) and HIV inhibition (100 µM) [20, 22] . Furthermore, we observed similar ZIKV inhibitory effects of chloroquine when tested on different ZIKV lineage infections (Figures 2 and 5) , supporting the idea that chloroquine could help to manage recent infections caused by Asian ZIKV lineage. Although chloroquine has shown antiviral activity against a large spectrum of viruses in vitro, few clinical studies have been performed to evaluate chloroquine effects on patients with viral infections. Two clinical trial studies of chloroquine have been conducted to assess chloroquine treatment in patients infected with DENV [37, 38] . One of the trials evaluated the benefits of chloroquine treatment for 3 days in patients infected with DENV and showed no reduction in the duration or intensity of DENV viremia or nonstructural 1 protein (NS1) antigenemia clearance [37] . However, a trend towards a reduction in the number of dengue hemorrhagic fever cases was noticed in the chloroquine-treated group [37] . A more recent clinical trial of chloroquine administration to DENV-infected patients, also for 3 days, showed that 60% of the patients in the chloroquine-treated group reported feeling less pain and showed improvement in the performance of daily chores during treatment [38] . Moreover, the symptoms returned after medication withdrawal. However, chloroquine treatment did not reduce the duration and intensity of the fever or duration of the disease [38] . The antiviral effect of chloroquine may be insufficient to produce a decrease in viral load or improvement of the disease progression when chloroquine/hydroxychloroquine is used in monotherapy. However, chloroquine may produce a significant antiflaviviral effect when used in combination therapies, as recently shown in a clinical trial of hydroxychloroquine plus ribavirin and interferon alpha in individuals infected with hepatitis C virus (HCV) [39] . In regard to the potential antiviral therapeutic combinations for Zika, a freshly published screening of drugs already approved for other clinical indications has resulted in the identification of more than 20 candidate drugs [40] . Of note, one of these is mefloquine, a compound related to chloroquine. In terms of safety for pregnant women, however, mefloquine is included in the B category, i.e., a drug for which the animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women. Be that as it may, the aforementioned study corroborates our results using chloroquine, and provides new anti-ZIKV drugs that could be tested in combination with chloroquine.
Different mechanisms for the chloroquine inhibition of viral infection have been described [49] [50] [51] . We observed a strong reduction in the release of ZIKV particles when the drug was added at 0 h post-infection (Figure 3) , suggesting a higher impact on early stages of infection, possibly during fusion of the envelope protein to the endosome membrane. Chloroquine inhibits acidification of the endosome, consequently inhibiting the low pH-induced conformational changes required for the fusion of the envelope protein of flaviviruses with the endosomal membrane [52] . Chloroquine was also effective in decreasing virus release, although less pronouncedly and not statistically significant, when added after the early stages of virus infection (from 0.5 to 24 h post-infection), suggesting that later stages of the ZIKV replication cycle might also be affected (Figure 3) .
Our results suggest that the chloroquine concentrations inhibiting ZIKV replication in vitro may overlap the highest drug concentrations detected in humans [56] . We therefore suggest that the therapeutic potential of chloroquine for Zika be subjected to further study.
13 section matches

Abstract

We report, however, that chloroquine has strong antiviral effects on SARS-CoV infection of primate cells. These inhibitory effects are observed when the cells are treated with the drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensinconverting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of infection and spread of SARS CoV at clinically admissible concentrations.

Background

Due to the severity of SARS-CoV infection, the potential for rapid spread of the disease, and the absence of proven effective and safe in vivo inhibitors of the virus, it is important to identify drugs that can effectively be used to treat or prevent potential SARS-CoV infections. Many novel therapeutic approaches have been evaluated in laboratory studies of SARS-CoV: notable among these approaches are those using siRNA [7] , passive antibody transfer [8] , DNA vaccination [9] , vaccinia or parainfluenza virus expressing the spike protein [10, 11] , interferons [12, 13] , and monoclonal antibody to the S1-subunit of the spike glycoprotein that blocks receptor binding [14] . In this report, we describe the identification of chloroquine as an effective pre-and post-infection antiviral agent for SARS-CoV. Chloroquine, a 9-aminoquinoline that was identified in 1934, is a weak base that increases the pH of acidic vesicles. When added extracellularly, the non-protonated portion of chloroquine enters the cell, where it becomes protonated and concentrated in acidic, low-pH organelles, such as endosomes, Golgi vesicles, and lysosomes. Chloroquine can affect virus infection in many ways, and the antiviral effect depends in part on the extent to which the virus utilizes endosomes for entry. Chloroquine has been widely used to treat human diseases, such as malaria, amoebiosis, HIV, and autoimmune diseases, without significant detrimental side effects [15] . Together with data presented here, showing virus inhibition in cell culture by chloroquine doses compatible with patient treatment, these features suggest that further evaluation of chloroquine in animal models of SARS-CoV infection would be warranted as we progress toward finding effective antivirals for prevention or treatment of the disease.

Postinfection chloroquine treatment is effective in preventing the spread of SARS-CoV infection

In order to investigate the antiviral properties of chloroquine on SARS-CoV after the initiation of infection, Vero E6 cells were infected with the virus and fresh medium supplemented with various concentrations of chloroquine was added immediately after virus adsorption. Infected cells were incubated for an additional 16-18 h, after which the presence of virus antigens was analyzed by indirect immunofluorescence analysis. When chloroquine was added after the initiation of infection, there was a dramatic dose-dependant decrease in the number of virus antigen-positive cells ( Fig. 2A) . As little as 0.1-1 µM chloroquine reduced the infection by 50% and up to 90-94% inhibition was observed with 33-100 µM concentrations ( Fig. 2B ). At concentrations of chloroquine in excess of 1 µM, only a small number of individual cells were initially infected, and the spread of the infection to adjacent cells was all but eliminated. A half-maximal inhibitory effect was estimated to occur at 4.4 ± 1.0 µM chloroquine (Fig. 2C ). These data clearly show that addition of chloroquine can effectively reduce the establishment of infection and spread of SARS-CoV if the drug is added immediately following virus adsorption.
Electron microscopic analysis indicated the appearance of significant amounts of extracellular virus particles 5-6 h after infection [16] . Since we observed antiviral effects by chloroquine immediately after virus adsorption, we further extended the analysis by adding chloroquine 3 and 5 h after virus adsorption and examined for the presence of virus antigens after 20 h. We found that chloroquine was still significantly effective even when added 5 h after infection (Fig. 3) ; however, to obtain equivalent antiviral effect, a higher concentration of chloroquine was required if the drug was added 3 or 5 h after adsorption.

Effect of chloroquine and NH 4 Cl on cell surface expression of ACE2

We performed additional experiments to elucidate the mechanism of SARS-CoV inhibition by chloroquine and NH 4 Cl. Since intra-vesicular acidic pH regulates cellular functions, including N-glycosylation trimming, cellular trafficking, and various enzymatic activities, it was of interest to characterize the effect of both drugs on the processing, glycosylation, and cellular sorting of SARS-CoV spike glycoprotein and its receptor, ACE2. Flow cytometry analysis was performed on Vero E6 cells that were either untreated or treated with highly effective anti-SARS-CoV concentrations of chloroquine or NH 4 Cl. The results revealed that neither drug caused a significant change in the levels of cell-surface ACE2, indicating that the observed inhibitory effects on SARS-CoV infection are not due to the lack of available cell-surface ACE2 (Fig. 5A ). We next analyzed the molecular forms of endog-Prophylactic effect of chloroquine enous ACE2 in untreated Vero E6 cells and in cells that were pre-incubated for 1 h with various concentrations of either NH 4 Cl (2.5-10 mM) or chloroquine (1 and 10 µM) and labeled with 35 S-(Met) for 3 h in the presence or absence of the drugs (Fig. 5B and 5C ). Under normal conditions, we observed two immunoreactive ACE2 forms, migrating at ~105 and ~113 kDa, respectively (Fig. 5B , lane 1). The ~105-kDa protein is endoglycosidase H sensitive, suggesting that it represents the endoplasmic reticulum (ER) localized form, whereas the ~113-kDa protein is endoglycosidase H resistant and represents the Golgimodified form of ACE2 [19] . The specificity of the antibody was confirmed by displacing the immunoreactive protein bands with excess cold-soluble human recombinant ACE2 (+ rhACE2; Fig. 5B , lane 2). When we analyzed ACE2 forms in the presence of NH 4 Cl, a clear stepwise increase in the migration of the ~113-kDa protein was observed with increasing concentrations of NH 4 Cl, with a maximal effect observed at 10 mM NH 4 Cl, resulting in only the ER form of ACE2 being visible on the gel (Fig. 5B , compare lanes [3] [4] [5] . This suggested that the trimming and/or terminal modifications of the N-glycosylated chains of ACE2 were affected by NH 4 Cl treatment.

Effect of chloroquine and NH 4 Cl on the biosynthesis and processing of SARS-CoV spike protein

We next addressed whether the lysosomotropic drugs (NH 4 Cl and chloroquine) affect the biosynthesis, glyco-sylation, and/or trafficking of the SARS-CoV spike glycoprotein. For this purpose, Vero E6 cells were infected with SARS-CoV for 18 h. Chloroquine or ammonium chloride was added to these cells during while they were being starved (1 h), labeled (30 min) or chased (3 h). The cell lysates were analyzed by immunoprecipitation with the SARS-specific polyclonal antibody (HMAF). The 30-min pulse results indicated that pro-spike (proS) was synthesized as a ~190-kDa precursor (proS-ER) and processed into ~125-, ~105-, and ~80-kDa proteins (Fig. 6A, lane 2) , a result identical to that in our previous analysis [6] . Except for the 100 µM chloroquine (Fig. 6A, lane 3) , there was no significant difference in the biosynthesis or processing of the virus spike protein in untreated or chloroquine-treated cells (Fig. 6A, lanes 4-6) . It should be noted that chloroquine at 100 µM resulted in an overall decrease in biosynthesis and in the levels of processed virus glycoprotein. In view of the lack of reduction in the Effects of NH 4 Cl and chloroquine (CQ) on the biosynthesis, processing, and glycosylation of SARS-CoV spike protein Figure 6 Effects of NH 4 Cl and chloroquine (CQ) on the biosynthesis, processing, and glycosylation of SARS-CoV spike protein. Vero E6 cells were infected with SARS-CoV as described in Fig. 2 . CQ or NH 4 Cl was added during the periods of starvation (1 h) and labeling (30 min) with 35 S-Cys and followed by chase for 3 h in the presence of unlabeled medium. Cells were lysed in RIPA buffer and immunoprecipitated with HMAF. Virus proteins were resolved using 3-8% NuPAGE gel (Invitrogen). The cells presented were labeled for 30 min (A) and chased for 3 h (B). The migration positions of the various spike molecular forms are indicated at the right side, and those of the molecular standards are shown to the left side. proS-ER and proS-Golgi are the pro-spike of SARS-Co in the ER and Golgi compartments, respectively and proS-ungly is the unglycosylated pro-spike ER.
biosynthesis and processing of the spike glycoprotein in the presence of chloroquine concentrations (10 and 50 µM) that caused large reductions in SARS-CoV replication and spread, we conclude that the antiviral effect is probably not due to alteration of virus glycoprotein biosynthesis and processing. Similar analyses were performed with NH 4 Cl, and the data suggested that the biosynthesis and processing of the spike protein were also not negatively affected by NH 4 Cl (Fig. 6A, lanes 7-12) . Consistent with our previous analysis [6] , we observed the presence of a larger protein, which is referred to here as oligomers. Recently, Song et al. [20] provided evidence that these are homotrimers of the SARS-CoV spike protein and were incorporated into the virions. Interestingly, the levels of the homotrimers in cells treated with 100 µM chloroquine and 40 and 20 mM NH 4 Cl (Fig. 6A, lanes 3, 9, and 10 ) were slightly lower than in control cells or cells treated with lower drug concentrations.
The data obtained from a 30-min pulse followed by a 3-h chase (Fig. 6B , lanes 2 and 8) confirmed our earlier observation that the SARS-CoV spike protein precursor (proS-ER) acquires Golgi-specific modifications (proS-Golgi) resulting in a ~210-kDa protein [6] . Chloroquine at 10, 25, and 50 µM had no substantial negative impact on the appearance of the Golgi form (Fig. 6B , compare lane 2 to lanes 4-6). Only at 100 µM chloroquine was a reduction in the level of the Golgi-modified pro-spike observed (lane 3). On the other hand, NH 4 Cl abrogated the appearance of Golgi-modified forms at ≥10 mM (compare lane 8 with 9-11) and had a milder effect at 1 mM (lane 12). These data clearly demonstrate that the biosynthesis and proteolytic processing of SARS-CoV spike protein are not affected at chloroquine (25 and 50 µM) and NH 4 Cl (1 mM) doses that cause virus inhibitory effects. In addition, with 40, 20, and 10 mM NH 4 Cl, there was an increased accumulation of proS-ER with a concomitant decrease in the amount of oligomers (Fig. 6B, lanes 9-11) . When we examined the homotrimers, we found that chloroquine at 100 µM and NH 4 Cl at 40 and 20 mM resulted in slightly faster mobility of the trimers (Fig. 6B, lanes 3, 9, and 10 ), but lower drug doses, which did exhibit significant antiviral effects, did not result in appreciable differences. These data suggest that the newly synthesized intracellular spike protein may not be a major target for chloroquine and NH 4 Cl antiviral action. The faster mobility of the trimer at certain higher concentration of the drugs might be due the effect of these drugs on the terminal glycosylation of the trimers.

Discussion

We have identified chloroquine as an effective antiviral agent for SARS-CoV in cell culture conditions, as evidenced by its inhibitory effect when the drug was added prior to infection or after the initiation and establishment of infection. The fact that chloroquine exerts an antiviral effect during pre-and post-infection conditions suggest that it is likely to have both prophylactic and therapeutic advantages. Recently, Keyaerts et al. [21] reported the antiviral properties of chloroquine and identified that the drug affects SARS-CoV replication in cell culture, as evidenced by quantitative RT-PCR. Taken together with the findings of Keyaerts et al. [21] , our analysis provides further evidence that chloroquine is effective against SARS-CoV Frankfurt and Urbani strains. We have provided evidence that chloroquine is effective in preventing SARS-CoV infection in cell culture if the drug is added to the cells 24 h prior to infection. In addition, chloroquine was significantly effective even when the drug was added 3-5 h after infection, suggesting an antiviral effect even after the establishment of infection. Since similar results were obtained by NH 4 Cl treatment of Vero E6 cells, the underlying mechanism(s) of action of these drugs might be similar.
Apart from the probable role of chloroquine on SARS-CoV replication, the mechanisms of action of chloroquine on SARS-CoV are not fully understood. Previous studies have suggested the elevation of pH as a mechanism by which chloroquine reduces the transduction of SARS-CoV pseudotype viruses [17, 18] . We examined the effect of chloroquine and NH 4 Cl on the SARS-CoV spike proteins and on its receptor, ACE2. Immunoprecipitation results of ACE2 clearly demonstrated that effective anti-SARS-CoV concentrations of chloroquine and NH 4 Cl also impaired the terminal glycosylation of ACE2. However, the flow cytometry data demonstrated that there are no significant differences in the cell surface expression of ACE2 in cells treated with chloroquine or NH 4 Cl. On the basis of these results, it is reasonable to suggest that the pre-treatment with NH 4 Cl or chloroquine has possibly resulted in the surface expression of the under-glycosylated ACE2. In the case of chloroquine treatment prior to infection, the impairment of terminal glycosylation of ACE2 may result in reduced binding affinities between ACE2 and SARS-CoV spike protein and negatively influence the initiation of SARS-CoV infection. Since the biosynthesis, processing, Golgi modification, and oligomerization of the newly synthesized spike protein were not appreciably affected by anti-SARS-CoV concentrations of either chloroquine or NH 4 Cl, we conclude that these events occur in the cell independent of the presence of the drugs. The potential contribution of these drugs in the elevation of endosomal pH and its impact on subsequent virus entry or exit could not be ruled out. A decrease in SARS-CoV pseudotype transduction in the presence of NH 4 Cl was observed and was attributed to the effect on intracellular pH [17, 18] . When chloroquine or NH 4 Cl are added after infection, these agents can rapidly raise the pH and subvert on-going fusion events between virus and endosomes, thus inhibiting the infection.
In addition, the mechanism of action of NH 4 Cl and chloroquine might depend on when they were added to the cells. When added after the initiation of infection, these drugs might affect the endosome-mediated fusion, subsequent virus replication, or assembly and release. Previous studies of chloroquine have demonstrated that it has multiple effects on mammalian cells in addition to the elevation of endosomal pH, including the prevention of terminal glycosyaltion of immunoglobulins [22] . When added to virus-infected cells, chloroquine inhibited later stages in vesicular stomatitis virus maturation by inhibiting the glycoprotein expression at the cell surface [23] , and it inhibited the production of infectious HIV-1 particles by interfering with terminal glycosylation of the glycoprotein [24, 25] . On the basis of these properties, we suggest that the cell surface expression of under-glycosylated ACE2 and its poor affinity to SARS-CoV spike protein may be the primary mechanism by which infection is prevented by drug pretreatment of cells prior to infection. On the other hand, rapid elevation of endosomal pH and abrogation of virus-endosome fusion may be the primary mechanism by which virus infection is prevented under post-treatment conditions. More detailed SARS CoV spike-ACE2 binding assays in the presence or absence of chloroquine will be performed to confirm our findings.
The infectivity of coronaviruses other than SARS-CoV are also affected by chloroquine, as exemplified by the human CoV-229E [15] . The inhibitory effects observed on SARS-CoV infectivity and cell spread occurred in the presence of 1-10 µM chloroquine, which are plasma concentrations achievable during the prophylaxis and treatment of malaria (varying from 1.6-12.5 µM) [26] and hence are well tolerated by patients. It recently was speculated that chloroquine might be effective against SARS and the authors suggested that this compound might block the production of TNFα, IL6, or IFNγ [15] . Our data provide evidence for the possibility of using the well-established drug chloroquine in the clinical management of SARS.

Conclusion

Chloroquine, a relatively safe, effective and cheap drug used for treating many human diseases including malaria, amoebiosis and human immunodeficiency virus is effective in inhibiting the infection and spread of SARS CoV in cell culture. The fact that the drug has significant inhibitory antiviral effect when the susceptible cells were treated either prior to or after infection suggests a possible prophylactic and therapeutic use.
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Abstract

The restoration of the immune system prompted by antiretroviral therapy (ART) has allowed drastically reducing the mortality and morbidity of HIV infection. However, one main source of clinical concern is the persistence of immune hyperactivation in individuals under ART. Chronically enhanced levels of T-cell activation are associated with several deleterious effects which lead to faster disease progression and slower CD4 + T-cell recovery during ART. In this article, we discuss the rationale, and review the results, of the use of antimalarial quinolines, such as chloroquine and its derivative hydroxychloroquine, to counteract immune activation in HIV infection. Despite the promising results of several pilot trials, the most recent clinical data indicate that antimalarial quinolines are unlikely to exert a marked beneficial effect on immune activation. Alternative approaches will likely be required to reproducibly decrease immune activation in the setting of HIV infection. If the quinoline-based strategies should nevertheless be pursued in future studies, particular care must be devoted to the dosage selection, in order to maximize the chances to obtain effective in vivo drug concentrations.

Background

In the beginning of the millennium, an article authored by one of us launched chloroquine as a tool to inhibit viral replication and the related malignant immune activation associated with some viral diseases [7] . This article sparked a new wave of studies, in that it extended a theory, previously designed for HIV/AIDS [8] , to other viral diseases characterized by excessive immune activation. As will be discussed below, by accumulating in the acidic organelles, chloroquine exerts both direct antiviral effects on enveloped viruses and decreases activation of several cell types involved in the immune response. Chloroquine has since shown promise in preclinical studies (both in vitro and in vivo), as a therapeutic agent against emerging viruses such as MERS CoV [9] . Of note, chloroquine has been indicated as a promising candidate for filovirus treatment [10] , especially during the latest Ebola epidemic [11, 12] . In two studies out of three, chloroquine showed antiviral activity in mice at the maximum tolerated dose [10, 13, 14] , thus rendering this drug an interesting agent for further testing of combination anti-Ebola therapies. However, the effects of chloroquine and its hydroxyl analogue hydroxychloroquine, on HIV infection, i.e. the initial target for the repurposing of these drugs, have remained controversial. On the one hand, based on the results of some earlier clinical trials, chloroquine/hydroxychloroquine has been recently resuggested as a promising candidate to restrict the HIVrelated immune activation [15, 16] . On the other hand, the results from the latest clinical trials indicate that chloroquine/hydroxychloroquine has no beneficial effect on immune activation [17, 18] .
We here provide a state of the art of the studies investigating the use of chloroquine/hydroxychloroquine as a therapeutic tool for HIV/AIDS and suggest the possible biological grounds for the clinical results obtained. Moreover, we describe the reasons why our group decided to proceed further with strategies based on another drug, i.e. auranofin, which shares with chloroquine an antirheumatic effect [19] .

Mechanisms of action of chloroquine

1. Chloroquine and its hydroxyl analogue hydroxychloroquine were shown in several studies to inhibit HIV-1 replication (reviewed in: [7] ). The effects of these quinolines, mainly due to the induction of a defect in the maturation of the viral envelope glycoprotein gp120 [35, 36] , might mimic the effects of broadly neutralizing antibodies directed against the viral envelope, although the effects of these antibodies are weaker than those directed against the CD4binding site [37] . These effects are additive to those of non-nucleosidic reverse transcriptase inhibitors (NNRTIs) and synergistic to those of protease inhibitors (PIs) [38] . As quinoline drugs accumulate in lymphoid tissues [39] , they might decrease ongoing viral replication during ART in anatomical sanctuaries and, consequently switch off one of the main drivers of immune activation. Chloroquine is also an inhibitor of P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs) [40, 41] , cell surface glycoproteins which extrude several antiretroviral drugs to the extracellular medium. In line with this evidence, chloroquine was shown to increase the intracellular levels of PIs [38] . The effects of chloroquine in com-bination with NRTIs are instead controversial: some reported an additive effect [42] , while others did not detect it [43] . The combined effects of chloroquine and integrase inhibitors are as yet unknown. 2. Chloroquine accumulates in phagosomes of pDCs and inhibits their HIV-induced activation [44] . It might therefore impact on innate immunity-induced immune hyperactivation. 3. A recent study showed that hydroxychloroquine selectively induces apoptosis in the memory T-cell compartment (CD45RA − CD45RO + ) [45] . As, upon activation, naïve T-cells (CD45RA + CD45RO − ) acquire a CD45RA − CD45RO + phenotype, the "antimemory" effect should limit immune activation (Figure 2 ) [46] . There is growing consensus that induction of apoptosis in the memory T-cell compartment might have a detrimental effect on the viral reservoir [47] [48] [49] . In this light, chloroquine/hydroxychloroquine should have an anti-reservoir potential. This view is supported by another recent study which shows that chloroquine sensitizes to apoptosis the latently infected cells upon viral reactivation, likely by removing the anti-apoptotic effect of the virus structural gag gene products [50] . These effects are potentially interesting, since it has been well demonstrated that viral reactivation from latency does not necessarily result in cell death [51] .

In vivo effects of chloroquine/hydroxychloroquine: clinical trials

Suppressive effects on immune activation by chloroquine were shown in the trial conducted by Murray et al. [55] . However, in this trial, the dosage administered was not the same for all individuals, some of them receiving 500 mg/die instead of 250 mg/die. It is thus possible that the statistical significance of the effects reported in this study was driven by the higher dosage of the drug. This view is supported by a later study which tested chloroquine at 250 mg/die and failed to show any effect of the drug [18] .
The hydroxychloroquine levels show high inter-subject variability and, although individuals receiving the higher hydroxychloroquine dosages (800 and 1,200 mg/day) also showed significantly higher blood levels of the drug than those receiving 400 mg/die, the range of the blood concentrations was in part overlapping in the different dosage groups [61] . Chloroquine has similar pharmacokinetics [62] ; therefore, not only the dosage but also individual differences in drug metabolism and distribution may explain the different conclusions of the aforementioned studies. A large clinical trial has recently been completed (ClinicalTrials.gov Identifier: NCT00819390) and its results can help to better represent the response of a population, thus abolishing the bias due to limited sample size. In this trial, however, chloroquine has been tested at 250 mg/day in the absence of ART; thus, in light of the results of the aforementioned clinical trials and considerations derived from basic science (see next paragraph), it is not surprising that the preliminary results released so far for this trial (https://clinicaltrials.gov/ct2/ show/NCT00819390) do not show any significant effect of chloroquine on immune activation, viral load and CD4 counts.

Lessons learnt from chloroquine/ hydroxychloroquine use in HIV infection

Chloroquine/hydroxychloroquine-treated individuals display blood concentrations that are highly variable and only rarely exceed 10 or 20 µM, respectively [61, 62] . Therefore, at the steady state levels, these blood concentrations only in part overlap those at which a therapeutic effect is expected. For example, the EC 50 of chloroquine on PBMC proliferation upon activation is, in general, ≥10 µM [63] , and this value can explain the varying results obtained in the different clinical trials, with clearer effects associated with the higher drug dosages. Similarly, the pro-apoptotic effect of hydroxychloroquine on the memory T-cells is only moderate at the concentrations reachable in blood, especially in the lower range [45, 61] . The pro-apoptotic effect of chloroquine described by Li et al. on latently infected cells upon viral reactivation is instead more marked, although still partial, at the upper range of clinically achievable blood concentrations (5-10 µM) [50] . This effect could therefore be visible in vivo in terms of viral reservoir reduction, but only treating with high chloroquine dosages in the presence of suppressive ART. Moreover, to maximize the chances to obtain viral reservoir reduction in vivo, chloroquine treatment should be prolonged, as the events of virus reactivation from latency are rather rare (estimated as one event of transition from latency to productive infection every 10 mL of blood each day) [64] .

Figure 3

Published clinical studies evaluating the effects of chloroquine/hydroxychloroquine administration, alone or in combination with other drugs, in HIV infected subjects. Highlighted in blue, red or white are the studies that have reported a positive, negative, or neutral outcome of the therapy respectively. CQ chloroquine, HCQ hydroxychloroquine. [65] . In this case, the in vitro effect is in line with the results of two in vivo studies [53, 60] . The use of chloroquine-related compounds with increased potency is yielding promising results in vitro [66] , and it will be interesting to test the best-performing candidates in the simian AIDS model. The effects of chloroquine/hydroxychloroquine on viral replication have been repeatedly shown in vitro at lower drug levels than those inducing the cellular effects [35, 36, 63, 65] . The blood concentration/EC 50 ratio is however much narrower than those shown by antiretroviral drugs [63] . The antiretroviral effects of chloroquine/hydroxychloroquine may though become visible in anatomical sanctuaries of those individuals treated with PI-containing antiretroviral regimens. In any case, we recommend that chloroquine/hydroxychloroquine be tested at the highest recommended dosages in future HIV clinical trials.
Another open question remains the influence of the duration of drug exposure, as it has been shown that chloroquine/hydroxychloroquine has cumulative effects [67] . As a proportion of HIV-infected patients in Africa may already be on chloroquine medication to prevent malaria, it might be worth examining the long-term effects of this treatment. In this regard, an ongoing phase III clinical trial will assess the long-term effects of chloroquine and trimethoprim-sulfamethoxazole phrophylaxis on survival and disease control in HIV-infected individuals with suppressed viral load and good clinical response to ART [68] .

Current and future directions: another approach based on antirheumatic therapy

Given the aforementioned problems in the pharmacokinetics of chloroquine/hydroxychloroquine, our group chose to follow a different, yet partly similar, approach to corroborate treatment of HIV/AIDS. Based on the feedback received from basic science studies and clinical trials that have been published throughout the years, we decided to use drugs the desired effects of which be striking in vitro at concentrations lower than the trough plasma concentrations in vivo. We also decided to redirect our research on the basis of the plasma concentrations rather than on whole-blood concentrations (widely used for chloroquine/hydroxychloroquine), because we thought that the former might better mimic the tissue culture concentrations. The drug that we selected is the gold-based compound auranofin, the pharmacodynamics and pharmacokinetics of which are well known, due to its decade-long employment for treatment of rheumatoid arthritis [69] .
The main rationale for the use of auranofin in our studies was its ability to target the central/transitional memory CD4 + T-cell compartment ( Figure 2 ) [48, 70] , which is known to harbor the main viral reservoir in patients receiving ART [33] . Auranofin is drastically active at submicromolar (i.e. ≤250 nM) concentrations, which are below those readily achievable in human plasma [71] . The administration of auranofin ultimately led to a reduction of the viral reservoir in ART-treated SIVmac251-infected macaques [70] . A review on our preclinical studies has recently been published [46] and the reader is addressed to it for further detail. Not surprisingly for a drug effective against an autoimmune disease such as rheumatoid arthritis, auranofin may as well be beneficial in terms of reduction of cell activation. In particular, the downregulation of the CD28 molecule induced by auranofin can disrupt the co-stimulatory signal often crucial for lymphocyte activation [48] . Moreover, apart from memory CD4 + T-cells, auranofin also targets the memory CD8 + T-cell compartment [48] , i.e. a cellular subset known to be hyperactivated during HIV infection [2] . Interestingly, as described for hydroxychloroquine [60] , auranofin was shown to disrupt in various cell lines the TLR-4 signaling [72] , which is activated by bacterial lipopolysaccharides and likely constitutes another source of immune hyperactivation. In vitro data indicate that the impact of auranofin on lymphocyte activation may be mediated, at least in part, by modulation of oxidative stress [48] . Of note, the addition of a potent pro-oxidant drug, such as buthionine sulfoximine (BSO), increases the potency of auranofin, decreasing phytohemagglutinin-induced activation and expression of the α-chain of the IL-2 receptor [73] . This is in line with our preliminary data in SIVmac251-infected macaques, in which a combined regimen of ART, auranofin and BSO induced a functional cure-like condition following suspension of all therapies [74] . These observations provide proof of concept that drastically decreasing immune hyperactivation arrests SIV disease progression and turns the virus/immune system balance in favor of the latter. Clinical trials will be required to assess the potential of auranofin to decrease immune activation in ART-treated subjects.
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Discussion

Chloroquine phosphate has shown better anti-SARS-CoV-2 effects in recent studies, but this drug has no clear target of action. In our docking results, chloroquine phosphate is predicted to possibly combine with