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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: preclinical models

As the immune activation set point is established during acute infection [4] , Vaccari et al. [54] treated with chloroquine (18.7 mg/day for 112 consecutive days) seven SIVmac 251 -infected rhesus macaques during the viral load peak that characterizes acute infection. Apart from an unexpected, although transient, increase in the Comparison of the susceptibility to chloroquine/hydroxychloroquine and auranofin of the cellular subsets involved in HIV production and persistence. Shown in the figure is a schematic depiction of a activation and b differentiation stages of CD4 + T-lymphocytes and their correlation with viral production, latency and viral reactivation. Both chloroquine/hydroxychloroquine and auranofin can influence these transitions by exerting a pro-apoptotic effect, the efficacy of which is graphically exemplified by the intensity of the blue color in the corresponding rectangles. Efficacy gradients are based on data derived from Refs. [45, 48, 50] . expression of interferon-regulated genes (perhaps not population-relevant as possibly driven by only one animal), no significant differences were reported in viral load and T-cell activation and proliferation (measured as expression of CD69 and Ki67, respectively) [54] . A trend was however noticed for maintenance of decreased levels of Ki67, CD69 and CCR5 in the gut of the chloroquinetreated animals, although the differences with values from the control group did not reach statistical significance. The effect of chloroquine in this simian model in the presence of ART is still unknown.

In vivo effects of chloroquine/hydroxychloroquine: clinical trials

Chloroquine and hydroxychloroquine have so far been tested in several HIV clinical trials. The results summarized in Figure 3 support the hypothesis that the chloroquine/hydroxychloroquine dosage may be an important driver of at least partial clinical success.
In two clinical trials conducted in the 1990s, Sperber et al. reported suppressive effects on immune activation (measured at that time as IL-6 production) and viral load in individuals treated with 800 mg of hydroxychloroquine/day (bioequivalent to 500 mg/day of chloroquine) [56, 57] . The other clinical trials testing hydroxychloroquine at a lower dosage (i.e. 400 mg/day) led to conflicting results. Earlier studies [58, 59] and the more recent study of Piconi et al. [60] reported significant effects on viral load [58] , CD4 counts [59] , and immune activation. [60] . Instead, a more recent clinical trial, randomized and double blind, showed disappointing results, even hinting at possibly deleterious effects of hydroxychloroquine on viral load and CD4 counts [17] . This trial was conducted in the absence of ART, and this might explain differences between this study and the study of Piconi et al., which was conducted on individuals under ART [60] . Another trial in ART-treated patients is currently ongoing and will provide more information on the effects of hydroxychloroquine (ClinicalTrials.gov identifier: NCT01232660).
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.
Alternative/complementary interpretations of the results so far obtained are possible. For example, the effectiveness of the ART regimen employed may play a role in determining the magnitude of the effects (if any) observed following chloroquine/hydroxichloroquine addition. The study of Piconi et al. [60] , showing some benefit in immunological non responders, may indicate that the effects of chloroquine may be visible only in some subsets of individuals with peculiar immunological characteristics, and that these effects can be hindered when immunologically non homogeneous cohorts are studied. In this regard, larger studies, with cohorts stratified according to immunological responsiveness to ART, could provide further information on the effects of chloroquine/hydroxychloroquine.
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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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) .

Therapeutically Exploitation as Lysosomotropic Property of Chloroquine Analogs

The increasing evidence suggests that the entry, replication and infection processes of several viruses such as Ebola, Marburg, dengue, Chikungunya, HIV etc. are highly dependent on endosomal-lysosomal acidification and the activities of several host endosomal proteaseswhich are also active in acidic pH environments (Sun and Tien 2012; Barrow et al. 2013) . By neutrality of acidic pH in endosomes, chloroquine analogs inhibit these viral entry and replication processes into the cytoplasm of susceptible cells and thereby abrogate their infections (Chiang et al. 1996; Savarino et al. 2003) . Furthermore, the dysfunction of various enzymes e.g. glycosylating enzymes, glycosyltransferases caused by increased acidic pH and/or structural changes in the Golgi apparatus with hydroxychloroquine or by specific interaction with chloroquine, have been shown to suppress not only glycosylation of SARS-coronaviruses (Vincent et al. 2005; Savarino et al. 2006) but also that of the HIV-1 gp120 envelope protein, resulting in structural changes in the gp120 glycoprotein, which in turn reduce the reactivity and infectivity of newly produced virions (Savarino et al. 2004; Naarding et al. 2007 ). Since the surface glycoproteins of filoviruses (Ebola and Marburg) involve in initiation of infection (Takada et al. 1997; Yang et al. 2000) , and cytotoxicity (Yang et al. 2000) , the inhibition of glycosylation by the analogs prevents the viral entries for a wide variety of host cells and leads to suppress their pathogenicity by producing of noninfectious or decreased infectivity viruses. This inhibited glycosylation will therefore allow time for the adaptive immune response to deal with the infection (Baize et al. 1999 ).

HIV

The productive entry and replication of AIDS causative agent, HIV-1 are dependent on the endocytic pathways and involve acidic organelles, such as endosomes, lysosomes and trans-Golgi network (Daecke et al. 2005; Chauhan et al. 2014 ). Many studies have focused on the anti-HIV activities of chloroquine analogs (e.g., chloroquine, hydroxychloroquine, pamaquine, plasmoquine or primaquine) against HIV (Leroux-Roels et al. 2014; Martinson et al. 2014; Mizuguchi et al. 2015; Savarino and Shytaj 2015) .
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) .

Enigma of clandestine association with failure of chloroquine analogs clinically

1 Chloroquine and its analogs such as hydroxychloroquine are ineffectiveness in treating low pH-dependent emerging viral infections due to failed to attain and sustain steady state concentrations in blood sufficient to increase and keep the pH of the acidic organelles to approximately neutral until patients' viremia becomes undetectable. Although there is a considerable intersubject variability in the steady state blood concentrations of chloroquine analogs, the maximum safe serum concentration of chloroquine diphosphate is 250-280 ng/mL at maximum safe dose of 4 mg/kg per day (Laaksonen et al. 1974) and whole blood concentration of hydroxychloroquine is 1.0-2.6 lg/L (Munster et al. 2002) . However, the doses and dose regimens should be adjusted to optimize the benefit/risk ratio on the rational basis of pharmacokinetics and pharmaco/toxicodynamics considerations. Moreover, the plasma levels of chloroquine analogs depend on some other factors such as methodology as well as storage conditions of samples because the analogs have been trapped in erythrocytes, lymphocytes and platelets. Chloroquine is also a racemic mixture. It has been reported that the kinetic behavior of separate enantiomers differs in humans (Augustijns et al. 1992 ). There is nothing to know about the relevance of stereospecificity in the therapy of emerging viral infections. 2 For pharmacokinetic parameter considerations, chloroquine has been disputed for its narrow therapeutic indexes and poor penetration in specific tissues (Augustijns et al. 1992) . It is reported that there are differences in efficacy and toxicity between chloroquine and its analog, hydroxychloroquine for long-term effectiveness in rheumatoid diseases (Avina-Zubieta et al. 1998 ). In rheumatoid arthritis therapy, it is reported that hydroxychloroquine is one half to two-thirds as effective as chloroquine but one half in the toxicity (Tobin et al. 1982; Mackenzie 1983) . Thus, hydroxychloroquine is generally regarded as a safe, reasonably effective and less toxic for the treatment of rheumatoid diseases, with a recommended daily dose of <6.4 mg/kg per day and a maximum dose of 400 mg/day. It is suggested that the blood concentration of desethylhydroxychloroquine, one of the oxidative metabolites of hydroxychloroquine is related to treatment efficacy, and that the blood hydroxychloroquine concentration is associated with toxicity (Munster et al. 2002) . Thus, selection of chloroquine analogs or its metabolite is also an important factor for successful treatment of viral diseases.
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Lysosome Alkalizers

Chloroquine, hydroxychloroquine, NH 4 Cl, and neutral red are chemicals that can rapidly neutralize the acidic environment of the lysosome; therefore, they are used to block autophagosome maturation. Chloroquine and hydroxychloroquine are repurposed drugs that have been used to treat malaria, SLE, and rheumatoid arthritis [470] ; however, higher hydroxychloroquine concentrations are required to induce active autophagy as a cancer treatment, which are usually not achievable in cancer patients. Chloroquine-mediated lysosomal dysfunction is thought to have increased anti-cancer functions when combined with nutrient deprivation [471] . Lys01, a dimeric form of chloroquine in which each molecule is separated by the spacer molecule N-bis(2-aminoethyl)-methylamine, has been reported to inhibit autophagy at a level 10-fold higher than chloroquine Lys05, a water-soluble salt of Lys01, effectively accumulates within the lysosome to concurrently deacidify and inhibit autophagy [472] .

Other Molecules

Compounds that inhibit and activate autophagy are depicted in Figure 7 . Compounds that inhibit and activate autophagy. Autophagy inhibitors: 3-methyladenine inhibits PI3K. Bafilomycin A1 causes dissociation of the Beclin 1-Vps34 complex and prevents the formation of autolysosome. Chloroquine/hydroxychloroquine, NH4Cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. Leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. Autophagy activators: rapamycin inhibits the mTOR. RAD001 and AP23573 are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. Trehalose causes LC3-I to LC3-II conversion in an mTOR-independent pathway. Valproic acid increases LC3-II and Beclin 1 concentrations.

Figure 7.

Compounds that inhibit and activate autophagy. Autophagy inhibitors: 3-methyladenine inhibits PI3K. Bafilomycin A1 causes dissociation of the Beclin 1-Vps34 complex and prevents the formation of autolysosome. Chloroquine/hydroxychloroquine, NH 4 Cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. Leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. Autophagy activators: rapamycin inhibits the mTOR. RAD001 and AP23573 are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. Trehalose causes LC3-I to LC3-II conversion in an mTOR-independent pathway. Valproic acid increases LC3-II and Beclin 1 concentrations.

Concluding Remarks and Future Perspectives

Efforts are being made to explore the positive aspects of autophagy to overcome various health and disease problems. Novel therapeutics and interventions are being investigated to counter autophagy-associated diseases, including apoptosis inhibitors and activators. Excessive autophagy stimulation may cause self-destruction which could be controlled with drugs such as vacuolar-type H (+)-ATPase inhibitors, cycloheximide, lysosome alkalizers (chloroquine, hydroxychloroquine, NH 4 Cl, and neutral red), and acidic protease inhibitors (E64d and pepstatin A), whilst knocking down Beclin 1 and Atg5 using MiR-30a could be used to inhibit the excessive cannibalism caused by autophagy. Upregulating autophagy could therapeutically benefit a range of diseases caused by reduced neuronal apoptosis, including the neurodegeneration-associated impairment of learning/memory capabilities, motor dysfunction, seizures, adult stroke, neonatal asphyxia, cardioskeletal myopathy, and cancers. Autophagy could also prevent various bacterial and viral diseases, inflammatory and autoimmune conditions, and also increase lifespan. Rapamycin, small-molecular enhancers of rapamycin (AUTEN-9), trehalose, IMPase inhibitors (carbamazepine and valproic acid), epigenetic modulators (anacardic acid, curcumin, garcinol, and spermidine), and chemicals (fluoxetine, penitrem A, and metformin) are all autophagy stimulators which help to ameliorate disorders associated with reduced autophagy.
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INTRODUCTION

http://e-aair.org history of cough and dyspnea. He had had a mild cough for 1 week before a sudden deterioration in his condition. The dyspnea increased despite treatment, and he was transferred to the Department of Pediatrics at the Asan Medical Center for further management. On arrival, he was apyrexial and tachypneic (respiratory rate, 66/min). Mild subcostal retraction was observed, and coarse breath sounds without crackles were noted on auscultation. Arterial blood gas analysis (ABGA) showed mild hypoxemia. His white blood cell (WBC) count was 14,300/ μL, with 55.8% neutrophils and 36.3% lymphocytes. Radiography and computed tomography (CT) of the chest revealed the presence of fine peribronchial ground-glass opacities in both lungs ( Fig. 1A and 1B) . Blood, bronchoalveolar lavage (BAL) fluid, and sputum cultures were negative for bacteria, viruses, and fungi. A lung biopsy performed on day 3 of admission showed the organizing phase of DAD distributed mainly in the centrilobular area, with destruction and obliteration of bronchioles by fibroblasts (Fig. 1E ). The patient was administered intravenous corticosteroids (2 mg/kg/day), followed by oral prednisolone (which was gradually tapered), hydroxychloroquine, and oral cyclophosphamide. His condition gradually improved, although exercise intolerance persisted. At the 1-year follow-up, a repeat CT scan of the chest revealed a decrease in the extent of ground-glass opacities in the affected areas of both lungs ( Fig. 1C and 1D ).

Family 2, Case 3

A previously healthy 35-month-old girl was referred to our medical center for severe dyspnea. Four weeks prior to admission, she had developed a mild cough, and her oral intake had become poor. Four days prior to admission, she had developed sudden onset dyspnea and subcutaneous emphysema. On admission, she presented with marked tachypnea (respiratory rate, 80/min) and chest retraction, and rales were audible in both lungs on auscultation. Radiography and a CT scan of the chest showed an extensive pneumomediastinum, a pneumothorax in the right hemithorax, and diffuse ground-glass opacities with consolidation in both lungs ( Fig. 2A and 2B ). Her total leukocyte count was 9,300/μL, with 66% neutrophils and 27% lymphocytes. Blood, sputum, and BAL cultures for bacteria and fungi were negative. Multiplex RT-PCR of nasopharyngeal aspirates revealed the presence of respiratory syncytial virus (RSV). She was admitted to the intensive care unit and treated with steroids, cyclophosphamide, hydroxychloroquine, and broad-spectrum antibiotics. Her response to treatment was poor, and her condition deteriorated rapidly. On the third day of admission, mechanical ventilation was required for impending respiratory failure. Repeated chest radiography revealed the presence of increased opacities in both lungs (Fig. 2C ). She then developed refractory hypoxemic and hypercarbic respiratory failure with subsequent multiorgan dysfunction. Despite the administration of antifibrotics and supportive therapy, she died 70 days after admission. A postmortem lung biopsy revealed the features of the fibrotic phase of DAD with occasional hyaline membrane and type II pneumocyte hyperplasia (Fig. 2D ).

Family 2, Case 5

A 17-month-old girl presented to the out-patient clinic at our medical center with a 30-day history of worsening cough. Her older sister (Case 3) had been admitted to the intensive care unit at the Asan Medical Center with respiratory failure 1 month previously, and her mother (Case 4) had had similar symptoms during the previous 45 days. On physical examination, the patient was apyrexial, tachycardic, and tachypneic (respiratory rate, 40/min). Her WBC count was 21,900/μL, with 48% neutrophils and 43% lymphocytes. Blood, sputum, and BAL fluid cultures were negative for bacteria, viruses and fungi. However, multiplex RT-PCR of nasopharyngeal aspirates revealed the presence of rhinovirus. Radiography and a CT scan of the chest showed the presence of centrilobular ground-glass opacities and septal thickening in both lungs. She was treated with steroids, cyclophosphamide, and hydroxychloroquine for 2 weeks. She showed significant clinical improvement, and repeat chest radiography 2 weeks after the initiation of treatment revealed that the ground-glass opacities, septal thickening, and consolidation had decreased. After discharge, she did not require oxygen, and her daily activities were not restricted, although she continued to experience dyspnea on exertion.

DISCUSSION

Although there is no proven effective therapy for unclassified interstitial pneumonia with fibrosis, recommended treatments include supportive care and immunosuppressive therapies such as corticosteroids, cyclophosphamide, and vincristine. 7,10 These treatments were found to be most effective during the early proliferative phase, i.e., before the development of fibrosis in the late fibrotic phase. 9 Early treatment with corticosteroids, cyclophosphamide, and hydroxychloroquine, a combination designed to inhibit fibrogenesis, might have contributed to the more favorable outcome in Case 1 of the present series. In addition, the presence of a pneumothorax and/or a pneumomediastinum on chest radiography (case 3) and the severity of the respiratory difficulty, both reflecting disease severity, may predict poor response to treatment.
3 section matches

Abstract

The present review is aimed at investigating whether chloroquine could combat the present Ebola virus epidemic, and also at exploring the main reasons for the reported lack of efficacy. Literature was sourced from PubMed, Scopus, Google Scholar, reference list of articles and textbooks -Fields Virology (Volumes 1and 2), the cytokine handbook, Pharmacology in Medicine: Principles and Practice, and hydroxychloroquine and chloroquine retinopathy.
The present analysis concludes that (1) chloroquine might find a place in the treatment of Ebola, either as a monotherapy or in combination therapies; (2) the ineffectiveness of chloroquine, or its analogue, hydroxychloroquine, at treating infections from low pH-dependent viruses is a result of the failure to attain and sustain a steady state concentration sufficient to increase and keep the pH of the acidic organelles to approximately neutral levels; (3) to successfully treat filoviral infectionsor other viral infections that emerge or emerged from low pH-dependent virusesa steady state chloroquine plasma concentration of at least 1 μg/mL(~3.125 μM/L) or a whole blood concentration of 16 μM/L must be achieved and be sustained until the patients' viraemia becomes undetectable. These concentrations, however, do not rule out the efficacy of other, higher, steady state concentrationsalthough such concentrations might be accompanied by severe adverse effects or toxicities. The feasibility of the conclusion in the preceding texts has recently been supported by a subsequent study that shows that amodiaquine, a derivative of CQ, is able to protect humans infected with Ebola from death.

INTRODUCTION

The CQ-increased pH of the acidic organelles has been shown to inhibit several virusesincluding influenza A and B, SARS coronavirus, hepatitis A virus and the Borna disease viruswhich all require a low pH for entry. 24, [34] [35] [36] [37] [38] [39] [40] This suggests that CQ could also inhibit filoviruses' entry into the cytoplasm of susceptible cells and thereby abrogate their infection, since this is dependent on endosomal acidification and the activities of several host endosomal proteases. CQ might also inhibit the assembly and budding of filoviruseswhich partly require the late endosome in their assembly. 1, 2 Accordingly, the dysfunction of enzymes, e.g. some glycosyltransferases, caused by CQ or hydroxychloroquine (HCQ), as a direct result of the increased pH and/or structural changes in the Golgi apparatus, has been shown to inhibit not only the glycosylation of SARS coronavirus, 34 but also that of HIV-1 gp120, thereby leading to the production of noninfectious virions or virus with decreased infectivity. 31, 33, 40, 41 This mechanism has been invoked to explain the decrease in viral load that was observed when patients with HIV-1 were orally administered HCQ at 800 mg/day for 8 weeks. 31, 42 These results, though obtained with nonrelated viruses, could suggest that CQ, if given at the correct dosage, might inhibit the glycosylation of EboV peplomers, which is more pronounced than that occurring with HIV-1. [1] [2] [3] 43 Since the GP of filoviruses is the only protein involved in initiating infection, 1,2,5,6,8 and cytotoxicity is dependent on its expression, 1,2,5 inhibiting its glycosylation could potentially (1) inhibit EboV tropism for a broad variety of host cells and organs; (2) lead to the production of noninfectious or decreased infectivity virus (as seen with HIV-1) 31,33 ; and (3) decrease Ebov pathogenicity. Impaired glycosylation could therefore save time for the adaptive immune response, which normally fails in fatal cases, 1,2,11 to be established and deal with the infection.
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Discussion

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.
Chloroquine is widely distributed to body tissues as well as its analogue hydroxychloroquine. The concentration of hydroxychloroquine in the brain is 4-30 times higher than in the plasma [46] . The concentration of chloroquine in the plasma reached 10 µM when a daily intake of 500 mg was prescribed to arthritis patients [47] . Chloroquine is able to cross the placental barrier and is supposed to reach similar concentrations in both maternal and fetal plasma [48] . Concentrations of chloroquine, similar to the EC50 values calculated here (Table 1) , are achieved in the plasma in current chloroquine administration protocols and might reach the brain.
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Antiparasitic Drugs

Among drugs correlated with chloroquine, amodiaquine, hydroxychloroquine, and aminoquinoline have been shown to inhibit filovirus infection in vitro using a pseudotyped virus assay and the authentic EBOV [75] . Although no in vivo experiments have been undertaken yet, a promising result was obtained by a retrospective analysis performed on patients treated in Liberia with artesunate-amodiaquine during the Western Africa outbreak of EVD. In fact, these patients showed a lower risk of death from EVD than patients treated with artemether-lumefantrine. Although this observation lacks of several controls, the clinical effect of the artesunate-amodiaquine treatment should be better investigated as a possible therapeutic option for patients with EVD [78] .

Antibiotics and Antifungal Drugs

Teicoplanin, a glycopeptide antibiotic, and its derivatives potently inhibit the entry of EBOV-GP-pseudotyped viruses in various cell types [83, 84] . Studies on the antiviral mechanism indicated that teicoplanin blocks EBOV entry by specifically inhibiting the activity of cathepsin L, thus avoiding the maturation of GP and the release of the viral genome into the cytoplasm [83] . The antibiotic azithromycin has been demonstrated to inhibit eVLP entry but further studies have not been performed and its mechanism of action is still largely uncharacterized [82] .
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Abstract

Dengue virus causes dengue fever, a debilitating disease with an increasing incidence in many tropical and subtropical territories. So far, there are no effective antivirals licensed to treat this virus. Here we describe the synthesis and antiviral activity evaluation of two compounds based on the quinoline scaffold, which has shown potential for the development of molecules with various biological activities. Two of the tested compounds showed dose-dependent inhibition of dengue virus serotype 2 in the low and sub micromolar range. The compounds 1 and 2 were also able to impair the accumulation of the viral envelope glycoprotein in infected cells, while showing no sign of direct virucidal activity and acting possibly through a mechanism involving the early stages of the infection. The results are congruent with previously reported data showing the potential of quinoline derivatives as a promising scaffold for the development of new antivirals against this important virus. Molecules 2018, 23, 672 2 of 11 antivirals might be very useful in the case of severe dengue, as high viremia has been shown to correlate with severity [7]. Quinolines are heterocyclic molecules composed of fused benzene and pyridine rings. The quinolines and their derivatives have shown a wide range of biological activities, including antiproliferative [8], antiviral [9], antibacterial [10], antifungal [11], anti-inflammatory [12], and antiparasitic [13]. Members of the quinoline family, such as chloroquine and hydroxychloroquine, have shown antiviral activity against several viruses, such as coronaviruses [9], human immunodeficiency virus [14], and respiratory syncytial virus [15]. Concerning Flavivirus, quinoline derivatives have proved active against the Hepatitis C virus [16], West Nile virus [17,18], Japanese Encephalitis virus [19], Zika virus [20], and dengue virus [21].

Introduction

Quinolines are heterocyclic molecules composed of fused benzene and pyridine rings. The quinolines and their derivatives have shown a wide range of biological activities, including antiproliferative [8] , antiviral [9] , antibacterial [10] , antifungal [11] , anti-inflammatory [12] , and antiparasitic [13] . Members of the quinoline family, such as chloroquine and hydroxychloroquine, have shown antiviral activity against several viruses, such as coronaviruses [9] , human immunodeficiency virus [14] , and respiratory syncytial virus [15] . Concerning Flavivirus, quinoline derivatives have proved active against the Hepatitis C virus [16] , West Nile virus [17, 18] , Japanese Encephalitis virus [19] , Zika virus [20] , and dengue virus [21] .
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ZIKV Replication Inhibitors

Li Y. et al. have found that a pyrazole ester derivative, 5-amino-1-((4-methoxyphenyl)sulfonyl) -1H-pyrazol-3-yl benzoate [ Figure 3(25) ], can inhibit ZIKV replication with an IC 50 of 1.5 µM when benzoyl-Nle-Lys-Arg-Arg-aminomethylcoumarin (BznKRR-AMC) was used as a substrate (Table 3 ; Li Y. et al., 2018) . The benzoyl group of this inhibitor forms a covalent bond with the side chain of catalytic residue S135 to stabilize the closed conformation of the ZIKV bZiPro construct of NS2B-NS3 protease . In addition, a derivative of pyrazole ester, 5-amino-1-((4-methoxyphenyl)sulfonyl)-1H-pyrazol-3-yl benzoate, with an IC 50 of 0.1 µM can interact in a manner similar to the compound [ Figure 3(25) ] and strongly inhibit binary ZIKV bZiPro construct of NS2B-NS3 protease . Hydroxychloroquine [ Figure 3(26) ], a drug already approved and used in pregnancy, can inhibit the bZiPro construct of NS2B-NS3 protease activity with an inhibition constant (Ki) of 92.34 ± 11.91 µM ( Table 3 ; Kumar et al., 2018) .
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Potential Therapeutics

Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60.