Preparing for the next viral threat with broad-spectrum antivirals

The cellular machineries co-opted to support the life cycle of viruses are often conserved across viral families, representing attractive targets for broad-spectrum antiviral strategies. With approximately 20,000 proteins, the human proteome offers a much larger repertoire of candidate targets than a viral proteome. Indeed, the discovery of such proviral factors required by multiple viruses has been the subject of fruitful research. Aided by breakthroughs in multi-omics approaches, these efforts have led to the discovery of numerous druggable proviral factors. Some examples are discussed below.

Beyond a larger target repertoire, an important advantage of the host-targeted approach is its higher barrier to viral resistance. Since cellular targets are not under genetic control of a virus, the likelihood that escape mutations will emerge is lower than with DAAs. This advantage was demonstrated in cell culture models, such as with inhibitors targeting various cellular kinases (9698), and in animal models, such as DENV-infected mice treated with α-glucosidase inhibitors (99). In patients, cyclophilin inhibitors and other host-targeted approaches have demonstrated longer time to resistance and lower levels of resistance than DAAs (100).

Targeting cellular functions can also provide opportunities not only to suppress viral replication but also to moderate deleterious host responses, which play key roles in the pathogenesis of multiple viral infections, including dengue, EVD, and COVID-19. Targeting p38 MAPK or ErbBs, for example, as we and others have demonstrated in preclinical models, can reduce inflammation and protect from tissue injury beyond suppression of viral replication (98, 101). Another example is enhancement of type I interferon responses contributing to the protective effect of tamoxifen treatment in vesicular stomatitis virus–infected (VSV-infected) mice (102). Lastly, since most approved drugs target cellular functions, there is an opportunity to repurpose existing drugs for antiviral indications, as was extensively explored during the COVID-19 outbreak (reviewed in ref. 103).

Below are examples of classes of host-targeted approaches that show some promise (Figure 3 and Table 2).

Approved and experimental host-targeted compounds with broad-spectrum antivFigure 3

Approved and experimental host-targeted compounds with broad-spectrum antiviral activity. Depicted here is a generic viral life cycle. Examples of classes of inhibitors with broad-spectrum antiviral activity are connected to the specific stage(s) of the viral life cycle or cellular process they target.

Table 2

Classification, cellular targets, and antiviral activity in vitro, in vivo, and in clinical models of host-directed compounds with broad-spectrum antiviral activity

Targeting protein folding and transport. Cyclosporin A (CsA) and experimental non-immunosuppressive inhibitors of cyclophilin A (CypA) — a cellular factor involved in protein folding — such as alisporivir (Debio-025) and SCY-635, suppress the replication of multiple viruses in vitro (104). Blockage of interactions between CypA and the HIV-1 nucleocapsid and HCV NS5A proteins is thought to mediate the antiviral effect (105, 106). Other mechanisms of antiviral action were reported, including suppression of HBV binding to its entry receptor (107), of coronaviral RNA synthesis (104), and of nuclear import of IAV genome (108). The effect of these compounds in mouse models has been variable (100), yet prevention of disease progression was demonstrated in mice infected with coronaviruses (109). Accordingly, transplant recipients receiving CsA treatment for their underlying condition experienced reduced morbidity and mortality upon SARS-CoV-2 infection (110). Whereas alisporivir significantly reduced viremia in chronically infected HCV patients, a phase III trial was terminated due to toxicity.

α-Glucosidase is another protein required for proper folding of proteins — including viral glycoproteins — that serves as a broad-spectrum antiviral target. Celgosivir and other iminosugars are competitive substrates for α-glucosidases with activity against multiple viruses in cultured cells (111). These inhibitors have demonstrated efficacy in murine models of RNA and DNA viruses (111, 112). The utility of celgosivir for the treatment of dengue infection is currently being explored, although safety but little or no efficacy have been documented to date in a dengue pilot study and in patients infected with HCV or HIV-1 (113115).

The molecular chaperones heat shock protein 70 (HSP70) and HSP90, involved in protein folding and transport, are also broadly required factors shown to function at temporally distinct stages of viral life cycles (116, 117). Stabilization and transport of viral proteins were among the proposed underlying mechanisms (117, 118). Pharmacological inhibition of HSP70 by TH3289 blocked replication of flaviviruses, coronaviruses, and Crimean-Congo hemorrhagic fever virus in vitro (116, 119). In murine models of ZIKV and Chikungunya virus (CHIKV) infections, small-molecule inhibitors of these chaperones reduced viral titers, inflammation, and/or mortality (120, 121). While thus far demonstrated with tool compounds only, these examples provide evidence for the potential utility of targeting HSPs.

Oligosaccharyltransferase (OST), an endoplasmic reticulum protein complex that catalyzes N-glycosylation, was discovered as a candidate antiviral target via CRISPR screens for flaviviral proviral factors (122). OST subunits interact with DENV nonstructural proteins and are required for viral RNA replication (122). NGI-1, a small-molecule inhibitor of OST, has shown antiviral activity against flaviviruses and more recently HSV-1 and SARS-CoV-2 (123125). Interestingly, whereas the anti-DENV activity is independent of the canonical role of OST in N-linked glycosylation, the anti-IAV effect is associated with reduced hemagglutinin (HA) and neuraminidase (NA) glycosylation (123). A concern was recently raised that glycome-modified viruses generated upon NGI-1 treatment can reduce antibody responses in IAV-infected mice and requires further investigation (126).

Targeting cellular kinases. Multiple cellular kinases are hijacked by viruses, representing candidate targets for broad-spectrum antivirals (127). The epidermal growth factor receptor family of tyrosine kinases (ErbB1, 2, 4) is one example. A requirement for ErbBs was documented in the entry and/or post-entry stages of multiple viruses (128). Several anticancer ErbB inhibitors, including gefitinib, demonstrate activity against HCV, human cytomegalovirus (HCMV), poxvirus, and Lassa virus in cultured cells (129133), and CMV in guinea pigs (132). In human lung and brain organoid models of SARS-CoV-2 and VEEV infections, respectively, we have recently shown that, beyond suppressing viral replication, lapatinib, an anticancer pan-ErbB inhibitor, protects from virus-induced activation of pathways implicated in non-infectious tissue injury downstream of ErbBs, proinflammatory cytokine production, and epithelial or blood-brain barrier injury (98). Moreover, we have validated ErbB inhibition as the mechanism of antiviral action (98). Remarkably, ibrutinib, a BTK inhibitor with potent pan-ErbB activity (134), has demonstrated protection from progression to severe COVID-19, albeit in a small number of patients (135), highlighting that clinical evaluation of these ErbB inhibitors is warranted.

The numb-associated (serine/threonine) kinases (NAKs) — AAK1, BIKE, GAK, and STK16 — have also been studied as targets for broad-spectrum antivirals. We have demonstrated a requirement for NAKs in the regulation of intracellular cotrafficking of specific cellular cargo adaptor proteins with viral particles during entry, assembly, and/or release of HCV, DENV, EBOV, and SARS-CoV-2 (96, 97, 136, 137). Approved anticancer drugs with potent anti-NAK activity, including sunitinib-erlotinib combinations, 5Z-7-oxozeaenol, and chemically distinct more selective inhibitors, demonstrate broad-spectrum antiviral activity against eight viral families in vitro (96, 97, 137139). A combination treatment with sunitinib-erlotinib was shown to protect mice from DENV and EBOV challenges (96, 138). Inhibition of intracellular membrane trafficking regulated by NAKs was validated as an important mechanism of antiviral action (96, 97, 140). The safety and efficacy of NAK inhibition for the treatment of viral infections in humans remain to be determined.

Lipid kinases have also been shown to be required for effective replication of multiple viruses. For example, the endosomal phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) (141) has been implicated in the entry of filoviruses, Lassa virus, and coronaviruses (142). The PIKfyve inhibitors apilimod and YM201636 suppress trafficking and maturation of endolysosomes, preventing viral fusion and/or egress (142, 143). Apilimod is currently being studied as a COVID-19 therapeutic (NCT04446377). Whereas a suboptimal pharmacokinetic profile (144, 145) limits its development, the excellent safety profile demonstrated with apilimod in clinical trials for inflammatory diseases has de-risked PIKfyve as a target (146, 147). While two chemically distinct small molecules with anti-PIKfyve activity were recently shown to increase SARS-CoV-2–induced pathology in a mouse model, since their selectivity has not been reported, it is possible that other targets have mediated this effect (148). Further evaluation of the potential of PIKfyve inhibition in other animal models and ideally human organoid models is therefore warranted. Pharmacological inhibition of other lipid and protein kinases by approved and investigational compounds has also shown promise in vitro with variable results in animal models (reviewed in ref. 149).

Targeting cellular proteases. Proteases are another group of cellular enzymes co-opted by viruses. Influenza viruses and coronaviruses, for example, rely on proteases, such as TMPRSS2 and cathepsins, for cleavage and activation of their surface glycoproteins (150, 151). Among cellular protease inhibitors showing antiviral activity, camostat mesylate and nafamostat mesylate, oral serine protease inhibitors approved for the treatment of chronic pancreatitis and other conditions (152), have shown TMPRSS2-dependent suppression of viral fusion in vitro (151) and protection in mouse models of IAV and coronaviral infections (153, 154). However, when studied for the treatment of COVID-19 patients, these compounds had no significant impact on clinical outcomes (155, 156). Thus, the evaluation of other strategies targeting cellular proteases for the treatment of viral infections is warranted.

Targeting lipid metabolism. Cholesterol-lowering drugs, like statins, have demonstrated in vitro activity against HCV, attributed to their effect on lipid biosynthesis. Indeed, antiviral activity in cells was reversed upon addition of mevalonate or geranylgeraniol, and resistance to these drugs coincided with an increase in HMG-CoA reductase level — statins’ target (157). Nevertheless, a variable, modest, and short-lived effect was demonstrated in HCV patients when statins were combined with peginterferon-ribavirin (158). Beyond HCV, statins have demonstrated efficacy in animal models of multiple viral infections, including respiratory viruses, CMV, HIV-1, and DENV (159, 160). Owing to their ability to restore endothelial stability, statins were used, albeit in a non-formal study, in combination with an angiotensin receptor blocker for treating EVD, an infection whose pathogenesis is associated with endothelial dysfunction — showing reduced mortality in 100 patients (161). Recently, reduced morbidity and mortality were documented also in COVID-19 patients with statin prescriptions, albeit in observational studies only (162). Inhibitors of proprotein convertase subtilisin kexin type 9 (PCSK9), such as the monoclonal antibodies alirocumab and evolocumab, represent another class of lipid-lowering agents shown to suppress DENV replication in vitro and reduce mortality and inflammation in severe COVID-19 patients (163, 164). Whereas statins showed no antiviral activity in dengue patients (165), PCSK9 inhibitors may offer greater protection given the recent discovery that PCSK9 expression is induced by DENV infection in cells residing in physiologically hypoxic conditions and is increased in severe dengue patients, reducing cholesterol uptake and dampening susceptibility to statins (163).

Host-targeted approaches with complex mechanisms of action. Metformin, an approved oral drug for the treatment of diabetes, has demonstrated potent antiviral activity against multiple viruses in vitro. Activation of AMP-activated protein kinase–dependent (AMPK-dependent) type I interferon signaling was proposed as an underlying mechanism in DENV and HCV infections (166, 167). Metformin reduced morbidity and mortality in mice infected with DENV and IAV, but not ZIKV (168, 169). Diabetic patients on metformin treatment were found to have lower morbidity and mortality upon influenza virus infection (170) and a trend toward reduced mortality when infected with SARS-CoV-2 (171). Contrastingly, metformin showed no clinical benefit in nondiabetic COVID-19 patients (172). The therapeutic potential of metformin in reducing HIV-1 reservoirs and combating DENV infection is currently being studied clinically (173, 174).

Tamoxifen and other inhibitors of the estrogen receptor (ER) approved for the treatment of breast cancer inhibit the replication of multiple RNA and DNA viruses in vitro (175). The proposed mechanisms of antiviral action include blockage of a chloride channel required for HSV-1 entry; endosomal/lysosomal proteins required for EBOV entry; SARS-CoV-2 spike-mediated membrane fusion (176, 177); and binding of ER to HCV and CHIKV polymerases (178, 179). In rodent models of VSV, EBOV, CHIKV, and SARS-CoV-2 infections, treatment with ER antagonists reduced viral titers, inflammation, and/or mortality (175, 179, 180). Treatment with ER antagonists in humans shortened the time of SARS-CoV-2 shedding (181), reduced HCV viremia but not the resulting liver inflammation (NCT00749138), and did not impact HIV-1 viremia (182). Thus, further studies are required to define the clinical utility of ER antagonists as antivirals.

Nitazoxanide, approved for the treatment of parasitic infections, is another candidate drug for repurposing with a complex mechanism of antiviral action. Nitazoxanide suppresses replication of multiple RNA viruses in vitro and in vivo (183, 184). While the precise target remains unknown, several mechanisms of action have been proposed, such as blocking of the maturation of the influenza hemagglutinin (185) and the coronaviral spike proteins (186, 187) and, in the case of HCV and HBV infections, blocking of protein kinase R–mediated phosphorylation of eIF2α (183, 188). Nitazoxanide modestly reduced the time to resolution of flu symptoms in a phase II trial and is currently being evaluated in a phase III trial for this indication (189). Whereas the addition of nitazoxanide to peginterferon-ribavirin improved sustained virologic responses in HCV patients in a phase II trial (190), no such improvement was observed in a phase III trial in genotype 4–infected patients (191). In a recent randomized, double-blind pilot study in 50 COVID-19 patients, nitazoxanide shortened hospitalization, accelerated viral clearance, and reduced inflammatory cytokine production (192), warranting a larger-scale study.

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