Back-Pocket Optimization of 2-Aminopyrimidine-Based Macrocycles Leads to Potent EPHA2/GAK Kinase Inhibitors.

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here we have optimized 2-aminopyrimidine-based macrocycles to use these compounds as chemical tools for the ephrin kinase family. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55, a potent inhibitor of EPHA2/A4 and GAK. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/A4 and GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells. However, further investigations are needed to determine whether this was a compound-specific effect or target-related.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for antiviral strategies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

Discovery of 3-phenyl- and 3-N-piperidinyl-isothiazolo[4,3-b]pyridines as highly potent inhibitors of cyclin G-associated kinase.

Structural modifications at position 3 of the isothiazolo[4,3-b]pyridine scaffold afforded a new series of cyclin G-associated kinase (GAK) inhibitors. It was shown that the insertion of a carboxamide residue at position 3 of a phenyl or piperidinyl moiety generated potent GAK inhibitors with IC50 values in a low nanomolar range. This potent GAK binding affinity was rationalized by molecular modelling demonstrating that the carboxamide moiety engages in an extra hydrogen bond with GAK. Moreover, this new series of compounds was also endowed with antiviral activity against dengue virus, highlighting the potential utility of GAK as a target for the development of antiviral drugs.

BIKE regulates dengue virus infection and is a cellular target for broad-spectrum antivirals.

Global health is threatened by emerging viruses, many of which lack approved therapies and effective vaccines, including dengue, Ebola, and Venezuelan equine encephalitis. We previously reported that AAK1 and GAK, two of the four members of the understudied Numb-associated kinases (NAK) family, control intracellular trafficking of RNA viruses. Nevertheless, the role of BIKE and STK16 in viral infection remained unknown. Here, we reveal a requirement for BIKE, but not STK-16, in dengue virus (DENV) infection. BIKE mediates both early (postinternalization) and late (assembly/egress) stages in the DENV life cycle, and this effect is mediated in part by phosphorylation of a threonine 156 (T156) residue in the µ subunit of the adaptor protein (AP) 2 complex. Pharmacological compounds with potent anti-BIKE activity, including the investigational anticancer drug 5Z-7-oxozeaenol and more selective inhibitors, suppress DENV infection both in vitro and ex vivo. BIKE overexpression reverses the antiviral activity, validating that the mechanism of antiviral action is, at least in part, mediated by BIKE. Lastly, 5Z-7-oxozeaenol exhibits antiviral activity against viruses from three unrelated RNA viral families with a high genetic barrier to resistance. These findings reveal regulation of poorly understood stages of the DENV life cycle via BIKE signaling and establish a proof-of-principle that pharmacological inhibition of BIKE can be potentially used as a broad-spectrum strategy against acute emerging viral infections.

Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics.

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified J9 and J8, two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies showed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against DENV with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested that J9 and J8 followed divergent somatic hypermutation pathways, and that a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a new epitope that can be exploited for vaccine design.

MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment.

The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design.

Feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment.

There is an urgent need for strategies to combat dengue virus (DENV) infection; a major global threat. We reported that the cellular kinases AAK1 and GAK regulate intracellular trafficking of multiple viruses and that sunitinib and erlotinib, approved anticancer drugs with potent activity against these kinases, protect DENV-infected mice from mortality. Nevertheless, further characterization of the therapeutic potential and underlying mechanism of this approach is required prior to clinical evaluation. Here, we demonstrate that sunitinib/erlotinib combination achieves sustained suppression of systemic infection at approved dose in DENV-infected IFN-α/ß and IFN-γ receptor-deficient mice. Nevertheless, treatment with these blood-brain barrier impermeable drugs delays, yet does not prevent, late-onset paralysis; a common manifestation in this immunodeficient mouse model but not in humans. Sunitinib and erlotinib treatment also demonstrates efficacy in human primary monocyte-derived dendritic cells. Additionally, DENV infection induces expression of AAK1 transcripts, but not GAK, via single-cell transcriptomics, and these kinases are important molecular targets underlying the anti-DENV effect of sunitinib and erlotinib. Lastly, sunitinib/erlotinib combination alters inflammatory cytokine responses in DENV-infected mice. These findings support feasibility of repurposing sunitinib/erlotinib combination as a host-targeted antiviral approach and contribute to understanding its mechanism of antiviral action.

Combating Intracellular Pathogens with Repurposed Host-Targeted Drugs.

There is a large, global unmet need for the development of countermeasures to combat intracellular pathogens. The development of novel antimicrobials is expensive and slow and typically focuses on selective inhibition of proteins encoded by a single pathogen, thereby providing a narrow spectrum of coverage. The repurposing of approved drugs targeting host functions required for microbial infections represents a promising alternative. This review summarizes progress and challenges in the repurposing of approved drugs as host-targeted broad-spectrum agents for the treatment of intracellular pathogens. These strategies include targeting both cellular factors required for infection by various viruses, intracellular bacteria, and/or protozoa as well as factors that modulate the host immune response to these microbial infections. The repurposed approach offers complementary means to develop therapeutics against existing and emerging intracellular microbial threats.

Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects.

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

Pathogen receptor discovery with a microfluidic human membrane protein array.

The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

Selective Inhibitors of Cyclin G Associated Kinase (GAK) as Anti-Hepatitis C Agents.

Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson's disease).

AP-2-associated protein kinase 1 and cyclin G-associated kinase regulate hepatitis C virus entry and are potential drug targets.

UNLABELLED: Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the µ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved anticancer drugs sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown-HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), an HCV entry cofactor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, each a substrate of AAK1 and/or GAK, or overexpression of either an AP2M1 or NUMB phosphorylation site mutant inhibited HCV entry. Last, in addition to affecting assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively. IMPORTANCE: Understanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1- and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV life cycle, AAK1 and GAK represent "master regulators" of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK inhibit HCV entry in addition to assembly. These results contribute to an understanding of the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications for other viruses that hijack clathrin-mediated pathways.

B-cell receptors expressed by lymphomas of hepatitis C virus (HCV)-infected patients rarely react with the viral proteins.

Chronic hepatitis C virus (HCV) infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this derives from clinical observations of tumor regressions upon antiviral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon antiviral treatments. We expressed the lymphoma B-cell receptors as soluble immunoglobulin Gs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus.

Identification and targeting of an interaction between a tyrosine motif within hepatitis C virus core protein and AP2M1 essential for viral assembly.

Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the µ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.

The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection.

UNLABELLED: Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular transformation are poorly understood. We hypothesized that the guanosine triphosphatase activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play a role in the transformation process. Here we report that NS4B can transform NIH-3T3 cells, leading to tumor formation in vivo. This transformation was independent of co-transfection with activated Ha-ras. Detailed analyses of NS4B mutants revealed that this transforming activity could be progressively inhibited and completely abrogated by increasing genetic impairment of the NS4B nucleotide binding motif. CONCLUSION: NS4B has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is indeed mediated by its NBM. Moreover, our results suggest that pharmacological inhibition of the latter might inhibit not only HCV replication but also the associated HCC.