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Research

Interests

We are a multi-disciplinary research group with broad interests in drug discovery and pharmacology. The goal is to develop first-in-class chemical probes for target validation and translational drug development. In addition, we are also interested in understanding the mechanism of drug resistance and aim to develop drugs with a high genetic barrier to drug resistance. A key component of our research program is developing biochemical and cellular assays for a diverse set of drug targets, including but not limited to ion channels, protein-protein interactions, enzymes, and capsid proteins. We exploit high-throughput screening and structure-based drug design to inform the lead optimization through iterative cycles of design, synthesis, and pharmacological characterization.

1. Drug Discovery and Pharmacology of SARS-CoV-2 Antivirals

To combat the COVID-19 pandemic, we are pursuing several drug targets including the main protease (Mpro or 3CLpro), the papain-like protease (PLpro), the nucleoprotein, and the polymerase. In addition, we have developed FlipGFP and protease-Glo assays for the validation and invalidation of literature reported SARS-CoV-2 Mpro and PLpro inhibitors.

  • Discovery of SARS-CoV-2 PLpro inhibitors. The papain-like protease (PLpro) of SARS-CoV-2 is a validated yet challenging antiviral drug target (ACS Cent Sci 2021). Our lab has been at the forefront of PLpro drug discovery, pioneering the development of first-in-class PLpro inhibitors with demonstrated in vivo antiviral efficacy, including Jun12682 and Jun13296 (Science 2024). This success was enabled by our identification of a novel binding site, Val70Ub, which opened new avenues for inhibitor design. We are currently focused on lead optimization to enhance potency, pharmacokinetics, and safety, with the goal of advancing PLpro inhibitors into IND-enabling studies for clinical development.
  • Discovery of novel SARS-CoV-2 Mpro inhibitors. At the onset of the COVID-19 pandemic, we identified several promising SARS-CoV-2 main protease (Mpro) inhibitors through screening, including boceprevir, GC-376, and calpain inhibitors II and XII. Notably, boceprevir and calpain inhibitors II and XII represent novel chemotypes distinct from existing GC-376 analogs. All four compounds exhibited potent enzymatic inhibition and robust cellular antiviral activity against Mpro and infectious SARS-CoV-2 (Cell Res 2020). In collaboration with Dr. Yu Chen’s group at the University of South Florida, we determined multiple high-resolution X-ray crystal structures of Mpro bound to these inhibitors (Sci Adv 2021), providing critical insights for the rational design of next-generation Mpro inhibitors (ACS Pharmacol Transl Sci 2021). The identification of boceprevir as an Mpro inhibitor also contributed to the design of nirmatrelvir. Our current efforts focus on developing novel Mpro inhibitors effective against SARS-CoV-2 variants with resistance to nirmatrelvir and/or ensitrelvir.
  • Rational design of non-covalent SARS-CoV-2 Mpro inhibitors. By integrating structure-based drug design with the one-pot Ugi four-component reaction, we developed one of the most potent noncovalent SARS-CoV-2 Mpro inhibitors, 23R (Jun8-76-3A), which features a scaffold distinct from the canonical inhibitor GC-376. Importantly, 23R exhibits high selectivity, particularly over host proteases, compared to covalent inhibitors like GC-376. The co-crystal structure of SARS-CoV-2 Mpro bound to 23R revealed a previously unexplored binding site situated between the S2 and S4 pockets, offering new opportunities for the design of next-generation noncovalent Mpro inhibitors. (J Med Chem 2021).
  • Development of assays for SARS-CoV-2 Mpro and PLpro hit validation/invalidation. A variety of structurally diverse compounds have been reported as SARS-CoV-2 Mpro inhibitors, raising concerns about their target specificity. To address this, we systematically evaluated the mechanism of action and cellular target engagement of these claimed inhibitors using a comprehensive panel of assays, including a cell-free FRET assay, thermal shift binding assay, cell lysate Protease-Glo luciferase assay, and cell-based FlipGFP assay. Our findings revealed that many repurposed compounds, such as ebselen, carmofur, disulfiram, and shikonin, act as promiscuous cysteine modifiers rather than specific Mpro inhibitors, while others—including chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole—showed no Mpro inhibition in any assay. This study underscores the critical importance of rigorous hit validation early in the drug discovery process. (ACS Pharmacol Transl Sci 2020, PNAS 2021, Acta Pharm Sin B 2021).

2. Drug Discovery and Pharmacology of Enterovirus Antivirals

Enteroviruses are important human pathogens with disease manifestations ranging from mild respiratory illness to more severe neurological complications such as encephalitis, aseptic meningitis, acute flaccid paralysis/myelitis, and even death. The enterovirus genera of the Picornaviridae family contains poliovirus, rhinovirus, enterovirus A71 (EV-A71), enterovirus D68 (EV-D68), and coxsackievirus. Although poliovirus was nearly eradicated by vaccination, there are currently no antivirals or vaccines for the remaining non-polio enteroviruses (NPEVs) (reviews ACS Infect Dis 2020, Acta Pharm Sin B 2021). We are particularly interested in EV-D68, which has been recently labeled as “new ploliovirus” due to its link with neurological complications such as acute flaccid myelitis (AFM). Contemporary EV-D68 viruses appear to become more virulent and there is a biannual pattern of EV-D68 outbreak the U.S.  As there is currently no antivirals available, we aim to develop antivirals against EV-D68 by targeting essential viral proteins such as the viral capsid protein VP1, the 2A protease, the 3C protease, the 2C protein, and the viral 3D polymerase.

  • Design non-structural 2C protein inhibitors. The enterovirus non-structural 2C protein is a multifunctional and highly conserved viral protein. Interestingly, structurally disparate compounds have been reported by us and others as 2C inhibitors with broad-spectrum antiviral activity against EV-D68, EV-A71, CVB3, and poliovirus (J Med Chem 2019, J Med Chem 2021, ACS Infect Dis 2020). We aim to solve the X-ray and cryo-EM structures of 2C with inhibitors to elucidate their mechanism of action and drug resistance.
  • Validation of EV-D68 2A protease as a novel drug target. We have shown that 2A is a cysteine protease that is critical for the digestion of viral polyproteins during viral replication. We also identified the first 2A protease inhibitor telaprevir with potent enzymatic inhibition and cellular antiviral activity (J Virol 2019). We solved the X-ray crystal structures of 2A and its mutants, paving the way for structure-based lead optimization.
  • Development of VP1 capsid inhibitors. The VP1 capsid is a validated antiviral drug target and we discovered several VP1 capsid inhibitors with potent antiviral activity (ACS Infect Dis 2019). Importantly, they do not have overlapping resistance with pleconaril and have synergistic antiviral activity with the polymerase inhibitors.

3. Drug Discovery and Pharmacology of Influenza Antivirals

The efficacy of current anti-influenza drugs is limited by the emergence of multidrug-resistant influenza viruses. Adamantanes (amantadine and rimantadine) are no longer recommended for prophylaxis and treatment of influenza infection. Resistance against neuraminidase inhibitors, oseltamivir, zanamivir, and paramivir, are continuously on the rise. As influenza viruses continuously undergo antigenic shift and antigenic drift, it is imperative to develop next generation of influenza antivirals with a novel mechanism of action. We are exploring both viral proteins and host factors as drug targets. Our initial efforts have yielded several promising drug candidates including viral M2 channel blockers, viral polymerase PA-PB1 inhibitors, host kinase inhibitors, receptor antagonists, and protease inhibitors. Representative host-targeting antivirals we have discovered include cyclosporine A, dapivirine, hesperadine, and NMS-873.

  • Design influenza A virus M2-S31N and M2-V27A channel blockers. M2-S31N and M2-V27A are the predominant amantadine-resistant mutants, and we have developed the first-in-class M2-S31N and M2-V27A channel blockers with both in vitro and in vivo antiviral efficacy (Emerg Microbes Infect 2021, Antiviral Res 2017).
  • Design influenza polymerase PA-PB1 inhibitors. By targeting the viral polymerase PA and PB1 subunit interactions, we have shown that PA-PB1 inhibitors have broad-spectrum antiviral activity with a high genetic barrier to drug resistance (ACS Chem Biol 2020, Sci Rep 2018).
  • Development of host-targeting antivirals. Targeting host factors that are essential for viral replication is an orthogonal approach in combating viral infection. We have shown that cyclosporine A, dapivirine, hesperadine, and NMS-873 are host-targeting antivirals with broad-spectrum antiviral activity and a high genetic barrier to drug resistance (Antiviral Res 2017, Antiviral Res 2016).

4. Drug resistance

Drug resistance is an unavoidable challenge in antiviral therapy—it is not a question of if, but when resistance will emerge. Viral populations continually evolve under therapeutic pressure, leading to the selection of resistant variants that can undermine drug efficacy. Predicting and validating resistance mutations in the laboratory provides an unbiased and rigorous method to confirm cellular target engagement, offering definitive proof of an antiviral’s on-target activity. Moreover, these studies help forecast clinically relevant resistance pathways, informing the design of next-generation therapeutics with improved resistance profiles. Proactively addressing resistance is essential for developing durable, long-lasting antiviral agents that remain effective against evolving viral threats.

  • Structure-based prediction of naturally occurring nirmatrelvir-resistant Mpro mutations . Using a structure-based approach, we systematically analyzed naturally occurring mutations at key residues within the drug-binding site. This work identified several drug-resistance hotspots, including S144, M165, E166, H172, and Q192. Notably, multiple mutations predicted by our study, such as E166V, have since been independently confirmed in clinical isolates, underscoring the relevance and predictive power of our findings (ACS Cent Sci 2023).