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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 novel SARS-CoV-2 Mpro inhibitors. We recently identified several promising Mpro inhibitors including boceprevir, GC-376, calpain inhibitors II and XII. Boceprevir, calpain inhibiotrs II and XII represent novel Mpro inhibitors that are distinct from previously reported Mpro inhibitors such as GC-376 analogs. All four compounds showed potent enzymatic inhibition and 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 solved multiple X-ray crystal structures of Mpro with the hits (Sci Adv 2021). These high-resolution X-ray crystal structures provides a foundation for the designing of next-generation of Mpro inhibitors (ACS Pharmacol Transl Sci 2021).
  • Rational design of non-covalent SARS-CoV-2 Mpro inhibitors. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we designed one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets (J Med Chem 2021).
  • Discovery of SARS-CoV-2 PLpro inhibitors. The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PLpro inhibitors including Jun9-72-2 and Jun9-75-4 with improved enzymatic inhibition and antiviral activity compared to GRL0617, which was reported as a SARS-CoV PLpro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PLpro inhibitors in the BSL-2 setting. X-ray crystal structure of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that Jun9-72-2 and Jun9-75-4 engaged in more extensive interactions than GRL0617 (ACS Cent Sci 2021).
  • Development of assays for SARS-CoV-2 Mpro and PLpro hit validation/invalidation. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery (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).