SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The approval of vaccines and small-molecule antivirals is vital in combating the pandemic. The viral polymerase inhibitors remdesivir and molnupiravir and the viral main protease inhibitor nirmatrelvir/ritonavir have been approved by the U.S. FDA. However, the emergence of variants of concern/interest calls for additional antivirals with novel mechanisms of action. The SARS-CoV-2 papain-like protease (PL pro ) mediates the cleavage of viral polyprotein and modulates the host’s innate immune response upon viral infection, rendering it a promising antiviral drug target. This Perspective highlights major achievements in structure-based design and high-throughput screening of SARS-CoV-2 PL pro inhibitors since the beginning of the pandemic. Encouraging progress includes the design of non-covalent PL pro inhibitors with favorable pharmacokinetic properties and the first-in-class covalent PL pro inhibitors. In addition, we offer our opinion on the knowledge gaps that need to be filled to advance PL pro inhibitors to the clinic.
Conspectus SARS-CoV-2 is the etiological pathogen of the COVID-19 pandemic, which led to more than 6.5 million deaths since the beginning of the outbreak in December 2019. The unprecedented disruption of social life and public health caused by COVID-19 calls for fast-track development of diagnostic kits, vaccines, and antiviral drugs. Small molecule antivirals are essential complements of vaccines and can be used for the treatment of SARS-CoV-2 infections. Currently, there are three FDA-approved antiviral drugs, remdesivir, molnupiravir, and paxlovid. Given the moderate clinical efficacy of remdesivir and molnupiravir, the drug–drug interaction of paxlovid, and the emergence of SARS-CoV-2 variants with potential drug-resistant mutations, there is a pressing need for additional antivirals to combat current and future coronavirus outbreaks. In this Account, we describe our efforts in developing covalent and noncovalent main protease (M pro ) inhibitors and the identification of nirmatrelvir-resistant mutants. We initially discovered GC376, calpain inhibitors II and XII, and boceprevir as dual inhibitors of M pro and host cathepsin L from a screening of a protease inhibitor library. Given the controversy of targeting cathepsin L, we subsequently shifted the focus to designing M pro -specific inhibitors. Specifically, guided by the X-ray crystal structures of these initial hits, we designed noncovalent M pro inhibitors such as Jun8-76-3R that are highly selective toward M pro over host cathepsin L. Using the same scaffold, we also designed covalent M pro inhibitors with novel cysteine reactive warheads containing di- and trihaloacetamides, which similarly had high target specificity. In parallel to our drug discovery efforts, we developed the cell-based FlipGFP M pro assay to characterize the cellular target engagement of our rationally designed M pro inhibitors. The FlipGFP assay was also applied to validate the structurally disparate M pro inhibitors reported in the literature. Lastly, we introduce recent progress in identifying naturally occurring M pro mutants that are resistant to nirmatrelvir from genome mining of the nsp5 sequences deposited in the GISAID database. Collectively, the covalent and noncovalent M pro inhibitors and the nirmatrelvir-resistant hot spot residues from our studies provide insightful guidance for future work aimed at developing orally bioavailable M pro inhibitors that do not have overlapping resistance profile with nirmatrelvir.
T-type calcium channels activate in response to subthreshold membrane depolarizations and represent an important source of Ca 2+ influx near the resting membrane potential. These channels regulate neuronal excitability and have been linked to pain. For this reason, T-type calcium channels are suitable molecular targets for the development of new non-opioid analgesics. Our previous work identified an analogue of benzimidazolonepiperidine, 5bk, that preferentially inhibited Ca V 3.2 channels and reversed mechanical allodynia. In this study, we synthesized and screened a small library of 47 compounds derived from 5bk. We found several compounds that inhibited the Ca 2+ influx in DRG neurons of all sizes. After separating the enantiomers of each active compound, we found two compounds, 3-25-R and 3-14-3-S, that potently inhibited the Ca 2+ influx. Whole-cell patch clamp recordings from small-to medium-sized DRG neurons revealed that both compounds decreased total Ca 2+ . Application of 3-14-3-S (but not 3-25-R) blocked transiently expressed Ca V 3.1-3.3 channels with a similar IC 50 value. 3-14-3-S decreased T-type, but not N-type, Ca 2+ currents in DRG neurons. Furthermore, intrathecal delivery of 3-14-3-S relieved tonic, neuropathic, and inflammatory pain in preclinical models. 3-14-3-S did not exhibit any activity against G protein-coupled opioid receptors. Preliminary docking studies also suggest that 3-14-3-S can bind to the central pore domain of T-type channels. Together, our chemical characterization and functional and behavioral data identify a novel T-type calcium channel blocker with in vivo efficacy in experimental models of tonic, neuropathic, and inflammatory pain.
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