Protein conformational dynamics play an important role in numerous biological processes. Markov State Models (MSMs) provide a powerful approach to study these dynamic processes by predicting long time scale dynamics based on many short molecular dynamics (MD) simulations. To improve the efficiency of MSMs, we recently developed quasi-MSM (qMSM) that encodes the non-Markovian dynamics in a generally time-dependent memory kernel. We successfully applied qMSMs to elucidate molecular mechanisms of DNA loading into a bacterial RNA polymerase complex via flexible loading gate (consisting of the clamp and β-lobe domain), a process occurs at millisecond. Using qMSMs, we showed that the opening of β-lobe is orders of magnitude faster than that of the clamp, which depends on the structure of the Switch 2 region. Strikingly, opening of the β-lobe is sufficient geometrically to accommodate DNA loading even when the clamp is partially closed. These two observations highlight β-lobe’s critical role allowing DNA loading during initiation. In my talk, I will also present our recent results in elucidating molecular mechanisms of 1′-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp).

COVID-19 pandemic is a great threat to the general and global public health and economy. The rapid development of new antiviral compounds and vaccines is needed to control the current pandemic as well as to prepare for the emergence of new variants. Among the proteins encoded by the SARS-CoV-2 genome, M^pro is one of the primary drug targets due to its essential role in maturation of the viral polyprotein. In this study, we describe a high-density acoustic droplet ejection (ADE) method for co-crystallization of M^pro-ligand complexes using only 40 nL M^pro solution. Also, we will briefly describe crystallographic data from crystals obtained using ADE and other methods as evidence that three clinically approved anti hepatitis C virus (HCV) drugs are capable of covalent binding to the M^pro Cys145 catalytic residue in the active site (Fig. 1). Activities of the National Virtual Biotechnology Laboratory (NVBL) for the design and development of new antiviral inhibitors for SARS-CoV-2 is briefly discussed.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness is responsible for more than half a million deaths in the United States alone. The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2’-OH of the first transcribed nucleotide (N₁ base) of the viral mRNA by covalently attaching a methyl group to it. This single RNA modification event converts the status of the mRNA cap from Cap-0 (^m7GpppA) to Cap-1(^m7GpppAm) and helps the virus evade immune surveillance in the host cell. Here, we report three high-resolution crystal structures of nsp16/nsp10 heterodimer representing substrate (Cap-0)-bound state, and pre- and post-release states of the RNA product (Cap-1). The binding of Cap-0 induces large conformational changes. This ‘induced fit’ model provides new mechanistic insights into the 2’-O methylation of the viral mRNA cap. We reveal the structural basis for the RNA specificity of nsp16/nsp10. We also discover an alternative ligand-binding site unique to SARS-CoV-2 [1]. We also observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate-binding region upon product release. These changes reset the enzyme for the next round of catalysis, and may be the structural basis of dissociation nsp10 from nsp16. The structures also identify a unique binding mode of a divalent metal ion in nsp16, which aligns the first two bases of the viral RNA in the catalytic pocket for efficient Cap-1 formation. Using LC/MS-based intact mass analysis, we show dramatic perturbations in Cap-1 formation by an emerging clinical variant of SARS-CoV-2, previous SARS-CoV outbreak strain, and their altered sensitivity to divalent metal ions [2]. Such reliance and preference for metals also suggests that an imbalance in cellular metal concentrations could differentially alter the RNA capping and thus, host innate immune response to infections by various CoVs. Altogether, our work provides a revised framework from which new therapeutic modalities may be designed for the treatment of COVID-19 and emerging coronavirus illnesses.

SARS-CoV-2 is a novel coronavirus and causative agent of the zoonotic disease Covid-19, which has been responsible for over 3 million deaths globally. Although the rapid development of several highly efficacious vaccines is proving effective in reducing the spread and severity of the disease, the development of novel, low cost and globally available anti-viral therapeutics remains an essential goal, both for this pandemic and for future outbreaks of related coronaviruses.

To identify starting points for such therapeutics, the XChem team at Diamond Light Source, in collaboration with various international colleagues, have performed large crystallographic fragment screens against 7 key SARS-CoV-2 proteins including the Main protease, the Nsp3 macrodomain and the helicase Nsp13 [1-3]. The expeditious collection and dissemination of data from these screens has been enabled by the well-established platform at Diamond and by the implementation of various new tools in the XChem pipeline.

This work has identified numerous starting points for the development of more potent inhibitors as exemplified by the ongoing work from the open science drug discovery project, the Covid Moonshot [4]. By merging fragment hits from the initial XChem screen and harnessing crowdsourced medicinal chemistry designs from the global community we have been able to rapidly develop potent inhibitors of the Main protease that exhibit promising antiviral activity.

[1] Douangamath, A., et al., Nature Communications, 11, 2020.

[2] Schuller, M., et al., Science Advances, 7, 2021.

[3] Newman, J., et al., BioRxiv, 2021.

[4] The COVID Moonshot Consortium, BioRxiv, 2021.

Human coronaviruses such as SARS-CoV, MERS and SARS-CoV-2 continue to emerge as significant threats to public health. Other human coronaviruses such as NL63, HKU1, 229E and OC43 continue to persist in the population but are significantly less deadly. Since the SARS-CoV epidemic emerged in 2003, we have worked to develop small-molecule inhibitors of coronavirus 3C-like protease (3CLpro, also known as main protease or Mpro) and the papain-like protease (PLP or PLpro). Initially, we focused on the proteases from SARS and then on NL63 and MERS. However, the differences in inhibitory potencies of our compounds and the taxonomic distance of the alpha and beta coronavirus genera taught us that approach of studying one virus at a time was too slow and provided to little molecular information to inhibit multiple coronaviruses. Moreover, it was not allowing us to predict how to inhibit emerging coronavirus pathogens. In the interest of pandemic preparedness, we are now taking what we call a taxonomically-driven approach to the structure-based design of coronavirus protease inhibitors. We targeted 12 different 3CLpros from the alpha-, beta- and gamma-coronavirus genera with a series of 50 compounds that we designed and synthesized using the Automated Synthesis and Purification platform at Eli Lilly. We identified inhibitor templates that potently inhibit the enzymes from the alpha and beta genera but not the gamma genus. To ascertain the structural basis of the selectivity, we utilized LS-CAT and LRL-CAT beamlines at the APS and performed a sparse-matrix sampling approach and determined multiple X-ray structures of 3CLpro from the different coronavirus genera in complex with different inhibitors. We identified precise structural regions that define inhibitor selectivity for different inhibitor scaffolds and we are now extending this approach to PLpro. We have been able to design and synthesize over 350 additional compounds against SARS-CoV-2 3CLpro. These compounds include potent non-covalent inhibitors, reversible-covalent and covalent inhibitors with low nanomolar to picomolar potency including inhibitors with broad-spectrum, i.e. pancoronavirus, activity against 12 different alpha, beta and gamma coronavirus.

This work was supported in part by funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201700060C.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) is essential for the virus replication and covers multiple functions (1,2). In this context, PLpro is an interesting drug target to identify compounds that inhibit the activity and can further be optimized towards drugs to cure Covid-19 in the future. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, in terms of drug discovery investigations PLpro is thus an excellent drug target allowing a two-fold strategy, to identify compounds that inhibit viral replication and strengthen the immune response of the host in parallel. To establish a framework allowing an efficient and high throughput screening of compounds to identify inhibitors, we first expressed, purified and crystallized PLpro (Fig.1), determined and refined the native crystal structure to atomic resolution of 1.42 Å (Fig.2, pdb code: 7NFV).

Further, we initiated screening via co-crystallization utilizing a library of 2.500 selected natural compounds, obtained from ICCBS Karachi, and identified first potential inhibitors binding to a site that has been previously shown to bind to the ISG15 molecule, refined structures were deposited with pdb codes: 7OFS, 7OFT, 7OFU. Comparing the PLpro-ligand complex structures with the PLpro-ISG15 complex crystal structure (pdb code: 6XAA) clearly shows that several regions of the Ubiquitin fold domain move dynamically, showing functional flexibility to accommodate the ligands (Fig. 3). Corresponding structural data and details, as well as on-going structural efforts to identify new antiviral compounds to combat the coronavirus spread will be presented.