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1. Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease

   https://doi.org/https://doi.org/10.1101/2020.09.07.286344  

   Pavlova, Anna ; Lynch, Diane L ; Daidone, Isabella ; Zanetti-Polzi, Laura ; Smith, Micholas D ; Chipot, Chris ; Kneller, Daniel W ; Kovalevsky, Andrey ; Coates, Leighton ; Golosov, Andrei A ; et al ( January 2020 , NA)

   The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored Nδ (HD) and Nϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably boundmore »in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.« less
2. Conformational dynamics of cathepsin D and binding to a small‐molecule BACE1 inhibitor

   https://doi.org/10.1002/jcc.24719  

   Ellis, Christopher R. ; Tsai, Cheng‐Chieh ; Lin, Fang‐Yu ; Shen, Jana ( April 2017 , Journal of Computational Chemistry)

   BACE1 is a major therapeutic target for prevention and treatment of Alzheimer's disease. Developing inhibitors that can selectively target BACE1 in favor of other proteases, especially cathepsin D (CatD), has presented significant challenges. Here, we investigate the conformational dynamics and protonation states of BACE1 and CatD using continuous constant pH molecular dynamics with pH replica‐exchange sampling protocol. Despite similar structure, BACE1 and CatD exhibit markedly different active site dynamics. BACE1 displays pH‐dependent flap dynamics that controls substrate accessibility, while the CatD flap is relatively rigid and remains open in the pH range 2.5–6. Interestingly, although each protease hydrolyzes peptide bonds, the protonation states of the catalytic dyads are different within the active pH range. The acidic and basic components of the BACE1 catalytic dyad are clear, while either aspartic acid of the CatD catalytic dyad could play the role of acid or base. Finally, we investigate binding of the inhibitor LY2811376 developed by Eli Lilly to BACE1 and CatD. Surprisingly, in the enzyme active pH range, LY2811376 forms a stronger salt bridge with the catalytic dyad in CatD than in BACE1, which might explain the retinal toxicity of the inhibitor related to off‐target inhibition of CatD. This work highlights themore »complexity and challenge in structure‐based drug design where receptor‐ligand binding induces protonation state change in both the protein and the inhibitor. © 2017 Wiley Periodicals, Inc.

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3. Unveiling the molecular mechanism of SARS-CoV-2 main protease inhibition from 137 crystal structures using algebraic topology and deep learning

   https://doi.org/10.1039/d0sc04641h  

   Nguyen, Duc Duy ; Gao, Kaifu ; Chen, Jiahui ; Wang, Rui ; Wei, Guo-Wei ( November 2020 , Chemical Science)

   Currently, there is neither effective antiviral drugs nor vaccine for coronavirus disease 2019 (COVID-19) caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to its high conservativeness and low similarity with human genes, SARS-CoV-2 main protease (M pro ) is one of the most favorable drug targets. However, the current understanding of the molecular mechanism of M pro inhibition is limited by the lack of reliable binding affinity ranking and prediction of existing structures of M pro –inhibitor complexes. This work integrates mathematics ( i.e. , algebraic topology) and deep learning (MathDL) to provide a reliable ranking of the binding affinities of 137 SARS-CoV-2 M pro inhibitor structures. We reveal that Gly143 residue in M pro is the most attractive site to form hydrogen bonds, followed by Glu166, Cys145, and His163. We also identify 71 targeted covalent bonding inhibitors. MathDL was validated on the PDBbind v2016 core set benchmark and a carefully curated SARS-CoV-2 inhibitor dataset to ensure the reliability of the present binding affinity prediction. The present binding affinity ranking, interaction analysis, and fragment decomposition offer a foundation for future drug discovery efforts.
4. Molecular characterization of ebselen binding activity to SARS-CoV-2 main protease

   https://doi.org/10.1126/sciadv.abd0345  

   Menéndez, Cintia A. ; Byléhn, Fabian ; Perez-Lemus, Gustavo R. ; Alvarado, Walter ; de Pablo, Juan J. ( September 2020 , Science Advances)

   There is an urgent need to repurpose drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent computational-experimental screenings have identified several existing drugs that could serve as effective inhibitors of the virus’ main protease, M pro , which is involved in gene expression and replication. Among these, ebselen (2-phenyl-1,2-benzoselenazol-3-one) appears to be particularly promising. Here, we examine, at a molecular level, the potential of ebselen to decrease M pro activity. We find that it exhibits a distinct affinity for the catalytic region. Our results reveal a higher-affinity, previously unknown binding site localized between the II and III domains of the protein. A detailed strain analysis indicates that, on such a site, ebselen exerts a pronounced allosteric effect that regulates catalytic site access through surface-loop interactions, thereby inducing a reconfiguration of water hotspots. Together, these findings highlight the promise of ebselen as a repurposed drug against SARS-CoV-2.
5. Rational design of thioamide peptides as selective inhibitors of cysteine protease cathepsin L

   https://doi.org/10.1039/D1SC00785H  

   Phan, Hoang Anh ; Giannakoulias, Sam G. ; Barrett, Taylor M. ; Liu, Chunxiao ; Petersson, E. James ( January 2021 , Chemical Science)

   Aberrant levels of cathepsin L (Cts L), a ubiquitously expressed endosomal cysteine protease, have been implicated in many diseases such as cancer and diabetes. Significantly, Cts L has been identified as a potential target for the treatment of COVID-19 due to its recently unveiled critical role in SARS-CoV-2 entry into the host cells. However, there are currently no clinically approved specific inhibitors of Cts L, as it is often challenging to obtain specificity against the many highly homologous cathepsin family cysteine proteases. Peptide-based agents are often promising protease inhibitors as they offer high selectivity and potency, but unfortunately are subject to degradation in vivo . Thioamide substitution, a single-atom O-to-S modification in the peptide backbone, has been shown to improve the proteolytic stability of peptides addressing this issue. Utilizing this approach, we demonstrate herein that good peptidyl substrates can be converted into sub-micromolar inhibitors of Cts L by a single thioamide substitution in the peptide backbone. We have designed and scanned several thioamide stabilized peptide scaffolds, in which one peptide, R S 1A , was stabilized against proteolysis by all five cathepsins (Cts L, Cts V, Cts K, Cts S, and Cts B) while inhibiting Cts L with >25-fold specificitymore »against the other cathepsins. We further showed that this stabilized R S 1A peptide could inhibit Cts L in human liver carcinoma lysates (IC 50 = 19 μM). Our study demonstrates that one can rationally design a stabilized, specific peptidyl protease inhibitor by strategic placement of a thioamide and reaffirms the place of this single-atom modification in the toolbox of peptide-based rational drug design.« less