Why is the Omicron main protease of SARS-CoV-2 less stable than its wild-type counterpart? A crystallographic, biophysical, and theoretical study of the free enzyme and its complex with inhibitor 13b-K (preprint)

During the continuing evolution of SARS-CoV-2, the Omicron variant of concern emerged in the second half of 2021 and has been dominant since November that year. Along with its sublineages, it has maintained a prominent role ever since. The Nsp5 main protease (Mpro) of the Omicron virus is characterized by a single dominant mutation, P132H. Here we determined the X-ray crystal structures of the P132H mutant (or O-Mpro) as free enzyme and in complex with the Mpro inhibitor, the alpha-ketoamide 13b-K, and we conducted enzymology, biophysical as well as theoretical studies to characterize the O-Mpro. We found that O-Mpro has a similar overall structure and binding with 13b-K; however, it displays lower enzymatic activity and lower thermal stability compared to the WT-Mpro (with \"WT\" referring to the original Wuhan-1 strain). Intriguingly, the imidazole ring of His132 and the carboxylate plane of Glu240 are in a stacked configuration in the X-ray structures determined here. The empirical folding free energy calculations suggest that the O-Mpro dimer is destabilized relative to the WT-Mpro due to the less favorable van der Waals interactions and backbone conformation in the individual protomers. The all-atom continuous constant pH molecular dynamics (MD) simulations reveal that His132 and Glu240 display coupled titration. At pH 7, His132 is predominantly neutral and in a stacked configuration with respect to Glu240 which is charged. In order to examine whether the Omicron mutation eases the emergence of further Mpro mutations, we also determined crystal structures of the relatively frequent P132H+T169S double mutant but found little evidence for a correlation between the two sites.

Drug resistance revealed by in silico deep mutational scanning and mutation tracker (preprint)

As COVID-19 enters its fifth year, it continues to pose a significant global health threat, with the constantly mutating SARS-CoV-2 virus challenging drug effectiveness. A comprehensive understanding of virus-drug interactions is essential for predicting and improving drug effectiveness, especially in combating drug resistance during the pandemic. In response, the Path Laplacian Transformer-based Prospective Analysis Framework (PLFormer-PAF) has been proposed, integrating historical data analysis and predictive modeling strategies. This dual-strategy approach utilizes path topology to transform protein-ligand complexes into topological sequences, enabling the use of advanced large language models for analyzing protein-ligand interactions and enhancing its reliability with factual insights garnered from historical data. It has shown unparalleled performance in predicting binding affinity tasks across various benchmarks, including specific evaluations related to SARS-CoV-2, and assesses the impact of virus mutations on drug efficacy, offering crucial insights into potential drug resistance. The predictions align with observed mutation patterns in SARS-CoV-2, indicating that the widespread use of the Pfizer drug has lead to viral evolution and reduced drug efficacy. PLFormer-PAF's capabilities extend beyond identifying drug-resistant strains, positioning it as a key tool in drug discovery research and the development of new therapeutic strategies against fast-mutating viruses like COVID-19.

H172Y mutation perturbs the S1 pocket and nirmatrelvir binding of SARS CoV-2 main protease through a nonnative hydrogen bond (preprint)

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID-19 patients. Although the prevalent Mpro mutants in the SARS-CoV-2 Variants of Concern (e.g., Omicron) are still susceptible to nirmatrelvir, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the selective pressure of antiviral therapy may favor resistance mutations, there is an urgent need to understand the effect of the H172Y mutation on Mpro's structure, function, and drug resistance. Here we report the molecular dynamics (MD) simulations as well as the measurements of stability, enzyme kinetics of H172Y Mpro, and IC50 value of nirmatrelvir. Simulations showed that mutation disrupts the interactions between the S1 pocket and N terminus of the opposite protomer. Intriguingly, a native hydrogen bond (H-bond) between Phe140 and the N terminus is replaced by a transient H-bond between Phe140 and Tyr172. In the ligand-free simulations, strengthening of this nonnative H-bond is correlated with disruption of the conserved aromatic stacking between Phe140 and His163, leading to a partial collapse of the oxyanion loop. In the nirmatrelvir-bound simulations, the nonnative H-bond is correlated with the loss of an important H-bond between Glu166 and nirmatrelvir's lactam nitrogen at P1 position. These results are consistent with the newly reported X-ray structures of H172Y Mpro and suggest a mechanism by which the H172Y substitution perturbs the S1 pocket, leading to the decreased structural stability and binding affinity, which in turn explains the drastic reduction in catalytic activity and antiviral susceptibility.

H172Y mutation perturbs the S1 pocket and nirmatrelvir binding of SARS-CoV-2 main protease through a nonnative hydrogen bond (preprint)

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID- 19 patients. Although the prevalent Mpro mutants in the SARS-CoV-2 Variants of Concern (e.g., Omicron) are still susceptible to nirmatrelvir, a rare mutation, H172Y, was found to significantly reduce nirmatrelvir’s inhibitory activity. As the selective pres- sure of antiviral therapy may favor resistance mutations, there is an urgent need to understand the effect of H172Y mutation on Mpro’s structure, function, and drug re- sistance. Here we report the molecular dynamics (MD) simulations as well as the measurements of stability, enzyme kinetics of H172Y Mpro, and IC50 value of nir- matrelvir. Simulations showed that mutation disrupts the interactions between the S1 pocket and N terminus of the opposite protomer. Intriguingly, a native hydrogen bond (H-bond) between Phe140 and the N terminus is replaced by a transient H- bond between Phe140 and Tyr172. In the ligand-free simulations, strengthening of this nonnative H-bond is correlated with disruption of the conserved aromatic stacking between Phe140 and His163, leading to a partial collapse of the oxyanion loop. In the nirmatrelvir-bound simulations, the nonnative H-bond is correlated with the loss of an important H-bond between Glu166 and nirmatrelvir’s lactam nitrogen at P1 position. These results are consistent with the newly reported X-ray structures of H172Y Mpro and suggest a mechanism by which the H172Y substitution perturbs the S1 pocket, leading to the decreased structural stability and binding affinity, which in turns explains the drastic reduction in catalytic activity and antiviral susceptibility.

How mu-Opioid Receptor Recognizes Fentanyl (preprint)

The opioid crisis has escalated during the COVID-19 pandemic. More than half of the overdose-related deaths are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, crystal structures of mOR complexed with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like synthetic opioids remains lacking. Exploiting the X-ray structure of mOR bound to a morphinan ligand and several state-of-the-art simulation techniques, including weighted ensemble and continuous constant pH molecular dynamics, we elucidated the detailed binding mechanism of fentanyl with mOR. Surprisingly, in addition to the orthosteric site common to morphinan opiates, fentanyl can move deeper and bind mOR through hydrogen bonding with a conserved histidine H297, which has been shown to modulate mOR's ligand affinity and pH dependence in mutagenesis experiments, but its precise role remains unclear. Intriguingly, the secondary binding mode is only accessible when H297 adopts a neutral HID tautomer. Alternative binding modes and involvement of tautomer states may represent general mechanisms in G protein-coupled receptor (GPCR)-ligand recognition. Our work provides a starting point for understanding mOR activation by fentanyl analogs that are emerging at a rapid pace and assisting the design of safer analgesics to combat the opioid crisis. Current protein simulation studies employ standard protonation and tautomer states; our work demonstrates the need to move beyond the practice to advance our understanding of protein-ligand recognition.

Howµ-Opioid Receptor Recognizes Fentanyl (preprint)

In 2019, drug overdose has claimed over 70,000 lives in the United States. More than half of the deaths are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, the crystal structures of mOR in complex with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like synthetic opioids remains lacking. Exploiting the X-ray structure of mOR bound to a morphinan ligand and several state-of-the-art simulation techniques, including weighted ensemble and continuous constant pH molecular dynamics, we elucidated the detailed binding mechanism of fentanyl with mOR. Surprisingly, in addition to forming a salt-bridge with Asp147 3.32 in the orthosteric site common to morphinan opiates, fentanyl can move deeper and bind mOR through hydrogen bonding with a conserved histidine His297 6.52 , which has been shown to modulate mOR’s ligand affinity and pH dependence in mutagenesis experiments, but its precise role remains unclear. Intriguingly, the secondary binding mode is only accessible when His297 adopts a neutral HID tautomer. Alternative binding modes and involvement of tautomer states may represent general mechanisms in G protein-coupled receptor (GPCR)-ligand recognition. Our work provides a starting point for understanding the molecular basis of mOR activation by fentanyl which has many analogs emerging at a rapid pace. The knowledge may also inform the design of safer analgesics to combat the opioid crisis. Current protein simulation studies employ standard protonation and tautomer states; our work demonstrates the need to move beyond the practice to advance our understanding of protein-ligand recognition.

Assessment of Proton-Coupled Conformational Dynamics of SARS and MERS Coronavirus Papain-like Proteases: Implication for Designing Broad-Spectrum Antiviral Inhibitors (preprint)

Broad-spectrum antiviral drugs are urgently needed to stop the COVID-19 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines and their protonation/deprotonation modulates catalysis and conformational plasticity. Here we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using a newly developed GPU-accelerated implicit-solvent continuous constant pH molecular dynamics method with an asynchronous replica-exchange scheme. The calculated pKa’s support the catalytic roles of the Cys-His-Asp triad. We also found that several residues can switch protonation states at physiological pH, among which is C270/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2/CoV PLpro. Simulations revealed that the BL2 conformational dynamics is coupled to the titration of C271/270, in agreement with the crystal structures of SARS-CoV-2 PLpro. Simulations also revealed that BL2 in MERS-CoV PLpro is very flexible, sampling both open and closed states despite the lack of an analogous cysteine. Our work provides a starting point for more detailed mechanistic studies to assist structure-based design of broad-spectrum inhibitors against CoV PLpros.Competing Interest StatementThe authors have declared no competing interest.View Full Text