Resistance mechanisms of SARS-CoV-2 3CLpro to the non-covalent inhibitor WU-04.

Drug resistance poses a significant challenge in the development of effective therapies against SARS-CoV-2. Here, we identified two double mutations, M49K/M165V and M49K/S301P, in the 3C-like protease (3CLpro) that confer resistance to a novel non-covalent inhibitor, WU-04, which is currently in phase III clinical trials (NCT06197217). Crystallographic analysis indicates that the M49K mutation destabilizes the WU-04-binding pocket, impacting the binding of WU-04 more significantly than the binding of 3CLpro substrates. The M165V mutation directly interferes with WU-04 binding. The S301P mutation, which is far from the WU-04-binding pocket, indirectly affects WU-04 binding by restricting the rotation of 3CLpro's C-terminal tail and impeding 3CLpro dimerization. We further explored 3CLpro mutations that confer resistance to two clinically used inhibitors: ensitrelvir and nirmatrelvir, and revealed a trade-off between the catalytic activity, thermostability, and drug resistance of 3CLpro. We found that mutations at the same residue (M49) can have distinct effects on the 3CLpro inhibitors, highlighting the importance of developing multiple antiviral agents with different skeletons for fighting SARS-CoV-2. These findings enhance our understanding of SARS-CoV-2 resistance mechanisms and inform the development of effective therapeutics.

Assessing the Performance of Traveling-salesman based Automated Path Searching (TAPS) on Complex Biomolecular Systems.

Though crucial for understanding the function of large biomolecular systems, locating the minimum free energy paths (MFEPs) between their key conformational states is far from trivial due to their high-dimensional nature. Most existing path-searching methods require a static collective variable space as input, encoding intuition or prior knowledge of the transition mechanism. Such information is, however, hardly available a priori and expensive to validate. To alleviate this issue, we have previously introduced a Traveling-salesman based Automated Path Searching method (TAPS) and demonstrated its efficiency on simple peptide systems. Having implemented a parallel version of this method, here we assess the performance of TAPS on three realistic systems (tens to hundreds of residues) in explicit solvents. We show that TAPS successfully located the MFEP for the ground/excited state transition of the T4 lysozyme L99A variant, consistent with previous findings. TAPS also helped identifying the important role of the two polar contacts in directing the loop-in/loop-out transition of the mitogen-activated protein kinase kinase (MEK1), which explained previous mutant experiments. Remarkably, at a minimal cost of 126 ns sampling, TAPS revealed that the Ltn40/Ltn10 transition of lymphotactin needs no complete unfolding/refolding of its ß-sheets and that five polar contacts are sufficient to stabilize the various partially unfolded intermediates along the MFEP. These results present TAPS as a general and promising tool for studying the functional dynamics of complex biomolecular systems.