Potential Resistance of SARS-CoV-2 Main Protease (Mpro) against Protease Inhibitors: Lessons Learned from HIV-1 Protease.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome 2 (SARS-CoV-2), has been one of the most devastating pandemics of recent times. The lack of potent novel antivirals had led to global health crises; however, emergence and approval of potent inhibitors of the viral main protease (Mpro), such as Pfizer's newly approved nirmatrelvir, offers hope not only in the therapeutic front but also in the context of prophylaxis against the infection. By their nature, RNA viruses including human immunodeficiency virus (HIV) have inherently high mutation rates, and lessons learnt from previous and currently ongoing pandemics have taught us that these viruses can easily escape selection pressure through mutation of vital target amino acid residues in monotherapeutic settings. In this paper, we review nirmatrelvir and its binding to SARS-CoV-2 Mpro and draw a comparison to inhibitors of HIV protease that were rendered obsolete by emergence of resistance mutations, emphasizing potential pitfalls in the design of inhibitors that may be of important relevance to the long-term use of novel inhibitors against SARS-CoV-2.

Flap structure within receptor binding domain of SARS-CoV-2 spike periodically obstructs hACE2 Binding subdomain bearing similarities to HIV-1 protease flap.

The SARS-CoV-2 prefusion spike protein is characterized by a high degree of flexibility and temporal transformations associated with its multifunctional behavior. In this study, we have examined the dynamics of the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein in detail. Its primary, binding subdomain with human Angiotensin Covering Enzyme II includes a highly conspicuous flap or loop that is part of a beta hairpin loop structural motif. Dynamic details of the RBD obtained through RMSF and Order Parameter calculations are consistent with structural details including the stability of "glue" points or dominant interaction energy residues of the RBD in the Up and Down states with its neighboring N-terminal domain (NTD) protomer. The RBD flap in the Up state protomer periodically obstructs the binding site on an approximate 70 ns time interval and is reminiscent of an HIV-1 protease polypeptide flap that opens and closes to modulate that enzymes activity. No claim is made here regarding the possible modulating role of the flap; however, the flap may be a potential site for therapeutic targeting aimed at keeping it in the closed state, as previously demonstrated in the inhibition of the HIV-1 protease polypeptide. The RBD primary binding subdomain is further shown to have not only similar dynamics but, also, an approximate 30% sequence similarity to the HIV-1 protease polypeptide.

In silico evaluation of atazanavir as a potential HIV main protease inhibitor and its comparison with new designed analogs.

Starting three decades ago and spreading rapidly around the world, acquired immunodeficiency syndrome (AIDS) is an infectious disease distinct from other contagious diseases by its unique ways of transmission. Over the past few decades, research into new drug compounds has been accompanied by extensive advances, and the design and manufacture of drugs that inhibit virus enzymes is one way to combat the AIDS virus. Since blocking enzyme activity can kill a pathogen or correct a metabolic imbalance, the design and use of enzyme inhibitors is a new approach against viruses. We carried out an in-depth analysis of the efficacy of atazanavir and its newly designed analogs as human immunodeficiency virus (HIV) protease inhibitors using molecular docking. The best-designed analogs were then compared with atazanavir by the molecular dynamics simulation. The most promising results were ultimately found based on the docking analysis for HIV protease. Several exhibited an estimated free binding energy lower than -9.45 kcal/mol, indicating better prediction results than the atazanavir. ATV7 inhibitor with antiviral action may be more beneficial for infected patients with HIV. Molecular dynamics analysis and binding energy also showed that the ATV7 drug had more inhibitory ability than the atazanavir drug.

Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model.

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR's specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

Computational Determination of Potential Inhibitors of SARS-CoV-2 Main Protease.

The novel coronavirus (SARS-CoV-2) has infected several million people and caused thousands of deaths worldwide since December 2019. As the disease is spreading rapidly all over the world, it is urgent to find effective drugs to treat the virus. The main protease (Mpro) of SARS-CoV-2 is one of the potential drug targets. Therefore, in this context, we used rigorous computational methods, including molecular docking, fast pulling of ligand (FPL), and free energy perturbation (FEP), to investigate potential inhibitors of SARS-CoV-2 Mpro. We first tested our approach with three reported inhibitors of SARS-CoV-2 Mpro, and our computational results are in good agreement with the respective experimental data. Subsequently, we applied our approach on a database of ∼4600 natural compounds, as well as 8 available HIV-1 protease (PR) inhibitors and an aza-peptide epoxide. Molecular docking resulted in a short list of 35 natural compounds, which was subsequently refined using the FPL scheme. FPL simulations resulted in five potential inhibitors, including three natural compounds and two available HIV-1 PR inhibitors. Finally, FEP, the most accurate and precise method, was used to determine the absolute binding free energy of these five compounds. FEP results indicate that two natural compounds, cannabisin A and isoacteoside, and an HIV-1 PR inhibitor, darunavir, exhibit a large binding free energy to SARS-CoV-2 Mpro, which is larger than that of 13b, the most reliable SARS-CoV-2 Mpro inhibitor recently reported. The binding free energy largely arises from van der Waals interaction. We also found that Glu166 forms H-bonds to all of the inhibitors. Replacing Glu166 by an alanine residue leads to ∼2.0 kcal/mol decreases in the affinity of darunavir to SARS-CoV-2 Mpro. Our results could contribute to the development of potential drugs inhibiting SARS-CoV-2.

Possibility of HIV-1 protease inhibitors-clinical trial drugs as repurposed drugs for SARS-CoV-2 main protease: a molecular docking, molecular dynamics and binding free energy simulation study.

Initially, the SARS-CoV-2 virus was emerged from Wuhan, China and rapidly spreading across the world and urges the scientific community to develop antiviral therapeutic agents. Among several strategies, drug repurposing will help to react immediately to overcome the COVID-19 pandemic. In the present study, we have chosen two clinical trial drugs against HIV-1 protease namely, TMB607 and TMC310911 to use as the inhibitors of SARS-CoV-2 main protease (Mpro) enzyme. To make use of these two inhibitors as the repurposed drugs for COVID-19, it is essential to know the molecular basis of the binding mechanism of these two molecules with the SARS-CoV-2 Mpro. To understand the binding mechanism, we have performed molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations against the SARS-CoV-2 Mpro. The docking results indicate that both molecules form intermolecular interactions with the active site amino acids of Mpro enzyme. However, during the MD simulations, TMB607 forms strong interaction with the key amino acids of Mpro, and remains intact. The RMSD and RMSF values of both complexes were stable throughout the MD simulations. The MM-GBSA binding free energy values of both complexes are -43.7 and -34.9 kcal/mol, respectively. This in silico study proves that the TMB607 molecule binds strongly with the SARS-CoV-2 Mpro enzyme and it may be suitable for the drug repurposing of COVID-19 and further drug designing.Communicated by Ramaswamy H. Sarma.