Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CL ^pro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CL ^pro , and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CL ^pro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

The prediction of ligand efficacy has long been linked to thermodynamic properties such as the equilibrium dissociation constant, which considers both the association and the dissociation rates of a defined protein-ligand complex. In the last 15 years, there has been a paradigm shift, with an increased interest in the determination of kinetic properties such as the drug-target residence time since they better correlate with ligand efficacy compared to other parameters. In this article, we present thermal titration molecular dynamics (TTMD), an alternative computational method that combines a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints for the qualitative estimation of protein-ligand-binding stability. The protocol has been applied to four different pharmaceutically relevant test cases, including protein kinase CK1δ, protein kinase CK2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease, on a variety of ligands with different sizes, structures, and experimentally determined affinity values. In all four cases, TTMD was successfully able to distinguish between high-affinity compounds (low nanomolar range) and low-affinity ones (micromolar), proving to be a useful screening tool for the prioritization of compounds in a drug discovery campaign.

Pyruvate dehydrogenase kinases (PDKs) are serine/threonine kinases, that are directly involved in altered cancer cell metabolism, resulting in cancer aggressiveness and resistance. Dichloroacetic acid (DCA) is the first PDK inhibitor that has entered phase II clinical; however, several side effects associated with weak anticancer activity and excessive drug dose (100 mg/kg) have led to its limitation in clinical application. Building upon a molecular hybridization approach, a small library of 3-amino-1,2,4-triazine derivatives has been designed, synthesized, and characterized for their PDK inhibitory activity using in silico, in vitro, and in vivo assays. Biochemical screenings showed that all synthesized compounds are potent and subtype-selective inhibitors of PDK. Accordingly, molecular modeling studies revealed that a lot of ligands can be properly placed inside the ATP-binding site of PDK1. Interestingly, 2D and 3D cell studies revealed their ability to induce cancer cell death at low micromolar doses, being extremely effective against human pancreatic KRAS mutated cancer cells. Cellular mechanistic studies confirm their ability to hamper the PDK/PDH axis, thus leading to metabolic/redox cellular impairment, and to ultimately trigger apoptotic cancer cell death. Remarkably, preliminary in vivo studies performed on a highly aggressive and metastatic Kras-mutant solid tumor model confirm the ability of the most representative compound 5i to target the PDH/PDK axis in vivo and highlighted its equal efficacy and better tolerability profile with respect to those elicited by the reference FDA approved drugs, cisplatin and gemcitabine. Collectively, the data highlights the promising anticancer potential of these novel PDK-targeting derivatives toward obtaining clinical candidates for combatting highly aggressive KRAS-mutant pancreatic ductal adenocarcinomas.

Molecular docking is one of the most widely used computational approaches in the field of rational drug design, thanks to its favorable balance between the rapidity of execution and the accuracy of provided results. Although very efficient in exploring the conformational degrees of freedom available to the ligand, docking programs can sometimes suffer from inaccurate scoring and ranking of generated poses. To address this issue, several post-docking filters and refinement protocols have been proposed throughout the years, including pharmacophore models and molecular dynamics simulations. In this work, we present the first application of Thermal Titration Molecular Dynamics (TTMD), a recently developed method for the qualitative estimation of protein-ligand unbinding kinetics, to the refinement of docking results. TTMD evaluates the conservation of the native binding mode throughout a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints. The protocol was successfully applied to retrieve the native-like binding pose among a set of decoy poses of drug-like ligands generated on four different pharmaceutically relevant biological targets, including casein kinase 1δ, casein kinase 2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease.

Pancreatic ductal adenocarcinoma (PDAC) is one of the main aggressive types of cancer, characterized by late prognosis and drug resistance. Among the main factors sustaining PDAC progression, the alteration of cell metabolism has emerged to have a key role in PDAC cell proliferation, invasion, and resistance to standard chemotherapeutic agents. Taking into account all these factors and the urgency in evaluating novel options to treat PDAC, in the present work we reported the synthesis of a new series of indolyl-7-azaindolyl triazine compounds inspired by marine bis-indolyl alkaloids. We first assessed the ability of the new triazine compounds to inhibit the enzymatic activity of pyruvate dehydrogenase kinases (PDKs). The results showed that most of derivatives totally inhibit PDK1 and PDK4. Molecular docking analysis was executed to predict the possible binding mode of these derivatives using ligand-based homology modeling technique. Evaluation of the capability of new triazines to inhibit the cell growth in 2D and 3D KRAS-wild-type (BxPC-3) and KRAS-mutant (PSN-1) PDAC cell line, was carried out. The results showed the capacity of the new derivatives to reduce cell growth with a major selectivity against KRAS-mutant PDAC PSN-1 on both cell models. These data demonstrated that the new triazine derivatives target PDK1 enzymatic activity and exhibit cytotoxic effects on 2D and 3D PDAC cell models, thus encouraging further structure manipulation for analogs development against PDAC.

The SARS-CoV-2 main protease (M^pro) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a promising target for drugs against the COVID-19 pandemic. Here, a crystal structure is presented in which M^pro adopts an inactive state that has never been observed before, called new-inactive. It is shown that the oxyanion loop, which is involved in substrate recognition and enzymatic activity, adopts a new catalytically incompetent conformation and that many of the key interactions of the active conformation of the enzyme around the active site are lost. Solvation/desolvation energetic contributions play an important role in the transition from the inactive to the active state, with Phe140 moving from an exposed to a buried environment and Asn142 moving from a buried environment to an exposed environment. In new-inactive M^pro a new cavity is present near the S2′ subsite, and the N-terminal and C-terminal tails, as well as the dimeric interface, are perturbed, with partial destabilization of the dimeric assembly. This novel conformation is relevant both for comprehension of the mechanism of action of M^pro within the catalytic cycle and for the successful structure-based drug design of antiviral drugs.