Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme-ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue.

Steviol glycoside (SG) is a potential natural sugar substitute. The taste of various SG structures differ significantly, while their mechanism has not been thoroughly investigated. To investigate the taste mechanism, molecular docking simulations of SGs with sweet taste receptor TAS1R2 and bitter taste receptor TAS2R4 were conducted. The result suggested that four flexible coils (regions) in TAS1R2 constructed a geometry open pocket in space responsible for the binding of sweeteners. Amino acids that form hydrogen bonds with sweeteners are located in different receptor regions. In bitterness simulation, fewer hydrogen bonds were formed with the increased size of SG molecules. Particularly, there was no interaction between RM and TAS2R4 due to its size, which explains the non-bitterness of RM. Molecular dynamics simulations further indicated that the number of hydrogen bonds between SGs and TAS1R2 was maintained during a simulation time of 50 ns, while sucrose was gradually released from the binding site, leading to the break of interaction. Conclusively, the high sweetness intensity of SG can be attributed to its durative concurrent interaction with the receptor\'s binding site, and such behavior was determined by the structure feature of SG.

Currently, the treatment of Proteus mirabilis infections is considered to be complicated as the organism has become resistant to numerous antibiotic classes. Therefore, new inhibitors should be developed, targeting bacterial molecular functions. Methionine tRNA synthetase (MetRS), a member of the aminoacyl-tRNA synthetase family, is essential for protein biosynthesis offering a promising target for novel antibiotics discovery. In the context of computer-aided drug design (CADD), the current research presents the construction and analysis of a comparative homology model for P. mirabilis MetRS, enabling development of novel inhibitors with greater selectivity. Molecular Operating Environment (MOE) software was used to build a homology model for P. mirabilis MetRS using Escherichia coli MetRS as a template. The model was evaluated, and the active site of the target protein predicted from its sequence using conservation analysis. Molecular dynamic simulations were performed to evaluate the stability of the modeled protein structure. In order to evaluate the predicted active site interactions, methionine (the natural substrate of MetRS) and several inhibitors of bacterial MetRS were docked into the constructed model using MOE. After validation of the model, pharmacophore-based virtual screening for a systemically prepared dataset of compounds was performed to prove the feasibility of the proposed model, identifying possible parent compounds for further development of MetRS inhibitors against P. mirabilis.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 μs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a \'molten globule\' state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain\'s rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism.

Evaluation of the structural perturbations introduced by a single amino acid mutation is the main issue for protein structural biology. We propose here to present some recent advances in methods, allowing the splitting of distortion between the actual substitution effect and the contribution of the local flexibility of the position where the mutation occurs. Its main drawback is the need of many structures with a single mutation in each of them. To bypass this difficulty, we propose to use molecular modeling tools, with several software enabling us to build a model from a template, given the sequence. As a proof of concept, we rely on a gold standard, the human lysozyme. Both wild-type and three mutant structures are available in the PDB. Two of these mutations result in amyloid fibril formation, and the last one is neutral. As a conclusion, irrespective of the algorithm used for modeling, side chain conformations at the site of mutation are reliable, although long-range effects are out of reach of these tools.

Xeroderma pigmentosum C (XPC) is a key initiator in the global genome nucleotide excision repair pathway in mammalian cells. Inherited mutations in the XPC gene can cause xeroderma pigmentosum (XP) cancer predisposition syndrome that dramatically increases the susceptibility to sunlight-induced cancers. Various genetic variants and mutations of the protein have been reported in cancer databases and literature. The current lack of a high-resolution 3-D structure of human XPC makes it difficult to assess the structural impact of the mutations/genetic variations. Using the available high-resolution crystal structure of its yeast ortholog, Rad4, we built a homology model of human XPC protein and compared it with a model generated by AlphaFold. The two models are largely consistent with each other in the structured domains. We have also assessed the degree of conservation for each residue using 966 sequences of XPC orthologs. Our structure- and sequence conservation-based assessments largely agree with the variant\'s impact on the protein\'s structural stability, computed by FoldX and SDM. Known XP missense mutations such as Y585C, W690S, and C771Y are consistently predicted to destabilize the protein\'s structure. Our analyses also reveal several highly conserved hydrophobic regions that are surface-exposed, which may indicate novel intermolecular interfaces that are yet to be characterized.Communicated by Ramaswamy H. Sarma.

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC50 ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

Muscle myosin inhibition could be used to treat many medical conditions involving hypercontractile states, including muscle spasticity, chronic musculoskeletal pain, and hypertrophic cardiomyopathy. A series of 13 advanced analogs of 3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one (BHC) were synthesized to explore extended imine nitrogen side chains and compare aldimines vs. ketimines. None of the new analogs inhibit nonmuscle myosin in a cytokinesis assay. ATPase structure-activity relationships reveal that selectivity for cardiac vs. skeletal myosin can be tuned with subtle structural changes. None of the compounds inhibited smooth muscle myosin II. Docking the compounds to homology models of cardiac and skeletal myosin II gave rationales for the effects of side arm length on inhibition selectivity and for cardiac vs. skeletal myosin. Properties including solubility, stability and toxicity, suggest that certain BHC analogs may be useful as candidates for preclinical studies or as lead compounds for advanced candidates for drugs with cardiac or skeletal muscle myosin selectivity.

Multidrug-resistant tuberculosis (MDR-TB) poses a significant threat to mankind and as such earned its place on the WHO list of priority pathogens. New antimycobacterials with a mechanism of action different to currently used agents are highly required. This study presents the design, synthesis, and biological evaluation of 3-acylaminopyrazine-2-carboxamides derived from a previously reported inhibitor of human prolyl-tRNA synthetase. Compounds were evaluated in vitro against various strains of mycobacteria, pathogenic bacteria, and fungi of clinical significance. In general, high activity against mycobacteria was noted, while the antibacterial and antifungal activity was minimal. The most active compounds were 4\'-substituted 3-(benzamido)pyrazine-2-carboxamides, exerting MIC (Minimum Inhibitory Concentration) from 1.95 to 31.25 µg/mL. Detailed structure-activity relationships were established and rationalized in silico with regard to mycobacterial ProRS as a probable target. The active compounds preserved their activity even against multidrug-resistant strains of Mycobacterium tuberculosis. At the same time, they were non-cytotoxic against HepG2 human hepatocellular carcinoma cells. This project is the first step in the successful repurposing of inhibitors of human ProRS to inhibitors of mycobacterial ProRS with antimycobacterial activity.

Mutation of p53 is the most common genetic alteration in human cancer. The vast majority of p53 mutations found in cancer are missense mutations, with some single nucleotide point mutations leading to the accumulation of mutant p53 protein with potential gain of oncogenic function. The mechanism for stabilization and accumulation of missense mutant p53 protein in malignant cells is not fully understood. It is thought that DNAJA1 plays a crucial role as a co-chaperone protein by stabilizing mutant p53 and amplifying oncogenic potential. As such, identifying small molecule inhibitors to disrupt the protein-protein interaction between mutant p53 and DNAJA1 may lead to an effective treatment for preventing carcinogenesis. Studying protein-protein interactions and identifying potential druggable hotspots has historically been limited-protein-protein binding sites require more complex characterization than those of single proteins and the crystal structures of many proteins have not been identified. Due to these issues, identifying salient druggable targets in protein-protein interactions through bench research may take years to complete. However, in silico modeling approaches allow for rapid characterization of protein-protein interfaces and the druggable binding sites they contain. In this chapter, we first review the oncogenic potential of mutant p53 and the crucial role of DNAJA1 in stabilizing missense mutant p53. We then detail our methodology for using in silico modeling and molecular biology to identify druggable protein-protein interaction sites/pockets between mutant p53 and DNAJA1. Finally, we discuss screening for and validating the utility of a small molecule inhibitor identified through our in silico framework. Specifically, we describe GY1-22, a unique compound with activity against mutant p53 that demonstrates therapeutic potential to inhibit cancer cell growth both in vivo and in vitro.