The overexpression of p21-activated kinase 4 (PAK4) is associated with a variety of cancers. In this paper, the binding modes and inhibitory mechanisms of four 7H-pyrrolo[2,3-d]pyrimidine competitive inhibitors of PAK4 were investigated at the molecular level, mainly using molecular dynamics simulations and binding free energy calculations. The results show that the inhibitors had strong interactions with the hinge region, the β-sheets, and the residues with charged side chains around the 4-substituent. The terminal amino group of the inhibitor 5n was different from the other three, which could cause the enhancement of hydrogen bonds or electrostatic interactions formed with the surrounding residues. Thus, inhibitor 5n had the strongest inhibition capacity. The different halogen atoms on the 2-substituents of the inhibitors 5h, 5g, and 5e caused differences in the positions of the 2-benzene rings and affected the interactions of the hinge region. It also affected to some extent the orientations of the 4-imino groups and consequently their affinities for the surrounding charged residues. The combined results lead to the weakest inhibitory capacity of inhibitor 5e.

E. coli thioesterase ‘TesA is an important enzyme in fatty acid production. Medium-chain fatty acids (MCFAs, C6-C10) are of great interest due to their similar physicochemical properties to petroleum-based oleo-chemicals. It has been shown that wild-type ‘TesA had better selectivity for long-chain acyl substrates (≥C16), while the two mutants ‘TesA^E142D/Y145G and ‘TesA^{M141L/E142D/Y145G} had better selectivity for medium-chain acyl substrates. However, it is difficult to obtain the selectivity mechanism of substrates for proteins by traditional experimental methods. In this study, in order to obtain more MCFAs, we analyzed the binding mode of proteins (‘TesA, ‘TesA^E142D/Y145G and ‘TesA^{M141L/E142D/Y145G}) and substrates (C16/C8-N-acetylcysteamine analogs, C16/C8-SNAC), the key residues and catalytic mechanisms through molecular docking, molecular dynamics simulations and the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA). The results showed that several main residues related to catalysis, including Ser10, Asn73 and His157, had a strong hydrogen bond interaction with the substrates. The mutant region (Met141-Tyr146) and loop_107–113 were mainly dominated by Van der Waals contributions to the substrates. For C16-SNAC, except for ‘TesA^{M141L/E142D/Y145G} with large conformational changes, there were strong interactions at both head and tail ends that distorted the substrate into a more favorable high-energy conformation for the catalytic reaction. For C8-SNAC, the head and tail found it difficult to bind to the enzyme at the same time due to insufficient chain length, which made the substrate binding sites more variable, so ‘TesA^{M141L/E142D/Y145G} with better binding sites had the strongest activity, and ‘TesA had the weakest activity, conversely. In short, the matching substrate chain and binding pocket length are the key factors affecting selectivity. This will be helpful for the further improvement of thioesterases.

Monoacylglycerol lipase (MAGL) can regulate the endocannabinoid system and thus becomes a target of antidepressant drugs. In this paper, molecular docking and molecular dynamics simulations, combined with binding free energy calculation, were employed to investigate the inhibitory mechanism and binding modes of four aryl formyl piperidine derivative inhibitors with different 1-substituents to MAGL. The results showed that in the four systems, the main four regions where the enzyme bound to the inhibitor included around the head aromatic ring, the head carbonyl oxygen, the tail amide bond, and the tail benzene ring. The significant conformational changes in the more flexible lid domain of the enzyme were caused by 1-substituted group differences of inhibitors and resulted in different degrees of flipping in the tail of the inhibitor. The flipping led to a different direction of the tail amide bond and made a greater variation in its interaction with some of the charged residues in the enzyme, which further contributed to a different swing of the tail benzene ring. If the swing is large enough, it can weaken the binding strength of the head carbonyl oxygen to its nearby residues, and even the whole inhibitor with the enzyme so that the inhibition decreases.