Steered Molecular Dynamics Simulations for Studying Protein–Ligand Interaction in Cyclin-Dependent Kinase 5

In this study, we applied steered molecular dynamics (SMD) simulations to investigate the unbinding mechanism of nine inhibitors of the enzyme cyclin-dependent kinase 5 (CDK5). The study had two major objectives: (i) to create a correlation between the unbinding force profiles and the inhibition activities of these compounds expressed as IC₅₀ values; (ii) to investigate the unbinding mechanism and to reveal atomistic insights, which could help identify accessory binding sites and transient interactions. Overall, we carried out 1.35 μs of cumulative SMD simulations. We showed that SMD could qualitatively discriminate binders from nonbinders, while it failed to properly rank series of inhibitors, particularly when IC₅₀ values were too similar. From a mechanistic standpoint, SMD provided useful insights related to transient and dynamical interactions, which could complement static description obtained by X-ray crystallography experiments. In conclusion, the present study represents a further step toward a systematic exploitation of SMD and other dynamical approaches in structure-based drug design and computational medicinal chemistry

Mutational Analysis of a Conserved Glutamate Reveals Unique Mechanistic and Structural Features of the Phosphatase PRL‑3

Phosphatase of regenerating liver (PRL)-3 (<i>PTP4A3</i>) has gained much attention in cancer research due to its involvement in tumor promoting and metastatic processes. It belongs to the protein tyrosine phosphatase (PTP) superfamily and is thought to follow the catalytic mechanism shared by this family, which aside from the conserved active-site amino acids includes a conserved glutamic acid residue that is usually required for the integrity of the active site in PTPs. We noted that in structures of PRL-3, PRL-1, and PTEN these residues do not clearly align and therefore we sought to investigate if the glutamic acid residue fulfills its usual function in these proteins. Although this residue was essential for PTEN’s catalytic activity, it was nonessential for PRL-1 and PRL-3. Surprisingly, the mutation E50R increased PRL-3 activity against all tested in vitro substrates and also enhanced PRL-3-promoted cell adhesion and migration. We show that the introduction of Arg50 leads to an enhancement of substrate turnover for both PRL-3 and, to a lesser extent, PRL-1, and that the stronger gain in activity correlates with a higher structural flexibility of PRL-3, likely allowing for conformational adaptation during catalysis. Thus, in contrast to its crucial functions in other PTPs, this conserved glutamic acid can be replaced in PRL-3 without impairing the structural integrity. The variant with enhanced activity might serve as a tool to study PRL-3 in the future

Predicting the Reactivity of Nitrile-Carrying Compounds with Cysteine: A Combined Computational and Experimental Study

Here, we report on a mechanistic investigation based on DFT calculations and kinetic measures aimed at determining the energetics related to the cysteine nucleophilic attack on nitrile-carrying compounds. Activation energies were found to correlate well with experimental kinetic measures of reactivity with cysteine in phosphate buffer. The agreement between computations and experiments points to this DFT-based approach as a tool for predicting both nitrile reactivity toward cysteines and the toxicity of nitriles as electrophile agents

Synthesis, Biological Evaluation, and 3D QSAR Study of 2‑Methyl-4-oxo-3-oxetanylcarbamic Acid Esters as <i>N</i>‑Acylethanolamine Acid Amidase (NAAA) Inhibitors

<i>N</i>-(2-Oxo-3-oxetanyl)­carbamic acid esters have recently been reported to be noncompetitive inhibitors of the <i>N</i>-acylethanolamine acid amidase (NAAA) potentially useful for the treatment of pain and inflammation. In the present study, we further explored the structure–activity relationships of the carbamic acid ester side chain of 2-methyl-4-oxo-3-oxetanylcarbamic acid ester derivatives. Additional favorable features in the design of potent NAAA inhibitors have been found together with the identification of a single digit nanomolar inhibitor. In addition, we devised a 3D QSAR using the atomic property field method. The model turned out to be able to account for the structural variability and was prospectively validated by designing, synthesizing, and testing novel inhibitors. The fairly good agreement between predictions and experimental potency values points to this 3D QSAR model as the first example of quantitative structure–activity relationships in the field of NAAA inhibitors

Synthesis and Structure–Activity Relationship (SAR) of 2‑Methyl-4-oxo-3-oxetanylcarbamic Acid Esters, a Class of Potent <i>N</i>‑Acylethanolamine Acid Amidase (NAAA) Inhibitors

<i>N</i>-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid agonists of peroxisome proliferator-activated receptor-α, which include oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). The β-lactone derivatives (<i>S</i>)-<i>N</i>-(2-oxo-3-oxetanyl)-3-phenylpropionamide (<b>2</b>) and (<i>S</i>)-<i>N</i>-(2-oxo-3-oxetanyl)-biphenyl-4-carboxamide (<b>3</b>) inhibit NAAA, prevent FAE hydrolysis in activated inflammatory cells, and reduce tissue reactions to pro-inflammatory stimuli. Recently, our group disclosed ARN077 (<b>4</b>), a potent NAAA inhibitor that is active in vivo by topical administration in rodent models of hyperalgesia and allodynia. In the present study, we investigated the structure–activity relationship (SAR) of threonine-derived β-lactone analogues of compound <b>4</b>. The main results of this work were an enhancement of the inhibitory potency of β-lactone carbamate derivatives for NAAA and the identification of (4-phenylphenyl)-methyl-<i>N</i>-[(2<i>S</i>,3<i>R</i>)-2-methyl-4-oxo-oxetan-3-yl]­carbamate (<b>14q</b>) as the first single-digit nanomolar inhibitor of intracellular NAAA activity (IC₅₀ = 7 nM on both rat NAAA and human NAAA)