In cancer, the programmed death-1 (PD-1) pathway suppresses T cell stimulation and mediates immune escape. Upon stimulation, PD-1 becomes phosphorylated at its immune receptor tyrosine–based inhibitory motif (ITIM) and immune receptor tyrosine–based switch motif (ITSM), which then bind the Src homology 2 (SH2) domains of SH2-containing phosphatase 2 (SHP2), initiating T cell inactivation. The SHP2–PD-1 complex structure and the exact functions of the two SH2 domains and phosphorylated motifs remain unknown. Here, we explain the structural basis and provide functional evidence for the mechanism of PD-1-mediated SHP2 activation. We demonstrate that full activation is obtained only upon phosphorylation of both ITIM and ITSM: ITSM binds C-SH2 with strong affinity, recruiting SHP2 to PD-1, while ITIM binds N-SH2, displacing it from the catalytic pocket and activating SHP2. This binding event requires the formation of a new inter-domain interface, offering opportunities for the development of novel immunotherapeutic approaches.

Kinase large-scale conformational rearrangement is an issue of enormous biological and pharmacological relevance. Atomistic simulations able to capture the dynamics and the energetics of kinase large-scale motions are still in their infancy. Here, we present a computational study in which the atomistic dynamics of the "open-to-closed" movement of the cyclin-dependent kinase 5 (CDK5) have been simulated. Simulations were carried out using a new sampling method that is able to find the lowest free-energy channel between an initial state and a final state. This large-scale movement has a two-step mechanism: first, the alphaC-helix rotates by approximately 45 degrees , allowing the interaction between Glu51 and Arg149; then the CDK5 activation loop refolds to assume the closed conformation. We have also estimated the free-energy profile associated with the global motion and identified a CDK5 intermediate, which could be exploited for drug-design purposes. Our new sampling method turned out to be well-suited for investigating at an atomistic level the energetics and dynamics of kinase large-scale conformational motions.

The study of biomolecular interactions between a drug and its biological target is of paramount importance for the design of novel bioactive compounds. In this paper, we report on the use of molecular dynamics simulations and machine learning to study the binding mechanism of a transition state analogue (DADMe-Immucillin-H) to the purine nucleoside phosphorylase enzyme. Microsecond-long molecular dynamics simulations allow us to observe several binding events, following different dynamical routes and reaching diverse binding configurations. These simulations are used to estimate kinetic and thermodynamic quantities, such as kon and binding free energy, obtaining a good agreement with available experimental data. In addition, we advance a hypothesis for the slow-onset inhibition mechanism of DADMe-Immucillin-H against purine nucleoside phosphorylase. Combining extensive molecular dynamics simulations with machine learning algorithms could therefore be a fruitful approach to capturing key aspects of drug-target recognition and binding.

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.

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.

N-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 (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropionamide (2) and (S)-N-(2-oxo-3-oxetanyl)-biphenyl-4-carboxamide (3) inhibit NAAA, prevent FAE hydrolysis in activated inflammatory cells, and reduce tissue reactions to pro-inflammatory stimuli. Recently, our group disclosed ARN077 (4), 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 4. 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-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate (14q) as the first single-digit nanomolar inhibitor of intracellular NAAA activity (IC 50 = 7 nM on both rat NAAA and human NAAA).

Despite the daily use of urea to influence protein folding and stability, the molecular mechanism with which urea acts is still not well understood. Here the use of combined parallel tempering and metadynamics simulation allows us to study the free-energy landscape associated with the folding/unfolding of β-hairpin GB1 equilibrium in 8 M urea and pure water. The nature of the unfolded state in both solutions has been analyzed: in urea solution the addition of denaturants acts to expand the denatured state, while in pure water solution the unfolded state is noticeably more compact. For what concerns the mechanism by which urea acts as a denaturant, a preferential direct interaction between urea molecules and protein backbone has been found. However, the bias toward urea solvation is largest at intermediate values of the gyration radius.

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.