Estimation of Protein-Ligand Unbinding Kinetics Using Non-Equilibrium Targeted Molecular Dynamics Simulations.

We here report on nonequilibrium targeted molecular dynamics simulations as a tool for the estimation of protein-ligand unbinding kinetics. Correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90, we show that the mean nonequilibrium work computed in an ensemble of trajectories of enforced ligand unbinding is a promising predictor for ligand unbinding rates. We furthermore investigate the molecular basis determining unbinding rates within the compound libraries. We propose ligand conformational changes and protein-ligand nonbonded interactions to impact on unbinding rates. Ligands may remain longer at the protein if they exhibit strong electrostatic and/or van der Waals interactions with the target. In the case of ligands with a rigid chemical scaffold that exhibit longer residence times, transient electrostatic interactions with the protein appear to facilitate unbinding. Our results imply that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding kinetics.

The use of virtual screening and differential scanning fluorimetry for the rapid identification of fragments active against MEK1.

We report the analysis of an in-house fragment screening campaign for the oncology target MEK1. The application of virtual screening (VS) as a primary fragment screening approach, followed by biophysical validation using differential screening fluorimetry (DSF), with resultant binding mode determination by X-ray crystallography (X-ray), is presented as the most time and cost-effective combination of in silico and in vitro methods to identify fragments. We demonstrate the effectiveness of the VS-DSF workflow for the early identification of fragments to both 'jump-start' the drug discovery project and to complement biochemical screening data.

KRAS G12C fragment screening renders new binding pockets.

KRAS genes belong to the most frequently mutated family of oncogenes in cancer. The G12C mutation, found in a third of lung, half of colorectal and pancreatic cancer cases, is believed to be responsible for a substantial number of cancer deaths. For 30 years, KRAS has been the subject of extensive drug-targeting efforts aimed at targeting KRAS protein itself, but also its post-translational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. So far, most KRAS targeting strategies have failed, and there are no KRAS-specific drugs available. However, clinical candidates targeting the KRAS G12C protein have recently been developed. MRTX849 and recently approved Sotorasib are covalent binders targeting the mutated cysteine 12, occupying Switch II pocket.Herein, we describe two fragment screening drug discovery campaigns that led to the identification of binding pockets on the KRAS G12C surface that have not previously been described. One screen focused on non-covalent binders to KRAS G12C, the other on covalent binders.

Estimation of Drug-Target Residence Times by τ-Random Acceleration Molecular Dynamics Simulations.

Drug-target residence time (τ), one of the main determinants of drug efficacy, remains highly challenging to predict computationally and, therefore, is usually not considered in the early stages of drug design. Here, we present an efficient computational method, τ-random acceleration molecular dynamics (τRAMD), for the ranking of drug candidates by their residence time and obtaining insights into ligand-target dissociation mechanisms. We assessed τRAMD on a data set of 70 diverse drug-like ligands of the N-terminal domain of HSP90α, a pharmaceutically important target with a highly flexible binding site, obtaining computed relative residence times with an accuracy of about 2.3τ for 78% of the compounds and less than 2.0τ within congeneric series. Analysis of dissociation trajectories reveals features that affect ligand unbinding rates, including transient polar interactions and steric hindrance. These results suggest that τRAMD will be widely applicable as a computationally efficient aid to improving drug residence times during lead optimization.

Discovery and Pharmacokinetic and Pharmacological Properties of the Potent and Selective MET Kinase Inhibitor 1-{6-[6-(4-Fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl]benzothiazol-2-yl}-3-(2-morpholin-4-ylethyl)urea (SAR125844).

The HGF/MET pathway is frequently activated in a variety of cancer types. Several selective small molecule inhibitors of the MET kinase are currently in clinical evaluation, in particular for NSCLC, liver, and gastric cancer patients. We report herein the discovery of a series of triazolopyridazines that are selective inhibitors of wild-type (WT) MET kinase and several clinically relevant mutants. We provide insight into their mode of binding and report unprecedented crystal structures of the Y1230H variant. A multiparametric chemical optimization approach allowed the identification of compound 12 (SAR125844) as a development candidate. In this chemical series, absence of CYP3A4 inhibition was obtained at the expense of satisfactory oral absorption. Compound 12, a promising parenteral agent for the treatment of MET-dependent cancers, promoted sustained target engagement at tolerated doses in a human xenograft tumor model. Preclinical pharmacokinetics conducted in several species were predictive for the observed pharmacokinetic behavior of 12 in cancer patients.

UMP kinase from Streptococcus pneumoniae: evidence for co-operative ATP binding and allosteric regulation.

UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl2 or MgCl2, the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (K(m)=0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S0.5=9.4+/-0.7 mM and n=1.9+/-0.1 in the presence of MgCl2). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient. The kcat (175+/-13 s(-1) in the presence of MgCl2) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP.