Discovery and Optimization of a Series of Benzofuran Selective ERAP1 Inhibitors: Biochemical and In Silico Studies.

ERAP1 is a key aminopeptidase involved in peptide trimming before major histocompatibility complex (MHC) presentation. A single nucleotide polymorphism (SNP) in the ERAP1 gene can lead to impaired trimming activity and affect ERAP1 function. ERAP1 genetic variations have been linked to an increased susceptibility to cancer and autoimmune disease. Here, we report the discovery of novel ERAP1 inhibitors using a high throughput screening approach. Due to ERAP1 broad substrate specificity, the hit finding strategy included testing inhibitors with a range of biochemical assays. Based on the hit potency, selectivity, and in vitro absorption, distribution, metabolism, excretion, and toxicity, the benzofuran series was selected. Fifteen derivatives were designed and synthesized, the compound potency was improved to the nanomolar range, and the structure-activity relationship supported by modeling studies.

Sensitivity of Oncogenic KRAS-Expressing Cells to CDK9 Inhibition.

Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAFV600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.

Retrospective molecular docking study of WY-25105 ligand to ß-secretase and bias of the three-dimensional structure flexibility.

ß-Secretase (BACE) is a very promising target in the search for a treatment for Alzheimer's disease using a protein-ligand inhibition approach. Given the many published X-ray structures of BACE protein, structure-based drug design has been used extensively to support new inhibitor discovery programs. Due to the high flexibility and large catalytic site of this protein, sampling of the huge conformational space of the binding site is the big challenge to overcome and is the main limitation of the most widely used docking programs. Incorrect treatment of these pitfalls can introduce bias into ligand docking and could affect the results. This is especially the case with the WY-25105 compound reported by the Wyeth Corporation as a BACE ligand that did not fit into any of the known crystal structures. In the present retrospective study, a set of available X-ray enzyme structures was selected and molecular dynamics simulations were conducted to generate more diverse representative BACE protein conformations. These conformations were then used for a docking study of the WY-25105 compound. The results confirmed the need to use an ensemble of structures in protein-ligand docking for identification of new binding modes in structure-based drug design of BACE inhibitors.

On the possibility of the observation of valence electron density for individual bonds in proteins in conventional difference maps.

In the last decade, high-resolution data have become available for macromolecular objects. Furthermore, ultrahigh-resolution diffraction data (resolution close to 0.6 A) have been collected for several protein crystals. This allows the study of fine details of the electron-density distribution such as the deformation density, i.e. the deviation of the experimentally determined electron density from the density composed of 'free' non-bonded atoms. This paper discusses the resolution and atomic temperature factors necessary to make the valence electron density visible at individual bonds in conventional difference maps for macromolecules. The study of theoretical maps calculated by quantum-chemistry methods allows estimation of these conditions; these results are confirmed by analysis of experimental maps for Leu-enkephalin and antifreeze protein RD1. A resolution limit close to 0.6 A was found to be highly important for refinement even when the maps were calculated at lower resolution. The refinement of the same models at near to 0.9 A resolution results in artificially increased values of the atomic displacement parameters and does not permit bond electron density to be visible in difference maps. To some extent, overestimation of the atomic displacement parameters may be restricted if dummy bond electrons are used in the refinement.

Electrostatic complementarity in an aldose reductase complex from ultra-high-resolution crystallography and first-principles calculations.

The electron density and electrostatic potential in an aldose reductase holoenzyme complex have been studied by density functional theory (DFT) and diffraction methods. Aldose reductase is involved in the reduction of glucose in the polyol pathway by using NADPH as a cofactor. The ultra-high resolution of the diffraction data and the low thermal-displacement parameters of the structure allow accurate atomic positions and an experimental charge density analysis. Based on the x-ray structural data, order-N DFT calculations have been performed on subsets of up to 711 atoms in the active site of the molecule. The charge density refinement of the protein was performed with the program MOPRO by using the transferability principle and our database of charge density parameters built from crystallographic analyses of peptides and amino acids. Electrostatic potentials calculated from the charge density database, the preliminary experimental electron density analysis, DFT computations, and atomic charges taken from the amber software dictionary are compared. The electrostatic complementarity between the cofactor NADP+ and the active site shows up clearly. The anchoring of the inhibitor is due mainly to hydrophobic forces and to only two polar interaction sites within the enzyme cavity. The potentials calculated by x-ray and DFT techniques agree reasonably well. At the present stage of the refinement, the potentials obtained directly from the database are in excellent agreement with the experimental ones. In addition, these results demonstrate the significant contribution of electron lone pairs and of atomic polarization effects to the host and guest mechanism.