Nanomole Scale Screening of Fluorescent RNA-Methyltransferase Probes Enables the Discovery of METTL1 Inhibitors.

RNA methylation is a metabolic process validated for its association with various diseases, and thus, RNA methyltransferases (MTases) have become increasingly important in drug discovery. Yet, most frequently utilized RNA MTase assays are limited in their throughput and hamper this rapidly evolving field of medicinal chemistry. In this study, we describe a modular nanomole scale building block system that allowed the identification of tailored fluorescent MTase probes to unlock a broad selection of MTase drug targets for fluorescence-based binding assays. Probe candidates were initially prepared on a 4 nanomole scale and could be tested directly from crude reaction mixtures to allow rapid probe identification and optimization. Using an alkyne-azide click late-stage functionalization strategy and in silico protein databank mining, we established a selection of fluorescent probes suitable for relevant drug targets from the METTL and NSUN families, as well as bacterial and viral MTases. Using this concept, a high-throughput screening on the unexplored drug target METTL1 discovered three hit compounds with micromolar potency providing a first-in-class starting point for METTL1 drug discovery.

Identification of Dual Inhibitors Targeting Main Protease (Mpro) and Cathepsin L as Potential Anti-SARS-CoV-2 Agents.

In this structure-activity relationship (SAR) study, we report the development of dual inhibitors with antiviral properties targeting the SARS-CoV-2 main protease (Mpro) and human cathepsin L (hCatL). The novel molecules differ in the aliphatic amino acids at the P2 site and the fluorine position on the phenyl ring at the P3 site. The identified dual inhibitors showed Ki values within 1.61 and 10.72 µM against SARS-CoV-2 Mpro; meanwhile, Ki values ranging from 0.004 to 0.701 µM toward hCatL were observed. A great interdependency between the nature of the side chain at the P2 site and the position of the fluorine atom was found. Three dual-targeting inhibitors exhibited antiviral activity in the low micromolar range with CC50 values >100 µM. Docking simulations were executed to gain a deeper understanding of the SAR profile. The findings herein collected should be taken into consideration for the future development of dual SARS-CoV-2 Mpro/hCatL inhibitors.

Improving binding entropy by higher ligand symmetry? - A case study with human matriptase.

Understanding different contributions to the binding entropy of ligands is of utmost interest to better predict affinity and the thermodynamic binding profiles of protein-ligand interactions and to develop new strategies for ligand optimization. To these means, the largely neglected effects of introducing higher ligand symmetry, thereby reducing the number of energetically distinguishable binding modes on binding entropy using the human matriptase as a model system, were investigated. A set of new trivalent phloroglucinol-based inhibitors that address the roughly symmetric binding site of the enzyme was designed, synthesized, and subjected to isothermal titration calorimetry. These highly symmetric ligands that can adopt multiple indistinguishable binding modes exhibited high entropy-driven affinity in line with affinity-change predictions.