Biochemical Characterization of Selective Inhibitors of Human Group IIA Secreted Phospholipase A₂ and Hyaluronic Acid-Linked Inhibitor Conjugates

We explored the inhibition mode of group IIA secreted phospholipase A₂ (GIIA sPLA₂) selective inhibitors and tested their ability to inhibit GIIA sPLA₂ activity as chemical conjugates with hyaluronic acid (HA). Analogues of a benzo-fused indole sPLA₂ inhibitor were developed in which the carboxylate group on the inhibitor scaffold, which has been shown to coordinate to a Ca²⁺ ligand in the enzyme active site, was replaced with other functionality. Replacing the carboxylate group with amine, amide, or hydroxyl groups had no effect on human GIIA (hGIIA) sPLA₂ inhibition potency but dramatically lowered inhibition potency against hGV and hGX sPLA₂s. An alkylation protection assay was used to probe active site binding of carboxylate and noncarboxylate inhibitors in the presence and absence of Ca²⁺ and/or lipid vesicles. We observed that carboxylate-containing inhibitors bind the hGIIA sPLA₂ active site with low nanomolar affinity, but only when Ca²⁺ is present. Noncarboxylate, GIIA sPLA₂ selective inhibitors also bind the hGIIA sPLA₂ active site in the nanomolar range. However, binding for GIIA sPLA₂ selective inhibitors was dependent on the presence of a lipid membrane and not Ca²⁺. These results indicate that GIIA sPLA₂ selective inhibitors exert their inhibitory effects by binding to the hGIIA sPLA₂ active site. An HA-linked GIIA inhibitor conjugate was developed using peptide coupling conditions and found to be less potent and selective against hGIIA sPLA₂ than the unconjugated inhibitor. Compounds reported in this study are some of the most potent and selective GIIA sPLA₂ active site binding inhibitors reported to date

The chemical biology of coronavirus host–cell interactions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic that has led to a global economic disruption and collapse. With several ongoing efforts to develop vaccines and treatments for COVID-19, understanding the molecular interaction between the coronavirus, host cells, and the immune system is critical for effective therapeutic interventions. Greater insight into these mechanisms will require the contribution and combination of multiple scientific disciplines including the techniques and strategies that have been successfully deployed by chemical biology to tease apart complex biological pathways. We highlight in this review well-established strategies and methods to study coronavirus–host biophysical interactions and discuss the impact chemical biology will have on understanding these interactions at the molecular level

A second-generation phosphohistidine analog for production of phosphohistidine antibodies

Protein histidine phosphorylation plays a crucial role in cell signaling and central metabolism. However, its detailed functions remain elusive due to technical challenges in detecting and isolating proteins bearing phosphohistidine (pHis), a labile posttranslational modification (PTM). To address this issue, we previously developed the first pHis-specific antibodies using stable, synthetic triazole-based pHis analogs. A second-generation, pyrazole-based pHis analog that enabled the development of a pan-pHis antibody with much improved pHis specificity is now reported.close2

Near-Infrared Photoredox Catalyzed Tryptophan Functionalization for Peptide Stapling and Protein Labeling in Complex Tissue Environments

The chemical transformation of aromatic amino acids has emerged as an attractive alternative to non-selective lysine or cysteine labeling for the modification of biomolecules. However, this strategy has largely been limited by the scope of functional groups and biocompatible reaction conditions available. Herein, we report the implementation of near-infrared-activatable photocatalysts, TTMAPP and n-Pr-DMQA+, capable of generating fluoroalkyl radicals for selective tryptophan functionalization within simple and complex biological systems. At the peptide level, a diverse set of iodo-perfluoroalkyl reagents were used to install bioorthogonal handles for downstream applications or link inter- or intramolecular tryptophan residues for peptide stapling. We also found this photoredox transformation amenable to biotinylation of intracellular proteins in live cells for downstream confocal imaging and mass spectrometry-based analysis. Given the inherent tissue penetrant nature of near-infrared light we further demonstrated the utility of this technology to achieve photocatalytic protein fluoroalkylation in physiologically relevant tissue and tumor environments

μMap Photoproximity Labeling Enables Small Molecule Binding Site Mapping

The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish μMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data