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.

Beyond Amphiphilic Balance: Changing Subunit Stereochemistry Alters the Pore-Forming Activity of Nylon-3 Polymers.

Amphiphilic nylon-3 polymers have been reported to mimic the biological activities of natural antimicrobial peptides, with high potency against bacteria and minimal toxicity toward eukaryotic cells. Amphiphilic balance, determined by the proportions of hydrophilic and lipophilic subunits, is considered one of the most important features for achieving this activity profile for nylon-3 polymers and many other antimicrobial polymers. Insufficient hydrophobicity often correlates with weak activities against bacteria, whereas excessive hydrophobicity correlates with high toxicity toward eukaryotic cells. To ask whether factors beyond amphiphilic balance influence polymer activities, we synthesized and evaluated new nylon-3 polymers with two stereoisomeric subunits, each bearing an ethyl side chain and an aminomethyl side chain. Subunits that differ only in stereochemistry are predicted to contribute equally to amphiphilic balance, but we observed that the stereochemical difference correlates with significant changes in biological activity profile. Antibacterial activities were not strongly affected by subunit stereochemistry, but the ability to disrupt eukaryotic cell membranes varied considerably. Experiments with planar lipid bilayers and synthetic liposomes suggested that eukaryotic membrane disruption results from polymer-mediated formation of large pores. Collectively, our results suggest that factors other than amphiphilic balance influence the membrane activity profile of synthetic polymers. Subunits that differ in stereochemistry are likely to have distinct conformational propensities, which could potentially lead to differences in the average shapes of polymer chains, even when the subunits are heterochiral. These findings highlight a dimension of polymer design that should be considered more broadly in efforts to improve specificity and efficacy of antimicrobial polymers.

Current advances in photocatalytic proximity labeling.

Understanding the intricate network of biomolecular interactions that govern cellular processes is a fundamental pursuit in biology. Over the past decade, photocatalytic proximity labeling has emerged as one of the most powerful and versatile techniques for studying these interactions as well as uncovering subcellular trafficking patterns, drug mechanisms of action, and basic cellular physiology. In this article, we review the basic principles, methodologies, and applications of photocatalytic proximity labeling as well as examine its modern development into currently available platforms. We also discuss recent key studies that have successfully leveraged these technologies and importantly highlight current challenges faced by the field. Together, this review seeks to underscore the potential of photocatalysis in proximity labeling for enhancing our understanding of cell biology while also providing perspective on technological advances needed for future discovery.