Harnessing Ionic Selectivity in Acetyltransferase Chemoproteomic Probes

Abstract

Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from the background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based sepharose pulldown resins differentiated by a single negatively charged residue and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of "probe selectivity" versus traditional "competitive chemical proteomics." This reveals that the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of preclinical therapeutic agents.

Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA

“…To extend our BRET sensing method into higher concentration regimes, we tested metabolite competition in the presence of 2, a CoA ligand with a higher affinity for NAA50. 30 As anticipated, competition of the tighter-binding CoA BRET acceptor requires higher concentrations of acetyl-CoA (IC 50 = 620 nM) and CoA (IC 50 = 7300 nM; Figure 4b). However, the selectivity for these metabolites remains unchanged.…”

“…CoA analogues were synthesized via a previously reported method (Scheme S1). 30 Chemoproteomic capture confirmed preferential binding of acetyl-CoA to the fusion protein (Figure S5). To assess this system's capabilities as a BRET reporter (Figure 3a), purified Nanoluc-NAA50 (Figure 3b) was incubated with the Nanoluc substrate furimazine in the presence of increasing Cy3-Ahx-CoA (1).…”

“…Prior studies have found both 1 and 2 capture NAA50 from cell extracts when immobilized on resin; however, enrichment by Ahx-D-CoA ( 2) is more efficient than 1 due to its higher affinity. 30 This is useful as high and low affinity BRET acceptor ligands may enable our sensing window to be shifted into different concentration regimes (e.g., micromolar versus nanomolar), increasing the utility of our approach.…”

Functionalised Cofactor Mimics for Interactome Discovery and Beyond

“…Although cofactors conjugated to solid supports, electrodes, or nanoparticles have been described e.g. for PQQ, [4] FAD, [5] heme, [6,7] and coenzyme A, [8] they are not the focus of this Review. In addition, synthetic cofactor analogues that do not incorporate additional functionality, such as those developed as inhibitors for medicinal chemistry or biochemical studies (see Table 1, for examples) are not discussed.…”

Funktionalisierte Cofaktor‐Analoga für die Erforschung von Interaktomen und darüber hinaus

“…Obwohl Cofaktoren, die an feste Trägerstoffe, Elektroden oder Nanopartikel konjugiert sind, z. B. für PQQ, [4] FAD, [5] Häm [6,7] und Coenzym A, [8] beschrieben wurden, stehen sie nicht im Mittelpunkt dieses Aufsatzes. Darüber hinaus werden synthetische Cofaktor-Analoga, die keine zusätzliche Funktionalität aufweisen, wie z.…”