HARNESSING ALLOSTERY TO MODULATE PROTEIN-PROTEIN INTERACTIONS AND ENZYMATIC ACTIVITY WITH SMALL MOLECULES

ACEBEDO MARTINEZ MACARENA; SACERDOTI MARIANA; GROSS LISSY Z F; DI LELLA SANTIAGO; GIRONACCI MARIELA; BIONDI RICARDO MIGUEL; LEROUX ALEJANDRO EZEQUIEL

Ciudad Autónoma de Buenos Aires

Congreso; VI International Congress In Translational Medicine; 2023

International Master Program in Biomedical Sciences

Background and Aims: Allostery refers to the process in which an interaction at one specific site on a protein (allosteric site) causes a conformational change that impacts the function of a distant site. This phenomenon is bidirectional, meaning that binding at the distant site can also induce a conformational change at the regulatory site. Based on our previous work on protein kinases regulation and the existing literature, this study aims to assess the allosteric regulation of angiotensin-converting enzyme II (ACE2). We are searching for small molecules that can modulate ACE2 enzyme activity or affect ACE2-Spike interaction as proof of concept for drug development projects to treat hypertension or coronavirus infection.Methods: Molecules identified as disruptors of the ACE2-Spike interaction through high-throughput screening (Hanson et al., 2020) and analogs of interest were tested in our own AlphaScreen interaction assay. The effect on enzyme activity was assessed using a fluorogenic assay. The molecules identified as activators or inhibitors were further evaluated using a HPLC-coupled enzymatic assay with the radioactive-labeled endogenous substrate. We validated compound binding by thermal shift with a differential scanning fluorimetry assay (DSF). We used molecular dynamics and docking studies to identify potential binding sites of the compounds.Results: Our in house alphascreen interaction assay failed to identify compounds thatspecifically disrupt the ACE2-Spike interaction. However, some of the tested compounds produce a 3-4 fold increase in the enzymatic activity of ACE2 strongly suggesting an allosteric regulation of enzyme activity. Furthermore, the ACE2 activator compound 53 generates a destabilization of ACE2 in DSF, confirming its binding to the protein. Lastly, in silico results using peptidase domain of ACE2 allowed us to identify a possible binding site of compound 39. Conclusions: There is evidence for an allosteric mechanism to break Spike-ACE2 interactions but so far, we could not identify potent compounds using our Alphascreen interaction assay. The identification of small molecules that reproducibly enhance ACE2 enzyme activity, allowed us to validate an allosteric regulation of ACE2.