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

ACEBEDO MARTINEZ, M; SACERDOTI, M; GROSS, LZF; DI LELLA, S; GIRONACCI, M; BIONDI, RM; LEROUX, AE

Buenos Aires

Congreso; International Program in Biomedical Sciences; 2023

Background and Aims: Allosteryrefers 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 adistant site. This phenomenon is bidirectional, meaning that binding at thedistant site can also induce a conformational change at the regulatory site.Based on our previous work on protein kinases regulation and the existingliterature, this study aims to assess the allosteric regulation ofangiotensin-converting enzyme II (ACE2). We are searching for small moleculesthat can modulate ACE2 enzyme activity or affect ACE2-Spike interaction asproof of concept for drug development projects to treat hypertension orcoronavirus infection. Methods: Molecules identified asdisruptors of the ACE2-Spike interaction through high-throughput screening(Hanson et al., 2020) and analogs of interest were tested in our ownAlphaScreen interaction assay. The effect on enzyme activity was assessed usinga fluorogenic assay. The molecules identified as activators or inhibitors werefurther evaluated using a HPLC-coupled enzymatic assay with theradioactive-labeled endogenous substrate. We validated compound binding bythermal shift with a differential scanning fluorimetry assay (DSF). We usedmolecular dynamics and docking studies to identify potential binding sites ofthe compounds. Results: Our in house alphascreeninteraction assay failed to identify compounds thatspecifically disrupt theACE2-Spike interaction. However, some of the tested compounds produce a 3-4fold increase in the enzymatic activity of ACE2 strongly suggesting anallosteric regulation of enzyme activity. Furthermore, the ACE2 activatorcompound 53 generates a destabilization of ACE2 in DSF, confirming its bindingto the protein. Lastly, in  silicoresults using peptidase domain of ACE2 allowed us to identify a possiblebinding site of compound 39.  Conclusions: There is evidence for an allosteric mechanismto break Spike-ACE2 interactions but so far, we could not identify potentcompounds using our Alphascreen interaction assay. The identification of smallmolecules that reproducibly enhance ACE2 enzyme activity, allowed us tovalidate an allosteric regulation of ACE2.