EXPLORING ALLOSTERY IN ACE2: CAN SMALL MOLECULES AFFECT ENZYME ACTIVITY AND PROTEIN-PROTEIN INTERACTIONS?

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

Rosario

Congreso; LIX Reunión Científica anual; 2023

Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular (SAIB)

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. Protein-protein interactions are very difficult to disrupt with small molecules, and therefore an enormous challenge for drug discovery. In previous work conducted in the lab on protein kinases we showed that protein-protein interactions can be enhanced or potently disrupted allosterically (Trends Biochem Sci 45(1):27-41, 2020). We then aimed to investigate the possibility to expand our chemical biology studies on allostery to other important biomedical proteins. The protein ACE2 catalyses the cleavage of Angiotensin II into Angiotensin (1-7). In addition, ACE2 is the receptor that is used for infection by coronaviruses such as SARS-CoV2. We analyzed the published information on ACE2 and concluded that it could be an allosteric protein; therefore small molecules could potentially allosterically disrupt interactions with Spike and could potentially serve as anti-infectives (ChemMedChem. 15(18):1682-1690, 2020).In the meantime, a group at the NIH performed high-throughput screening using AlphaScreen and deposited the raw data of the screening online. To date, the NIH group and others have identified small compounds that disrupt the ACE2-Spike interaction. However, the mechanism of action of the compounds have not been investigated. Here, we describe our initial studies on the modulation of ACE2 by small compounds. Firstly, we independently analyzed the NIH screening data and investigated the effect of hits and structural analogs of interest in our own AlphaScreen interaction assay and on the effect on enzyme activity. We analyzed the enzyme activity using a fluorogenic assay. Notably, we identified compounds that produced a 3-4 fold increase in the enzymatic activity of ACE2 suggesting an allosteric mechanism. Those molecules identified as activators or inhibitors were further evaluated using a radioactive assay with the endogenous substrate (HPLC). Additionally, we setted-up a differential scanning fluorimetry assay to indirectly validate compound binding. The hit compound 53, an activator, generates a destabilization of ACE2 in our thermal stability assay, confirming its binding to the protein. Lastly, docking results using peptidase domain of ACE2 allowed us to identify a possible binding site for the activator compound 39. Our ongoing work supports the model where ACE2 is an allosteric protein. Most notably, our results indicate the existence of an allosteric regulation of the enzymatic activity of ACE2. This finding suggests that ACE2 activity could be physiologically regulated. Moreover, the findings open the possibility that ACE2 activity could be modulated pharmacologically, for example, as an anti-hypertensive treatment.