A CHEMICAL BIOLOGY APPROACH TO UNDERSTAND THE REGULATION OF FULL-LENGTH PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE 1 (PDK1)

GROSS, LISSY; SACERDOTI, MARIANA; LEROUX, ALEJANDRO E.; CAPPELLARI, MV; ARAMENDIA, PF; GHODE, PF; ANAND, GS; KLINKE, S; BIONDI, RICARDO M.

Salta

Congreso; LV Reunión Científica anual SAIB y XIV congreso de PABMB; 2019

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Phosphoinositide-dependent protein kinase 1 (PDK1) is a master AGC kinase that phosphorylates at least other 23 AGC kinases, being PKB/Akt the most relevant substrate downstream of PI3-kinase, important for growth and cell survival and a drug target for cancer treatment. Over the years, our laboratory studied and characterized in detail the catalytic domain of PDK1, as well as the selective activation of substrates such as SGK or S6K, which in order to be phosphorylated require a docking interaction with a hydrophobic site in PDK1 termed the PIF-Pocket. However, this is not the case of Akt/PKB, since it can be activated in a PIF-Pocket independent way. On the other hand, up to date little is known about the mechanistic and structural details of PDK1 full length. Therefore, we are using an interdisciplinary approach to understand how the full length protein is regulated and how this regulation mechanism can be manipulated to specifically inhibit the activation of PKB/Akt. As a result of a medium-scale screening of small compounds we carried out using AlphaScreen technology, we validated a series of small compounds ?hits? that modulate PDK1 structure by interaction at different sites on PDK1. We performed hydrogen/deuterium exchange (HDX) experiments and crystallization screenings to understand the structure of full length PDK1 and how it can be modulated with small compounds. We here present a series of results obtained using HDX on full length PDK1 and the crystal structure of the catalytic domain of PDK1 bound to a small compound identified in our screening. We also present our in vitro studies to understand the oligomerization of PDK1 visualizing single particles using STORM fluorescence microscopy. Finally, we integrate our data to present an updated model on the molecular mechanism of regulation of PDK1.