Towards the identification of lactate dehydrogenase B inhibitors

: crystallization assays and enzymatic characterization of thiosemicarbazide derivatives

Abstract

Deregulation of cellular energetics is a well-admitted hallmark of cancer. One of the most important deregulations is called the Warburg effect: even in the presence of oxygen, cancer cells rely mostly on glycolysis to get ATP and pyruvate is converted into lactate. This phenomenon is called “aerobic” glycolysis. The enzyme catalyzing the interconversion of lactate to pyruvate is a tetrameric enzyme called lactate dehydrogenase (LDH) which exists as two main isoforms: LDH-A and LDH-B. Produced lactate will then control lysosomal activity and promote autophagy in cancer cells, leading to an increased tumor proliferation. More specifically, LDH-B has been found to be implicated in these two mechanisms. Since targeting active site suffers from lack of selectivity due to the presence of the NAD+-binding site, a new approach consists in targeting the oligomeric state of the enzyme. Indeed, it has been proven that the catalytic activity depends on the tetrameric conformation. The key-core of this tetramerization process is the tetramerization arm. It was identified that the N-terminal ⍺-helix is essential for tetramerization to occur.
This master thesis aims to find out compounds that could inhibit LDH-B by targeting the active or the tetrameric state, rendering it inactive. Among the molecules of the “Namur Medicine & Drug Innovation Center” (NAMEDIC) chemolibrary, a special focus will be devoted to four thiosemicarbazides hits previously identified.
To evaluate the therapeutic interest of those compounds, those thiosemicarbazides hits were screened on a full-length form of LDH-B and a form lacking the tetramerization arm, leading to an inactive dimer. The goal is to find out those ligands that stabilize the inactive dimer while destabilizing the active tetramer. From the thermal shift assay, we isolated compound T1 since it is the one that showed a stabilization effect on the dimeric form of LDH-B. Then, localization of compound T1 was assessed by molecular docking in the tetramerization site and in the active site. From the interaction profile established by compound T1, it is more prone to localize in the active site and act as an inhibitor. To confirm this, co-crystallization assays and enzymatic characterization were performed. It was shown that compound T1 inhibits LDH-B but precise location has not been resolved yet.
Compound T1 has been identified as an LDH-B inhibitor, rendering it promising in the context of cancer drugs development. In perspectives, the same methodology should be used on LDH- A since the inhibition of one isoenzyme could be overcome by the overexpression of the other. Furthermore, compound T1 derivates should be tested and IC50 must be determined to find out the most potent inhibitor.

Date of Award	18 Jan 2022
Original language	English
Awarding Institution	University of Namur
Supervisor	Johan Wouters (Supervisor) & Megane Van Gysel (Co-Supervisor)