A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity

Atilio Reyes Romero,Tad A Holak,Alexander S S Dömling,Matteo Gentili,Maciej Kozak,Jacek Plewka,Michael Sattler,Grzegorz M Popowicz,Vito Calderone,Serjey Lunev,Michał Taube,Matthew R Groves

doi:10.1038/s42003-021-02442-1

Abstract

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site.

Highlights

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum
While cytoplasmic malate dehydrogenase (MDH) supports the malate–aspartate shuttle—allowing reducing equivalents to pass through the inner mitochondrial membrane—mitochondrial MDH is involved in NADH and citrate production to support the electron-transport chain within the TCA cycle[1]
Comparison of the results reveals that seven fragments binding to V190W PfMDH are bound to WT PfMDH—suggesting 30 potential candidates interacting with the oligomeric interface

Summary

Introduction

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. In the presence of LDH antagonists like oxamate, cancer cells adopt an evasion mechanism involving MDH which is recruited in order to support glycolysis in the absence of oxygen and production of NAD4. Given the ubiquity of the dinucleotide binding domain (Rossman fold) in NADH binding enzymes[10] and its presence in 155 reviewed enzymatic activities within human cells (Interpro ID: IPR03629111), it is perhaps not surprising that many of these antagonists possess cytotoxic effects. 3 h exposure to 5 μM of the alkaloid staurosporine aglycone led to an 80% decrease in cell viability via caspase activation mechanisms and oxidative DNA damage through reactive oxygen species formation[13,14]

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Results

Conclusion