Pyruvate kinase (PK) plays an essential role in the final step of glycolysis, catalyzing the transfer of phosphate from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to form pyruvate and adenosine triphosphate (ATP), respectively. PK has four distinct isoenzymes in humans, including L, R, M1, and M2, with different tissue specificities. The highly homologous L and R isoenzymes (PKLR) are expressed by the gene PKLR. PKLR has been implicated in several diseases. In erythrocytes, activation of PKLR reduces the levels of 2,3-Bisphosphoglycertae (2,3-BPG), which is an allosteric effector of hemoglobin, and a critical contributor to sickle cell disease pathogenesis. Cancer cells rewire their metabolism to promote growth, survival, proliferation, and long-term maintenance through increased glucose uptake and fermentation to lactate. This phenomenon is known as the Warburg effect. Being a crucial enzyme in the glycolytic cycle, PKLR inhibition has been shown to suppress the Warburg effect in hepatocellular carcinoma (HCC). Therefore, finding both activators and inhibitors of PKLR have potential uses as anti-sickling and anticancer agents, respectively. A virtual screening campaign was initiated to identify potential allosteric modulators of PKLR using AtomNet® technology, a unique artificial intelligence platform that applies deep convolutional neural networks to drug discovery. Seventy compounds were obtained and tested for their modulating effects on human PKLR. For the assay, the PKLR protein carrying an N-terminal His-tag was expressed in E. coli and purified using a Ni-NTA column. The enzyme activity was determined at 37 °C by lactate dehydrogenase (LDH)–coupled assay. This assay measures the change in UV/VIS absorbance at 340 nm due to the oxidation of NADH by LDH in the course of converting pyruvate (the product of PKLR enzymatic reaction) to lactate. The screening was performed at a 1 mM concentration of PEP (PKLR substrate). Promising compounds were studied in more detail with different PEP concentrations (0.25-3.0 mM) to estimate kinetic parameters and the potency of the compounds. Several potential hits were found, including activators and inhibitors. The results will be presented.

Artificial intelligence aided drug discovery of pyruvate kinase modulators

ACS Fall 2021

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