Journal Article

Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets

Artigues, Antonio, Tang, Qingling, Villar, Maria T., Peterson, Kenneth R., Thyfault, John P., Fenton, Aron W., Apte, Udayan, Zhu, Hao

2019

Overview

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Amino acid susbstitutions that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model. Inhibition of hLPYK was the first desired outcome of this study. We identified individual point mutations that: 1) mimicked allosteric inhibition by alanine, 2) mimicked inhibition by protein phosphorylation, and 3) prevented binding of fructose-1,6-bisphosphate (Fru-1,6-BP). Our second desired outcome was activation of hLPYK. We identified individual point mutations that: 1) prevented hLPYK from binding alanine, the allosteric inhibitor, 2) prevented inhibitory protein phosphorylation, or 3) mimicked allosteric activation by Fru-1,6-BP. Combining the three activating point mutations produced a constitutively activated enzyme that was unresponsive to regulators. Expression of a mutant hLPYK transgene containing these three mutations in a mouse model was not lethal. Thus, mutational mimics of allosteric effectors will be useful to confirm whether allosteric activation of hLPYK will control glycolytic flux in the diabetic liver to reduce hepatic glucose production and, in turn, reduce or prevent hyperglycemia.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 42 / 42/41 / 631/45/320 / 631/45/607/275 / 82 / 82/58 / 82/83 / Alanine / Allosteric properties / Allosteric Regulation - drug effects / Amino acids / Animals / Diabetes mellitus / Drug development / Enzymes / Fructose / Fructose-1,6-diphosphate / Fructosephosphates - metabolism / Gene Editing / Genome editing / Genomes / Glycolysis / Humanities and Social Sciences / Humans / Hyperglycemia / Kinases / Liver / Liver - enzymology / Medical treatment / Mice / Models, Animal / multidisciplinary / Mutation / Phosphorylation / Pyruvate kinase / Pyruvate Kinase - genetics / Pyruvate Kinase - metabolism / Pyruvic acid / Science / Science (multidisciplinary)