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

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

doi:10.1038/s41598-019-45202-2

Qingling Tang, Antonio Artigues + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-45202-2

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Journal: Scientific Reports	Publication Date: Jun 21, 2019
Citations: 6	License type: open-access

Affiliation: University of Kansas Medical Center

Abstract

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.

Highlights

An emerging class of drugs that allosterically modulate enzymatic activity is generating considerable excitement because of the many reported advantages associated with this approach for the treatment of metabolic diseases[1,2,3,4,5,6]
We focus on “allosterically engineering” human liver pyruvate kinase (LPYK for liver PYK and hLPYK for the human isozyme) to demonstrate that mutations can mimic allosteric outcomes in vivo, the first step needed in our proposed model system design
Like other PYK isozymes, hLPYK catalyzes the final step of glycolysis, the conversion of phosphoenolpyruvate (PEP) and MgADP to pyruvate and MgATP

Summary

Introduction

An emerging class of drugs that allosterically modulate enzymatic activity is generating considerable excitement because of the many reported advantages associated with this approach for the treatment of metabolic diseases[1,2,3,4,5,6]. We propose to introduce mutations that mimic allosteric regulation by genome-editing into cell or animal models to verify allosteric drug targets in vivo. We focus on “allosterically engineering” human liver pyruvate kinase (LPYK for liver PYK and hLPYK for the human isozyme) to demonstrate that mutations can mimic allosteric outcomes in vivo, the first step needed in our proposed model system design. These regulatory mechanisms result in tight control of PYK activity in the liver This control is critical since 90% of blood glucose produced by gluconeogenesis is generated in the liver. For the treatment of hyperglycemia, these regulatory mechanisms offer a variety of potential targets for rational drug design aimed at increasing hLPYK activity, either through activation or preventing inhibition, and increasing hepatic glycolysis. We anticipate that activation of liver pyruvate kinase under conditions that favor hepatic gluconeogenesis, such as occurs in the diabetic liver, will create a futile cycle that will waste energy and reduce/prevent hepatic production of glucose (Fig. 1)

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