Hyperglycemia results in many of the chronic complications associated with Type 2 Diabetes. Controlling hyperglycemia attenuates these complications;however, current antihyperglycemic agents are limited in their efficacy. Activating human liver pyruvate kinase (hL-PYK) increases glucose catabolism by glycolysis and is therefore an antihyperglycemic drug target, a conclusion supported by Dr. David Robbins, former Medical Director for Diabetes and Obesity Drug Development at Ely Lily and Co. At the enzyme level, hL-PYK activity is regulated via allosteric mechanisms and hormonally-controlled phosphorylation. The locations of ligand binding sites on the protein structure have been identified for Ala (inhibitor) and Fru-1,6-BP (activator), but not for ATP (inhibitor). In the N-terminus, Ser12 is the site of phosphorylation. Modulating allosteric activation and/or preventing inhibition are promising targets to increase PYK activity and thereby decrease serum glucose. Based on preliminary data, we hypothesize that specific subsets of protein/ligand interactions are important for allostery. Understanding which protein/activator interactions contribute to ligand binding and which contribute to eliciting activation will facilitate the design of allosteric drugs. Likewise, in order to design a drug that both 1) binds competitively with an allosteric inhibitor and 2) does not elicit an allosteric response, it is first necessary to understand which protein/inhibitor interactions contribute to allosteric function. The focus of this study is to identify allosteric-specific interactions (i.e., the interactions that elicit an allosteric response) between hL-PYK and effectors or the phosphorylated N-terminus. To identify allosteric- specific protein/ligand interactions, Specific Aim 1 will use linkage-analysis to determine binding and allosteric properties for: 1) allosteric effector analogs and the wild type hL-PYK, and 2) native effectors and hL-PYK with mutations introduced in ligand binding sites. Since little is understood about the interactions between the phosphorylated N-terminus and the main body of the protein, these interactions will be detected in Specific Aim 2 using X-ray crystallography and/or cross-linking/mass spectrometry. Phosphorylation dependent changes in the dynamic motions of the N-terminus will be monitored with dynamic fluorescence anisotropy.
In Specific Aim 3 novel lead ligand/drug compounds that do not resemble physiological effectors will be identified by screening a chemical library. X-ray crystallography will be used to characterize how identified library compounds bind to hL-PYK. Project Narrative Activation of liver glycolysis, the major glucose utilizing pathway, offers a potentially effective strategy to treat the hyperglycemia associated with Type 2 Diabetes. Allosteric activation of pyruvate kinase, a major regulatory enzyme in glycolysis, increases the rate of glucose metabolism. The current studies will facilitate drug development targeting the activation of human liver pyruvate kinase to increase glucose metabolism in the treatment of hyperglycemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK078076-05
Application #
8217215
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Sechi, Salvatore
Project Start
2008-02-19
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
5
Fiscal Year
2012
Total Cost
$288,149
Indirect Cost
$92,129
Name
University of Kansas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
Holyoak, Todd; Zhang, Bing; Deng, Junpeng et al. (2013) Energetic coupling between an oxidizable cysteine and the phosphorylatable N-terminus of human liver pyruvate kinase. Biochemistry 52:466-76
Prasannan, Charulata B; Villar, Maria T; Artigues, Antonio et al. (2013) Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry. Biochemistry 52:1998-2006
Urness, James M; Clapp, Kelly M; Timmons, J Cody et al. (2013) Distinguishing the chemical moiety of phosphoenolpyruvate that contributes to allostery in muscle pyruvate kinase. Biochemistry 52:1-3
Fenton, Aron W (2013) Are all regions of folded proteins that undergo ligand-dependent order-disorder transitions targets for allosteric peptide mimetics? Biopolymers :
Prasannan, Charulata B; Tang, Qingling; Fenton, Aron W (2012) Allosteric regulation of human liver pyruvate kinase by peptides that mimic the phosphorylated/dephosphorylated N-terminus. Methods Mol Biol 796:335-49
Fenton, Aron W (2012) Identification of allosteric-activating drug leads for human liver pyruvate kinase. Methods Mol Biol 796:369-82
Miller, Danny E; Prasannan, Charulata B; Villar, Maria T et al. (2012) HDXFinder: automated analysis and data reporting of deuterium/hydrogen exchange mass spectrometry. J Am Soc Mass Spectrom 23:425-9
Prasannan, Charulata B; Artigues, Antonio; Fenton, Aron W (2011) Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation. Anal Bioanal Chem 401:1083-6
Alontaga, Aileen Y; Fenton, Aron W (2011) Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes. Biochemistry 50:1934-9
Fenton, Aron W; Johnson, Troy A; Holyoak, Todd (2010) The pyruvate kinase model system, a cautionary tale for the use of osmolyte perturbations to support conformational equilibria in allostery. Protein Sci 19:1796-800

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