Insulin is a key hormone required for metabolic homeostasis. Relative lack of insulin results in diabetes mellitus (DM), a condition marked by hyperglycemia and associated metabolic abnormalities. Long-term complications include retinopathy and nephropathy. Current insulin therapies are designed to achieve normoglycemia by mimicking pancreatic basal/bolus secretion of insulin. Classical clinical trials in Type 1 DM and new-onset Type 2 DM have shown that intensive treatment of blood glucose concentration leads to a significant reduction in microvascular complications at the cost of increased hypoglycemic events. We seek to design novel insulin analogs to enhance the safety and efficacy of insulin replacement therapy. Current insulin analog formulations (although representing a significant advance in the 1990s) are sub- optimal with respect to pharmacokinetics (PK) and physical stability. In brief, short-acting analogs are too slow relative to post-prandial pancreatic secretion whereas basal analogs do not offer true 24-hour PK. The goal of my thesis is to design and characterize a novel ultra-short-acting insulin analog for (i) prandial control in muli- injection regiments and (ii) use in a closed-loop system ("smart pumps"). The essential idea underlying my research plan is exploitation of an expanded genetic code to enable recombinant expression of non-standard insulin analogs. Preliminary studies of insulin analogs containing unnatural amino-acid substitutions (obtained by chemical synthesis) suggest the utility of a para-Cl-PheB24 (in place of PheB24) at the potential dimer interface of insulin lispro (the active component of HumalogR). We hypothesize that the chloro-aromatic substitution leads to accelerated disassembly of the zinc insulin analog hexamer due to the electronegativity or size of the halogen substitution (relative to Phe). Pilot studies in anesthetized pigs indicate that para-Cl-PheB24-insulin lispro exhibits accelerated pharmacodynamics relative to HumalogR. In my thesis I plan to express this non-standard analog in the yeast Pichia pastoris, determine its three-dimensional structure by X-ray crystallography, and characterize its physical stability under conditions of potential formulation. To my knowledge, this will be the first structure to be determined of a protein containing a chloro-aromatic substitution and the first example of how such a modification can enhance the therapeutic properties of a protein.

Public Health Relevance

Introduction of non-standard amino acids in both a specific and highly reproducible manner in insulin using recent advances in recombinant protein expression can drastically improve the properties of insulin. One such candidate analog incorporating a chlorine atom shows promise as an ultra-fast acting insulin analog. These second generation insulin analogs will be safer and display improved physical characteristics leading to better patient treatment and efficacy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30DK094685-03
Application #
8517712
Study Section
Special Emphasis Panel (ZDK1-GRB-9 (O1))
Program Officer
Castle, Arthur
Project Start
2011-09-05
Project End
2015-09-04
Budget Start
2013-09-05
Budget End
2014-09-04
Support Year
3
Fiscal Year
2013
Total Cost
$47,232
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Menting, John G; Yang, Yanwu; Chan, Shu Jin et al. (2014) Protective hinge in insulin opens to enable its receptor engagement. Proc Natl Acad Sci U S A 111:E3395-404
Pandyarajan, Vijay; Phillips, Nelson B; Cox, Gabriela P et al. (2014) Biophysical optimization of a therapeutic protein by nonstandard mutagenesis: studies of an iodo-insulin derivative. J Biol Chem 289:23367-81