Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: Molecular yardsticks for analyzing catalytic mechanism and inhibitor design

Aliya C. Hines, Keykavous Parang, Ronald A. Kohanski, Stevan R. Hubbard, Philip A. Cole

Research output: Contribution to journalArticle

Abstract

Bisubstrate analogs have the potential to provide enhanced specificity for protein kinase inhibition and tools to understand catalytic mechanism. Previous efforts led to the design of a peptide-ATP conjugate bisubstrate analog utilizing aminophenylalanine in place of tyrosine and a thioacetyl linker to the γ-phosphate of ATP which was a potent inhibitor of the insulin receptor kinase (IRK). In this study, we have examined the contributions of various electrostatic and structural elements in the bisubstrate analog to IRK binding affinity. Three types of changes (seven specific analogs in all) were introduced: a Tyr isostere of the previous aminophenylalanine moiety, modifications of the spacer between the adenine and the peptide, and deletions and substitutions within the peptide moiety. These studies allowed a direct evaluation of the hydrogen bond strength between the anilino nitrogen of the bisubstrate analog and the enzyme catalytic base Asp and showed that it contributes 2.5 kcal/mol of binding energy, in good agreement with previous predictions. Modifications of the linker length resulted in weakened inhibitory affinity, consistent with the geometric requirements of an enzyme-catalyzed dissociative transition state. Alterations in the peptide motif generally led to diminished inhibitory potency, and only some of these effects could be rationalized based on prior kinetic and structural studies. Taken together, these results suggest that a combination of mechanism-based design and empirical synthetic manipulation will be necessary in producing optimized protein kinase bisubstrate analog inhibitors.

Original languageEnglish (US)
Pages (from-to)285-297
Number of pages13
JournalBioorganic Chemistry
Volume33
Issue number4
DOIs
StatePublished - Aug 2005

Fingerprint

Insulin Receptor
Peptides
Protein Kinases
Phosphotransferases
Adenosine Triphosphate
Adenine
Enzymes
Viperidae
Binding energy
Static Electricity
Tyrosine
Hydrogen
Electrostatics
Hydrogen bonds
Substitution reactions
Nitrogen
Phosphates
Kinetics

Keywords

  • Inhibitor
  • Kinase
  • Nucleotide
  • Peptide
  • Protein

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Organic Chemistry
  • Drug Discovery

Cite this

Bisubstrate analog probes for the insulin receptor protein tyrosine kinase : Molecular yardsticks for analyzing catalytic mechanism and inhibitor design. / Hines, Aliya C.; Parang, Keykavous; Kohanski, Ronald A.; Hubbard, Stevan R.; Cole, Philip A.

In: Bioorganic Chemistry, Vol. 33, No. 4, 08.2005, p. 285-297.

Research output: Contribution to journalArticle

Hines, Aliya C. ; Parang, Keykavous ; Kohanski, Ronald A. ; Hubbard, Stevan R. ; Cole, Philip A. / Bisubstrate analog probes for the insulin receptor protein tyrosine kinase : Molecular yardsticks for analyzing catalytic mechanism and inhibitor design. In: Bioorganic Chemistry. 2005 ; Vol. 33, No. 4. pp. 285-297.
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