Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: molecular yardsticks for analyzing catalytic mechanism and inhibitor design.
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 gamma-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.
Hines
Aliya C
AC
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Parang
Keykavous
K
Kohanski
Ronald A
RA
Hubbard
Stevan R
SR
Cole
Philip A
PA
D000595:Amino Acid Sequence; D002384:Catalysis / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry; D015195:Drug Design; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000378:metabolism*; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D009711:Nucleotides; D047428:Protein Kinase Inhibitors; D011972:Receptor, Insulin