STRUCTURE-BASED DESIGN OF A POTENT, SELECTIVE, AND IRREVERSIBLE INHIBITOR OF THE CATALYTIC DOMAIN OF THE ERBB RECEPTOR SUBFAMILY OF PROTEIN-TYROSINE KINASES

We report the use of structure-based drug design to create a selective erbB-1 (a.k.a. epidermal growth factor receptor) and erbB-2 (a.k.a. n eu/her2 growth factor receptor) tyrosine kinase inhibitor. Using the X -ray crystal structure of the ternary complex of the cAMP-dependent Se r/Thr kinase(1) together with a sequence alignment of the catalytic do mains of a representative set of Ser/Thr and Tyr protein kinases, we h ave examined the nucleotide binding site for potential positions to at tach an irreversible inhibitor. This information, combined with homolo gy modeling of the erbB-1 and erbB-2 tyrosine kinase catalytic domains , has led to the identification of Cys797 of erbB1 and Cys805 of erbB2 , which are structurally equivalent to Glu127 in the cAMP dependant Se r/Thr kinase as potential target residues. The X-ray structure of the cAMP Ser/Thr kinase shows Glu127 to be involved in a hydrogen-bonding interaction with the 2'-OH of the ribose portion of ATP. Using molecul ar modeling, it was predicted that the Cys side chains in erbB-1 and e rbB-2 performed an analogous role, and it was postulated that the repl acement of the 2'-OH of adenosine with a thiol might allow for a coval ent bond to form. Since only erbB-1 and erbB-2 have a Cys at this posi tion, the inhibitor should be selective. This model was subsequently t ested experimentally by chemical synthesis of 2'-thioadenosine and ass ayed against the full length erbB-1 receptor and the catalytic domains of erbB-2, insulin receptor, beta-PDGF receptor, and the FGF receptor . Our results show that thioadenosine covalently inactivates erbB-1 wi th a second-order rate constant of k(max)/K-S = 2000 +/- 500 M(-1) s(- 1). Inactivation is fully reversed by 1 mM dithiothreitol, suggesting that inactivation involves the modification of a cysteine residue at t he active site, presumably Cys797. The rate of inactivation saturates with increasing thioadenosine concentrations, suggesting that inactiva tion occurs through initial formation of a noncovalent complex with K- D = 1.0 +/- 0.3 mu M, followed by the slow formation of a disulfide bo nd with a rate constant of k(max) = (2.3 +/- 0.2) x 10(-3) s(-1). This approach may have application in the design of selective irreversible inhibitors against other members of the kinase family.