Structural Basis of Src Tyrosine Kinase Inhibition with a New Class of Potent and Selective Trisubstituted Purine‐based Compounds

Abstract

The tyrosine kinase pp60src (Src) is the prototypical member of a family of proteins that participate in a broad array of cellular signal transduction processes, including cell growth, differentiation, survival, adhesion, and migration. Abnormal Src family kinase (SFK) signaling has been linked to several disease states, including osteoporosis and cancer metastases. Src has thus emerged as a molecular target for the discovery of small-molecule inhibitors that regulate Src kinase activity by binding to the ATP pocket within the catalytic domain. Here, we present crystal structures of the kinase domain of Src in complex with two purine-based inhibitors: AP23451, a small-molecule inhibitor designed to inhibit Src-dependent bone resorption, and AP23464, a small-molecule inhibitor designed to inhibit the Src-dependent metastatic spread of cancer. In each case, a trisubstituted purine template core was elaborated using structure-based drug design to yield a potent Src kinase inhibitor. These structures represent early examples of high affinity purine-based Src family kinase-inhibitor complexes, and they provide a detailed view of the specific protein-ligand interactions that lead to potent inhibition of Src. In particular, the 3-hydroxyphenethyl N9 substituent of AP23464 forms unique interactions with the protein that are critical to the picomolar affinity of this compound for Src. The comparison of these new structures with two relevant kinase-inhibitor complexes provides a structural basis for the observed kinase inhibitory selectivity. Further comparisons reveal a concerted induced-fit movement between the N-and C-terminal lobes of the kinase that correlates with the affinity of the ligand. Binding of the most potent inhibitor, AP23464, results in the largest inducedfit movement, which can be directly linked to interactions of the hydrophenethyl N9 substituent with a region at the interface between the two lobes. A less pronounced induced-fit movement is also observed in the Src-AP23451 complex. These new structures illustrate how the combination of structural, computational, and medicinal chemistry can be used to rationalize the process of developing high affinity, selective tyrosine kinase inhibitors as potential therapeutic agents. Received 2 October 2005, revised and accepted for publication 11 October 2005The protein tyrosine kinase pp60src (Src) is the prototypical member of a family of non-receptor kinases, termed the Src family kinases (SFKs), which participate in a broad spectrum of cellular processes, including cell growth, differentiation, survival, adhesion, and migration (1-4). Src was identified as the transforming element of the Rous oncogenic retrovirus, exhibiting its transforming properties through a constitutively active mutant form of the protein effecting hyperactivation of kinase-dependent substrate protein phosphorylation and subsequent aberrant cellular signaling processes (5). Not surprisingly, SFK-dependent signal transduction mechanisms have been studied in a wide variety of cell types. S...

Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum

“…It is, however, possible to build selectivity into ATP-competitive inhibitors by extending the interaction of the inhibitor molecules to non-conserved allosteric sites located within a hydrophobic pocket adjacent to the ATP-binding site 38,39 . This strategy has been successfully employed for a number of protein kinase inhibitors designed to target human kinases in the treatment of cancer 40 including the clinically approved Bcr-Abl inhibitor Imatinib (Gleevec) 41 . That a similar approach to drug design may be effective in the search for molecules with selectivity against malaria is supported by the significant phylogenetic diversity between the human and plasmodial kinomes 4 .…”

Activation of c-Src and Fyn Kinases by Protein-tyrosine Phosphatase RPTPα Is Substrate-specific and Compatible with Lipid Raft Localization

“…Osteoporosis and stroke are examples of additional pathologies where pharmacological modulation of SFK activity may be relevant (43,44). Although SFK inhibitors are currently under development, side effects and toxicity constitute a concern given the many physiological processes in which SFKs participate (45).…”

Synthesis, biological evaluation and docking studies of 4-amino-tetrahydroquinazolino[3,2-e]purine derivatives

“…All the calculations were realized under the Sybyl 6.9.1 molecular modelling package [37]. The protein cocrystallized with the purine inhibitor AP-23464, was retrieved from the Protein Data Bank [38] under the entry 2BDJ [39], the binding site being defined as a sphere of 10 Å around the cocrystallized inhibitor. The studied compounds were built from the internal fragments library of Sybyl then their geometry was optimized by the Powell method available in the Maximin2 procedure to a gradient of 0.001 kcal/mol Å using the Tripos force field [40].…”