REVIEW Protein Kinase Inhibitors: Insights into Drug Design from Structure Martin E. M. Noble,* Jane A. Endicott,* Louise N. Johnson*† Protein kinases are targets for treatment of a number of diseases. This review focuses on kinase inhibitors that are in the clinic or in clinical trials and for which structural information is available. Structures have informed drug design and have illuminated the mechanism of inhibition. We review progress with the receptor tyrosine kinases (growth factor receptors EGFR, VEGFR, and FGFR) and nonreceptor tyrosine kinases (Bcr-Abl), where advances have been made with cancer therapeutic agents such as Herceptin and Gleevec. Among the serine-threonine kinases, p38, Rho-kinase, cyclin- dependent kinases, and Chk1 have been targeted with productive results for inflam- mation and cancer. Structures have provided insights into targeting the inactive or active form of the kinase, for targeting the global constellation of residues at the ATP site or less conserved additional pockets or single residues, and into targeting noncatalytic domains. A number of diseases, including cancer, dia- betes, and inflammation, are linked to pertur- bation of protein kinase–mediated cell signal- ing pathways. The human genome encodes some 518 protein kinases (1) that share a catalytic domain conserved in sequence and structure but which are notably different in how their catalysis is regulated. The ATP- binding pocket is between the two lobes of the kinase fold (Fig. 1). This site, together with less conserved surrounding pockets, has been the focus of inhibitor design that has exploited differences in kinase structure and pliability in order to achieve selectivity. Drugs are in clinical trials that target all stages of signal transduction: from the recep- tor tyrosine kinases that initiate intracellular signaling, through second-messenger genera- tors and kinases involved in signaling cas- cades, to the kinases that regulate the cell cycle that governs cellular fate (2–5 ). Protein Kinases as Targets for Inhibitor Design Receptor tyrosine kinases. Dysregulation of growth factor signaling networks has been reported in multiple human cancers. Binding of growth factors to extracellular domains of receptor tyrosine kinases activates the intra- cellular kinase domain. The epidermal growth factor receptor (EGFR) is normally activated by oligomerization in response to ligand binding, but in cancer cells, family members [EGFR (ErbB1, HER1) and its ho- mologs HER2, HER3, HER4] are frequently overactive. To block the EGFR signal, differ- ent therapeutic agents have been developed that target the extracellular ligand-binding and intracellular kinase domains. The HER2/Neu gene product is up- regulated in the tumor cells of about 30% of breast cancer patients (6 ). This finding pro- vided the rationale for the development of Herceptin, a humanized monoclonal antibody that binds the HER2 receptor and induces receptor internalization. In clinical trials, Herceptin alone proved effective in treatment for 15% of patients with HER2-overexpressing metastatic breast cancer and was more effective when used in combination with chemotherapy agents such as paclitaxel (7 ). Iressa (8) and Tarceva (9) (Fig. 2) are small-molecule inhibitors that bind to the EGFR tyrosine kinase domain. Iressa has been registered for treatment of metastatic non–small cell lung cancer where other treat- ments have failed, and Tarceva is currently in phase III clinical trials for several tumor types such as non-small cell lung cancer and pancreatic cancer. Inhibitors that bind irre- versibly to the EGFR through covalent bond formation with a cysteine residue in the ATP pocket are even more effective as kinase inhibitors (10, 11). Their clinical efficacy is being evaluated. A second class of receptor tyrosine ki- nases rationally targeted in anticancer drug development are those that promote angio- genesis (12), particularly the vascular endo- thelial growth factor receptor (VEGFR). This strategy is based on the rationale that forma- tion of a blood supply is required for progres- sion of solid tumors (13). Agents that target VEGFR signaling include the VEGF-specific antibody bevacizumab, the small-molecule tyrosine kinase inhibitors such as SU5416 (Fig. 2) and the anilino-phthalazine PTK787. Some of these agents have met with limited success in monotherapy against solid tumors [e.g., (14 )], but may be more effective in combination with other agents. The fibroblast growth factor receptor (FGFR) is a further target for rational inhibitor design, particular- ly for angiogenesis in a spectrum of pathol- ogies that include cancer, rheumatoid arthri- tis, diabetic retinopathy, and atherosclerosis [see references in (15 )]. Nonreceptor tyrosine kinases. About one- third of tyrosine kinases are classed as non- receptor tyrosine kinases. They are found in the cytoplasm, lack a transmembrane section, and generally function downstream of the receptor tyrosine kinases. Chronic myeloid leukemia (CML) is a relatively rare cancer (5000 cases per year in the United States), often associated with reciprocal translocation of chromosomes 9 and 22. This event fuses Laboratory of Molecular Biophysics, Department of Biochemistry, Rex Richards Building, University of Ox- ford, Oxford 3X2 3QU, UK. *These authors contributed equally to this work. †To whom correspondence should be addressed. E- mail: louise@biop.ox.ac.uk Fig. 1. The structure of the catalytic domain of cAbl in complex with Gleevec (51). The N-terminal lobe consists of a  sheet and one conserved  helix (helix C). The C-terminal lobe is largely helical and contains a segment, the activation segment, which includes resi- due(s) that in many kinases are phosphoryl- ated for activity (72). The hinge region con- nects the two lobes. The protein structure is color ramped so that residues close to the N terminus are blue, and those close to the C terminus are red. Gleevec is shown bound to the ATP-binding site, from which it extends under the C helix. Thr 315 , the “gatekeeper” residue, and Phe 382 , the conserved phenylal- anine that marks the beginning of the acti- vation segment, are labeled. D RUG D ISCOVERY 19 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org 1800 S PECIAL S ECTION