GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the activelike structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly, we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor, ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures.

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.

Orotidine 5 -phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme. The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5 -phosphate, at a resolution of 2.1 Å and 2.4 Å, respectively. Orotidine 5 -phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel. The binding of 6-hydroxyuridine 5 -phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product. In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state. Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation.O rotidine 5Ј-phosphate decarboxylase (ODCase) (EC 4.1.1.23) is responsible for de novo synthesis of uridine 5Ј-phosphate, an essential precursor of RNA and DNA. In neutral solution, orotidine 5Ј-monophosphate (OMP) undergoes spontaneous decarboxylation to uridine 5Ј-phosphate with a half-time of 78 million years (1). At the ODCase active site, the same reaction proceeds with a half-time of 18 msec (2). Comparison of k cat ͞K m with k non indicates that ODCase surpasses other enzymes in its proficiency ʈ as a catalyst, achieving a remarkable affinity for the altered substrate in the transition state (1). In addition to surmounting this formidable kinetic barrier, the ODCase reaction is of special interest in view of its lack of precedent in biological chemistry. The substrate is devoid of an effective repository for the negative charge that is generated at C-6 when CO 2 is eliminated, yet the enzyme functions without metals or other cofactors. Enzymatic decarboxylation of OMP is also remarkable in the importance (for catalysis) of a seemingly irrelevant part of the substrate. By its presence, the 5Ј-phosphoryl group contributes a factor of Ϸ10 8 -fold to k cat ͞K m , in spite of its considerable distance from the site of chemical transformation of the substrate (3). As a first step toward understanding these unusual properties, we have investigated the crystal structure of recombinant Saccharomyces cerevisiae ODCase alone and in complex with a postulated transition state analogue, 6-hydroxyuridine 5Ј-phosphate (BMP) (K i ϭ 9 ϫ 10 Ϫ12 M (4)]. Materials and MethodsRecombinant yeast ODCase was expressed in Escherichia coli SS6130 (pBGM88) and was purified as described (5). Crystals of the native enzyme were grown a...

Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.