More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1a and HIF2a, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2a that allosterically blocks its dimerization with the

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effect...

The crystal structure of 4-oxalocrotonate tautomerase (4-OT) inactivated by the active site-directed irreversible inhibitor 2-oxo-3-pentynoate (2-OP) has been determined to 2.4 A resolution. The structure of the enzyme covalently modified at Pro-1 by the resulting 2-oxo-3-pentenoate adduct is nearly superimposable on that of the free enzyme and confirms that the active site is located in a hydrophobic region surrounding Pro-1. Both structures can be described as a trimer of dimers where each dimer consists of a four-stranded beta-sheet with two antiparallel alpha-helices on one side. Examination of the structure also reveals noncovalent interactions between the adduct and two residues in the active site. The epsilon and eta nitrogens of the guanidinium side chain of Arg-39" from a neighboring dimer interact respectively with the C-2 carbonyl oxygen and one C-1 carboxylate oxygen of the adduct while the side chain of Arg-61' from the same dimer as the modified Pro-1 interacts with the C-1 carboxylate group in a bidentate fashion. An additional interaction to the 2-oxo group of the adduct is provided by one of the two ordered water molecules within the active site region. These interactions coupled with the observation that 2-oxo-3-butynoate is a more potent irreversible inhibitor of 4-oxalocrotonate tautomerase than is 2-OP suggest that Arg-39" and the ordered water molecule polarize the carbonyl group of 2-OP which facilitates a Michael reaction between Pro-1 and the acetylene compound. On the basis of the crystal structure, a mechanism for the enzyme-catalyzed reaction is proposed.

The catalytic general base, Pro-1, of the enzyme 4-oxalocrotonate tautomerase has been mutated to Gly, Ala, Val, and Leu, residues with aliphatic side chains. The Val mutant was partially (55%) processed by removal of the amino-terminal methionine to yield P1V/M1P2V, while the Leu mutant was not processed and completely retained methionine (M1P2L). The M1P2L mutant lost 2300-fold in kcat with no change in Km, and the residual activity of the unresolvable P1V/M1P2V mixture could be explained by the summation of two activities, one equal to that of M1P2L and the other equal to that of the P1G mutant. The P1G and P1A mutants showed 76- and 58-fold decreases in kcat and much smaller decreases in Km of 4- and 2.8-fold, respectively. The dissociation constant of the substrate analog cis,cis-muconate decreased 1.7-fold in the P1G mutant as determined by NMR titration. 2D 1H-15N HSQC spectra and 3D 1H-15N NOESY HSQC spectra of the 15N-labeled P1G mutant showed no structural differences from the wild-type enzyme except for small changes in backbone 15N and NH chemical shifts at the active site. Both the P1G and P1A mutants showed no change in overall conformation by circular dichroic spectroscopy. Both mutants and the wild-type enzyme generate the S-enantiomer of the product [5-2H]-2-oxo-3-hexenedioate with comparable stereoselectivities indicating a largely intact active site. The P1G and P1A mutants showed 10- and 4-fold decreases, respectively, in catalysis of exchange of the C3 proton of the substrate 2-oxo-1,6-hexanedioate, consistent with the lower basicities of Gly-1 and Ala-1 compared to Pro-1. The pH dependences of kcat/Km for the P1G and P1A mutants revealed pKa values of the general base of 5.3 and 5.9, respectively. NMR titration of the uniformly 15N-labeled P1G mutant showed the pKa of Gly-1 to be < or = 5.6, in agreement with the kinetic data. As with the wild-type enzyme, the active site environments on the P1G and P1A mutants lower the pKa of the general base by at least 2.5 units. It is concluded that the 2 order of magnitude decreases in kcat in the P1G and P1A mutants result from both a decrease in basicity and an increase in flexibility of the general base. The greater 10(3.4)-fold decrease in kcat found with the presence of an additional residue at the amino-terminus is ascribed to either the complete blockage or the drastically altered position of the general base.

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-Å resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads. Inhibitors of PKC1 are currently being used in clinical trials for various types of cancer, and a PKC␤ inhibitor is being used in clinical trials for diabetes-related retinopathy (1).PKC and PKB/AKT kinase domains are related by sequence homology; however, there are key structural differences in the regulatory domains and second messenger cofactor requirements. PKB/AKT contains an N-terminal pleckstrin homology domain regulated by phosphoinositide second messengers, a central catalytic kinase domain, and a C-terminal regulatory region facilitating key protein-protein interactions with signal-