Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K(m) and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N(epsilon)-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K(m) values ranged from 60 to 110000 M(-1) s(-1). The pH profiles for both k(cat) and k(cat)/K(m) were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K(i) for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.

The B 2 bradykinin receptor, a seven-helix transmembrane receptor, binds the inflammatory mediator bradykinin (BK) and the structurally related peptide antagonist HOE-140. The binding of HOE-140 and the binding of bradykinin are mutually exclusive and competitive. Fifty-four site-specific receptor mutations were made. BK's affinity is reduced 2200-fold by F261A, 490-fold by T265A, 60-fold by D286A, and 3-10-fold by N200A, D268A, and Q290A. In contrast, HOE-140 affinity is reduced less than 7-fold by F254A, F261A, Y297A, and Q262A. Bradykinin is a central initiator of acute and chronic inflammation and the associated pain and edema. Most of the acute and many of the chronic responses to bradykinin are mediated by B 2 bradykinin receptors (1, 2), while some of the chronic responses to bradykinin are mediated by B 1 bradykinin receptors (3). B 1 and B 2 bradykinin receptors are members of the G-protein-coupled seven-transmembrane (GPC-7TM) 1 receptor superfamily (4 -7). For B 1 receptors, [des-Arg 9 ]bradykinin is a more potent agonist than bradykinin, whereas B 2 receptors bind and respond to bradykinin about 10,000-fold more effectively than [des-Arg 9 ]bradykinin. Several peptidic B 2 bradykinin antagonists have been identified; these compounds reduce pain and inflammation (8 -12). Peptidic antagonists also reduce death from experimental shock (13-15). Bradykinin receptor antagonists are potentially useful in the treatment of pain, acute and chronic inflammation, shock, allergic or infectious rhinitis, and asthma. The peptidic antagonists are useful tools; but, to date, these compounds have made poor human therapeutic agents because of their poor bioavailability and formulation difficulties (16,17). The discovery of a nonpeptide antagonist of bradykinin would improve the prospects of treating bradykinin-instigated inflammation, pain, or edema. Thus, we have focused on a molecular understanding of the bradykinin receptor ligand binding site, believing that this information may help in the discovery and design of nonpeptidic antagonists.We used the results from molecular modeling studies of B 2 bradykinin receptors and NMR studies of peptidic agonists and antagonists to generate a number of models for agonist binding to the B 2 BKR. These models were tested by site-directed mutagenesis of the receptor and by making single amino acid changes in agonist and antagonist peptides. The data reveal a disparity between the way peptidic agonists and antagonists bind to the BKR. We attempt to reconcile the disparities by proposing new models of the BKR-ligand complex. -140 (18, 19). The product was HPLC-purified and then characterized by HPLC and mass spectrometry as to its chemical purity (Ն96%), identity, and specific activity (56.5 Ci/mmol). Media and other cell culture additives were from Life Technologies, Inc. Biochemicals and enzymes were from Boehringer Mannheim. Common reagents were from Sigma. EXPERIMENTAL PROCEDURES Materials MethodsStandard molecular biological and cell culture methods were used except as speci...

Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones (VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome. Variations at the key P3 position confirmed the importance of a long side chain capped with a hydrophobic group for optimal potency, consistent with a model of binding to the S3 subsite. The tert-butyl glutamic ester initially used at P3 gave plasma unstable, insoluble compounds and was replaced with the better isostere, N-beta-neopentyl asparagine. The inhibitors were shortened by replacing the N-terminal Cbz-isoleucine with a p-tosyl group without loss of potency. Small l-amino acids were used at P2, where d-substitution was not tolerated. The resulting optimized P4-P3-P2 sequence was grafted onto a novel proteasome inhibitor warhead, 2-keto-1,3,4-oxadiazoles (KOD), to produce reversible, subnanomolar proteasome inhibitors that were 1000-fold selective versus cathepsin B (CatB), cathepsin S (CatS), and trypsin-like as well as PGPH-like proteasome activity. A number of compounds in both the VS and the KOD series exhibited growth inhibitory effects against the human prostate cancer cell line PC3 at submicromolar concentrations.

Isomeric nonconjugated diene structures are ruled out for 12 because they would require 3 vinyl protons. Anal. (Ci9H2eN2): C, , N.Acknowledgment.-We wish to thank Mr. L. Brancone and staff for microanalyses, Mr. W. Fulmor and staff for spectra, and Mr. C. Pidacks and staff for partition chromatographic separations. We also thank Mr. R. Vessey and Mr. E. Markely for conducting the biological assays.

The incorporation of Arg residues into position 6 of gonadotropin releasing hormone antagonists had resulted in compounds with increased in vivo potency but also made these analogues potent mast cell degranulators. We have focused on the substitution of position 8 by hArg(R)2 (NG,NG'-dialkylhomoarginine) substitutions, based on the hypotheses that the Arg-Pro sequence is of major importance for this side effect and that shielding of the charge may be an effective way to block degranulation. Analogues in four series were evaluated: (A) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal-(3)3,6,Arg5,hArg(R)2(8),D-+ ++Ala10]GnRH, (B) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(R)2(5,8),D-Ala10 ]-GnRH, (C) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(R)2(8),D-Ala10]G nRH, (D) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-hArg(R)2(6),hArg(R)2( 8),D-Ala10]GnRH. Although substitution by hArg(Et)2, hArg(Bu), hArg(CH2)3, and hArg(CH2CF3)2 was tested, in each series the hArg(Et)2 residue was superior. Two compounds were considered for clinical evaluation: [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(Et)2(8),D-Ala10] GnRH and [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-hArg(Et)2(6),hArg(Et) 2(8),D- Ala10]GnRH (ganirelix acetate). These compounds had high potency for ovulation suppression and low histamine-releasing potency in vitro (ED50 = 0.6, 0.29 microgram/rat and EC50 = 196, 13 micrograms/mL, respectively). Ganirelix is currently in Phase II clinical trials and appears to be the most potent GnRH antagonist tested in humans (based upon ED50 for 24-h suppression of testosterone levels).