A rational drug design approach, capitalizing on structure-activity relationships and involving transposition of functional groups from somatotropin release inhibitory factor (SRIF) into a reduced size cyclohexapeptide template, has led to the discovery of SOM230 (25), a novel, stable cyclohexapeptide somatostatin mimic that exhibits unique high-affinity binding to human somatostatin receptors (subtypes sst1-sst5). SOM230 has potent, long-lasting inhibitory effects on growth hormone and insulin-like growth factor-1 release and is a promising development candidate currently under evaluation in phase I clinical trials.

We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48 -72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G 2 -M phase of the cell cycle after compound treatment. However, an increased sub-G 1 population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.

ABSTRACT3-Hydroxyaspartic acid and 3-hydroxyasparagine are two rare amino acids that are present in domains homologous to the epidermal growth factor precursor in vitamin K-dependent plasma proteins as well as in proteins that do not require vitamin K for normal biosynthesis. They are formed by posttranslational hydroxylation of aspartic acid and asparagine, respectively. The first epidermal growth factorlike domain in factor IX (residues 45-87) was synthesized with aspartic acid in position 64, replacing 3-hydroxyaspartic acid. It was used as substrate in a hydroxylase assay with rat liver microsomes as the source of enzyme and reaction conditions that satisfy the requirements of 2-oxoglutarate-dependent dioxygenases. The synthetic peptide stimulated the 2-oxoglutarate decarboxylation in contrast to synthetic, modified epidermal growth factor (Met-21 and His-22 deleted and Glu-24 replaced by Asp) and synthetic peptides corresponding to residues 60-71 in human factor IX. This indicates that the hydroxylase is a 2-oxoglutarate-dependent dioxygenase with a selective substrate requirement.

A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC). These compounds were potent enzyme inhibitors, having IC(50)s < 400 nM in a partially purified enzyme assay. However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines. Selected compounds having cellular IC(50) < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay. Four compounds having an MTD > or = 100 mg/kg were selected for dose-response studies in the HCT116 xenograft model. One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity. On the basis, in part, of these properties, 9 has entered human clinical trials in 2002.

Biotinidase catalyzes the hydrolysis of N epsilon-biotinyllysine (biocytin) to form biotin and free lysine. The enzyme has been purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of (76 +/- 2) X 10(3). The same molecular weight was found by molecular sieve chromatography under nondenaturing conditions, indicating biotinidase is a monomer. This value is in contrast to a molecular weight of 115 000 determined by Pispa [Pispa, J. (1965) Ann. Med. Exp. Biol. Fenn., Suppl. 5, 5-39] with an impure biotinidase. The Km for biocytin was 6.2 X 10(-6) M, and biotinidase was found to be sensitive to phenylmethanesulfonamide and iodoacetamide in agreement with earlier studies by Knappe and co-workers [Knappe, J., Brümmer, W., & Bierderbick, K. (1963) Biochem. Z. 338, 599-613], who suggested that serine hydroxyl groups and sulfhydryl groups are essential for enzymatic activity. The specificity of biotinidase was examined by using synthetic and natural biotinyl peptides isolated by specific proteolytic cleavage of the biotinyl subunit of transcarboxylase. It was found that the rate of hydrolysis of biocytin was 83-fold higher than that found for biotin-containing peptides 5-13 residues in length. Removal of methionine from either side of the conserved region around the biocytin did not greatly alter the rate of cleavage. Increasing the peptide to 65-123 residues in length decreased the rate 1200-fold compared to that of biocytin.(ABSTRACT TRUNCATED AT 250 WORDS)