Dean Hickman

Genotyping human polymorphic arylamine N-acetyltransferase: identification of new slow allotypic variants.

Arylamine N-acetyltransferase catalyses the N-acetylation of primary arylamine and hydrazine drugs and chemicals. N-acetylation is subject to a polymorphism and humans can be categorized as either fast or slow acetylators according to their ability to N-acetylate polymorphic substrates in vivo. Previously, slow acetylation has been linked to four distinct polymorphic N-acetyltransferase (pnat) alleles each of which contains one or more point mutations within the coding region of the pnat gene. One new rare slow variant of pnat has been identified by cloning and sequencing the pnat DNA from an individual whose NAT phenotype was determined by in vivo acetylation of the polymorphic substrate sulphamethazine. This allele, designated S1c, differs from the wild type fast allele at nucleotide positions 341 and 803. A second new rare slow allotypic variant, designated S3, has been identified by resistance of the pnat specific DNA to digestion with the restriction enzymes Fok I and Bam HI. A method of genotyping individuals for the arylamine N-acetyltransferase (NAT) polymorphism is presented which correctly predicts the phenotype of greater than 95% (21 of 22) of individuals as measured by the extent of acetylation of sulphamethazine in urine. This refined genotyping method was applied to a clinical population of 48 Caucasians with classical or definite rheumatoid arthritis each receiving daily between 150 and 500 mg of the anti-rheumatic drug, D-penicillamine. There is no difference in the N-acetyltransferase phenotype of the individuals who developed proteinuria and the control group with no adverse effects.

Immunochemical detection of arylamine N-acetyltransferase in normal and neoplastic bladder.

The N-acetyltransferase (NAT) phenotype is an important determinant of individual susceptibility to occupational bladder cancer. N-Acetyltransferases arc known to metabolize aromatic amine bladder carcinogens, but the functional significance of NAT expression in the target organ is unclear. To resolve this issue, polygonal antisera against purified recombinant enzymes and C-terminal peptides of human NAT Type 1 (NAT1) and Type 2 (NAT2) were generated. Western blot analysis of exfoliated cells from human urine, pig bladder homogenate, and human bladder tumor-derived cell lines showed that NAT1 was expressed in all three systems, whereas NAT2 did not appear to be expressed in the bladder. Immunohistochemical analysis of human bladder tumor sections indicated that well-differentiated tumor cells expressed NAT1, with the highest level of expression being found in the umbrella cells that line the bladder lumen. Poorly differentiated tumor regions appeared to express NAT1 at lower levels than did well-differentiated areas. These findings support the hypothesis that aromatic amines are metabolized in the bladder epithelium by NAT1.

Enzyme kinetic properties of human recombinant arylamine n-acetyltransferase 2 allotypic variants expressed in Escherichia coli

Arylamine N-acetyltransferase (NAT2) catalyses the N-acetylation of primary arylamine and hydrazine drugs and chemicals. N-Acetylation is subject to polymorphism, and humans can be categorized as either fast or slow acetylators according to their ability to N-acetylate certain arylamine substrates in vivo. Genetic variants at the polymorphic NAT2 locus have been described. We expressed five of the most common NAT2 variants (NAT2 4, NAT2 5A, NAT2 5B, NAT2 6A and NAT2 7B) in Escherichia coli as a convenient source of the human variants. The apparent Km values (at 100 microM acetyl CoA as co-substrate) of the different NAT2 variants for sulphamethazine, dapsone, p-anisidine, 2-aminofluorene, procainamide and isoniazid were determined. Data show that the apparent Km of the slow variant NAT2 7B for the arylamine sulphamethazine was 10-fold lower than all the other allotypes. The apparent Km for the structurally related sulphone antibiotic dapsone was 5-fold lower for the slow variant NAT2 7B when compared with the wild-type NAT2 4. These results indicate that the NAT2 7B specific amino acid substitution, Gly286-Glu, is important in promoting the binding of sulphamethazine and dapsone to the active site.

2-amino-1,3-thiazol-4(5H)-ones as potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitors: enzyme-ligand co-crystal structure and demonstration of pharmacodynamic effects in C57Bl/6 mice.

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has attracted considerable attention during the past few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our ongoing work on 11beta-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5 H)-ones were explored. By inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position of the thiazolone, several potent 11beta-HSD1 inhibitors were identified. An X-ray cocrystal structure of human 11beta-HSD1 with compound 6d (Ki=28 nM) revealed a large lipophilic pocket accessible by substitution off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of human 11beta-HSD1 (Ki=3 nM) and also inhibited 11beta-HSD1 activity in lean C57Bl/6 mice when evaluated in an ex vivo adipose and liver cortisone to cortisol conversion assay.

Design and synthesis of potent, orally efficacious hydroxyethylamine derived β-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-β peptide (Aβ) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aβ levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.

Further Studies with the 2-Amino-1,3-thiazol-4(5H)-one Class of 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors: Reducing Pregnane X Receptor Activity and Exploring Activity in a Monkey Pharmacodynamic Model

A series of compounds containing the 2-amino-1,3-thiazol-4(5H)-one core were found to be potent inhibitors of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). One of our lead compounds from this series activated the human nuclear xenobiotic receptor, pregnane X receptor (PXR). To try and mitigate the PXR activity, we prepared analogues of our lead series that contained polar groups on the right-hand side of the thiazolone. Several analogues containing amides or alcohols appended to the C-5 position of the thiazolone showed a significant reduction in PXR activity. Through these structure-activity efforts, a compound containing a tert-alcohol group off the C-5 position, analogue (S)-33a, was found to have an 11beta-HSD1 Ki = 35 nM and negligible PXR activity. Compound (S)-33a was advanced into a pharmacodynamic model in cynomolgus monkeys, where it inhibited adipose 11beta-HSD1 activity after being orally administered.

A Potent and Orally Efficacious, Hydroxyethylamine-Based Inhibitor of β-Secretase.

β-Secretase inhibitors are potentially disease-modifying treatments for Alzheimers disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against β-site amyloid precursor protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to significantly reduce Aβ40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally efficacious BACE inhibitor.

In Vitro Metabolism of the Novel, Highly Selective Oral Angiogenesis Inhibitor Motesanib Diphosphate in Preclinical Species and in Humans

Motesanib diphosphate is a novel, investigational, highly selective oral inhibitor of the receptor tyrosine kinases vascular endothelial growth factor receptors 1, 2, and 3, the platelet-derived growth factor receptor, and the stem cell factor receptor (Kit). The in vitro metabolic profiles of [14C]motesanib were examined by using microsomes and hepatocytes from preclinical species and humans. Several oxidative metabolites were observed and characterized by tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and coinjection with authentic standards. Cytochrome P450 (P450) 3A4 is the major isozyme involved in the oxidative biotransformation of motesanib, but the CYP2D6 and CYP1A isozymes also make minor contributions. In hepatocyte incubations, oxidative and conjugative pathways were observed for all species examined, and indoline N-glucuronidation was the dominant pathway. Three less common and novel phase II conjugates of the indoline nitrogen were detected in hepatocytes and in microsomes supplemented with specific cofactors, including N-carbamoyl glucuronide, N-glucose, and N-linked β-N-acetylglucosamine. An N-glucuronide metabolite was the most frequently observed phase II conjugate in liver microsomes of all species, whereas the N-acetylglucosamine conjugate was observed only in monkey liver microsomes. Incubations with recombinant human UDP-glucuronosyltransferases (UGTs) and inhibition by the UGT1A4 and UGT1A1 substrates/inhibitors imipramine and bilirubin suggested that UGT1A4 is the major UGT isozyme catalyzing the N-glucuronidation of motesanib, with a minor contribution from UGT1A1. The in vitro metabolic profiles were similar between the human and preclinical species examined. All metabolites found in humans were also detected in other species.

Oxopyrido[2,3-d]pyrimidines as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors

In nonsmall cell lung cancer (NSCLC), the threonine(790)-methionine(790) (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound 24 which potently inhibits gefitinib-resistant EGFR(L858R,T790M) with 100-fold selectivity over wild-type EGFR. Compound 24 displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR(L858R,T790M) driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR.

An Orally Available BACE1 Inhibitor That Affords Robust CNS Aβ Reduction without Cardiovascular Liabilities

BACE1 inhibition to prevent Aβ peptide formation is considered to be a potential route to a disease-modifying treatment for Alzheimers disease. Previous efforts in our laboratory using a combined structure- and property-based approach have resulted in the identification of aminooxazoline xanthenes as potent BACE1 inhibitors. Herein, we report further optimization leading to the discovery of inhibitor 15 as an orally available and highly efficacious BACE1 inhibitor that robustly reduces CSF and brain Aβ levels in both rats and nonhuman primates. In addition, compound 15 exhibited low activity on the hERG ion channel and was well tolerated in an integrated cardiovascular safety model.