Gool F. Patel

Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates.

Abstract Sensitive fluorogenic substrates for trypsin, chymotrypsin, and elastase were prepared. These substrates are amides of an acyl amino acid or peptide with 7-amino-4-methylcoumarin (AMC). The substrates with their respective K cat K m ratios given in parentheses are: for chymotrypsin, glutaryl-Phe-AMC (78) and Ala-Ala-Phe-AMC · TFA (1660); for trypsin, benzoyl- dl -Arg-AMC (800) and Carbobenzoxy (Cbz)- l -Arg-AMC (5300); for elastase, N-acetyl-Ala-Ala-Pro-Ala-AMC (15,000). The detection limits obtained by using the best substrates and short incubation times are: chymotrypsin, 25 ng; trypsin, 5 ng; and elastase, 2 ng.

A new fluorogenic substrate for chymotrypsin

A new sensitive assay for amidase activity of chymotrypsin has been developed using 7-glutarylphenylalaninamido-4-methylcoumarin as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. As little as 0.5 μg/ml of chymotrypsin could be detected; at pH 8.0, K m = 0.7 m m . The substrate was not hydrolyzed by either trypsin or elastase and was capable of measuring chymotrypsin-like activity in tissue extracts. Hydrolysis of the substrate by chymotrypsin was blocked by specific inhibitors of the enzyme.

Phenylcyclobutyl triazoles as selective inhibitors of 11β-hydroxysteroid dehydrogenase type I

3-(Phenylcyclobutyl)-1,2,4-triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). These were active both in vitro and in an in vivo mouse pharmacodynamic (PD) model. Fluorine substitution of the cyclobutane ring improved the pharmacokinetic profile significantly. The synthesis and structure-activity relationships are presented.

Design and synthesis of novel antibacterial agents with inhibitory activity against DNA polymerase III.

4-Substituted 2-amino-6-(anilino)pyrimidines have been found to be selective inhibitors of DNA polymerase III, a replicative enzyme known to be essential in the DNA synthesis of Gram-positive bacteria. Among the analogues, 18 displayed an IC(50) of 10 microM against DNA polymerase III from Staphylococcus aureus.

Substituted penta- and hexapeptides as potent inhibitors of herpes simplex virus type 2 ribonucleotide reductase

Abstract Structural modifications of the Tyr, Asn, and Leu residues of YVVNDL, a peptide which is equipotent to YAGAVVNDL in the inhibition of herpes simplex virus type 2 ribonucleotide reductase (HSV-2 RR), have produced peptides which are as much as 90- to 120-times as potent as YAGAVVNDL in vitro against HSV-2 RR. The chemistry and the structure activity relationships of these inhibitors are described. For the inhibition of herpes simplex virus type 2 ribonucleotide reductase (HSV-2 RR), structure-activity relationship studies on Y, N, and/or L of YVVNDL (equipotent to YAGAVVNDL on HSV-2 RR) using synthetic peptides are reported. The most potent of these, YVV-N(Nγ-Me2)-D-L(γ-Me), and (Bzl)2CHCO-VVND-L(γ-Me) had relative potencies of 110 and 120, respectively, relative to YAGAVVNDL.

4-methyl-3-oxo-4-aza-5α-androst-1-ene-17β-n-aryl-carboxamides: an approach to combined androgen blockade [5α-reductase inhibition with androgen receptor binding in vitro]

Abstract 4-Aza-5α-androstan-3-one 17β-(N-substituted carboxamides) are potent human type 2 5α-reductase (5aR) inhibitors with generally poor binding to the human androgen receptor (hAR). When the 17-amide N-substituent included an aromatic residue, potent dual inhibitors of both type 1 and 2 5aR are produced, but hAR binding remained poor. Tertiary-substituted-17-amides have reduced inhibition of both 5aR isozymes. The addition of an N4-methyl substitutent to the A-ring profoundly increased hAR affinity and the addition of unsaturation to the A-ring (Δ1) modestly augmented hAR binding. The unsubstituted carbanilides in the Δ1-N4-methyl series show some selectivity for type 1 5aR over the type 2 isozyme, whereas addition of aryl substituents, particularly at the 2-position, increased type 2 5aR binding to provide dual inhibitors with excellent hAR binding, e.g. N-(2-chlorophenyl)-3-oxo-4-methyl-4-aza-5α-androst-1-ene-17β-carboxamide (9c). Compounds of this type exhibit low nanomolar IC50s for both human 5aR isozymes as well as the human androgen receptor. Kinetic analysis confirms that the prototype 9c displays reversible, competitive inhibition of both human isozymes of 5aR with Ki values of less than 10 nM. Furthermore, this compound binds to the androgen receptor with an IC50 equal to 8 nM. Compounds in this series are projected to be powerful antagonists of testosterone and dihydrotestosterone action in vivo, with potential utility in the treatment of prostatic carcinoma (PC).