Charles A. Kettner

Rational design of boropeptide thrombin inhibitors: β,β-dialkyl-phenethylglycine P2 analogs of DuP 714 with greater selectivity over complement factor I and an improved safety profile

The potent boropeptide thrombin inhibitor DuP 714 caused side effects in laboratory animals that appear to be related to its ability to inhibit complement factor I, thereby activating the complement cascade. Using X-ray crystal structure information, we have designed compounds that have greater selectivity for thrombin over factor I and that have reduced tendency to produce these side effects.

Pyrazoles, 1,2,4-triazoles, and tetrazoles as surrogates for cis-amide bonds in boronate ester thrombin inhibitors.

Substituted pyrazoles, 1,2,4-triazoles, and tetrazoles are good surrogates for cis-amide bonds in a series of boronate ester thrombin inhibitors.

DESIGN AND SYNTHESIS OF RING-CONSTRAINED BOROPEPTIDE THROMBIN INHIBITORS

Abstract Ring-constrained boropeptide thrombin inhibitors were designed using information from the X-ray crystal structure of 1 (3-Phenylpropionyl-Pro-boroLys-OH ·HCl) bound to thrombin. The constraints utilized cyclohexane and pyrrolidine rings to preorganize an aromatic ring in an orientation allowing optimum edge-to-face interaction with the tryptophan 215 side chain located in the S3 specificity pocket of thrombin.

Anticoagulant activity of a peptide boronic acid thrombin inhibitor by various routes of administration in rats

The peptide boronic acid analog Ac-(D)Phe-Pro-boroArg-OH (I) is a potent and selective inhibitor of thrombin. The objective of this study was to determine whether I is active orally or when administered by alternative transmucosal routes. The measured effect was the time for clotting of plasma after initiation with thrombin. With this assay there was a narrow window from no measurable effect to the maximal effect, a clotting time greater than 300 seconds. Intravenous I at a 0.15 mg/kg dose in rats, a nasal 0.45 mg/kg dose, and 3 mg/kg doses administered orally, colonically, or rectally all produced maximal effects. Therefore, although bioavailability cannot be estimated, it is demonstrated that this peptide analog was absorbed by each of these routes.

Synthesis of conformationally-restricted boropeptide thrombin inhibitors

Abstract A series of boropeptide thrombin inhibitors was prepared in which the P3 residues of 2 (Ac-( D )-Phe-Pro-boroLys-OH · HCl) and 3 (3-Phenylpropionyl-Pro-boroLys-OH · HCl) were replaced by conformationally-restricted, benzoic acid-derived residues 4 . The potent binding affinity of the resulting inhibitors such as 10 may be due in part to a unique mode of binding in the thrombin active site.

Hepatitis C Virus NS3 Protease Requires Its NS4A Cofactor Peptide for Optimal Binding of a Boronic Acid Inhibitor as Shown by NMR

NMR spectroscopy was used to characterize the hepatitis C virus (HCV) NS3 protease in a complex with the 24 residue peptide cofactor from NS4A and a boronic acid inhibitor, Ac-Asp-Glu-Val-Val-Pro-boroAlg-OH. Secondary-structure information, NOE constraints between protease and cofactor, and hydrogen-deuterium exchange rates revealed that the cofactor was an integral strand in the N-terminal beta-sheet of the complex as observed in X-ray crystal structures. Based upon chemical-shift perturbations, inhibitor-protein NOEs, and the protonation state of the catalytic histidine, the boronic acid inhibitor was bound in the substrate binding site as a transition state mimic. In the absence of cofactor, the inhibitor had a lower affinity for the protease. Although the inhibitor binds in the same location, differences were observed at the catalytic site of the protease.

Biaryl substituted alkylboronate esters as thrombin inhibitors

Abstract Thrombin is a serine protease that plays an important role in the blood coagulation cascade, and is a target enzyme for new therapeutic agents. Ac-(D)-Phe-Pro-boroArg-OH (DuP 714) was found to be a highly effective thrombin inhibitor. In order to reduce the peptidic nature of DuP 714, we have designed a series of novel biaryl substituted alkylboronate esters as potent thrombin inhibitors. The most potent compounds have subnanomolar affinity for thrombin.

(N-acyl-N-alkyl)glycyl borolysine analogs: A new class of potent thrombin inhibitors

Abstract In this report the structure-activity relationships of a series of novel (N-acyl-N-alkyl)glycyl borolysine thrombin inhibitors are described. This work culminates in the discovery of (N-3-phenylpropanoyl-N-phenethyl)glycyl borolysine (12j), a potent, orally active inhibitor with a binding conformation in which the N-phenethyl group occupies the aryl binding pocket of thrombin.

Resolution of α-aminoboronic esters by diastereoselective crystallization with pinanediols. Confirmation by X-ray analysis

Abstract A resolution method for α-aminoboronic esters 4a,b using chiral pinanediol is described.

In situ characterization of nasal leucine enkephalin degrading aminopeptidase susceptibility of the nasal enzyme to boronic acids and phosphorus-containing peptide and amino acid isosteres

Abstract The N -terminal Tyr-Gly bond of leucine-enkephalin is specifically hydrolyzed during exposure to the nasal mucosa. Kinetic properties in situ indicate that this activity is due to a single enzyme which has a K M app of 0.4 mM for leucine-enkephalin. Analysis of initial rates of hydrolysis from earlier inhibition studies using boroalanine, boroleucine, and borovaline indicated that these inhibitors bind the nasal enzyme with K m app values of 0.009–0.02 μM. In addition, we have evaluated borophenylalanine ( K app i = 0.004 μM) in this study. Similarly, H-PheΨP(O)(OH)CH 2 Phe-OMe binds the nasal aminopeptidase with a K app i of 0.2 μM. Comparison of these K i values with those of cytosolic aminopeptidase and microsomal aminopeptidase derived from porcine kidney, indicates that the nasal enzyme closely resembles the microsomal enzyme in properties. Major distinctions between the enzymes are: (1) the greater dependence of the cytosolic enzyme on the nature of the amino acid residue in the primary site (2) a much greater preference of both the microsomal and nasal enzyme for HPheΨ[P(O)(OH)CH 2 ]Phe-OMe over H-PheΨ[P(O)(OH) 2 ].