Daniel H. Rich

Episelection: novel Ki approximately nanomolar inhibitors of serine proteases selected by binding or chemistry on an enzyme surface.

A novel class of mechanism-based inhibitors of the serine proteases is developed using epitaxial selection. Tripeptide boronates esterified by an alcohol or alcohols at the boron retain the tight binding to trypsin-like enzymes associated with transition-state analogs and incorporate additional groups that can be utilized for selectivity between proteases. Formed by reaction of a series of alcohols with the inhibitor boronate oxygen(s), the most structurally compatible alcohol-derivatized inhibitors are either selected by binding to the enzyme (epitaxial selection) or assembled by epitaxial reaction on the enzyme surface. Mass spectrometry of the derivatized boronates and X-ray crystallography of the complexes identify the chemical structures and the three-dimensional interactions of inhibitors generated. This scheme also engineers novel, potent (Ki approximately 7 nM), and more specific inhibitors of individual serine proteases, by derivitizations of compounds obtained by epitaxial selection.

Synthesis of a novel polyurethane co-polymer containing covalently attached RGD peptide.

The synthesis of a novel polyurethane block co-polymer containing a covalently attached, well-oriented RGD (Arg-Gly-Asp) peptide was explored. A poly(tetramethylene oxide) (PTMO)-based polyurethane was synthesized, and a bimolecular nucleophilic substitution reaction was then employed to incorporate ethyl carboxylate groups onto the polymer backbone (i.e. carboxylated polyurethane). Elemental analysis was used to determine the extent of carboxylation. The hexapeptide H-Gly-Arg-Gly-Asp-Ser-Tyr-OH was coupled to the carboxylated polyurethane via the formation of an amide bond. The attachment of the peptide was controlled by a protection-deprotection scheme. Nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies were used to monitor the reactions. Sakaguchi assay and amino acid analysis confirmed that the RGD-containing peptide was successfully grafted onto the carboxylated polyurethane. This reaction scheme provides a new route for grafting end-linked, bioactive peptides onto polyurethanes.

Addition of Allylic Metals to α-Aminoaldehydes. Application to the Synthesis of Statine, Ketomethylene and Hydroxyethylene Dipeptide Isosteres

Abstract A general and stereoselective method to statine, ketomethylene and hydroxyethylene dipeptide isosteres is described. The key reaction is the diastereoselective allyl metal addition to α-aminoaldehydes.

Mechanism of inhibition of pepsin by pepstatin: Effect of inhibitor structure on dissociation constan and time-dependent inhibition

Abstract Investigations were made of the inhibition of pepsin by pepstatin and nine synthetic analogs of pepstatin. Analysis of progress curve data indicated that the inhibition produced by pepstatin (1) conforms to a mechanism in which a rapidly formed enzyme-inhibitor complex (collision complex) is transformed slowly to a more tightly bound complex (tightened complex). Dissociation constants for the collision complex k2/k1 and first-order rate constants for formation (k3) and reversal (k4) of the tightened complex are reported for pepstatin and nine pepstatin analogs. The data show that at least three structural parameters are needed to induce the lag transient characteristic of the tight-binding inhibition of pepsin by pepstatin. These are: a 3(S)-hydroxyl group in the third residue of pepstatin, an isopropyl group or its equivalent in the first residue, and some portion of the C-terminal dipeptidyl group -Ala-Sta. The data indicate that these groups interacted cooperatively to induce the timedependent increase in inhibition. Removal of any of these binding groups affected, predominatly, k4, the first-order rate constant for return of tightened complex to collision complex. The proposal that pepstatin is an analog of the transition state for pepsin catalyzed hydrolysis of amide bonds is discussed.

Comparative studies of the coupling of N-methylated, sterically hindered amino acids during solid-phase peptide synthesis

Abstract Comparison of different coupling reagents for effective coupling of N-methylated, sterically hindered amino acids under solid-phase peptide synthesis (SPPS) conditions is described. Superior results were obtained with the coupling additive 1-hydroxy-7-azabenzotriazole (HOAt), as well as its uronium salt derivative (HATU), which both produced quantitative couplings. Application of these reagents to the synthesis of the 2–7 sequence found in cyclosporin is reported.

The structure and conformation of HC-toxin

Abstract Difference nuclear magnetic resonance studies and amino acid oxidase experiments establish the sequence and configuration of amino acids in HC-toxin as cyclo(L-Aoe-D-Pro-L-Ala-D-Ala). HC-toxin adopts the bis-γ-turn conformation in solution previously found for the cytostatic cyclic tetrapeptide chlamydocin.

Vitamin K-dependent carboxylase: Requirements for carboxylation of soluble peptide substrates and substrate specificity

Abstract Rat liver microsomes contain a triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of precursor proteins to γ-carboxyglutamyl residues. This activity has been studied utilizing synthetic peptides as substrates for the enzyme. When compared to the carboxylation of the endogenous microsomal precursors, the peptide carboxylase activity is more sensitive to the action of various inhibitors, and requires a higher concentration of vitamin K for maximal activity. The apparent Km for the peptide Phe-Leu-Glu-Glu-Leu was found to be 4 mM. Substrate specificity depends on residues adjacent to the carboxylated Glu residues and macromolecular recognition sites.

Synthesis of hydroxyethylene and ketomethylene dipeptide isosteres

A general stereo-directed synthesis of “ketomethylene” and “hydroxyethylene” dipeptide isosteres is reported. Peptides containing the amino acids, (2R, 5S)-5-amino-4-oxo-2,8-dimethyloctanoic acid (the “ketomethylene” analog of L-Leu-L-Ala), and (2R, 5S-5-amino-4-hydroxy-2,8-dimethyloctanoic acid (the “hydroxyethylene” analog of L-Leu-L-Ala) were synthesized as analogs of pepstatin with statine replaced by the Leu-Ala isosteres.

Peptide inhibitors of hiv protease

Abstract Mass screening of our compound collection, utilizing the Amersham SPA technology, afforded pyrone and coumarin non-peptide templates as initial lead structures. X-ray cocrystallization and structure-based design were utilized to assist in the design of more potent inhibitors. These efforts resulted in the design of the 5,6-dihydropyrones, which afforded a more flexible template from which to fill the internal pockets of the enzyme. Optimization of the dihydropyrone series afforded a potent antiviral agent, PD 178390 (EC 50 =0.20 μM, TD 50 =>100 μM). PD 178390 retained antiviral potency in the presence of serum proteins with a modest three-to fivefold drop in antiviral activity in the presence of 40% human serum. The antiviral activity, in PBMCs, was unchanged against clinical strains of resistant HIV virus. In addition, PD 178390 showed excellent bioavailability in mice, rats, and dogs as well as a low level of P450 inhibition in microsomal assays. This combination of good antiviral efficacy, good pharmacokinetics, and low P450 inhibition make PD 178390 a promising agent for the treatment of HIV infection.

Tentoxin An uncompetitive inhibitor of lettuce chloroplast coupling factor 1

The interaction of tentoxin [cyclo-(-L-leucyl-N-methyl-(Z)-dehydrophenylalanyl-glycyl-N-methyl-L-alanyl-)] with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca2+ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 . 10(8) M-1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.