John DiMaio

A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

A synthetic hirudin peptide analog corresponding to N′‐acetyl [D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin55–65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow‐tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human α‐thrombin (K i=3.7±0.3 × 10−10 M) and bovine α‐thrombin (1.8±0.7 × 10−9 M). P79 showed saturable inhibition of plasma APTT. The P1 subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site‐directed tripeptide [D‐Phe‐Pro‐ArgΨ(COCH2)CH3COOCH3, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α‐thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K 1=17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α‐thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α‐thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N???‐acetyl[D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH2‐ and COOH‐terminal recognition sites.

A proposal for the molecular basis of μ and δ opiate receptor differentiation based on modeling of two types of cyclic enkephalins and a narcotic alkaloid

SummaryThe molecular basis underlying the divergent receptor selectivity of two cyclic opioid peptides Tyr-c[Nδ-d-Orn2-Gly-Phe-Leu-] (c-ORN) and [d-Pen2, l-Cys5]-enkephalinamide (c-PEN) was investigated using a molecular modeling approach. Ring closure and conformational searching procedures were used to determine low-energy cyclic backbone conformers. Following reinsertion of amino acid side chains, the narcotic alkaloid 7α-[(1R)-1-methyl-1-hydroxy-3-phenylpropyl]-6,14-endoethenotetrahydro oripavine (PEO) was used as a flexible template for bimolecular superpositions with each of the determined peptide ring conformers using the coplanarity and cocentricity of the phenolic rings as the minimum constraint. A vector space of PEO, accounting for all possible orientations for the C21-aromatic ring of PEO served as a geometrical locus for the aromatic ring of the Phe4 residue in the opioid peptides. Although a vast number of polypeptide conformations satisfied the criteria of the opiate pharmacophore, they could be grouped into three classes differing in magnitude and sign of the torsional angle values of the tyrosyl side chain. Only class III conformers for both c-ORN and c-PEN, having tyramine dihedral angles χ1 =−150° ± 30° and χ2=−155° ± 20°, had significant structural and conformational properties that were mutually compatible while respecting the PEO vector space. Comparison of these properties in the context of the divergent receptor selectivity of the studied opioid peptides suggests that the increased distortion of the peptide backbone in the closure region of c-PEN together with the pendant β,β-dimethyl group, combine to generate a steric volume which is absent in c-ORN and that may be incompatible with a restrictive topography of the μ receptor. The nature and stereo-chemistry of substituents adjacent to the closure region of the peptides could also modulate receptor selection by interacting with a charged (δ) or neutral (μ) subsite.

Insertion of the methylene-oxy surrogate of the amide bond into Boc-Val-Leu-OH: X-ray crystal structure, solution conformation and molecular modelling study

The conformational features associated with the introduction of the methylene-oxy surrogate of the amide bond were explored by studying the crystal and solution conformation of the related model peptides Boc-Val-Leu-OH (1) and Boc-Val-ψ(CH2O)-Leu-OH (2). Two independent molecular conformations were found for 1 in the crystal state whereas one was found for its congener 2. In compound 2 the dihedral angle defined by Cα–CH2–O–Cα(ω′) adopts a value of –165.7(3)°, close to the situation encountered in the classical amide bond. 2D NOE NMR studies suggest that the preferred backbone conformation of 2 in [2H6]DMSO correlates with the crystal structure whereas the preferred backbone conformation of 1 in [2H6]DMSO showed a departure from its crystalline conformation. Molecular mechanics computations demonstrate the effect of the short C(sp3)–O(sp3) bond in compound 2 in dictating the preferred dihedral angle values adopted by ω′ and φLeu.

S-p-methylbenzyl-β-phenylcysteine: a potential tool for probing receptor topologies

erythro and threo N-Acetyl-S-p-methylbenzyl-β-phenyl-DL-cysteine methyl esters were obtained by addition of p-methyltoluene-α-thiol to 4-benzylidene-2-methyloxazol-5-one in methanol–tetrahydrofuran under basic conditions. The diastereoisomers were separated as their methyl esters by fractional crystallization and the relative configuration assigned by X-ray crystallography. Both diastereoisomers were converted into their N-trifluoroacetyl derivatives and resolved using Carboxypeptidase A. Hydrazinolysis of the unchanged N-trifluoroacetyl amino acids gave the other enantiomers of the free amino-acids. Optical purity was determined using Eu(hfc)3 chemical shift reagent on N-trifluoroacetyl-D-amino acid methyl ester by both 1H and 19F NMR spectra. We discuss the conformation of this unique amino acid based on X-ray data and molecular mechanics calculations. Its usefulness in probing the opiate receptor site is also demonstrated.