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The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.

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A new synthesis of the 3,8-diazabicyclo[3.2.1]octan-2-one framework is described. Transannular enolate alkylation of piperazinone derivatives provides a flexible route to highly constrained bicyclic peptidomimetic synthons with substitution at the Calpha position. The chemistry was used to produce a conformationally constrained farnesyltransferase inhibitor, which aided the elucidation of enzyme-bound conformation.

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Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, D- and noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverse-turn conformation most closely approximating a type II' beta-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Ras-competitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists.

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PURPOSE: To elucidate the structure of a degradation product arising from a lyophilized formulation of a cyclic heptapeptide, and to provide a mechanism to account for its formation. METHODS: Preparative HPLC was used to isolate the degradate in quantities sufficient for structural studies. A structure assignment was made on the basis of the compounds spectroscopic properties (UV, MS, NMR) and the results of amino acid analysis. RESULTS: The degradate was identified as a benzaldehyde derivative arising from the oxidative deamination of an aminomethyl phenylalanine moiety. The extent of formation of this product is influenced by the amount of mannitol used as an excipient in the formulation. A mechanism is proposed whereby reducing sugar impurities in mannitol act as an oxidizing agent via the intermediacy of Schiff base adducts which subsequently undergo tautomerization and hydrolysis. CONCLUSIONS: Reducing sugar impurities in mannitol are responsible for the oxidative degradation of the peptide via a mechanism that involves Schiff base intermediates. This mechanism may be a potential route of degradation of other arylmethyl amines in mannitol-based formulations.

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We report structure-activity investigations in a series of tripeptide amide inhibitors of thrombin, and the development of a series of highly potent active site directed alpha-keto carbonyl inhibitors having the side chain of lysine at P1. Compounds of this class are unstable by virtue of reactivity at the electrophilic carbonyl and racemization at the adjacent carbon (CH). Modifications of prototype alpha-keto-ester 8a have afforded analogs retaining nanomolar Ki. Optimal potency and stability have been realized in alpha-keto-amides 11b (Ki = 2.8 nM) and 11c (Ki = 0.25 nM).

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Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase-bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L-739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse-turn conformations most closely approximating a type III beta-turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.

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A preliminary characterization is provided of a naturally occurring cyclic peptide with interesting and potent biological activity. A 31-residue cyclic peptide, designated cyclopsychotride A [1], was obtained from the organic extract of the tropical plant, Psychotria longipes. Compound 1 inhibited [125I] neurotensin (NT) binding to HT-29 cell membranes (IC50 3 microM) and also stimulated increased levels of cytosolic Ca2+ in two unrelated cell lines that do not express NT receptors. The peptide was found to dose-dependently increase intracellular Ca2+ at concentrations ranging from 3 to 30 microM, and this response was not blocked by a known NT antagonist. Cyclopsychotride A [1] possesses three disulfide linkages and is thought to be the largest cyclic peptide isolated from a natural source. Both 1H-nmr and cd spectroscopy showed 1 to be highly structured.

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The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist, was characterized in DMSO-d6 by the combination of nmr and molecular modeling. The conformational space available to the peptide was explored using a distance geometry algorithm with distance constraints derived from 1H-nmr spectra. The dynamics of the peptide were examined by relaxation time measurements and low temperature studies. The results from the low temperature studies suggest that the peptide backbone does not exist in a single, well-defined conformation but undergoes exchange between multiple conformers. This result is consistent with the inability to find a single structure that satisfies all the nmr-derived constraints. The constraints could only be satisfied by considering pairs of conformers to represent the experimental data. The low energy conformers comprise type II' or type V beta-turns with distinct side-chain directionality. The Arg-Gly-Asp portion of the ring is flexible and can be described by amide-plane rotations of the Arg-Gly and Gly-Asp peptide bonds. Although some backbone flexibility is evident, the incorporation of beta,beta-dimethyl cysteine imparted greater conformational rigidity as compared to the previously studied cyclic pentapeptide, Ac-Cys-Arg-Gly-Asp-Cys-OH.

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The endothelin family of polypeptides are known to exert potent physiological effects which include cardiovascular regulation. The solution conformation and dynamics of c(D-Trp-D-Cys(SO3-Na+)-Pro-D-Val-Leu), a potent endothelin-A receptor-selective antagonist, were characterized in aqueous solution by NMR spectroscopy and molecular modeling. NMR-derived conformational constraints were combined with computer-assisted molecular modeling using distance geometry calculations and energy minimization. The pentapeptide backbone is shown to adopt a single conformation in solution comprising a type II beta-turn and an inverse gamma-turn, with each residue in the trans conformation. Molecular dynamics were explored using relaxation measurements and low-temperature studies, and indicate that the peptide backbone is highly constrained with little conformational mobility present.

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The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.

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The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.

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Human urinary-type plasminogen activator (urokinase) and proteolytic fragments corresponding to the kringle, EGF-kringle, and protease domains have been examined by 1H NMR spectroscopy. The intact protein shows a very well-resolved spectrum for a molecule of this size (MW 54,000), with resonance line widths not greatly increased from those of the isolated domains. On increasing the temperature, the protein at pH values close to 4 was found to undergo two distinct and reversible conformational transitions. These were identified, by comparison with spectra of the proteolytic fragments, as the unfolding of the kringle (and EGF) domains (at approximately 42 degrees C) and of a segment of the protease domain (at approximately 60 degrees C). The remaining segment of the protease domain showed persistent structure to at least 85 degrees C at pH 4; only at lower pH values could complete unfolding be achieved. The results indicate that the structures and stabilities of the isolated domains are closely similar to those in the intact protein and suggest that there is a degree of independent motion at least between the kringle and protease domains.

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The recent demonstrations that thrombolytic therapy with plasminogen activators can result in substantial reductions in mortality from coronary thrombosis have generated considerable interest in the properties of fibrinolytic enzymes. Examination of the primary sequence of these proteins (which include tissue plasminogen activator, plasminogen, and urokinase) reveals that each is composed of a mosaic of domains which appear to be spatially distinct and connected by short peptide linkers. There is, however, little experimental information about the three-dimensional structure of any of the proteins, although several X-ray diffraction and NMR studies of isolated domains have been reported. Here we report two-dimensional NMR spectra of intact urokinase which are remarkably well resolved for a protein of this molecular weight. This effect is a consequence of substantial independent motion between individual domains of the protein, which overcomes the broadening effects anticipated for the slow overall tumbling rate of the intact molecule. As well as having significance for the physiological role of the protein, these results provide a direct means for the comparison of structural features determined for the isolated domains with those of the intact protein and may provide a basis for proposing or evaluating models for the overall structure of fibrinolytic proteins. Preliminary results with other proteins indicate that this approach may be generally applicable to other multidomain proteins of the fibrinolytic family.

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1H nuclear magnetic resonance experiments have shown that the amide hydrogens of residues 30 to 40 of bacteriophage Pf1 coat protein in micelles undergo very slow exchange with solvent deuterons. The amide 1H resonances from these residues were used to monitor the structural stability of the membrane-spanning helix of the coat protein during the transition of the coat protein from its structural form, in the virus particle, to the membrane-bound form, in micelles. The helix was found to remain folded on the 10(-3) second time-scale of the experiment, which indicates that no major disruption or rearrangement of the central part of the protein structure occurs during the process of coat protein solubilization by detergent. The results also suggest that a helical peptide can associate with lipids without reorganization of its secondary structure. However, a general model for the insertion of proteins into membranes cannot be established from these results, because the mechanism of the detergent solubilization process may differ somewhat from that of the membrane insertion process.

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Solid-state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane-bound form of the protein. The residues in the N- and C-terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C-terminal region of the coat protein in micelles; at 25 degrees C only the last two residues are mobile on the 10(9)-Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C-terminal residues are immobile in the virus particles, the mobility of these residues in the membrane-bound form of the protein may be important for the formation of protein-DNA interactions in the assembly process.

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The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation in detergent micelles. High-resolution 1H and 15N NMR experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pf1 coat protein in sodium dodecyl sulfate micelles. Uniform and specific-site 15N labels enable the immobile backbone sites to be identified by their 1H/15N heteronuclear nuclear Overhauser effect and allow the assignment of 1H and 15N resonances. About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments. The combination of results from 1H/15N heteronuclear correlation, 1H homonuclear correlation, and 1H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure.

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High resolution 15N NMR studies of proteins in solution can be performed efficiently by combining the use of isotopically enriched proteins and pulse sequences that generate polarization transfer from protons and result in two-dimensional heteronuclear chemical shift correlation spectra. The coat proteins of the filamentous bacteriophages fd and Pf1 solubilized in detergent micelles give one- and two- dimensional NMR spectra with resolved resonances for nearly all of the nitrogen sites in the proteins. The resonances from the amide sites with slowly exchanging protons can be obtained as a subset of the resonances of all amide sites by comparing the spectra of proteins in D2O and H2O solutions at pH = 4.0.

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