The discovery of novel small molecule non-peptide gonadotropin releasing hormone (GnRH) receptor antagonists

A novel series of non-peptide derivatives 1, 14, and 15 that bind with high affinity to the human GnRH receptors is discussed. The discovery was made from screening our in-house libraries that contained the active structure 2 along with a trace amount of a second active structure 1 that was derived from an acid-induced rearrangement. From this structure type 1, a series of guanidine and non-guanidine containing analogues were prepared and tested as GnRH receptor antagonists. Compounds derived from this series bind to both human and rat GnRH receptors and antagonize GnRH-mediated increases in inositol phosphate production in cells containing recombinant human receptors. These compounds or their analogues may be useful as therapeutic agents for the treatment of hormone-dependent pathologies including prostate, breast and ovarian cancers.     

A critical interpretation of experiments of binding of peptide hormone to specific receptors by computer modelling

Many investigations dealing with the interaction of peptide hormones and specific cell membrane receptors imply the existence of two classes of independent binding sites. One class is characterized by high affinity and low capacity, the other one by low affinity and high capacity. This conclusion has been derived from the fact that the Scatchard plots of binding data show a significant upward curvature. Using a more precise and critical method of evaluation these findings probably must be revised in some cases. Other conclusions taken from linear plots concerning the regulation of hormone receptors should be discussed more carefully. Some sources of errors caused by an uncritical interpretation of Scatchard plots are demonstrated.     

Immunization of cattle against modified peptides of gonadotropin releasing hormone conjugated to carriers: Effectiveness of Freund's and alternative adjuvants

Two gonadotropin-releasing hormone (GnRH) peptides with a cystein substitution of the first (C1-GnRH) or tenth (C10-GnRH) amino acid were conjugated to ovalbumin and equine serum albumin, respectively, via the sulfhydryl group of the introduced cysteine. Animals were immunized three times at 3-wk intervals with both conjugates in either saline (n = 5), Freund's complete adjuvant (FCA; n = 5), Havlogen (n = 6), Ribi adjuvant system (RAS; n = 5), dimethyl dioctadecyl ammonium bromide (DDA; n = 4), Alhydrogel (n = 5) or Regressin (n = 5). Animals immunized with conjugates in saline or RAS did not produce anti-GnRH titers. The highest anti-GnRH titers were produced by animals treated with FCA. The Alhydrogel and DDA treatments stimulated the production of GnRH antibodies in all animals treated, but titers were lower than in animals immunized with FCA. When vaccines were formulated with Havlogen or Regressin, anti-GnRH titers were low or absent. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were depressed in FCA and in Alhydrogel treated animals. The antisera raised were predominantly directed against either the carboxy- or the amino-terminal end of the GnRH peptide, or directed equally against both, depending on the individual animal. Results suggest that no epitope of GnRH dominates the immune response in cattle and show that the best alternative to FCA is Alhydrogel.     

Influences of hydrocarbon linkers on the receptor binding affinities of gonadotropin-releasing hormone peptides

Three new DOTA-conjugated GnRH peptides with various hydrocarbon linkers were synthesized to evaluate the influences of the linkers on their receptor binding affinities. The hydrocarbon linker displayed a profound impact on the receptor binding affinities of DOTA-conjugated GnRH peptides. The Aun linker was better than Gaba, Ahx and Aoc linkers in retaining strong receptor binding affinity of the GnRH peptide. DOTA-Aun-(d-Lys⁶-GnRH) displayed 22.8 nM GnRH receptor binding affinity. ¹¹¹In-DOTA-Aun-(d-Lys⁶-GnRH) exhibited fast tumor uptake and urinary clearance in MDA-MB-231 human breast cancer-xenografted nude mice. The cellular and biological results provided an insight into the design of new GnRH peptides in the future.     

The A14 position of insulin tolerates considerable structural alterations with modest effects on the biological behavior of the hormone

As part of our aim to investigate the contribution of the tyrosine residue found in the 14 position of the A-chain to the biological activity of insulin, we have synthesized six insulin analogues in which the A14 Tyr has been substituted by a variety of amino acid residues. We have selected three hydrophilic and charged residues—glutamic acid, histidine, and lysine—as well as three hydrophobic residues—cycloleucine, cyclohexylalanine, and naphthyl-(1)-alanine—to replace the A14 Tyr. All six analogues exhibit full agonist activity, reaching the same maximum stimulation of lipogenesis as is achieved with procine insulin. The potency for five of the six analogues, [A14 Glu]-, [A14 His]-, [A14 Lys]-, [A14 cycloleucine]-, and [A14 naphthyl-(1)-alanine]-insulins in receptor binding assays ranges from 40–71% and in stimulation of lipogenesis ranges from 35-120% relative to porcine insulin. In contrast, the potency of the sixth analogue, [A14 cyclohexylalanine]insulin, in both types of assays is less than 1% of the natural hormone. The retention time on reversed-phase high-performance liquid chromatography for the first five analogues is similar to that of bovine insulin, whereas for the sixth analogue, [A14 cyclohexylalanine]insulin, it is approximately 11 min longer than that of the natural hormone. This suggests a profound change in conformation of the latter analogue. Apparently, the A14 position of insulin can tolerate a wide latitude of structural alterations without substantial decrease in potency. This suggests that the A14 position does not participate directly in insulin receptor interaction. Only when a substitution which has the potential to disrupt the conformation of the molecule is made at this position, is the affinity for the receptor, and hence the biological potency, greatly reduced.     

A novel computational approach "BP-STOCH" to study ligand binding to finite lattice

We report a novel computational algorithm "BP-STOCH" to be used for studying single-type ligand binding with biopolymers of finite lengths, such as DNA oligonucleotides or oligopeptides. It is based on an idea to represent any type of ligand-biopolymer complex in a form of binary number, where "0" and "1" bits stand for vacant and engaged monomers of the biopolymer, respectively. Cycling over all binary numbers from the lowest 0 up to the highest 2(N) - 1 means a sequential generating of all possible configurations of vacant/engaged monomers, which, after proper filtering, results in a full set of possible types of complexes in solution between the ligand and the N-site lattice. The principal advantage of BP-STOCH algorithm is the possibility to incorporate into this cycle any conditions on computation of the concentrations and observed experimental parameters of the complexes in solution, and programmatic access to each monomer of the biopolymer within each binding site of every binding configuration. The latter is equivalent to unlimited extension of the basic reaction scheme and allows to use BP-STOCH algorithm as an alternative to conventional computational approaches.     

Synthesis and characterization of novel biotinylated carboxyl-terminal parathyroid hormone peptides that specifically crosslink to the CPTH-receptor

Parathyroid hormone (PTH) regulates calcium, phosphorous and skeletal homeostasis via interaction with the G protein-coupled PTH/PTHrP receptor, which is fully activated by the amino-terminal 34 amino-acid portion of the hormone. Recent evidence points to the existence of another class of receptors for PTH that recognize the carboxyl (C)-terminal region of intact PTH (1–84) (CPTHRs) and are highly expressed by osteocytes. Here we report the synthesis and characterization of two novel bifunctional CPTH ligands that include benzoylphenylalanine (Bpa) substitutions near their amino-termini and carboxyl-terminal biotin moieties, as well as a tyrosine³⁴ substitution to enable radioiodination. These peptides are shown to bind to CPTHRs with affinity similar to that of PTH (1–84) and to be specifically and covalently crosslinked to CPTHRs upon exposure to ultraviolet light. Crosslinking to osteocytes or osteoblastic cells generates complexes of 80 and 220 kDa, of which the larger form represents an aggregate that can be resolved into the 80 kDa. The crosslinked products can be further purified using immunoaffinity and avidin-based affinity procedures. While the molecular structure of the CPTHR(s) remains undefined, these bifunctional ligands represent powerful new tools for use in isolating and characterizing CPTHR protein(s).     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

A novel approach to the generation of high affinity class II-binding peptides

We have found that if core regions crucial for class II binding are incorporated in multiple copies in the same peptide molecule ("reiterative motifs"), marked enhancement of the binding capacity occurs. Isotype specificity (IAd vs IEd binding capacities) is retained in all three antigenic determinants so far analyzed (lambda rep 12-26, OVA 323-339, and hen egg lysozyme 105-120). The mechanism involved in such an effect is not clear, but experiments involving introduction of a peptide spacer between two repeated core regions do not support the notion that the effect is mediated by cross-linking of more than one MHC molecule, favoring the possibility that conformational effects or distinct subsites of interaction on the MHC molecule may be involved. Based on reiterative structures, a peptide molecule composed of only two different amino acids (Ala and His) has been produced that still retains a very high binding affinity. An 125I-radiolabeled form of this peptide has been used to demonstrate that the high binding detected is mediated by the same binding site involved in the interaction of IAd and OVA 323-339. Inhibition of Ag presentation studies further supports the immunologic relevance of the phenomena observed. Finally, we observed naturally occurring clustered binding sites in proximity of immunodominant protein regions, raising the possibility that the phenomenon might have a physiologic counterpart.     

Molecular modeling of the structures of human and rat pancreatic cholesterol esterases.

Structural models have been generated for rat and human cholesterol esterases by molecular modeling. For rat cholesterol esterase, three separate models were generated according to the following procedure: (1) the cholesterol esterase sequence was aligned with those of three template enzymes: Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase; (2) the X-ray structure coordinates of the three template enzymes were used to construct cholesterol esterase models by amino acid replacements of matched sequence positions and by making sequence insertions and deletions as required; (3) bad contracts in each of the cholesterol esterase models were relaxed by molecular dynamics and mechanics; (4) the three cholesterol esterase models were merged into one by arithmetic averaging of atomic coordinates; (5) Ramachandran analysis indicated that the model generated from the AChE template possessed the best set of phi/psi angles. Therefore, this model was subjected to molecular dynamics, with harmonic constraints imposed on the C(alpha) coordinates to drive them toward the coordinates of the averaged model. (6) Subsequent relaxation by molecular mechanics produced the final rat cholesterol esterase model. A model for human cholesterol esterase was produced by repeating steps 1-3 above, albeit with the rat cholesterol esterase model as the template. Hydrophobic and electrostatic analyses of the rat and human cholesterol esterase models suggest the structural origins of molecular recognition of hydrophobic substrates and interfaces, of charged interfaces, and of bile salt activators.     

A proposed bioactive form of peptide T and the design of peptidomimetics

Peptide T is a non-natural octapeptide of sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, taken from the sequence of the protein gp120 of HIV. The peptide has been shown to bind competitively to the CD4 receptors of the helper/inducer lymphocytes T. The peptide is presently used for the treatment of AIDS-associated dementia and has been proven useful for the treatment of psoriasis. Using molecular modeling procedures, we studied the conformational profile of this peptide as well as those of several active and inactive analogs. The analysis of these results gave rise to the proposal of a bioactive conformation of the peptide, which can be described as a pseudo -turn structure, involving the last four residues at the C-terminus of the peptide. The secondary structure is stabilized by a hydrogen bond between the hydroxyl hydrogen of the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷. From the bioactive form and different structure–activity relationship studies, a pharmacophore was proposed. This hypothesis was used to search on several 3D data bases. One of the hits obtained was the natural compound amigdalin, which was tested and exhibited moderate activity.     

Successional state dynamics: A novel approach to modeling nonequilibrium foodweb dynamics

Communities and ecosystems are often far from equilibrium, but our understanding of nonequilibrium dynamics has been hampered by a paucity of analytical tools. Here I describe a novel approach to modeling seasonally forced food webs, called “successional state dynamics” (SSD). It is applicable to communities where species dynamics are fast relative to the external forcing, such as plankton and other microbes, diseases, and some insect communities. The approach treats succession as a series of state transitions driven by both the internal dynamics of species interactions and external forcing. First, I motivate the approach with numerical solutions of a seasonally forced predator-prey model. Second, I describe how to set up and analyze an SSD model. Finally, I apply the techniques to three additional models of two-species interactions: resource competition (r-K selection), facilitation, and flip-flop competition (where the competitive hierarchy alternates over time). This approach allows easy and thorough exploration of how dynamics depend on the environmental forcing regime, and uncovers unexpected phenomena such as multiple stable annual trajectories and year-to-year irregularity in successional trajectories (chaos).     

A proposed bioactive form of peptide T and the design of peptidomimetics

Summary Peptide T is a non-natural octapeptide of sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, taken from the sequence of the protein gp 120 of HIV. The peptide has been shown to bind competitively to the CD4 receptors of the helper/inducer lymphocytes T. The peptide is presently used for the treatment of AIDS-associated dementia and has been proven useful for the treatment of psoriasis. Using molecular modeling procedures, we studied the conformational profile of this peptide as well as those of several active and inactive analogs. The analysis of these results gave rise to the proposal of a bioactive conformation of the peptide, which can be described as a pseudo β-turn structure, involving the last four residues at the C-terminus of the peptide. The secondary structure is stabilized by a hydrogen bond between the hydroxyl hydrogen of the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷. From the bioactive form and different structure-activity relationship studies, a pharmacophore was proposed. This hypothesis was used to search on several 3D data bases. One of the hits obtained was the natural compound amigdalin, which was tested and exhibited moderate activity.     

Multiple mapping method: a novel approach to the sequence-to-structure alignment problem in comparative protein structure modeling

A major bottleneck in comparative protein structure modeling is the quality of input alignment between the target sequence and the template structure. A number of alignment methods are available, but none of these techniques produce consistently good solutions for all cases. Alignments produced by alternative methods may be superior in certain segments but inferior in others when compared to each other; therefore, an accurate solution often requires an optimal combination of them. To address this problem, we have developed a new approach, Multiple Mapping Method (MMM). The algorithm first identifies the alternatively aligned regions from a set of input alignments. These alternatively aligned segments are scored using a composite scoring function, which determines their fitness within the structural environment of the template. The best scoring regions from a set of alternative segments are combined with the core part of the alignments to produce the final MMM alignment. The algorithm was tested on a dataset of 1400 protein pairs using 11 combinations of two to four alignment methods. In all cases MMM showed statistically significant improvement by reducing alignment errors in the range of 3 to 17%. MMM also compared favorably over two alignment meta-servers. The algorithm is computationally efficient; therefore, it is a suitable tool for genome scale modeling studies.     

Mapping of ligand binding sites of the cholecystokinin-B/gastrin receptor with lipo-gastrin peptides and molecular modeling

Double-tailed lipo-tetragastrin derivatives of increasing fatty acid chain length were used to identify the minimum size of the fatty acid moieties (≥C10) that restricts the access to the CCK-B/gastrin (CCK: cholecystokinin) receptor via a membrane-bound pathway. Then dimyristoyl-mercaptoglycerol/maleoyl-gastrin adducts of increasing peptide chain length were synthesized to define the minimal peptide size required for receptor binding affinities comparable to those of underivatized gastrin peptides despite anchorage of the lipid tails in the membrane bilayer. The experimental results indicated that most of the little-gastrin sequence, i.e., 2–17, is needed for optimal interaction of the molecule with the binding cleft of the receptor. From these data experimentally based restraints could be derived for docking of lipo-gastrin onto a CCK-B/gastrin receptor model applying molecular dynamics simulations and energy minimizations. In the receptor-bound state some of the secondary structure elements of gastrin as determined by nmr analysis of gastrin-peptides in low dielectric constant media are retained. The N-terminal gastrin portion interacts in a more or less extended conformation with the receptor surface, and upon a sharp kink at the Ala-Tyr dipeptide portion the C-terminal pentapeptide amide part inserts deeply into the helix bundle. Besides Arg-57 on top of helix 1 of the receptor, for which no potential interaction with the ligand could be detected, the other amino acid residues identified by mutagenesis studies as involved in gastrin recognition were found to interact with the C-terminal portion of gastrin. Even taking into account the strong limitations of such a model system, it represents an interesting tool for rationalizing the experimental results of the extensive structure-function studies performed previously on gastrin and to delineate more precisely the putative ligand binding site on the extracellular face of the receptor. © 1997 John Wiley & Sons, Inc. Biopoly 41: 799–817, 1997     

The sequence of an atriopeptigen: A precursor of the bioactive atrial peptides

The high molecular weight fraction (atriopeptigen-APG) obtained by gel filtration chromatography of rat atrial extracts was fractionated by isoelectric focusing and reverse phase HPLC to obtain a pure APG. Purification of cyanogen bromide digests of the crude high molecular weight fraction resulted in the isolation of a single biologically active cyanogen bromide cleavage peptide. Sequence analyses of these peptides coupled with recent reports of sequence analyses of intermediate molecular weight atrial peptides (Thibault, et al. (1984) FEBS Letters 167, 352–356, and Kangwa, et al., Biochem. Biophys. Res. Commun 119, 933–940) provide the complete primary structure of an 111 residue APG.     

Crystal structure of the Drosophila peptidoglycan recognition protein (PGRP)-SA at 1.56 A resolution

Peptidoglycan recognition proteins (PGRPs) form a recently discovered protein family, which is conserved from insect to mammals and is implicated in the innate immune system by interacting with/or degrading microbial peptidoglycans (PGNs). Drosophila PGRP-SA is a member of this family of pattern recognition receptors and is involved in insect Toll activation. We report here the crystal structure of PGRP-SA at 1.56 A resolution, which represents the first example of a "recognition" PGRP. Comparison with the catalytic Drosophila PGRP-LB reveals an overall structure conservation with an L-shaped hydrophilic groove that is likely the PGN carbohydrate core binding site, but further suggests some possible functional homology between recognition and catalytic PGRPs. Consistent with sequence analysis, PGRP-SA does not contain the canonical zinc-binding residues found in catalytic PGRPs. However, substitution of the zinc-binding cysteine residue by serine, along with an altered coordinating histidine residue, assembles a constellation of residues that resembles a modified catalytic triad. The serine/histidine juxtaposition to a threonine residue and a carbonyl oxygen atom, along with conservation of the catalytic water molecule found in PGRP-LB, tantalizingly suggests some hydrolytic function for this member of receptor PGRPs.     

N-terminal [Glu]3 moiety of γ-glutamyl peptides contributes largely to the activation of human calcium-sensing receptor,a kokumi receptor

ABSTRACT

γ-glutamyl peptides have been suggested to impart kokumi properties to foods by activating human calcium-sensing receptor (hCaSR). In this study, the relationship between γ-glutamyl peptide structure and hCaSR activity was systematically analyzed using γ-[Glu]_(n=0-4)-α-[Glu]_(n=0-3)-Tyr. Our results suggest that N-terminal [Glu]₃ moiety is very important for hCaSR activities of γ-glutamyl peptides.     

A structural and free energy analysis of Ag complexes to five small peptides

The complexes of Ag⁺ with the peptides MetGly, ProGly, GlyPro, GlyHis and GlyProAla were investigated using hybrid density functional theory at the B3LYP/DZVP level. The silver ion binding free energies at 298 K to each of these peptides was calculated to be 60.8, 52.0, 54.3, 71.2 and 63.3 kcal mol⁻¹, respectively. Structural information and relative free energies are presented for several isomers for each of the five complexes. Each of the global minima found for the five complexes is a charge-solvated ion. An important finding is that the Ag⁺-ProGly is the only complex where a salt bridge structure is energetically favored occurring at 4.0 kcal mol⁻¹ higher in free energy than the global minimum. The Ag⁺ ion in this salt bridge structure is attached to the carboxylate anion of zwitterionic ProGly in which the terminal amino nitrogen is protonated. For all the other complexes studied, the salt bridge structure occurs at much higher energies. All the dipeptide complexes with Ag⁺, but one, exhibit a di- or tri-coordinate metal where the sites of attachment are amino and carbonyl groups. However, the highest coordination numbers are not always the global minima due to steric costs. The global minimum of the Ag⁺-GlyProAla complex is the only structure found in this study where the metal is tetra-coordinated, binding to the terminal amino nitrogen and all three carbonyl oxygen atoms. Silver binding to sulphur and imidazole nitrogen atoms of MetGly and GlyHis, respectively, are present in the three most energetically favored species in each of these cases.