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V J Hruby

Publications and source records attributed to V J Hruby.

At least 73 records · Page 4Linked to original sources
PubMed1998

Sensitivity- and gradient-enhanced hetero (omega1) half-filtered TOCSY experiment for measuring long-range heteronuclear coupling constants.

An enhanced version of the X (omega1) half-filtered TOCSY experiment for measurement of long-range heteronuclear coupling constants is proposed which yields high-quality spectra with substantially increased sensitivity and resolution. The modified method features gradient-enhanced X filtering sequences, broadband homonuclear decoupling during t1, optional 1JXH scaling in the F1 domain, and gradient coherence selection in combination with the sensitivity-enhanced protocol for the TOCSY transfer. These modifications extend the applicability of the method--coupling constants can be measured accurately for natural abundance samples at low concentrations and for compounds yielding complex spectra. Computer-aided analysis of E.COSY-type multiplets is applied for the determination of heteronuclear long-range coupling constants.

Magnetic Resonance Spectroscopy↗
PubMed1998

Gradient- and sensitivity-enhanced TOCSY experiments.

A pulsed field gradient version of the sensitivity-enhanced 2D TOCSY experiment is proposed which yields high-quality spectra with improved sensitivity and a minimum of two scans per t1 increment. For rapid acquisition of 1D TOCSY spectra, the 1D DPFGSE-TOCSY experiment was modified to include phase-encoded multiple-selective excitation followed by a simple spectral editing. Combination of these two building blocks is used in a sensitivity-enhanced 2D analog of the 3D TOCSY-TOCSY experiment which provides an efficient tool for resolving severely overlapped signals of oligomers in short experimental time.

Analgesics, Opioid↗
PubMed1998

Peptide and peptidomimetic libraries. Molecular diversity and drug design.

Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split-couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.

Amino Acid Sequence↗
PubMed1998

[125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes.

Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2], both nonradioactive [I-Tyr1]biphalin and radioactive [125I-Tyr1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I-Tyr1]biphalin to the delta and mu opioid receptors are not independent. [125I-Tyr1]Biphalin binds to delta receptors as shown in NG108-15 cell membranes. Nevertheless, [125I]biphalin binding to delta receptors in rat brain membranes was hardly evident and mu receptor binding predominated or at least was much more readily detectable in this preparation.

Animals↗
PubMed1998

The efficacy of delta-opioid receptor-selective drugs.

Delta-opioid receptor-selective drugs may provide an alternative to mu-opioid-selective drugs currently used for the relief of pain. To develop improved delta-opioid receptor-selective drugs, better measures of drug activity are necessary. In this review we suggest that efficacy calculations provide a superior measure of drug activity as compared to dissociation constants and drug potencies in functional assays. Efficacy, as discussed in this review, is defined as a quantitative measurement of the ability of a drug to stimulate second messenger systems or measurable functional responses in cells or tissues under standard conditions. Efficacy values will allow medicinal chemists to understand the contributions of both the coupling efficiency and dissociation constant to drug potencies in the development of new delta-opioid receptor-selective drugs.

Analgesics↗
PubMed1998

NMR and quenched molecular dynamics studies of superpotent linear and cyclic alpha-melanotropins.

Conformational searching, computer simulations, synthesis and NMR are used on a variety of alpha melanocyte-stimulating hormone (alpha-MSH) analogues to understand the physical characteristics required for biological potency. Peptides I (Ac-[Nle4,Asp5,D-Phe7,Lys10]alpha-MSH(4-10)-NH2), II (Ac-c[Nle4,Asp5,D-Phe7,Lys10]alpha-MSH(4-10)-NH2) and III (Ac-[Nle4,Asp5,D-Phe7,Dap10]alpha-MSH(4-10)-NH2 all show very similar conformational properties (backbone and side-chain torsional angles), and all display high biological potencies. The modeling results for these compounds are supported by the NMR data. Peptide IV (Ac-c[Nle4,Asp5,D-Phe7,Dap10]alpha-MSH(4-10)-NH2) appears to have a markedly different conformation and has decreased biological potency.

Animals↗
PubMed1998

Prevention of reflex natriuresis after acute unilateral nephrectomy by melanocortin receptor antagonists.

gamma-Melanocyte-stimulating hormone (gamma-MSH), atrial natriuretic peptide (ANP), and oxytocin have been identified as candidate hormonal mediators of the reflex natriuresis that follows acute unilateral nephrectomy (AUN). Pharmacological characterization of the third melanocortin receptor (MC3-R) indicates that it uniquely responds to physiological concentrations of gamma-MSH. We tested the roles of gamma-MSH, ANP, and oxytocin in the postnephrectomy natriuresis by carrying out AUN during continuous intrarenal infusion of specific antagonists for their cognate receptors. In anesthetized Sprague-Dawley rats, urinary sodium excretion (UNaV) increased from 0.34 +/- 0.04 to 1.12 +/- 0.11 mu eq/min 90 min after AUN (P < 0.001). No change in UNaV occurred in rats undergoing a sham AUN procedure. Plasma immunoreactive gamma-MSH concentration was 53 +/- 8 fmol/ml after sham AUN but 112 +/- 17 fmol/ml after AUN (P < 0.01). SHU-9119 and SHU-9005 are substituted derivatives of alpha-MSH with potent antagonism at the MC3-R in vitro. Infusion of these compounds at 5 pmol/min completely blocked the natriuretic response to AUN despite a similar elevation in plasma gamma-MSH (111 +/- 12 vs. 49 +/- 8 fmol/ml in sham rats, P < 0.01). Intrarenal infusion of the ANP receptor antagonist A-71915 (5 pmol/min) or the oxytocin receptor antagonist [d(CH2)(5)1, Tyr(Me)2,Orn8] vasotocin (10 pmol/min) effectively inhibited the natriuresis induced by intravenous infusion of ANP or oxytocin (each at 1 pmol/min), respectively, but did not block the natriuresis after AUN. Plasma immunoreactivity of these peptides was not increased after AUN. These results indicate that reflex natriuresis after AUN is accompanied by an increase in plasma gamma-MSH but not ANP or oxytocin concentration and is prevented by intrarenal infusion of receptor antagonists with selectivity for MC3-R. The data indicate that gamma-MSH or a closely related peptide mediates postnephrectomy natriuresis and provide further support for the possibility that gamma-MSH may play a wider role in sodium homeostasis.

Animals↗
PubMed1998

Systemic alpha-MSH suppresses LPS fever via central melanocortin receptors independently of its suppression of corticosterone and IL-6 release.

Systemically administered alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits endotoxin (lipopolysaccharide; LPS)- or interleukin (IL)-1-induced fever and adrenocortical activation, but the sites of these actions and the mechanisms involved are unknown. The aims of this study were, first, to determine whether melanocortin receptors (MCR) located within the central nervous system mediate the suppressive effects of peripherally administered alpha-MSH on LPS-induced fever and activation of the pituitary-adrenal axis and, second, to determine whether systemic alpha-MSH suppresses the LPS-induced rise in plasma IL-6 levels, potentially contributing to its antipyretic effect. Male rats received Escherichia coli LPS (25 microg/kg ip). Core body temperatures (Tb) were determined hourly by radiotelemetry (0-8 h), and blood was withdrawn via venous catheters for plasma hormone immunoassays (0-2 h) and IL-6 bioassay (0-8 h). alpha-MSH (100 microg/kg ip) completely prevented the onset of LPS-induced fever during the first 3-4 h after LPS and suppressed fever throughout the next 4 h but did not affect Tb in afebrile rats treated with intraperitoneal saline rather than LPS. Intraperitoneal alpha-MSH also suppressed the LPS-induced rise in plasma IL-6, ACTH, and corticosterone (CS) levels. Intracerebroventricular injection of SHU-9119, a potent melanocortin-4 receptor (MC4-R)/MC3-R antagonist, completely blocked the antipyretic effect of intraperitoneal alpha-MSH during the first 4 h after LPS but had no effect on alpha-MSH-induced suppression of LPS-stimulated plasma IL-6 and CS levels. Taken together, the results indicate that the antipyretic effect of peripherally administered alpha-MSH during the early phase of fever is mediated by MCR within the brain. In contrast, the inhibition of LPS-induced increases in plasma CS and IL-6 levels by intraperitoneal alpha-MSH appears to be mediated by a different mechanism(s), and these effects do not contribute to its antipyretic action.

Adrenocorticotropic Hormone↗
PubMed1998

Synthetic melanotropic peptide initiates erections in men with psychogenic erectile dysfunction: double-blind, placebo controlled crossover study.

PURPOSE: We evaluated the erectogenic properties of a new cyclic alpha-melanocyte-stimulating hormone analogue, Melanotan-II, to treat men with psychogenic erectile dysfunction. MATERIALS AND METHODS: Ten men with erectile dysfunction of no known organic cause were entered in a double-blind, placebo controlled crossover study in which the erectogenic properties of Melanotan-II and a vehicle placebo were compared using real-time RigiScan monitoring. The presence, duration and rigidity of erections were recorded during a 6-hour period. RESULTS: In 8 of 10 men treated with Melanotan-II clinically apparent erections developed. Mean duration of tip rigidity greater than 80% was 38.0 minutes with Melanotan-II and 3.0 with placebo (p=0.0045). Transient side effects of nausea, stretching and yawning, and decreased appetite were reported more frequently after injections of Melanotan-II than placebo but none required treatment. CONCLUSIONS: Melanotan-II is a potent initiator of erections in men with psychogenic erectile dysfunction and has manageable side effects at a dose of 0.025 mg./kg.

Abdominal Muscles↗
PubMed1997

Cyclic enkephalin analogues with exceptional potency and selectivity for delta-opioid receptors.

Superpotent and highly delta-opioid receptor selective cyclic peptides of the general formula H-Tyr-c[D-Pen-Gly-Phe(p-X)-Pen]-Phe-OH (where X = hydrogen or halogen) have been synthesized. In the binding assays the most selective and most potent compound is the p-bromophenyl-alanine-4 analogue (IC50 value = 0.19 nM, selectivity ratio = 21,000 for delta vs mu). In the GPI and MVD bioassays the most selective and most potent analogue is the p-fluoro-substituted analogue Tyr-[D-Pen-Gly-Phe(p-F)-Pen]-Phe-OH. In the MVD assay it has an exceptionally low IC50 value of 0.016 nM and a delta vs mu selectivity ratio of 45,000.

Amino Acid Sequence↗
PubMed1997

Interaction of a highly potent dimeric enkephalin analog, biphalin, with model membranes.

Biphalin, (Tyr-D-Ala-Gly-Phe-NH)2, is a highly potent dimeric analog of enkephalin. Its analgesic efficacy is due in part to its ability to permeate the blood-brain barrier. To aid in understanding the mechanism of the transmembrane movement we determined and analyzed the permeability and partition coefficients of biphalin and a series of analogues where F, Cl, I, NO2, or NH2 were placed in the para position of the aromatic rings of Phe4,4'. Liposomes composed of neutral phospholipids and cholesterol were used as the model membrane. The overall good correlation between permeability and water-membrane partition coefficients suggests that the movement of biphalins across the model membrane is controlled by diffusion and depends on the water-membrane partition coefficient. To explain the observed correlation between permeability and the electron withdrawing/donating character of the substituents in the phenylalanine ring, we examined various folding patterns of Leu-enkephalin, an endogenous pentapeptide that exhibits affinities toward the same classes of opioid receptors (delta and mu). The observed permeabilities and partition coefficients of biphalin and analogues, as well as the tyrosine side chain accessibility, are consistent with the presence of the type of folding where the tyrosine and phenylalanine side chains are in a close contact. We propose that the aromatic ring interaction can promote the peptide permeability by stabilizing a more compact structure of biphalin that would minimize the number of hydrogen bonds with water and therefore enhances partitioning into the model membrane.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

beta-Methylation of the Phe7 and Trp9 melanotropin side chain pharmacophores affects ligand-receptor interactions and prolonged biological activity.

Topographically modified melanotropin side chain pharmacophore residues Phe7 and Trp9 in a cyclic peptide template (Ac-Nle4-c[Asp-His-Xaa7-Arg-Yaa9-Lys]-NH2) and Phe7 in a linear peptide template (Ac-Ser-Tyr-Ser-Nle4-Glu-His-Xaa7-Arg-Trp-Gly-Lys-Pro-Val-NH2) result in differences in potency and prolonged biological activity in the frog and lizard skin bioassays. These topographic modifications included the four isomers of beta-methylphenylalanine (beta-MePhe)7 and beta-methyltryptophan (beta-MeTrp)9 and the two isomers of 1,2,3,4-tetrahydro-beta-carboline (Tca)9 Modifications in the cyclic template resulted in up to a 1000-fold difference in potency for the beta-MePhe7 stereoisomeric peptides; up to a 476-fold difference in potency resulted for the beta-MeTrp9 peptides, and about a 50-fold difference between the Tca9-containing peptides. Up to a 40-fold difference in potency resulted for the beta-MePhe7 stereoisomeric peptides using the linear template in these assays. The relative potency ranking for modifications in the cyclic template of beta-MePhe7 were 2R,3S > 2S,3S = 2S,3R > 2R,3R in the frog assay and 2S,3R > 2R,3S > 2S,3S > 2R,3R in the lizard assay. The relative potencies for modifications in the cyclic template of beta-MeTrp9 were 2R,3S > 2R,3R > 2S,3S > > 2S,3R in the frog assay and 2S,3S = 2R,3R > 2R,3S > 2S,3R in the lizard assay. The relative potencies for modifications in the cyclic template of Tca9 were DTca > LTca in both assays. Significant differences in prolonged (residual) activities were also observed for these modified peptides and were dependent upon stereochemistry of the beta-methyl amino acid, peptide template, and bioassay system. Furthermore, comparisons of beta-MeTrp9 stereoisomeric peptides on the frog, lizard, and human MC1 receptors suggest that structure-activity relationships on both the classical frog and lizard skin bioassays do not necessarily predict corresponding SAR profiles for the human melanocortin receptors, indicating a remarkable species specificity of the MC1 receptor requirements.

Animals↗
PubMed1997

The role of phenylalanine at position 6 in glucagon's mechanism of biological action: multiple replacement analogues of glucagon.

Extensive evidence gathered from structure-activity relationship analysis has identified and confirmed specific positions in the glucagon sequence that are important either for binding to its receptor or for signal transduction. Fifteen glucagon analogues have been designed and synthesized by incorporating structural changes in the N-terminal region of glucagon, in particular histidine-1, phenylalanine-6, and aspartic acid-9. This investigation was conducted to study the role of phenylalanine at position 6 on the glucagon mechanism of action. These glucagon analogues have been made by either deleting or substituting hydrophobic groups, hydrophilic groups, aromatic amino acids, or a D-phenylalanine residue at this position. The structures of the new analogues are as follows: [des-His1, des-Phe6, Glu9]glucagon-NH2 (1); [des-His1,Ala6,Glu9]glucagon-NH2 (2); [des-His1,Tyr6,Glu9]glucagon-NH2 (3); [des-His1,Trp6,Glu9]-glucagon-NH2 (4); [des-His1,D-Phe6,Glu9]glucagon-NH2 (5); [des-His1,Nle6,Glu9]glucagon-NH2 (6); [des-His1,Asp6,Glu9]glucagon-NH2 (7); [des-His1,des-Gly4,Glu9]glucagon-NH2 (8); [desPhe6,-Glu9]glucagon-NH2 (9); [des-Phe6]glucagon-NH2 (10); [des-His1, des-Phe6]glucagon-NH2 (11); [des-His1, des-Phe6,Glu9]glucagon (12); [des-Phe6,Glu9]glucagon (13); [des-Phe6]glucagon (14); and [des-His1, des-Phe6]glucagon (15). The receptor binding potencies IC50 values are 48 (1), 126 (2), 40 (3), 19 (4), 100 (5), 48 (6), 2000 (7), 52 (8), 113 (9), 512 (10), 128 (11), 1000 (12), 2000 (13), 500 (14), and 200 nM (15). All analogues were found to be antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10(-5) M except for analogues 6 and 8, which were found to be weak partial agonists/partial antagonists with maximum stimulation between 6-12%. In competitive inhibition experiments, all the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA2 values were 8.20 (1), 6.40 (2), 6.20 (3), 6.25 (4), 6.30 (5), 6.30 (7), 6.05 (8), 6.20 (9), 6.30 (10), 6.25 (11), 6.10 (12), 6.20 (13), 6.20 (14), and 6.35 (15).

Adenylyl Cyclases↗
PubMed1997

Structural model of a cyclic dynorphin A analog bound to dodecylphosphocholine micelles by NMR and restrained molecular dynamics.

The compound c[Cys5,11]dynorphin A-(1-11)-NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the kappa-opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 omega bond contains either a beta-turn from residues Arg6 to Arg9 or an alpha-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 omega bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III beta-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a beta-turn from residues Cys5 and Arg8. In this report we describe the generation of three-dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as kappa-opioid agonists.

Amino Acid Sequence↗
PubMed1997

Discovery of prototype peptidomimetic agonists at the human melanocortin receptors MC1R and MC4R.

[Nle4, DPhe7]-alpha-MSH (NDP-MSH), a highly potent analogue of alpha-melanocyte-stimulating hormone (alpha-MSH), possesses nanomolar efficacies at all the melanocortin receptor subtypes except the MC2R. Evaluation of the melanocortin "message" sequence of [Nle4, DPhe7]-alpha-MSH was performed on the human melanocortin receptor subtypes designated hMC1, hMC3R, hMC4R, and hMC5R. Tetrapeptides and tripeptides were stereochemically modified to explore topochemical preferences at these receptors and to identify lead peptides possessing agonist activity and subtype selectivity. Four peptides were discovered to only bind to the hMC1 and hMC4 receptor subtypes. The tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 (1) possessed 0.6 microM binding affinity at the hMC1R, 1.2 microM binding affinity at the hMC4R, and agonist activity at both receptors. The tripeptides Ac-DPhe-Arg-Trp-NH2 (6) and Ac-DPhe-Arg-DTrp-NH2 (7) possessed 2.0 and 9.1 microM binding affinities, respectively, only at the hMC4R, and both compounds effected agonist activity. The tetrapeptide Ac-His-Phe-Arg-DTrp-NH2 (4) possessed 6.3 microM affinity and full agonist activity at the hMC1R, while only binding 7% at the hMC3R, 36% at the hMC4R, and 11% at the hMC5R at a maximal concentration of 10 microM. These data demonstrate that the His-Phe-Arg-Trp message sequence of the melanocortin peptides does not bind and stimulate each melanocortin receptor in a similar fashion, as previously hypothesized. Additionally, this study identified the simplest structural agonists for the hMC1R and hMC4R receptors reported to date.

Adrenocorticotropic Hormone↗
PubMed1997

Biological and conformational examination of stereochemical modifications using the template melanotropin peptide, Ac-Nle-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2, on human melanocortin receptors.

Examination of conformationally constrained melanotropin peptide (Ac-Nle4-c[Asp5-His-Phe7-Arg-Trp9-Ala-Lys]-NH2) on four human melanotropin receptors (hMC1R, hMC3R, hMC4R, and hMC5R) resulted in identifying the importance of ligand stereochemistry at positions 5, 7, and 9 for agonist binding affinity and receptor selectivity. A trend in ligand structure-activity relationships emerged for these peptides, with the hMC1R and hMC4R possessing similar tendencies, as did the hMC3R and hMC5R. alpha-MSH (Ac-Ser-Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-D-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), and MTII (Ac-Nle4-c[Asp5,D-Phe7,Lys10]-alpha-MSH(4-10)-NH2) were also examined at each of these melanocortin receptors. Interestingly, the linear NDP-MSH possessed greater binding affinity for the hMC3R and hMC5R than did the cyclic analogue MTII. The peptide Ac-Nle-c[Asp-His-Phe-Arg-D-Trp9-Ala-Lys]-NH2 demonstrated the greatest differentiation in binding affinity between the hMC1R and hMC4R (78-fold). Analogue Ac-Nle-c[Asp-His-Phe7-Arg-Trp-Ala-Lys]-NH2 resulted in micromolar binding affinity (or greater) at the hMC3R and hMC5R, demonstrating the importance of D-Phe7 for ligand binding potency at these receptors. Ac-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2 resulted in loss of binding affinity at the hMC5R, implicating the importance of Nle4 (or a hydrophobic residue in this position) for binding to this receptor. Ac-Nle-c[D-Asp5-His-Phe-Arg-Trp-Ala-Lys]-NH2 was unable to competitively displace [125I]NDP-MSH binding at micromolar concentrations on the hMC3R and hMC5R, suggesting the importance of chirality of Asp5 either for ligand-receptor interactions or for orientation of the side chain lactam bridge and the structural integrity of the peptide conformation. Energy calculations performed for these peptides resulted in the identification of a low-energy ligand conformer family that is common to all the ligands. The differences in ligand binding affinities observed in this study are postulated to be a result of different ligand-receptor complexed interactions and not solely to the ligand structure.

Amino Acid Sequence↗
PubMed1997

Relative efficacies of delta-opioid receptor agonists at the cloned human delta-opioid receptor.

The present study was conducted to determine the relative efficacies of the selective delta-opioid receptor agonists SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPgammaS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human delta-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/Emax)(A'/A + 1) X 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of (-)-TAN67.

Animals↗