Biomedical subjects

V J Hruby

Publications and source records attributed to V J Hruby.

At least 91 records · Page 5Linked to original sources

PubMed1997

Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever.

Bacterial infection causes fever, an adaptive but potentially self-destructive response, in the host. Also activated are counterregulatory systems such as the pituitary-adrenal axis. Antipyretic roles have also been postulated for certain endogenous central neuropeptides, including the melanocortins (alpha-MSH-related peptides). To test the hypothesis that endogenous central melanocortins have antipyretic effects mediated by central melanocortin receptors (MCRs), we determined the effect of intracerebroventricular injection of a synthetic MCR antagonist, Ac-Nle4,c-[Asp5,DNal(2')7,Lys10]alpha-MSH(4-10)-NH2 (SHU-9119) in endotoxin-challenged rats. The efficacy and specificity of SHU-9119 as an MCR antagonist in the rat was first validated in vitro and in vivo. In vitro, in heterologous cells expressing either rat MC3-R or MC4-R, the major MCR subtypes expressed in brain, SHU-9119 showed no intrinsic agonism, but it inhibited alpha-MSH-induced cAMP accumulation (IC50 = 0.48 +/- 0.19 and 0.41 +/- 0.28 nM, respectively) and [125I]-[Nle4,DPhe7]-alpha-MSH binding (IC50 = 1.0 +/- 0.1 and 0.9 +/- 0.3 nM, respectively). In vivo, exogenous alpha-MSH (180 pmol) inhibited fever in rats when administered intracerebroventricularly 30 min after Escherichia coli lipopolysaccharide (LPS) (25 microg/kg, i.p.). When co-injected with alpha-MSH, SHU-9119 (168 pmol, i.c.v.) prevented the antipyretic action of exogenous alpha-MSH. In contrast, neither alpha-MSH nor SHU-9119, alone or in combination, affected body temperatures in afebrile rats. In LPS-treated rats, intracerebroventricular injection of SHU-9119 significantly increased fever, whereas intravenous injection of the same dose of SHU-9119 had no effect. Neither intracerebroventricular nor intravenous SHU-9119 significantly affected LPS-stimulated plasma ACTH or corticosterone levels. The results indicate that endogenous central melanocortins exert an antipyretic influence during fever by acting on MCRs located within the brain, independent of any modulation of the activity of the pituitary-adrenal axis.

Analgesics, Non-Narcotic↗

PubMed1997

Characterisation of D117A and H260A mutations in the melanocortin 1 receptor.

Recent site directed mutagenesis studies on the melanocortin 1 (MC1) receptor have indicated the importance of D117 and H260 amino acid residues for the binding of alpha-MSH (melanocyte stimulating hormone). Here, we report the testing of 12 cyclic and linear MSH peptides on the D117A and H260A mutant receptors. Moreover, we constructed a double mutant which displayed a major loss in affinity for [Nle4, D-Phe7]alpha-MSH. Our new data of His6 and Phe7 substituted MSH peptides are compared with previous results and the hypothesis of putative interactions of D117 and H260 with single amino acids in the MSH peptide. Our conclusions are that the D117A and the H260A mutations may cause conformational changes in the receptor which can not be linked to any specific amino acid in the MSH-peptides.

Animals↗

PubMed1997

Role of melanocortinergic neurons in feeding and the agouti obesity syndrome.

Dominant alleles at the agouti locus (A) cause an obesity syndrome in the mouse, as a consequence of ectopic expression of the agouti peptide. This peptide, normally only found in the skin, is a high-affinity antagonist of the melanocyte-stimulating hormone receptor (MC1-R), thus explaining the inhibitory effect of agouti on eumelanin pigment synthesis. The agouti peptide is also an antagonist of the hypothalamic melanocortin-4 receptor (MC4-R). To test the hypothesis that agouti causes obesity by antagonism of hypothalamic melanocortin receptors, we identified cyclic melanocortin analogues that are potent agonists or antagonists of the neural MC3 (refs 11, 12) and MC4 receptors. Intracerebroventricular administration of the agonist, MTII, inhibited feeding in four models of hyperphagia: fasted C57BL/6J, ob/ob, and A(Y) mice, and mice injected with neuropeptide Y. Co-administration of the specific melanocortin antagonist and agouti-mimetic SHU9119 completely blocked this inhibition. Furthermore, administration of SHU9119 significantly enhanced nocturnal feeding, or feeding stimulated by a prior fast. Our data show that melanocortinergic neurons exert a tonic inhibition of feeding behaviour. Chronic disruption of this inhibitory signal is a likely explanation of the agouti obesity syndrome.

Agouti Signaling Protein↗

PubMed1997

Design of peptides, proteins, and peptidomimetics in chi space.

Peptide and protein biological activities depend on their three dimensionals structures in the free state and when interacting with their receptors/acceptors. The backbone conformations such as alpha-helix, beta-sheet, beta-turn, and so forth provide critical templates for the three-dimensional structure, but the overall shape and intrinsic stereoelectronic properties of the peptide or protein important for molecular recognition, signal transduction, enzymatic specificity, immunomodulation, and other biological effects depend on arrangement of the side chain groups in three-dimensional chi space (their chi 1, chi 2, etc. torsional angles). In this paper we explore approaches to the de novo design of polypeptides and peptidomimetics with biased or specific conformational/topographical properties in chi space. We consider computational and experimental methods that can be used to examine the effects of specific structural modifications in constraining side chain groups of amino acid residues and their similarities in chi space to the natural amino acids to evaluate what sort of mimetics are likely to mimic normal amino acids. We then examine some of the asymmetric synthetic methods that are being developed to obtain the amino acid mimetics. Finally, we consider selected examples in the literature where these specialized amino acids have been incorporated in biologically active peptides and the specific insights they have provided regarding the topographical requirements for bioactive peptide potency, selectivity, and other biochemical and pharmacological properties. Constraints in chi space show great promise as useful tools in peptide, protein, and peptidomimetic de novo design of structures and pharmacophores with specific stereostructural, biochemical and biological properties.

Drug Design↗

PubMed1997

Skin pigmentation and pharmacokinetics of melanotan-I in humans.

A comparative pharmacokinetic trial was performed with a superpotent synthetic melanotropic peptide, [Nle4-D-Phe7]-alpha-MSHi-13 (melanotan-I or MT-I) given by three routes of administration. Plasma levels were measured by RIA and tanning was quantiated using serial reflectometry. Doses of 0.16 mgkg-1 were administered intravenously (IV) and orally (PO), and doses from 0.08 to 0.21 mg kg-1 subcutaneously (SC), in a randomized crossover fashion to three male volunteers over five consecutive days for 2 weeks (ten doses). The results indicate that the SC dose is completely bioavailable compared to the IV dose. No detectable drug levels were observed following PO dosing. The plasma half-lives following SC dosing ranged from 0.07 to 0.79 h for the absorption phase and from 0.8 to 1.7 h for the beta-phase. Clearance ranged from 0.12 to 0.19 L kg-1 h-1 and 3.9% or less of the dose was recovered in the urine. Side-effects were minimal, consisting of occasional gastrointestinal upset and facial flushing. Significant tanning of the forehead, arms, and neck was noted following IV or SC dosing. This effect peaked at 1 week following drug administration but was still present 3 weeks after completing the ten-dose regimen. It is concluded that SC administration is an efficacious method of delivering melanotan-I.

Administration, Oral↗

PubMed1997

Comparative biological activities of alpha-MSH antagonists in vertebrate pigment cells.

We have previously reported that melatonin was an effective lightening agonist in the teleost Synbranchus marmoratus, the amphibians Rana pipiens and Bufo ictericus, and in the lizard Anolis carolinensis. The hormone, previously applied to the preparations, effectively inhibited alpha-MSH darkening activity in a dose-independent manner, and was also able to reverse MSH-induced darkening. We presently describe the inhibitory effect of the indoleamine on the murine melanoma cell proliferation. Interestingly, the hormone also stimulated tyrosinase activity, with a correlated increase in melanin content. We also demonstrate that in a diverse lizard species, Urosaurus ornatus, the indoleamine was totally ineffective. The competitive MSH antagonistic activity of H-His-D-Arg-Ala-Trp-D-Phe-Lys-NH2 has been demonstrated previously in R. pipiens and U. ornatus. Herein, its inhibitory activity is also reported in another lizard species, A. carolinensis. However, this MSH analogue was inactive in S. marmoratus, and in murine melanoma cells. On the other hand, the 7 thru 10 alpha-MSH fragment, Ac-Phe-Arg-Trp-Gly-NH2, although ineffective in S. marmoratus and R. pipiens, was an alpha-MSH antagonist in A. carolinensis. Surprisingly, in the melanoma cell line, the MSH fragment exhibited no agonist or antagonist activity, but dramatically potentiated the MSH-induced increase in tyrosinase activity. These data might suggest that the fragment is participating either in the process of facilitation or in positive cooperativity. The present results, taken together with our previously reported data, demonstrate a major interspecies diversity of the MC1 subtype of melanocortin receptor, and point out the relevance of the membrane microenvironment for the final receptor configuration.

Amino Acid Sequence↗

PubMed1997

Sensitivity- and gradient-enhanced heteronuclear coupled/decoupled HSQC-TOCSY experiments for measuring long-range heteronuclear coupling constants.

A pulsed field gradient version of the sensitivity-enhanced 2D HSQC-TOCSY experiment is proposed for measurement of long-range heteronuclear coupling constants. The coupling constants are obtained by computer-aided analysis of mixed-phase multiplets with and without the heteronuclear splitting. Generation of pure phase data is not required. Since large 1JXH and JHH couplings are used for coherence transfer, small nJXH can be measured accurately, which could be difficult to obtain from purely heteronuclear polarization transfer experiments.

Data Interpretation, Statistical↗

PubMed1997

Structure-activity relationship of biphalin. The synthesis and biological activities of new analogues with modifications in positions 3 and 4.

New analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2] with modifications of amino acid residues in positions 3,3' and 4,4' have been synthesized. The potency and selectivity of these analogues were evaluated by competitive radioreceptor binding assay in the rat brain using [3H]CTOP (mu ligand) and [3H][p-Cl-Phe4]DPDPE (delta ligand) as ligands, and by bioassay in the mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The symmetrical substitution of phenylalanine in positions 4 and 4' with p-fluorophenylalanine or p-nitrophenylalanine resulted in an enhancement of the affinity at both delta and mu receptors, with some increase of the selectivity for delta opioid receptors. The analogue containing p-chlorophenylalanine in positions 4 and 4' is the most selective to the delta receptors in this series, with a selectivity ratio about 5. The symmetrical substitution of the glycine-3 residue with phenylalanine resulted in a decrease of binding affinities and biological potencies at both mu & delta receptors.

Analgesics↗

PubMed1997

Extraordinary potency of a novel delta opioid receptor agonist is due in part to increased efficacy.

A new cyclic opioid peptide of sequence Tyr-D-Pen-Gly-Phe-Cys-Phe (HBP2) was examined in the mouse isolated vas deferens (MVD) bioassay. Studies with receptor-selective opioid antagonists showed the peptide to be highly selective for delta opioid receptors. HBP2 and the standard delta agonist DPDPE were simultaneously compared using the technique of partial irreversible receptor blockade; data were analyzed using the operational model of pharmacologic agonism. HBP2 was approximately 160 times as potent as DPDPE; estimation of the affinity and efficacy of the two peptides revealed that the potency increase was due to a 5.3-fold increase in efficacy, as well as a 37-fold increase affinity. This contrasts with our previous findings with other cyclic enkephalin analogs, in which increased affinity was achieved without a change in apparent efficacy. Analysis of concentration-response curve shape suggested in addition the possibility of heterogeneity in transduction mechanisms for MVD delta receptors.

Animals↗

PubMed1997

Opioid peptides: simultaneous delta agonism and mu antagonism in somatostatin analogues.

Four isomers of the Somatostatin analogue H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were made with beta-MePhe in position 1 and assayed for opioid binding in rat brain, biological activity in MVD and GPI bioassays, and antinociception in mouse warm-water tail flick assays. The analogues displayed varying potencies and biological activities including: simultaneous delta receptor agonism/mu receptor antagonism, mu receptor antagonism, and delta receptor agonism. These analogues demonstrated that the N-terminal residue is important for receptor potency/selectivity and signal transduction. These analogues my represent leads to therapeutic agents that yield analgesia via delta agonist effects, yet lack side effects associated with mu activity.

Analgesics↗

PubMed1997

Selectivity of cyclic [D-Nal7] and [D-Phe7] substituted MSH analogues for the melanocortin receptor subtypes.

The binding of the 2 cyclic lactam MSH (4-10) analogues (MTII, SHU9119), and 5 cyclic [Cys4, Cys10] alpha-MSH analogues were tested on cells transiently expressing the human MC1, MC3, MC4 and MC5 receptors. The results indicate a differential importance of the C-terminal (Lys-Pro-Val) and N-terminal (Ser-Tyr-Ser) of cyclic [Cys4, Cys10] alpha-MSH analogues in binding to the MC receptor subtypes. Substitution of D-Phe7 by D-Nal(2')7 in both the cyclic lactam MSH (4-10) and the cyclic disulphide MSH (4-10) analogues resulted in a shift in favour of selectivity for the MC4 receptor; the disulphide analogue, [Cys4, D-Nal(2')7 Cys10] alpha-MSH (4-10) (HS9510), showing the highest selectivity for the MC4 receptor among all the substances tested. However, the cyclic lactams displayed an over all higher affinity for the MC receptors, than any of the cyclic disulphide MSH (4-10) analogues.

Animals↗

PubMed1997

Pure glucagon antagonists: biological activities and cAMP accumulation using phosphodiesterase inhibitors.

Five new glucagon analogues have been designed, synthesized, characterized and their biological activities tested. The investigation was centered on modifications in the N-terminal region in particular, residues at Thr5, Phe6 and Tyr10 positions, with the goal of obtaining pure glucagon antagonists in our newly developed high sensitivity cAMP accumulation assay. The structures of the designed compounds are: [des-His1, des-Phe6, Glu9] glucagon-NH2 (1); [des-His1, des-Phe6, Glu9, Phe10]glucagon-NH2 (2); [des-His1, Tyr5, des-Phe6, Glu9]glucagon-NH2 (3); [des-His1, Phe5, des-Phe6, Glu9]glucagon-NH2 (4) and [des-His1, des-Phe6, Glu9, D-Arg18]glucagon-NH2 (5). The binding potencies IC50 values in (nM) were 48.0, 27.4, 26.0, 20.0 and 416.0, respectively. All of these analogues when tested in the classical adenylate cyclase assay demonstrate antagonist properties, and in competition experiments, all caused a rightward-shift of the glucagon stimulated adenylate cyclase dose-response curve. The pA2 values for these analogues were 8.20 (1); 6.25 (2); 6.10 (3); 6.25 (4); and 6.08 (5), respectively. A newly revised assay has been developed to determine the intracellular cAMP accumulation levels in hepatocytes at the highest possible sensitivity. Four of the five glucagon analogues in this report (analogues 1, 2, 4 and 5), did not activate the adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 microM, and thus are pure antagonists.

Adenylyl Cyclases↗

PubMed1997

Synthesis of oligopeptide and peptidomimetic libraries.

The elaboration of peptide libraries prepared by either chemical or biological methods in a format useful for discovery of peptides with specific biological activities was first introduced in the 1980s. A virtual explosion of activity in this area has occurred recently, and the basic approaches have been applied to a wide variety of chemistries and for all manner of biological, chemical and physical targets. Recent advances include new synthetic methodologies, new analytical methods, new design methods and new assay procedures.

Bacteriophages↗

PubMed1997

Brain and spinal cord distribution of biphalin: correlation with opioid receptor density and mechanism of CNS entry.

Biphalin [(Tyr-D-Ala-Gly-Phe-NH)2] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [125I-Tyr1]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [125I-Tyr1]biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of delta- and mu-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [125I-Tyr1] biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [125I-Tyr1]biphalin was decreased with naloxone, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to delta- and mu-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [125I-Tyr1]biphalin, which could be described by Michaelis-Menten kinetics with a Km of 2.6 +/- 4.8 microM, Vmax of 14.6 +/- 2.89 pmol(-1) x min(-1) x g(-1), and Kd of 0.568 +/- 0.157 microl x min(-1) x g(-1). Brain entry of [125I-Tyr1]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and L-phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.

Analgesics↗

PubMed1997

Synthesis and biological properties of beta-MePhe3 analogues of deltorphin I and dermenkephalin: influence of biased chi 1 Phe3 residues on peptide recognition for delta-opioid receptors.

Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I and dermenkephalin containing each of the four stereoisomers (2S,3S; 2S,3R; 2R,3S; 2R,3R) of the unusual amino acid beta-methylphenylalanine in position three. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (mu-ligand) and[3H]p-C1Phe4]DPDPE (delta-ligand), and by bioassay using the mouse vas deferens (delta-receptor assay) and guinea pig ileum (mu-receptor assay) assays. The substitution of a beta-MePhe for Phe3 in deltorphin I and dermenkephalin has a large and variable effect on the bioactivities of the synthesized analogues. The synthesized analogues are somewhat less potent than the native peptides. Both [(2S,3R)-beta-MePhe3]deltorphin and [(2S,3R)-beta-MePhe3] dermenkephalin are more selective, however, and interact essentially specifically with the receptor in the binding assays and bioassays. The bioassay data in vitro of the synthesized analogues of deltorphin I and dermenkephalin follow the same general trends as the receptor binding data. These results demonstrate that topographical modifications of the side-chain conformation of critical structural moieties in a ligand can significantly modulate both the potency and receptor selectivity for ligands that have multiple sites of biological activity, and they illustrate that this approach has general application to peptide and peptidomimetic ligand design.

Aminobutyrates↗

PubMed1997

Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon.

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis1, Val9. Ile11,16] glucagon amide, (3) [desHis1, Val9,11,16] glucagon amide, (4) [desHis1, Val9, Leu11,16]glucagon amide, (5) [desHis1, Nle9, Ile11,16]glucagon amide, (6) [desHis1, Nle9, Val11,16] glucagon amide, (7) [desHis1,-Nle9, Leu11,16] glucagon amide, (8) [desHis1, Val9, Leu11,16, Lys17,18, Glu21] glucagon amide and (9) [desHis1, Nle9, Leu11,16, Lys17,18, Glu21] glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have IC50 values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with pA2 values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively.

Adenylyl Cyclase Inhibitors↗

PubMed1997

Para-substituted phenylalanine-4 analogues of [L-Ala3]DPDPE: highly selective delta opioid receptor ligands.

Several para-substituted Phe4 analogues of the delta 1-selective antagonist [L-Ala3]DPDPE (DPADPE) were prepared and evaluated for their brain-binding and in vitro pharmacological effects. Unlike the p-haloPhe4 analogues of DPDPE and the deltorphins, similar analogues of DPADPE with electron-withdrawing groups substituted at the para-position of the Phe4 aromatic ring did not all have increased potency and selectivity for delta opioid receptors, but all retained high potency and selectivity for delta opioid receptors greater than DPDPE.

Animals↗

PubMed1997

Human epidermal melanocyte and keratinocyte melanotropin receptors: visualization by melanotropic peptide conjugated macrospheres (polyamide beads).

The objectives of this research were to determine whether melanotropin receptors are characteristic membrane markers of human epidermal melanocytes. Methodologies were developed to visualize these receptors by light microscopy. Multiple copies (up to a thousand) of [Nle4,D-Phe7] alpha-MSH, a superpotent analog of alpha-melanocyte stimulating hormone (alpha-MSH), were conjugated to a macromolecular carrier, large polyamide beads (macrospheres). Incubation in the presence of the I conjugated macrospheres resulted in binding of human epidermal melanocytes to the macrospheres. Specificity of the binding of melanocytes to the melanotropin-conjugated macrospheres was demonstrated by several studies: (i) Binding of melanocytes to the conjugate was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; (ii) The macrospheres after removal of the bound ligand did not bind to the melanocytes; (iii) Another peptide hormone ligand (e.g., a substance-P analog) attached to the macrospheres failed to bind to the melanocytes; (iv) B16/F10 mouse melanoma cells known to express melanotropin receptors bound to the macrospheres; (v) Cells of nonmelanocyte origin (e.g., mammary cancer cells, lung cancer cells, fibroblasts) did not bind to the macrospheres. One exception was that human epidermal keratinocytes also expressed melanotropin receptors as determined by all the criteria established for epidermal melanocytes. Thus, cell specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.

Animals↗