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V J Hruby

Publications and source records attributed to V J Hruby.

At least 163 records · Page 9Linked to original sources
PubMed1994

Binding and cAMP studies of melanotropin peptides with the cloned human peripheral melanocortin receptor, hMC1R.

Binding and stimulation of cAMP by the melanotropin peptides alpha-MSH (alpha-melanocyte-stimulating hormone) and its superpotent analogues [Nle4, DPhe7]alpha-MSH (MT-I) and Ac-[Nle4,[formula: see text]alpha-MSH4-10-NH2 (MT-II) were undertaken to examine their respective properties on the human peripheral melanocyte melanocortin receptor, hMC1R. alpha-MSH was found to possess a binding IC50 value of 6.5 +/- 0.9 x 10(-9) M and cAMP EC50 value of 2.0 +/- 0.6 x 10(-9) M. MT-I possesses a binding IC50 value of 1.2 +/- 0.3 x 10(-9) M and a cAMP EC50 of 0.5 +/- 0.03 x 10(-9) M. MT-II possesses a binding IC50 of 0.57 +/- 0.08 x 10(-9) M and cAMP EC50 value of 0.20 +/- 0.05 x 10(-9) M.

Affinity Labels↗
PubMed1994

Design, synthesis, and biological properties of highly potent cyclic dynorphin A analogues. Analogues cyclized between positions 5 and 11.

We have recently reported the synthesis of several cyclic disulfide bridge-containing peptide analogues of dynorphin A (Dyn A), which were conformationally constrained in the putative address segment of the opioid ligand. Several of these analogues, bridged between positions 5 and 11 of Dyn A1-11-NH2, exhibited unexpected selectivities for the kappa and mu receptors of the central over the peripheral nervous systems. In order to further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, we have synthesized a systematic series of Dyn A1-11-NH2 analogues incorporating the sulfydryl containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11, thus producing 16 cyclic peptides. In addition, Dyn A1-11-NH2, [D-Leu5]Dyn A1-11-NH2, and [D-Lys11]Dyn A1-11-NH2 were synthesized as standards. Several of these cyclic analogues, especially c[Cys5,D-Cys11] Dyn A1-11-NH2, c[Cys5, L- or D-Pen11]Dyn A1-11-NH2, c[Pen5, L-Pen11]Dyn A1-11-NH2 and c[Pen5, L- or D-Cys11]Dyn A1-11-NH2, retained the same affinity and selectivity (vs the mu and delta receptors) as the parent compound Dyn A1-11-NH2 in the guinea pig brain (GPB). These same analogues and most others exhibited a much lower activity in the guinea pig ileum (GPI), thus leading to centrally vs peripherally selective peptides, but showed a different structure-activity relationship than found previously. In a wider scope, this series of analogues also provided new insights into which amino acids (and their configurations) may be used in positions 5 and 11 of Dyn A analogues for high potency and good selectivity at kappa opioid receptors. The results obtained in the GPB suggest that requirements for binding are not the same for the kappa, mu, or delta central receptors.

Amino Acid Sequence↗
PubMed1994

[N-methylnorleucine-(28,31)]cholecystokinin-(26-33) (SNF 8702) activity at a cloned rat CCKB receptor.

[N-methyl-Nle 28,31)]cholecystokinin-(26-33) (SNF 8702) is a highly selective ligand for the CCKB type of receptor present in the vertebrate central nervous system. Radioligand binding data showing that SNF 8702 binding affinity is reduced by the GTP analog guanylyl-imidodiphosphate suggest that SNF 8702 is an agonist but the ability of SNF 8702 to activate CCKB receptors has not been demonstrated. The present study shows that SNF 8702 is a highly potent agonist at rat CCKB receptors expressed on COS-7 cells and that these receptors are coupled to the mobilization of intracellular calcium. The A50 measured for SNF 8702-induced calcium mobilization (66 pM) is over 6-fold less than that of cholecytstokinin octapeptide (420 pM). Data are also presented showing that SNF 8702 has high binding affinity for these receptors with a Kd value (760 pM) consistent with previous measurements using guinea pig brain tissue preparations.

Animals↗
PubMed1994

Glycopeptide enkephalin analogues produce analgesia in mice: evidence for penetration of the blood-brain barrier.

Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain. Intraperitoneally administered L-serinyl beta-D-glucoside analogues of [Met5]enkephalin (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted mu- and delta-opioid receptors in the mouse brain. The opioid nature of the binding has been demonstrated with intracerebroventricularly administered naloxone. Paradoxically, glucosylation decreases the lipophilicity of the peptides while promoting transport across the lipophilic endothelial layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting analgesia has been observed in mice (tail-flick and hot-plate assays) with two of the glycopeptide analogues when administered intraperitoneally.

Amino Acid Sequence↗
PubMed1994

Interaction of [D-Pen2,D-Pen5]enkephalin and [D-Ala2,Glu4]deltorphin with delta-opioid receptor subtypes in vivo.

The interaction of [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2,Glu4]deltorphin with delta-opioid receptor subtypes was investigated. Pretreatment of mice with the delta 1-opioid receptor antagonist, [D-Ala2,Leu5,Cys6]enkephalin (DALCE), produced a virtually complete antagonism of the antinociceptive actions of DPDPE, but had no effect on those of [D-Ala2,Glu4]deltorphin. In DALCE pretreated mice (i.e., delta 1-opioid receptors blocked), DPDPE was able to significantly antagonize the antinociceptive effects of [D-Ala2,Glu4]deltorphin. Pretreatment of mice with the delta 2-opioid receptor antagonist, naltrindole-5'-isothiocyanate (5'-NTII) produced a virtually complete antagonism of the antinociceptive effects of [D-Ala2,Glu4]deltorphin, but had no effect on the antinociception produced by DPDPE. In 5'-NTII pretreated mice (i.e., delta 2-opioid receptors blocked), [D-Ala2,Glu4]deltorphin had no effect on the antinociception produced by DPDPE. These data suggest that [D-Ala2,Glu4]deltorphin is highly selective for the delta 2-opioid receptor in vivo, and that neither agonist nor antagonist actions can be demonstrated at delta 1-opioid receptors for this peptide. In contrast, under appropriate conditions, DPDPE can be shown to interact with both delta 1- and delta 2-opioid receptor subtypes; DPDPE may have limited efficacy (i.e., is a partial agonist) at the delta 2-opioid receptor.

Amino Acid Sequence↗
PubMed1994

Design of cyclic deltorphins and dermenkephalins with a disulfide bridge leads to analogues with high selectivity for delta-opioid receptors.

We earlier suggested that the low receptor selectivity observed for previously synthesized constrained analogues of deltorphin I (DT I) was the result of a reduction in the lipophilic surface of the C-terminal of the peptide. To confirm this prediction and to further test a previously proposed conformational model for bioactivity at delta opioid receptors, we have synthesized several new cyclic analogues with the general structure [D-Xaa2,Yaa5]deltorphin I and II in which Xaa2 is D-cysteine or D-penicillamine (D-Pen), and Yaa5 is an L- or D-penicillamine residue. Additional substitutions at positions 4, 6, and 7 also were examined. The analogues were tested for binding to mu- and delta-opioid receptors and in mouse vas deferens and guinea pig ileum biological assays. The introduction of a lipophilic L-Pen in position 5 and D-Cys or D-Pen in position 2 resulted in a highly delta-selective series of analogues, which fully confirmed our prediction. The cyclic analogues formula; see text: DT I are among the most delta-selective analogues described thus far.

Amino Acid Sequence↗
PubMed1994

Cyclic enkephalin analogs with exceptional potency at peripheral delta opioid receptors.

A series of super potent and delta-opioid-receptor-selective cyclic hexapeptides of the general formula [formula: see text] (where X is hydrogen or halogen) has been synthesized. The unsubstituted hexapeptide formula; see text: [Phe6]DPLCE) has extremely high potency at peripheral delta opioid receptors (IC50 value in the MVD assay is 0.016 nM) and in bioassays is the most selective compound in this series. The introduction of halogens in the phenyl ring of phenylalanine at position 4 led to significant changes in the selectivity and affinities at peripheral and central opioid receptors. In the binding studies, the most potent compound is the p-fluoro analog, whereas the most selective analog is the p-iodo-substituted peptide.

Amino Acid Sequence↗
PubMed1994

Prediction of the conformational requirements for binding to the kappa-opioid receptor and its subtypes. I. Novel alpha-helical cyclic peptides and their role in receptor selectivity.

A conformational search of two similar kappa-selective cyclic Dynorphin A (Dyn A) analogues is presented. [Cys5, Cys11] Dyn A1-11-NH2 (1) and [Cys5, D-Ala8, Cys11] Dyn A1-11-NH2 (2) are not only highly potent kappa-selective peptides but they also exhibit exceptional selectivity for kappa receptors in the central (brain) vs. the peripheral (ileum) systems. Molecular mechanics systematic searching of the conformational preferences of the cyclic moieties of 1 and 2 produced 741 and 1003 starting ring structures, which were minimized at two dielectric constants of 2.0 and 80.0 in the AMBER force field. By rms superimposition, these low energy structures were grouped into conformational families for each ring system minimized at each dielectric. Comparison of the lowest energy structure of each of these families demonstrated that two (labeled A and B) were found as low energy ring systems for both 1 and 2 after minimization at either dielectric constant. These two structures are thus predicted to be the putative binding conformations for Dynorphin A at receptors in the brain. Interestingly, one of these putative binding structures exhibited an alpha-helical conformation in the disulfide bridged ring that has not been observed for small cyclic peptides of this nature before. Molecular dynamics simulation of the helical binding structures indicated that the helical configuration in 2 is lower in energy and is more conformationally stable than that of 1. We correlate this with the increased selectivity and potency of 2 for kappa receptors in the brain compared to the periphery, implying that this may be due to an alpha-helical conformation in the cyclized address or helical induction in the message sequence.

Amino Acid Sequence↗
PubMed1994

Preformulation studies with melanotan-II: a potential skin cancer chemopreventive peptide.

Melanotan-II (1) is a cyclic heptapeptide analogue of alpha-melanocyte-stimulating hormone (alpha-MSH) which tans the skin and is currently being evaluated for the prevention of sunlight-induced skin cancers. The dissociation constants of 1 were determined using potentiometric titration and ultraviolet spectrophotometry. The pKa1 (histidine) and pKa2 (arginine) were estimated to be 6.54 and 11.72, respectively. The apparent partition coefficient (PC) was measured at three pH values using both n-octanol and isooctane as the nonpolar phase. The PC(octanol) and delta log PC at pH 7.35 were 2.82 and 1.05, respectively. These data, together with the observance of a bioavailability of 4.6% in the rat, indicate that 1 may be a suitable candidate for oral delivery. The data presented here are useful in developing an appropriate dosage form for 1.

Amino Acid Sequence↗
PubMed1994

Selective inhibition of [D-Ala2, Glu4]deltorphin antinociception by supraspinal, but not spinal, administration of an antisense oligodeoxynucleotide to an opioid delta receptor.

Evidence in vivo has suggested the existence of subtypes of the delta opioid receptor (DOR), which have been termed delta 1 and delta 2. These proposed DOR subtypes are thought to be activated by [D-Pen2, D-Pen5]enkephalin (DPDPE, delta 1) and [D-Ala2, Glu4]deltorphin (delta 2). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin, but not of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO, mu agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonists selective for delta, mu and kappa receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin but not of i.th. DAMGO or U69,593 (kappa agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, selectively inhibited the antinociceptive response to i.c.v. [D-Ala2, Glu4]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as delta 2 and further, suggest that this delta receptor subtype may play a major role in eliciting spinal delta-mediated antinociception.

Analgesics↗
PubMed1994

Identification of a human delta opioid receptor: cloning and expression.

The delta opioid receptor is an important target for analgesic drug development. This report describes the identification of delta opioid receptor clones from human cDNA libraries and the preparation of a human delta receptor cDNA in the pcDNA3 expression vector for transfection studies. The cDNA encodes a 372 amino acid protein that has 93% amino acid identity to mouse and rat delta receptors. COS-7 cells transfected with this clone express over 1.0 pmol receptor/mg protein when measured by saturation binding with [3H]naltrindole. The delta receptor selective ligands NTB, BNTX, [4'-Cl-Phe4]DPDPE and [D-Ala2,Glu4]deltorphin all have Ki values under 10 nM while the affinities of the mu and kappa opioid receptor ligands CTAP and U-69593, respectively, are over 4.0 microM. Agonists show binding to multiple affinity states of the receptor consistent with the presence of G-protein coupled and uncoupled forms of the expressed receptor. The 8-fold higher affinity of NTB relative to BNTX suggests that the human delta receptor is of the delta 2 subtype.

Amino Acid Sequence↗
PubMed1994

The actions of melanin-concentrating hormone (MCH) on passive avoidance in rats: a preliminary study.

Melanin-concentrating hormone (MCH) is a hepadecapeptide hormone that is synthesized in the CNS and is responsible for melanosome aggregation in the teleost fish. Recent evidence suggests that this peptide hormone has a unique distribution in the mammalian brain, which leads to the speculation that it may serve as a neuromodulator. The present study was undertaken to explore the comparative effects of MCH to those of alpha-melanocyte-stimulating Hormone (MSH) (a neuropeptide that is known to influence learning) on the rate of extinction of a passive avoidance response in rats. Both MCH and MSH were administered SC at 10 micrograms per animal. Treatment with MCH appeared to hasten, whereas treatment with MSH appeared to delay, extinction of the passive avoidance response.

Animals↗
PubMed1994

Delta opioid receptor selective ligands; DPLPE-deltorphin chimeric peptide analogues.

Further efforts to correlate the topography of the bioactive structures of DPDPE and the deltorphins, two delta-opioid receptor active peptide families, are reported. A number of DPLPE-deltorphin chimeric peptides have been synthesized in which the C-terminal dipeptide delta-address of the deltorphins (-Val-GlyNH2, -Nle-GlyNH2) have been linked to the highly delta-opioid selective cyclic peptides DPDPE or DPLPE. These studies demonstrate that a major structural feature determining high potency of hybrid analogues is the chirality of the amino acid residue in position 5. The radioligand binding assays have revealed a decrease in potency (compared to DPDPE) at delta-receptors when the C-terminal dipeptides were added to DPDPE. On the other hand, chimeric peptides of DPLPE with these same C-terminal dipeptides retained high delta-selectivity and affinity. Similar results were obtained using the mouse vas deferens (MVD) and guinea pig ileum (GPI) bioassays. The importance of the hydrophilicity of amino acids in positions 2 and 5 for delta-selectivity is consistent with the previous finding for DPLPE and DPDPE. On the other hand, the replacement of phenylalanine-4 with p-chlorophenylalanine-4 did not increase delta-selectivity as in DPDPE. These findings suggest that the delta-receptor interacts with hybridized enkephalins and deltorphins somewhat differently than with DPDPE.

Amino Acid Sequence↗