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Biomedical subjects

Victor J Hruby

Publications and source records attributed to Victor J Hruby.

At least 73 records · Page 4Linked to original sources
PubMed2003

Converging protein kinase pathways mediate adenylyl cyclase superactivation upon chronic delta-opioid agonist treatment.

Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic delta-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 +/- 91, 399 +/- 2, and 433 +/- 73% after chronic treatment with the delta-opioid agonists (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide (SNC 80), [d-Pen2,d-Pen5]-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 micro M, 4-h) treatment augmented 32P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 +/- 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic delta-opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.

Adenylyl Cyclases↗
PubMed2003

NMR solution structure of the glucagon antagonist [desHis1, desPhe6, Glu9]glucagon amide in the presence of perdeuterated dodecylphosphocholine micelles.

Glucagon, a 29-residue peptide hormone, plays an important role in glucose homeostasis and in diabetes mellitus. Several glucagon antagonists and agonists have been developed, but limited structural information is available to clarify the basis of their biological activity. The solution structure of the potent glucagon antagonist, [desHis1, desPhe6, Glu9]glucagon amide, was determined by homonuclear 2D NMR spectroscopy at pH 6.0 and 37 degrees C in perdeuterated dodecylphosphocholine micelles. The overall backbone root-mean-square deviation (rmsd) for the structured portion (residues 7-29, glucagon numbering) of the micelle-bound 27-residue peptide is 1.36 A for the 15 lowest-energy structures, after restrained molecular dynamics simulation. The structure consists of four regions (segment backbone rmsd in A): an unstructured N-terminal segment between residues 2 and 5 (1.68), an irregular helix between residues 7 and 14 (0.79), a hinge region between residues 15 and 18 (0.54), and a well-defined alpha-helix between residues 19 and 29 (0.33). The two helices form an L-shaped structure with an angle of about 90 degrees between the helix axes. There is an extended hydrophobic cluster, which runs along the inner surface of the L-structure and incorporates the side chains of the hydrophobic residues of each of the amphipathic helices. The outer surface contains the hydrophilic side chains, with two salt bridges (D15-R18 and R17-D21) implied from close approach of the charged groups. This result is the first clear indication of an overall tertiary fold for a glucagon analogue in the micelle-bound state. The relationship of the two helical structural elements may have important implications for the biological activity of the glucagon antagonist.

Amino Acid Sequence↗
PubMed2003

Agonist-specific down-regulation of the human delta-opioid receptor.

Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.

Amino Acid Sequence↗
PubMed2003

Solution structures of cyclic melanocortin agonists and antagonists by NMR.

The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets for drugs to treat obesity, sexual dysfunction, etc. Understanding the conformational basis of the receptor-ligand interactions is crucial to the design of potent and selective ligands for these receptors. The solution structures of the cyclic melanocortin agonists, partial agonist, and antagonists MTII, VJH085, SHU9119, MK5, and MK9 were determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy at pH 4.5 and 25 degrees C in water (90% H(2)O/10% D(2)O). The overall backbone structures of these cyclic alpha-melanocyte-stimulating hormone (alpha-MSH) analogues around the message sequence (His(6)-D-Phe(7)/D-Nal(2')(7)-Arg(8)-Trp(9)) were similar and reasonably well defined. beta-Turns spanning His(6) and D-Phe(7)/D-Nal(2')(7) were identified in all analogues, and an amphiphilic molecular surface was obtained for the message sequence residues in most structures within the NMR ensembles. The beta-turn, which most closely resembles a type II beta-turn, leads to stacking between the aromatic rings of His(6) and D-Phe(7) in MTII and VJH085. However, no aromatic stacking between His(6) and D-Nal(2')(7) was found in structures of the D-Nal(2')(7)-containing analogues. The difference in the side-chain dispositions of His(6) and D-Nal(2')(7) may be responsible for the reduced potency or antagonist activity of the D-Nal(2')(7)-containing analogues. In addition, our results suggest that the side-chain orientations may also modulate the receptor selectivity. The information found in this study will be useful for the further design of ligands for melanocortin receptors.

Melanocyte-Stimulating Hormones↗
PubMed2003

Techniques: plasmon-waveguide resonance (PWR) spectroscopy as a tool to study ligand-GPCR interactions.

Plasmon-waveguide resonance (PWR) spectroscopy can be applied to integral membrane proteins incorporated into a supported lipid bilayer without the need for labeling. With high sensitivity and wide dynamic range, this technique can be used to characterize the kinetics and thermodynamics of conformational events associated with the binding of ligands to G-protein-coupled receptors (GPCRs), and to directly examine the interactions of GPCRs with G proteins and other downstream effectors in signal transduction. This allows an easy distinction to be made between agonists, antagonists and inverse agonists, and provides a powerful new tool for studying membrane signaling and for drug development.

Ligands↗
PubMed2003

Exploring the stereostructural requirements of peptide ligands for the melanocortin receptors.

The melanotropin peptides alpha-MSH, gamma-MSH, and beta-MSH are believed to be the natural ligands for the four melanocortin receptors, MC1R, MC3R, MC4R, and MC5R. However, these peptides generally have low selectivity for these receptors. We report on some approaches to the development of selective agonists and antagonists peptide ligands for these receptors.

Amino Acid Sequence↗
PubMed2003

MT-II induces penile erection via brain and spinal mechanisms.

alpha-Melanocyte-stimulating hormone induces penile erection via melanocortin (MC) receptors in areas surrounding the third ventricle, but spinal and peripheral mechanisms have not been demonstrated. We used pharmacological strategies to localize the site of the proerectile action of the melanocortin receptor agonist MT-II. We administered MT-II intracerebroventribularly (i.c.v.), intrathecally (i.th.), and intravenously (i.v.) and scored penile erection and yawning for 90 min in awake male rats. In some animals i.c.v. or i.th. SHU-9119 was injected 10 minutes before i.c.v. and i.th. MT-II to confirm the MC receptor action of the agonist and to distinguish spinal from supraspinal effects. To exclude a site of action in the penis, we recorded intracorporal pressure responses to intracavernosal injection of MT-II in the anesthetized rat. MT-II induced penile erections in a dose-dependent fashion, with optimal response at 1 microg for both i.c.v. and i.th. routes. Supraspinal MT-II-induced erections were completely suppressed by 1 microg SHU-9119 i.c.v. Yawning was observed with i.c.v. and i.v. MT-II, whereas spinal injection did not produce this behavior. SHU-9119 blocked the erectile responses to i.th. MT-II when injected i.th. but not i.c.v. Intracavernosal MT-II neither increased intracorporal pressure nor augmented neurostimulated erectile responses. The lumbosacral spinal cord contains MC receptors that can initiate penile erection independent of higher centers. We confirmed the supraspinal proerectile action of MT-II. These results provide insight into the central melanocortinergic pathways that mediate penile erection and may allow for more efficacious melanotropin-based therapy for erectile dysfunction.

Animals↗
PubMed2003

(2S,3R) beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] is a potent, selective delta-opioid receptor antagonist in mouse brain.

The constrained opioid peptide (2S,3R)beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] exhibits high affinity and selectivity for the delta-opioid receptors (). In the present study, we examined the pharmacological properties of (2S,3R)TMT-L-Tic-OH in mouse brain. A 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTP gamma S) binding assay was used to determine the effect of (2S,3R)TMT-L-Tic-OH on G protein activity in vitro, in mouse brain membranes. delta- (SNC80; (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl-benzamide) or mu- (DAMGO; [D-Ala(2), Me-Phe(4),Gly(ol)(5)]enkephalin) selective opioid full agonists stimulated [(35)S]GTP gamma S binding in mouse brain membranes 150 +/- 4.5% and 152 +/- 5.7% over the basal level, respectively. (2S,3R)TMT-L-Tic-OH did not influence basal [(35)S]GTP gamma S binding in mouse brain membranes but dose dependently shifted the dose-response curve of SNC80 to the right, with a K(e) value of 3.6 +/- 0.7 nM. In contrast, (2S,3R)TMT-L-Tic-OH had no effect on the dose-response curve of the mu-selective opioid agonist, DAMGO. Warm water (55 degrees C) tail-flick and radiant heat paw-withdrawal tests were used to determine the in vivo nociceptive properties of (2S,3R)TMT-L-Tic-OH in the mouse. Intracerebroventricular injection of (2S,3R)TMT-L-Tic-OH had no significant effect on withdrawal latencies in either nociceptive tests. (2S,3R)TMT-L-Tic-OH (30 nmol/mouse) attenuated deltorphin II- but not DAMGO-mediated antinociception (40 +/- 13 and 100% of maximal possible effect, respectively) when administered intracerebroventricularly 10 min before the agonist. Taken together these results suggest that (2S,3R)TMT-L-Tic-OH is a potent highly selective neutral delta-opioid antagonist in mouse brain.

Analgesics, Opioid↗
PubMed2003

Expression-driven reverse engineering of targeted imaging and therapeutic agents.

Cancer is a particularly daunting disease to treat because, in its most insidious forms, cells are disseminated throughout the body, rendering surgery and local therapies ineffective. Hence, systemic disease must be targeted by its metabolic, physiological or molecular phenotype rather than by its location. Both tumour metabolism and physiology have been exploited to target therapies with some success. There is every reason to expect that these approaches will continue to advance therapies that discriminate tumour metabolism from that of normal tissues. As an alternative, molecular phenotyping (pharmacogenomics) is a relatively new science, and holds great promise for development of novel therapies and approaches. The discipline of pharmacogenomics has, to date, involved segmentation of patients according to their protein expression patterns in order to direct existing therapies to those populations who stand to benefit the most. In this communication, the authors propose a further application of this technology to develop agents that are reverse-engineered to explicitly target a patient's expressed protein patterns.

Antigen-Antibody Reactions↗
PubMed2002

Structure-activity studies of the melanocortin peptides: discovery of potent and selective affinity antagonists for the hMC3 and hMC4 receptors.

We have designed and synthesized several novel cyclic SHU9119 analogues (Ac-Nle4-[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH2) modified in position 6 with nonconventional amino acids. SHU9119 is a high affinity nonselective antagonist at hMC3R and hMC4R with potent agonist activity at hMC1R and hMC5R. We measured the binding affinity and agonist potency of the novel analogues at cloned hMC3R, hMC4R, and hMC5R receptors and identified several selective, high affinity hMC3R and hMC4R antagonists. Compound 4 containing Che substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.48 nM) with 100-fold selectivity over hMC3R antagonist. Analogue 7 with a Cpe substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.51 nM) with a 200-fold selectivity vs the hMC3R. Interestingly, analogue 9 with an Acpc residue in position 6 is a high affinity hMC3R antagonist (IC50 = 2.5 nM) with 100-fold selectivity vs the hMC4R antagonist based on its binding affinities. This compound represents the first cyclic lactam antagonist with high selectivity for the hMC3R vs hMC4R. To understand the possible structural basis responsible for selectivity of these peptides at hMCR3 and hMCR4, we have carried out a molecular modeling study in order to examine the conformational properties of the cyclic peptides modified in position 6 with conformationally restricted amino acids.

Animals↗
PubMed2002

Stereoselective bromination-suzuki cross-coupling of dehydroamino acids to form novel reverse-turn peptidomimetics: substituted unsaturated and saturated indolizidinone amino acids.

A general and efficient methodology has been developed to prepare the C4-substituted dipeptide reverse-turn mimetics unsaturated (9a, 10a) and saturated (11a) azabicyclo[4.3.0] alkane amino acid derivatives. The side chain was introduced by bromination of dehydroamino acid intermediates followed by Suzuki coupling. Hydrogenation of the bicyclic dehydroamino acid 9a afforded saturated bicyclic lactam 11a. This approach can be further explored for the synthesis of a variety of such beta-turn mimetics with aryl and alkyl side chain functionalities. [reaction: see text]

Amino Acids↗
PubMed2002

Stereoselective synthesis of dipeptide beta-turn mimetics: 7-benzyl and 8-phenyl substituted azabicyclo[4.3.0]nonane amino acid esters.

A stereoselective method has been developed for the synthesis of 7- and 8-substituted dipeptide beta-turn mimetic azabicyclo[4.3.0]nonane amino acid esters. The allyl groups were introduced in high diastereoselectivity, controlled by 3-phenyl or 4-benzyl groups in pyroglutamic acid derivatives 3 or 9, respectively. The precursors, dehydroamino acids 7 and 13 derived from 5 or 11, underwent asymmetric hydrogenations with Burk's DuPHOS Rh(I)-based catalysts to furnish alpha-amino acid derivatives in high stereoselectivity. The resulting amino acids 8 and 14 were converted to the beta-turn mimetics 6,5-bicyclic lactams 1a-d in high yields.

Amino Acids↗
PubMed2002

Novel cyclic templates of alpha-MSH give highly selective and potent antagonists/agonists for human melanocortin-3/4 receptors.

In an effort to develop highly selective and potent agonists and/or antagonists for the hMC3 and hMC4 receptors, a new approach involving the use of linker arms and a backbone to side chain cyclization strategy was employed. Three key analogues were identified to have the required selectivity and potency at the hMC3 or hMC4 receptors, implicated to play pivotal roles in energy homeostasis and other biological effects. The novel cyclic peptide (O)C-CH(2)-CH(2)-C(O)-c-[His(6)-D-Phe(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2) (1) was found to be a highly selective and potent agonist of the hMC4 receptor. Structure-activity studies have shown that replacing the succinyl linker arm of 1 by an o-phthalic acid group and substituting a D-Nal(2')(7) residue in place of D-Phe(7) results in a potent antagonist 7 at the hMC4 receptor. Furthermore, increasing the 23-membered lactam ring of 1 by one carbon atom (succinyl --> glutaric acid linker) gives a highly selective and potent antagonist 9 for the hMC3 receptor. Analogues 1, 7, and 9 therefore represent the first examples of a class of cyclic melanotropin ligands with high selectivity and defined biological activities at the physiologically important hMC3 and hMC4 receptors.

Adenylyl Cyclases↗
PubMed2002

A general asymmetric synthesis of syn- and anti-beta-substituted cysteine and serine derivatives.

A stereodivergent synthetic route has been developed to make the optically pure anti- and syn-beta-substituted cysteine and serine derivatives. In this approach, the key intermediates, > 94% enantiomerically pure cyclic sulfates 3 and aziridines 7, were prepared from alpha,beta-unsaturated esters 1, employing the Sharpless asymmetric dihydroxylation. The high regio- and stereoselective ring-opening reactions of cyclic sulfates and aziridines provided enantiomerically pure beta-substituted cysteine and serine derivatives.

Amino Acid Sequence↗
PubMed2002

Design and synthesis of highly potent and selective melanotropin analogues of SHU9119 modified at position 6.

The melanocortin receptors are involved in several important physiological functions. The potent and enzymatically stable analogues MT-II (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH(2)) and SHU9119 (Ac-Nle-c[Asp-His-DNal(2')-Arg-Trp-Lys]-NH(2)) are important ligands of these receptors but are relatively nonselective. To differentiate between the physiological functions of these receptors, agonists, and antagonists with improved receptor selectivities are needed. We report here analogues of the well-characterized antagonist SHU9119 in which we replaced His(6) with conformationally constrained amino acids. By this structure-activity study we discovered two important compounds, PG-901 (Ac-Nle(4)-c[Asp(5)-Pro(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)) and PG-911 (Ac-Nle(4)-c[Asp(5)-Hyp(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)), characterized to be full agonists at the hMC5R (EC(50) = 0.072 nM and 0.031 nM, respectively), but full antagonists at the hMC3R and the hMC4R. We also demonstrated that the relative stereochemistry of the amino acid at the 6-position is critical for activity, and could play an important role in potency as well as in selectivity for the melanocortin receptors.

Amino Acid Substitution↗
PubMed2002

Regioselective and stereoselective nucleophilic ring opening reactions of a phenyl-substituted aziridine: enantioselective synthesis of beta-substituted tryptophan, cysteine, and serine derivatives.

The asymmetric synthesis of beta-phenyl-substituted cysteine, tryptophan, and serine derivatives was successfully developed. In this approach, the key intermediate, enantiomerically pure 3-phenylaziridine-2-carboxylic ester 7, was prepared from alpha,beta-unsaturated ester 1 by employing the Sharpless asymmetric dihydroxylation. The aziridine 7 was treated with 4-methoxybenzylthiol, indole, and acetic acid to give beta-phenyl-substituted cysteine, tryptophan, and serine, respectively, in a clean S(N)2 type ring opening at the C3 position. This general approach can be used to synthesize a variety of beta-substituted novel amino acids.

Amino Acids↗
PubMed2002

Biological properties of a new fluorescent biphalin fragment analogue.

Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH<--X, where X=Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X=DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both mu and delta opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation.

Analgesia↗