Search PubMed⌕ Search

Biomedical subjects

V J Hruby

Publications and source records attributed to V J Hruby.

At least 235 records · Page 13Linked to original sources

A new type of synthetic peptide library for identifying ligand-binding activity.

Our aim was to improve techniques for drug development by facilitating the identification of small molecules that bind with high affinity to acceptor molecules (for example, cell-surface receptors, enzymes, antibodies) and so to mimic or block their interaction with the natural ligand. Previously such small molecules have been characterized individually on a serial basis. The systematic synthesis and screening of peptide libraries of defined structure represents a new approach. For relatively small libraries, predetermined sequence variations on solid-phase supports have been used, and large libraries have been produced using a bacteriophage vector into which random oligodeoxynucleotide sequences have been introduced, but these techniques have severe limitations. Here we investigate an alternative approach to synthesis and screening of peptide libraries. Our simple methodology greatly enhances the production and rapid evaluation of random libraries of millions of peptides so that acceptor-binding ligands of high affinity can be rapidly identified and sequenced, on the basis of a 'one-bead, one-peptide' approach.

Amino Acid Sequence↗

Opioid receptor binding properties of analgesic analogues of cholecystokinin octapeptide.

Four analogues of cholecystokinin (CCK) octapeptide having analgesic activity after i.c.v. administration and high affinity for CCK-B receptors were studied for their ability to displace specific ligands, [3H]D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. [3H][D-Pen2, 4'-Cl-Phe4,D-Pen5]enkephalin and [3H]U-69,593, for mu-, delta- and kappa-opioid receptors, respectively. None of the analogues tested have high affinity for either mu- or kappa-receptors (IC50 values greater than 0.7 microM), but their IC50 values for delta-receptors range from 29 to 1023 nM. The results suggest a relationship between the ligand requirements of CCK-B and delta-opioid receptors which further implies a possible structural relationship between these receptors.

Amino Acid Sequence↗

Topographical requirements for delta-selective opioid peptides.

The conformational possibilities of three different delta-selective opioid peptides, which are DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen), DCFPE (Tyr-D-Cys-Phe-D-Pen), and DRE (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, dermenkephalin), were explored using energy calculations. Sets of low-energy conformers were obtained for each of these peptides. The sets consisted of 61 structures for DPDPE, 32 for DCFPE, and 38 for DRE, including various types of rotamers of the Tyr and Phe side-chain groups. Comparison of the geometrical shapes of the conformers was performed for these sets using topographical considerations, i.e., examination of the mutual spatial arrangement of the N-terminal alpha-amino group, and of the Tyr and Phe side-chain groups. The results obtained suggest a model for the delta-receptor-bound conformer(s) for opioid peptides. The model suggests the placement of the Phe side chain in a definite position in space corresponding to the g- rotamer of Phe for peptides containing Phe4 and to the t rotamer for peptides containing Phe. The position of the Tyr1 side chain cannot be specified so precisely. The proposed model is in a good agreement with the results of biological testing of beta-Me-Phe4-substituted DPDPE analogues that were not considered in the process of model construction.

Amino Acid Sequence↗

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro.

Several peptides of diverse structure, reported to possess high affinity and selectivity for the delta opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5] enkephalin, the cyclic enkephalin analogs [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,p-F-Phe4,D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5,Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 x 10(-10) molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.

Amino Acid Sequence↗

Delta opioid receptor-selective ligands: [D-Pen2,D-Pen5]enkephalin-dermenkephalin chimeric peptides.

A number of DPDPE-dermenkephalin chimeric peptides have been synthesized in which the putative C-terminal delta-address of dermenkephalin has been linked to the highly delta opioid selective cyclic peptide [D-Pen2,D-Pen5]enkephalin (DPDPE). Asp, Met-Asp and Leu-Met-Asp have been added to the C-terminus of DPDPE and both the carboxyl terminal and the carboxamide terminal series have been prepared. The bioassays using the mouse vas deferens and guinea pig ileum preparations have revealed a steady decrease in potency (compared to DPDPE) at delta and mu receptors as the dermenkephalin sequences were added. Some of the analogues, however, retained high delta selectivity. Similar results were obtained using radioligand binding assays. These findings suggest that the C-terminal amino acid sequence of dermenkephalin plays a role of delta-address which is specific to dermenkephalin itself, and is not additive with another delta selective ligand such as DPDPE.

Amino Acid Sequence↗

Designing peptide and protein ligands for biological receptors.

A clearer understanding of structure-function relationships of protein hormone-receptor systems is emerging from the increased use of molecular biology approaches. On the other hand, the introduction of rationally designed conformation constraints into peptide hormones and neurotransmitters is leading to the development of highly receptor-selective ligands that allow further investigations into the topographic modulation of their bioactivities and rational design principles for peptide antagonists.

Amino Acid Sequence↗

Dynamical search for bis-penicillamine enkephalin conformations.

Quenched molecular dynamics is used as a conformational search technique for the constrained cyclic analog [D-Pen2,D-Pen5]enkephalin (DPDPE) in a continuum solvent. The results show a Gaussianlike distribution of conformations as a function of energy, unlike the distributions found for simple liquids which have sharp bands for different crystal forms and broad glasslike states are found. The lowest energy conformers have structural features in common with those obtained from constrained searches based on energy minimization. (Hruby, V. J., L-.F. Kao, B. M. Pettitt, and M. Karplus. 1988. J. Am. Chem. Soc. 110:3351-3359). Many of the low energy configurations are amphiphilic with the carbonyl groups on one surface and the hydrophobic groups on the other. This supports the conclusions from the previous modeling study, which yielded amphiphilic structures as the most probable conformations of DPDPE when NOE data were included.

Calorimetry↗

Topographically designed analogues of [D-Pen,D-Pen5]enkephalin.

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5]enkephalin (1, DPDPE) was systematically modified topographically by addition of a methyl group at either the pro-S or pro-R position of the beta carbon of an L-Phe4 or D-Phe4 residue to give [(2S,3S)-beta-MePhe4]DPDPE (2), [(2R,3R)-beta-MePhe4]DPDPE (3), [(2S,3R)-beta-MePhe4]DPDPE (4), and [(2R,3S)-beta-MePhe4]DPDPE (5). The four corresponding isomers were prepared in which the beta-methylphenylalanine residue was p-nitro substituted, that is with a beta-methyl-p-nitrophenylalanine (beta-Me-p-NO2Phe) residue, to give [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (6), [(2R,3R)-beta-Me-p-NO2Phe4]DPDPE (7), [(2S,3R)-beta-Me-p-NO2Phe4] DPDPE (8), and [(2R,3S)-beta-Me-p-NO2Phe4]DPDPE (9), respectively. The potency and selectivity (delta vs mu opioid receptor) were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H]DPDPE (delta ligand) and by bioassay with mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The eight analogues of DPDPE showed highly variable binding and bioassay activities particularly at the delta opioid receptor (4 orders of magnitude), but also at the mu opioid receptor, which led to large differences (3 orders of magnitude) in receptor selectivity. For example, [(2S,3S)-beta-MePhe4]DPDPE (2) is 1800-fold selective in binding to the delta vs mu receptor, making it one of the most selective delta opioid receptor ligands in the enkephalin series as assessed by the rat brain binding assay, whereas the corresponding (2R,3R)-beta-Me-p-NO2Phe-containing analogue 9 is only 4.5-fold selective (nonselective) in this same assay. On the other hand, in the bioassay systems, [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (5) is more potent than DPDPE and 8800-fold selective for the MVD (delta receptor) vs the GPI (mu receptor), making it the most highly selective ligand in this series for the delta opioid receptor on the basis of these bioassays. In these assay systems, the (2R,3S)-beta-MePhe4-containing analogue 5 had very weak potency and virtually no receptor selectivity (4.4-fold). These results demonstrate that topographical modification alone in a conformationally restricted peptide ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity and suggest that this approach may have general application to peptide ligand design.

Amino Acid Sequence↗

Oxytocin antagonists with changes in the Asn5 position shed light on hormone-oxytocin receptor interactions.

Since oxytocin agonists and antagonists have different structure-activity relationships, we have investigated the stereostructural and stereoelectronic requirements of the Asn5 residue in oxytocin antagonists by the synthesis of four analogues of the potent, prolonged acting oxytocin antagonist [Pen1,D-Phe2,Thr4,Orn8]-oxytocin (I) in which Asn5 was replaced respectively with Thr (II), Leu5 (III), Asp5 (IV) and Tyr5 (V). These analogues had pA2 values in the antioxytocic in vitro rat uterine assay of 7.23 (I), 7.16 (II), 6.67 (III), 7.21 (IV), and 6.76 (IV), respectively. All were also found to be weakly potent in the in vivo anti-vasopressor assay in the rat. These studies demonstrate very different structural and stereoelectronic requirements for oxytocin agonists and antagonists when they interact with the oxytocin uterine receptor.

Amino Acid Sequence↗

Synthetic glucagon antagonists and partial agonists.

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17,18,Glu21]glucagon,1 a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [D-Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17,18,Glu21]glucagon (2); [Tip1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (3); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (4); [dHis1,Asp3,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21+ ++]glucagon (5); des-Phe6-[Tip1,D-Phe4,Tyr5,Arg12,Glu21]glucagon (6); des-Phe6-[Asp3,D-Phe4,Tyr5,Arg12,Glu21]glucagon (7). The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10(-5) M. In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

Protein-kinase C mediates MCH signal transduction in teleost, synbranchus marmoratus, melanocytes.

MCH (melanin concentrating hormone) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which stimulates melanosome (melanin granule) aggregation to a perinuclear position within teleost fish integumental melanocytes, resulting in lightening of the skin. The mechanisms of action of MCH are unknown. Drugs that affect the diacylglycerol/inositol triphosphate pathway were used to investigate the possible roles of this pathway in the mechanisms of action of MCH on Synbranchus marmoratus (teleost) melanocytes. The shift of the dose-response curve to MCH in the presence of various concentrations of 4-bromophenacyl bromide and neomycin sulphate, phospholipase C inhibitors, suggests that phospholipase C is stimulated after MCH receptor activation. Low concentrations (10(-9) to 10(-8) M) of the phorbol ester TPA exhibited MCH-like activity, eliciting a dose-dependent melanosome aggregation. Higher doses, however, displaced to the right the dose-response curve to MCH, as did the protein kinase C inhibitors, dibucaine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). These results support the assumption that protein kinase C mediates the pigment aggregating activity of MCH. Both MCH and norepinephrine lightening actions were abolished by beta-glycerophosphate, a phosphatase inhibitor, suggesting that a protein dephosphorylation occurs during melanosome aggregation, and is, therefore, a common event triggered by MCH and norepinephrine, although both agonists act through separate receptors and exhibit different transduction mechanisms.

Amino Acid Sequence↗

Biological activities of melanotropic peptide fatty acid conjugates.

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the alpha-MSH fragment analog, H-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a "creeping" potency in the lizard skin bioassay-that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10-100 times more active than alpha-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Asp5, D-Phe7, Lys10]alpha-MSH5-10-NH2, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.

Amino Acid Sequence↗

Effects of inactivation of oxytocin receptor and inhibition of prostaglandin synthesis on uterine oxytocin receptor and gap junction formation and labor in the rat.

Normal term labor is associated with a surge in myometrial oxytocin receptor formation and gap junction development. We have previously shown that inhibition of prostaglandin synthesis by naproxen sodium, 2.0 mg/day, suppressed oxytocin receptor formation but not gap junction formation and prolonged gestation. In this study, we investigated the effects of a specific oxytocin antagonist on oxytocin receptor formation, gap junction formation, and labor in the rat. [Pen1,Phe(Me)2,Thr4,Orn8]oxytocin, a specific oxytocin antagonist, was infused subcutaneously during the last 3 days of pregnancy at 300 micrograms/day. Measurements of myometrial oxytocin receptor concentrations and gap junction formation on days 21 and 22 and days 22-23 (in labor) pregnant uteri showed no significant differences in the Bmax and Kd values between the control and the treated group. Gestation period was not prolonged by the oxytocin antagonist. However, in a separate group of day 23 pregnant rats, the uterine contractile response to 60 mU of oxytocin i.v. was found completely blocked by 10 micrograms of the oxytocin antagonist. These findings suggest that although functional oxytocin receptors did not appear to be essential for the initiation of labor, oxytocin antagonists may still be effective in the prevention of premature contractions. We also examined the effects of a higher dose of naproxen sodium, 5.0 mg/day, on gap junction formation. At this dose, naproxen sodium suppressed both oxytocin receptor and gap junction formation, prolonged gestation, and delayed parturition by 24 h or longer. Prostaglandin appears to be an important regulator or mediator of oxytocin receptor and gap junction formation and plays a critical role in the initiation of labor.

Angiotensin Receptor Antagonists↗

Distribution and analgesia of [3H][D-Pen2, D-Pen5]enkephalin and two halogenated analogs after intravenous administration.

To improve pharmacological characteristics of the delta-selective, cyclic peptide [D-Pen2, D-Pen5]enkephalin (DPDPE), modification by halogenation at the Phe4 residue was undertaken. The present study was to determine the extent [3H]DPDPE, [3H][p-Cl-Phe4]DPDPE and [p-125IPhe4]DPDPE crosses the blood-brain barrier, elicits analgesia and to characterize selective organ distribution and stability after i.v. administration. A significantly greater percentage of total [3H][p-Cl-Phe4]DPDPE reached the brain after 10, 20 and 40 min as compared to [3H]DPDPE and both peptides were significantly displaced by pretreatment with naloxone or naltrindole. The amount of [3H]DPDPE detected in the brain was greater than that of [p-125IPhe4]DPDPE. Distribution results revealed large amounts of the administered peptides were sequestered rapidly in the gall bladder and secreted into the small intestine. Hot-plate antinociception tests 5 min after i.v. administration (30 and 60 mg/kg) revealed [p-Cl-Phe4]DPDPE to elicit a much greater analgesic effect as compared to DPDPE or [p-125IPhe4]DPDPE. These results provide evidence that [p-Cl-Phe4]DPDPE has a greater apparent distribution to the brain and has a greater effect on the antinociception threshold as tested on the hot-plate than DPDPE or [p-125IPhe4]DPDPE. Stability of unlabeled and tritiated DPDPE and [p-Cl-Phe4]DPDPE was determined both in vitro and in vivo; both unlabeled and tritiated DPDPE and [p-Cl-Phe4]DPDPE remain intact.

Analgesics↗

Examination of the conformational meaning of "delta-address" in the dermenkephalin sequence.

Comprehensive energy calculations were applied to four opioid-related peptides with different receptor selectivities, namely the delta-selective dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, DRE), the mu-selective dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DRM) and their "hybrid" peptides DRM/DRE (Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH2) and DRE/DRM (Tyr-D-Met-Phe-His-Tyr-Pro-Ser-NH2). It was shown that the N-terminal tripeptide "mu-messages" in the delta-selective ligands DRE and DRM/DRE can possess similar low energy space arrangements of their functionally important elements (the N-terminal alpha-amino group and the aromatic moieties of Tyr and Phe), but that these are different from the space arrangement of these moieties in mu-selective DRM and DRE/DRM. These results suggest that the C-terminal tripeptide "delta-address" in DRE may influence the conformation of the "mu-message" in DRM. A refined model for the delta-receptor-bound conformation of DRE is proposed based on these calculations which is similar to that previously suggested for the cyclic delta-selective peptide [D-Pen2, D-Pen5]enkephalin (DPDPE). This model also has partial correspondence with the structure of the delta-selective alkaloid naltrindole.

Amino Acid Sequence↗