Biomedical subjects

Victor J Hruby

Publications and source records attributed to Victor J Hruby.

At least 19 recordsLinked to original sources

PubMed2007

Partial retro-inverso, retro, and inverso modifications of hydrazide linked bifunctional peptides for opioid and cholecystokinin (CCK) receptors.

Partially modified retro-inverso, retro, and inverso isomers of hydrazide linked bifunctional peptides were designed, synthesized, and evaluated for bioactivities at delta/mu opioid receptors and CCK-1/CCK-2 receptors. All modifications of the CCK pharmacophore moiety affected bioactivities for the CCK-1 and CCK-2 receptors (up to 180-fold increase in the binding affinity with higher selectivity) and for the delta and mu opioid receptors. The results indicate that the opioid and CCK pharmacophores in one molecule interact with each other to induce topographical changes for both pharmacophores.

Animals↗

PubMed2006

Design, synthesis, and biological evaluation of new cyclic melanotropin peptide analogues selective for the human melanocortin-4 receptor.

Intensive efforts have been made to develop potent and selective ligands for certain human melanocortin receptors as possible treatments for obesity and sexual dysfunction due to the role of these receptors in feeding behavior, energy homeostasis, sexual function, etc. A number of novel alpha-MSH analogues were designed and synthesized primarily on the basis of our previous MTII NMR structure. In these peptide analogues, a disulfide or lactam bridge between residues at positions 5 and 8 was used as a conformational constraint to enhance the beta-turn spanning His6 and D-Phe7, while the pharmacophore group in Arg8 was mimicked via Nalpha-alkylation of residues 8 or 9 with the guanidinylbutyl group. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues have good binding affinity for the hMC4R (0.7-4.1 nM), but have no binding affinity up to 10 microM at the other three melanocortin receptors. Interestingly, the three hMC4R selective analogues display only 50% binding efficiency, suggesting there is allosteric modulation of the melanocortin-4 receptor. These analogues were found to act as antagonists of the hMC4R. This result represents a discovery of very selective peptide-based antagonists for the hMC4R. The high selectivity may be due to the strong conformational constraint via ring contraction as compared to MTII, and the rigid conformation preferred by these new ligands allows them to recognize only the hMC4R, but not to activate the second messenger. The MTII NMR structure-based design thus not only examined the structural model of melanocortin ligands, but also yielded new biologically unique alpha-MSH analogues.

Adenylyl Cyclases↗

PubMed2006

Antihyperalgesic effects of loperamide in a model of rat neuropathic pain are mediated by peripheral delta-opioid receptors.

The possible antihyperalgesic and antiallodynic activity of loperamide, an opioid agonist which does not readily penetrate the blood-brain barrier, were examined in the spinal nerve ligation model of experimental neuropathic pain. Intraperitoneal (i.p.) injection of loperamide effectively reversed thermal hyperalgesia. In contrast, loperamide had minimal effects on cold allodynia and no effects on mechanical allodynia. The antihyperalgesic action of loperamide against noxious heat was antagonized by naltrindole, a delta-opioid receptor selective antagonist, but not by pretreatment with beta-funaltrexamine, a mu-opioid receptor selective antagonist, or administration of nor-binaltorphimine, a kappa-opioid receptor selective antagonist. Furthermore, i.p. injection of [d-Ala(2), Glu(4)]-deltorphin II, a delta-opioid receptor selective peptide agonist, also reversed thermal hyperalgesia. The present results suggest that thermal hyperalgesia in experimental neuropathic pain can be reduced through activation of peripheral delta-opioid receptors. The data suggest the possible application of peripherally restricted and delta-opioid receptor selective agonists in the treatment of some aspects of neuropathic pain without many of the side effects associated with centrally acting opioids and without the peripheral side effects of opioid agonists acting at mu-receptors.

Analgesics↗

PubMed2006

Synthesis of Anti-beta-substituted gamma,delta-unsaturated amino acids via Eschenmoser-Claisen rearrangement.

Anti-beta-substituted gamma,delta-unsaturated amino acids have been synthesized via a novel design of the Eschenmoser-Claisen rearrangement. The rearrangement gives good isolated yields and excellent diastereoselectivity due to (Z)-N,O-ketene acetal formation and the pseudochairlike conformations of the reaction intermediates. Upon reductive hydrolysis, important novel amino acids and amino alcohols were synthesized for the first time via this methodology.

Amino Acids↗

PubMed2006

Design, synthesis, and biological evaluation of a new class of small molecule peptide mimetics targeting the melanocortin receptors.

A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, starting from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biological activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics. All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported.

Binding Sites↗

PubMed2006

New paradigms and tools in drug design for pain and addiction.

New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at mu and delta opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the delta opioid receptor.

Behavior, Addictive↗

PubMed2006

Synthesis and evaluation of 3-aminopropionyl substituted fentanyl analogues for opioid activity.

An enkephalin analogue coupled to 'aminofentanyl' has been synthesized and tested for biological activities at the mu and delta opioid receptors. Aminofentanyl which represents a structural derivative of fentanyl has been synthesized by acylation of 1-(2-phenethyl)-4-(N-anilino)piperidine with phthaloyl protected beta-alaninyl chloride in the presence of DIPEA, followed by deprotection with hydrazine hydrate. Aminofentanyl has also been successfully acylated with ethyl isocyanate, various acid anhydrides, to further investigate structure-activity relationships of these new fentanyl derivatives. Among the new derivatives compound 7 which carries a Tyr-D-Ala-Gly-Phe opioid message sequence showed good opioid affinity (1 nM at both delta and mu opioid receptors) and bioactivity (34.9 nM in MVD and 42 nM in GPI/LMMP bioassays).

Animals↗

PubMed2006

Structure-activity relationships of bifunctional peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors.

Cholecystokinin (CCK) has been identified as a pronociceptive endogenous peptide which also possesses antiopioid actions. CCK may be upregulated in conditions of chronic pain or during sustained morphine administration resulting in attenuation of opioid-mediated pain relief. These complex interactions between opioids and endogenous CCK receptor systems have suggested the need for a new paradigm in drug design for some states of chronic pain. In these circumstances the rational design of potential drugs for the treatment of these conditions must be based on one ligand for multiple targets. We have designed a single peptide which can interact with delta and mu opioid receptors as agonists and with CCK receptors as antagonists. The ligands were designed based on a model of overlapping pharmacophores of opioid and CCK peptide ligands, which incorporates opioid pharmacophores at the N-terminal and CCK tetrapeptide pharmacophores at the C-terminal of the designed ligands. We measured binding and activities of our bifunctional peptides at opioid and CCK receptors. Compound 11 (Tyr-d-Ala-Gly-d-Trp-NMeNle-Asp-Phe-NH(2)) demonstrated opioid agonist properties at delta and mu receptors (IC(50) = 63 +/- 27 nM and 150 +/- 65 nM, respectively in MVD and GPI tissue assays) and high binding affinity at CCK-1 and CCK-2 receptors (K(i) = 320 and 1.5 nM, respectively). Compound 9 (Tyr-d-Nle-Gly-Trp-Nle-Asp-Phe-NH(2)) displayed potent agonist activity at delta and mu receptors (IC(50) = 23 +/-10 nM and 210 +/- 52 nM, respectively in MVD and GPI tissue assays), with a balanced binding affinity for CCK-1 and CCK-2 receptors (K(i) = 9.6 and 15 nM, respectively). These results provide evidence supporting the concept that opioid and CCK receptors have overlapping pharmacophores required for binding affinity and biological activity and that designing overlapping pharmacophores of two peptides into a single peptide is a valid drug design approach.

Analgesics↗

PubMed2006

The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy.

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O(2))(11)]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(11)]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).

Animals↗

PubMed2006

Synthesis of constrained analogues of cholecystokinin/opioid chimeric peptides.

In our ongoing research on the synthesis of constrained analogues of CCK/opioid chimeric peptides, a bicyclic dipeptide mimetic for Nle-Asp was designed and synthesized. Starting from β-allyl substituted aspartic acids, the terminal double bond was oxidized resulting in spontaneous cyclization to form racemic hemiaminals. Allylation of the hemiaminals afforded 5-allyl substituted proline analogues, which on oxidation, Horner-Emmons olefination, asymmetric hydrogenation, and bicyclization afforded bicyclic dipeptide mimetics for Nle-Asp. Constrained CCK/opioid peptide analogues containing bicyclic dipeptide mimetics for Nle-Gly, Nle-Asp, and homoPhe-Gly were then synthesized and analyzed at both the CCK and opioid receptors.

Journal Article↗

PubMed2006

Development of cyclic gamma-MSH analogues with selective hMC3R agonist and hMC3R/hMC5R antagonist activities.

A series of cyclic lactam analogues of gamma-MSH (H-Tyr1-Val2-Met3-Gly4-His5-Phe6-Arg7-Trp8-Asp9-Arg10-Phe11-Gly12-OH) with a bulky hydrophobic residue in the direct proximity to the pharmacophore (Xaa-D-Phe/D-Nal(2')-Arg-Trp) were designed and synthesized by solid-phase methods. A variety of amino acids with a broad range of hydrophobic/hydrophilic properties was introduced in position 5 to further explore their complementary role in receptor selectivity. Biological evaluation of these peptides revealed several analogues with potent hMC3R agonist and hMC3R/hMC5R antagonist activities, and good receptor selectivity. Analogue 4, c[Nle-Arg-D-Phe-Arg-Trp-Glu]-NH2, was found to be a very potent and selective hMC3R agonist (EC50=1.2 nM, 112% act). In addition, analogue 13, c[Nle-Val-D-Nal(2')-Arg-Trp-Glu]-NH2, was identified as an hMC3R/hMC5R antagonist with the best selectivity against the hMC4R in this series (pA2(hMC3R)=8.4; pA2(hMC5R)=8.7). These results indicate the significance of steric factors in melanocortin receptor selectivity and suggest that introduction of bulky residues in the direct proximity to the melanocortin pharmacophore is an effective approach to design of novel hMC3R and hMC5R selective ligands.

Adenylyl Cyclases↗

PubMed2006

Design and synthesis of novel hydrazide-linked bifunctional peptides as delta/mu opioid receptor agonists and CCK-1/CCK-2 receptor antagonists.

A series of hydrazide-linked bifunctional peptides designed to act as agonists for delta/mu opioid receptors and antagonists for CCK-1/CCK-2 receptors was prepared and tested for binding to both opioid and CCK receptors and in functional assays. SAR studies in the CCK region examined the structural requirements for the side chain groups at positions 1', 2', and 4' and for the N-terminal protecting group, which are related to interactions not only with CCK, but also with opioid receptors. Most peptide ligands that showed high binding affinities (0.1-10 nM) for both delta and mu opioid receptors generally showed lower binding affinities (micromolar range) at CCK-1 and CCK-2 receptors, but were potent CCK receptor antagonists in the GPI/LMMP assay (up to Ke = 6.5 nM). The results indicate that it is reasonable to design chimeric bifunctional peptide ligands for different G-protein coupled receptors in a single molecule.

Animals↗

PubMed2006

Effects of macrocycle size and rigidity on melanocortin receptor-1 and -5 selectivity in cyclic lactam alpha-melanocyte-stimulating hormone analogs.

The effects of the linker arm rigidity and size on melanocortin receptor selectivity were explored in a series of compounds using cyclic lactam alpha-melanocyte-stimulating hormone template. A variety of dicarboxylic acid linkers introduced between the alpha-amino group of His(6) and the epsilon-amino group of Lys(10) lead to high-affinity, selective human melanocortin receptor-1 and -5 (hMC1R and hMC5R) antagonists. The incorporation of hydrophilic functions into the linker arm was found to be unfavorable for both binding potency and receptor selectivity. Analogs 8 and 9 containing highly conformationally constrained hydrophobic linkers (m- and p-phthalic acids) were found to be selective nanomolar range hMC1R antagonists (IC(50) = 7 and 4 nm, respectively), whereas the employment of a small conformationally constrained linker (maleic acid) resulted in a high-affinity (IC(50) = 19 nm) and selective hMC5R antagonist (analog 12). These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin-1 and -5 receptors.

Adenylyl Cyclases↗

PubMed2006

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit beta-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin-mediated clathrin-coated pits, whereas, beta-arrestin-2 conjugated green fluorescence protein (beta-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

Arrestins↗

PubMed2006

The melanocortin circuit in obese and lean strains of chicks.

Agonists of membranal melanocortin 3 and 4 receptors (MC3/4Rs) are known to take part in the complex control mechanism of energy balance. In this study, we compared the physiological response to an exogenous MC3/4R agonist and the hypothalamic expression of proopic melanocortin (POMC) gene, encoding few MC3/4R ligands, between broiler and layer chicken strains. These strains, representing the two most prominent commercial strains of chickens grown for meat (broilers) and egg production (layers), differ in their food intake, fat accumulation, and reproductive performance and, therefore, form a good model of obese and lean phenotypes, respectively. A single i.v. injection of the synthetic peptide melanotan-II (MT-II; 1 mg/kg body weight) into the wing vein of feed-restricted birds led to attenuation of food intake upon exposure to feeding ad libitum in both broiler and layer chickens. A study of the POMC mRNA encoding the two prominent natural MC3/4R agonists, alpha-MSH and ACTH, also revealed a general similarity between the strains. Under feeding conditions ad libitum, POMC mRNA levels were highly similar in chicks of both strains and this level was significantly reduced upon feed restriction. However, POMC mRNA down-regulation upon feed restriction was more pronounced in layers than in broilers. These results suggest: (i) a role for MC3/4R agonists in the control of appetite; (ii) that the physiological differences between broilers and layers are not related to unresponsiveness of broiler chickens to the satiety signal of MC3/4R ligands. Therefore, these findings suggest that artificial activation of this circuit in broiler chicks could help to accommodate with their agricultural shortcomings of overeating, fattening, and impaired reproduction.

Animals↗

PubMed2005

Structure-activity studies of new melanocortin peptides containing an aromatic amino acid at the N-terminal position.

Cyclic melanotropin peptides, designed with an aromatic amino acid substitution at the N-terminal position of the MT-II-type scaffold, were prepared by solid-phase peptide synthesis and evaluated for their ability to bind to and activate human melanocortin-1, -3, -4, and -5 receptors. The structure-activity studies of these MT-II analogues have identified a selective antagonist at the hMC4R (H-Phe-c[Asp-Pro-d-Nal(2')-Arg-Trp-Gly-Lys]-NH(2), pA(2)=8.7), a selective partial agonist at the hMC4R (H-d-Nal(2')-c[Asp-Pro-d-Phe-Arg-Trp-Gly-Lys]-NH(2), IC(50)=11nM, EC(50)=56nM), and a selective partial agonist at the hMC3R (H-d-Phe-c[Asp-Pro-d-Phe-Arg-Trp-Lys]-NH(2), IC(50)=3.7nM, EC(50)=4.9nM). Aromatic amino acid substitution at the N-terminus in conjuction with the expansion of the 23-membered cyclic lactam MT-II scaffold to a 26-membered scaffold by addition of a Gly residue in position 10 leads to melanotropin peptides with enhanced receptor selectivity.

Amino Acid Sequence↗

PubMed2005

Synthesis and biological activity of the first cyclic biphalin analogues.

Biphalin is a linear octapeptide with strong opioid activity. Its structure is based on two identical sequences derived from enkephalins joined C-terminal to C-terminal by an hydrazide bridge (Tyr-D-Ala-Gly-Phe-NH-NH<--Phe<--Gly<--D-Ala<--Tyr). In this study we present the design, synthesis, and biological evaluation of the first cyclic biphalin analogues. d-Alanine residues in positions 2, 2' of the parent peptide were replaced by d- and l-cysteine and an intramolecular disulfide bond between the cysteine thiol groups was introduced. We obtained two cyclic analogues with quite different biological profiles.

Binding Sites↗

PubMed2005

Michael addition reactions between chiral equivalents of a nucleophilic glycine and (S)- or (R)-3-[(E)-enoyl]-4-phenyl-1,3-oxazolidin-2-ones as a general method for efficient preparation of beta-substituted pyroglutamic acids. Case of topographically controlled stereoselectivity.

This paper describes a systematic study of addition reactions between the chiral Ni(II) complex of the Schiff base of glycine with (S)-o-[N-(N-benzylprolyl)amino]benzophenone and (S)- or (R)-3-[(E)-enoyl]-4-phenyl-1,3-oxazolidin-2-ones as a general and synthetically efficient approach to beta-substituted pyroglutamic acids and relevant compounds. These reactions were shown to occur at room temperature in the presence of nonchelating organic bases and, most notably, with very high (>98% diastereomeric excess (de)) stereoselectivity at both newly formed stereogenic centers. The stereochemical outcome of the reactions was found to be overwhelmingly controlled by the stereochemical preferences of the Michael acceptors, and the chirality of the glycine complex influenced only the reaction rate. Thus, in the reactions of both the (S)-configured Ni(II) complex and the Michael acceptors, the reaction rates were exceptionally high, allowing preparation of the corresponding products with virtually quantitative (>98%) chemical and stereochemical yields. In contrast, reactions of the (S)-configured Ni(II) complex and (R)-configured Michael acceptors proceeded at noticeably lower rates, but the addition products were obtained in high diastereo- and enantiomeric purity. To rationalize the remarkably high and robust stereoselectivity observed in these reactions, we consider an enzyme-substrate-like mode of interaction involving a topographical match or mismatch of two geometric figures. Excellent chemical and stereochemical yields, combined with the simplicity and operational convenience of the experimental procedures, render the present method of immediate use for preparing various beta-substituted pyroglutamic acids and related compounds.

Catalysis↗