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Structure-activity studies of delta-selective opioid analogues
No full textThe two structurally different peptides DPDPE and Dermenkephalin show a similar remarkably high affinity and selectivity for the delta opioid receptor subtype. An effort has been made to gain some insight into the factors responsible for the recognition ability of these two molecules by synthesizing some DPDPE-Dermenkephalin peptide hybrids and some conformationally restricted Dermenkephalin analogues. The results of the binding and the in-vitro bioassays have been compared with those of the parent peptides. A general decrease in receptor affinity has been observed in the peptide hybrids while the dermenkephalin analogues have shown a wider range of affinities and selectivities. The above findings contribute to the understanding of the structural requirements of the delta receptor, provide information about the sensitivity of Dermenkephalin to enzymatic degradation, and indicate directions for future research
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Design and synthesis of conformationally and topographically constrained amino acids as peptidomimetics
No full textA major goal of peptide research has been to elucidate or understand the relationships between a peptide's three-dimensional structure and its biological activity. De Novo design of peptide mimetics requires assembling all components necessary for molecular recognition and transduction, which needs the proper choice of a template that can place the key side chain residues in 3D space. Two widely used methods are novel β-substituted amino acids and conformationally constrained secondary structure mimetics. In this thesis, we report our efforts to fulfill the aforementioned criteria in synthesizing β-isopropyl aromatic amino acids and constrained reverse turn dipeptide mimetics. Through asymmetric Michael addition reaction, highly topographically constrained β-isopropyl aromatic amino acids have been synthesized. In order to develop a general approach to synthesize these novel amino acids, we re-examined the reaction conditions for Evans' diastereoselective 1,4-addition, and found conditions which gave excellent diastereoselectivities and good chemical yields. A concise and straightforward five-step synthesis of [5.5]-bicyclic reverse turn dipeptide mimetic scaffolds with side chain functionality at the i+1 and i+2 positions has been developed. In the bicyclic structure, two dihedral angles (ψ₂ and φ₃) are greatly restricted. Further development of this synthesis will enable us to prepare various types of reverse turns with different backbone geometry and side chain topography. Enantiomerically pure (S)-trans-cinnamylglycine and (S)-α-trans-cinnamyl-α-alanine have been prepared via reaction of chiral Ni (II)-complexes of glycine and alanine respectively, with cinnamyl halides. Inexpensive and readily available reagents and solvents are used, including a recyclable chiral ligand. The simplicity of the experimental procedures and high stereochemical outcome make this method synthetically attractive for preparing the target amino acids on multi-gram scales. Further studies by incorporating these mimetics into potent peptide analogues will greatly help us to understand the bioactive conformation of the parent peptides
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The design and synthesis of novel beta-substituted amino acids, bicyclic dipeptide mimetics, and their incorporation into cholecystokinin/opioidchimeric peptides
No full textPeptide ligands and protein receptors play critical roles in the regulation of nearly every biological system. However, peptides are characteristically highly flexible and thus identifying the basic conformational elements necessary for recognition between a peptide ligand and it's receptor at the molecular level remains a formidable task. Great emphasis in peptide research has thus focused on the determination of the receptor-bound conformation adopted by bioactive peptides by synthesizing constrained analogues of the peptides. Knowledge of the three dimensional interaction between a peptide ligand and a receptor could be invaluable in understanding bioactivity and in the design of therapeutics. To determine the bioactive conformation of our novel chimeric peptides for the opioid and cholecystokinin receptors, constrained analogues were designed to limit the conformations that the peptides would adopt. In this regard, [5,5]- and [6,5]-bicyclic dipeptide mimetics were designed and synthesized to constrain a dipeptide unit and by extension limit the flexibility of the peptide. The bicyclic dipeptide mimetics were synthesized from precursors obtained by the beta-alkylation of aspartic acid and from the Kazmaier-Claisen rearrangement reaction. A protocol for the alkylkation of aspartic acid with allyl bromide, benzyl bromide, and benzyl disulfide was developed. The bicyclic dipeptide mimetics were then introduced into the peptides whose biological activity was evaluated at both the opioid and cholecystokinin receptors. The peptides showed good binding and functional activities at the CCK receptors, but low activities at the opioid receptors
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Synthesis of Novel Biologically Active Peptide Analogues that are Agonists at Opioid Receptors and Antagonists at Cholecystokinin Receptors
No full textWe know that many disease states lead to changes in expressed proteins. Therefore, drug design and discovery based on normal states and single targets often is inadequate. The "system changes" that occurs must be considered in any treatment for the disease, clearly evident in neuropathic pain where opioids can actually heighten pain. To effectively treat diseases involving "systems changes" a new paradigm was recently introduced. In this new approach single peptide molecules are designed to interact with multiple receptor targets. For the treatment of pain, a series of linear and cyclic peptides were designed based on the overlapping pharmacophores of opioid and CCK ligands. The opioid/CCK analogues were synthesized and evaluated for their biological activities. Several analogues were found to simultaneously interact with opioid receptors as agonists and CCK receptors as antagonists. This study further modifies the RSA analogues to improve on the bioassays of the previous peptides
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DESIGN AND SYNTHESIS OF STRUCTURAL, STEREOISOMERIC AND CONFORMATIONALLY RESTRICTED ANALOGUES OF ALPHA-MELANOTROPIN: COMPARATIVE BIOLOGICAL PROPERTIES ON MELANOPHORES AND MELANOMA CELLS
No full textSeveral chemically-modified analogues of α-melanotropin (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) were prepared by solid-phase peptide synthesis, including [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH₄₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH₁₋₆-NH₂, Ac-α-MSH₇₋₁₀-NH₂, Ac-α-MSH₁₁₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH(,4-10)-NH₂, Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂, [Nle⁴, D-Phe⁷]-α-MSH, Ac-α-MSH₄₋₁₀-NH₂, Ac-[Tyr⁴]-α-MSH₄₋₁₀-NH₂ and [half-Cys⁴, half-Cys¹⁰]-α-MSH. The synthetic strategy involved: (1) p-methylbenzhydrylamine resin as a solid support, (2) N,N'-dicyclohexylcarbodiimide as a coupling reagent, (3) acetylation of the N-terminus and HF cleavage and deprotection (except for Nⁱ-For-Trp) of the fully assembled peptide-resin and (4) alkaline hydrolysis to deformylate Nⁱ-For-Trp. In the preparation of [half-Cys⁴, half-Cys¹⁰]-α-MSH, oxidative-cyclization provided formation of an intramolecular disulfide bridge. A comparative biological analysis in vitro of these above structural, stereoisomeric and conformationally-restricted analogues of α-MSH on several different vertebrate pigment cell systems provided the following results: (1) The [Nle⁴, D-Phe⁷]-α-MSH effected high melanotropic potency (> 60 times relative to α-MSH), ultralong biological activity and unprecedented metabolic stability. (2) Utilizing [Nle⁴, D-Phe⁷]-α-MSH as a molecular probe, two melanotropic receptor types were demonstrated which were mechanistically different in terms of calcium dependency and apparent hormone-receptor complex reversibility. (3) The Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂ was a highly potent active site (Met-Glu-His-Phe-Arg-Trp-Gly) analogue of α-MSH (ranging from 0.2- to 10-times relative to α-MSH) without the ultralong melanotropic activity possessed by the parent stereostructural tridecapeptide. (4) The [half-Cys4, half-Cys10]-α-MSH exhibited superpotency on frog (Rana pipiens) melanophores (≥ 10,000 times relative to α-MSH), and provided experimental evidence that a pseudocyclic conformation of the native hormone containing a β-turn structural requirement at His-Phe-Arg-Trp might be related to its biological activity at the pigment cell receptor. The [Nle⁴, D-Phe⁷]-α-MSH may be suitable for use as a radio-labeled tracer or drug-delivery agent for the localization or treatment of human melanoma in vivo.This item was digitized from a paper original and/or a microfilm copy. If you need
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Design and synthesis of topographically constrained amino acids, and bioactive peptides for studies of ligand-receptor interaction, and for de novo design of delta-opioid selective non-peptide mimetics as potential therapeutics
No full textTopographical constraint is the most powerful approach for the design of bioactive peptides to explore the bioactive conformation of crucial side-chain pharmacophores of amino acid residues in peptide-receptor recognition and signal transduction. Novel topographically constrained amino acids β-isopropylphenylalanine and 2',6'-dimethyl-2,3-methanophenylalanine have been designed and synthesized. Incorporation of the four optically pure β-isopropylphenylalanine stereoisomers into deltorphin I produced four peptide analogues of [β-iPrPhe]Deltorphin I with differentiated bioactivities. The most potent and selective analogue, [(2S,3R)-β-iPrPhe]Deltorphin I showed an IC₅₀ nM binding affinity, and a 29000 fold selectivity for the δ-opioid receptor over the μ opioid receptor. Combined molecular modeling and NMR studies indicated that the (2S,3R)-β-iPrPhe³ residue in the analogue favors the trans rotamer, and can induce the linear peptide to form a low-energy folded conformation which was proposed as the bioactive conformation for the δ-opioid receptor. Coupling four optically pure, conformationally constrained β-methyl-2',6'-dimethyltyrosine (TMT) with L-Tic formed four dipeptide analogues of TMT-L-Tic. The most potent and selective analogue, (2S,3R)-TMT-L-Tic showed 9 nM binding affinity and 4000 fold selectivity to the δ vs μ opioid receptor. The lowest-energy conformation of (2S,3R)-TMT-L-Tic was suggested to be the bioactive one in which TMT side chain is trans and Tic side chain is in a gauche (+) conformation. Bicyclic oxytocin antagonist [dPen¹, cyclo(Glu⁴ Lys⁸)]OT (BC-OT) (pA₂ = 8.10) is an excellent template to examine further topographical ideas. Substitution of Tyr² with the topographically constrained para-methoxy-β-methyl-2',6'-dimethyltyrosine (p-MeOTMT) amino acids produced two very potent antagonists [(2S,3S)-p-MeOTMT²]BC-OT (pA₂ = 8.26) and [(2R,3R)-p-MeOTMT²]BC-OT(pA₂ = 7.80), and two inactive analogues [(2S,3R)-p-MeOTMT²]BC-OT and [(2R,3S)-p-MeOTMT²]BC-OT. These interesting results can be attributed to the biased side-chain conformation, gauche(+) and gauche(-) in (2S,3S)-p-MeOTMT and (2R,3R)-p-MeOTMT respectively, and trans in both (2S,3R)-p-MeOTMT and (2R,3S)-p-MeOTMT residues. Rational design of non-peptide mimetics from peptide leads is still elusive. Based on the δ-opioid selective lead [(2S,3R)-TMT¹]DPDPE and SAR of δ-opioid selective ligands, the first generation of non-peptide mimetics have been designed and synthesized. The new lead SL-3111 showed binding affinity IC₅₀ = 8 nM, and over 2000 fold selectivity for the δ-opioid receptor over the μ receptor.This item was digitized from a paper original and/or a microfilm copy. If you need higher-resolution images for any content in this item, please contact us at [email protected] file replaced with corrected file October 2023
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Development of new conformationally and topographically constrained p60(c-src) PTK inhibitors. Solution and solid-phase approaches for the synthesis of delta-opioid receptor peptidomimetic ligands
No full textBased on the efficient substrate for p60ᶜ⁻ˢʳᶜ protein tyrosine kinase (PTK) YIYGSFK (1) (K(m) = 55 μM) obtained by combinatorial methods, we have designed and synthesized a series of conformationally and topographically constrained substrate-based peptide inhibitors for this enzyme. The inhibitors showed IC₅₀ values in low micromolar range (0.1-3 μM). A "rotamer scan" was performed by introducing four stereoisomers of β-Me(2')Nal in the postulated interaction site of peptide inhibitor (23) Y-c[D-Pen-(2')Nal-GSFC]KR-NH₂ (IC₅₀ = 1.6 μM). We found that the χ¹ space constraints imposed by the specialized amino acids, introduced at position 3 of peptide 23, were not as important as the configuration of the Cᵅ of that residue to recognize the active site of Src and Lck PTK, as reflected on the observed selectivity ratios. Cocrystallization studies between Lck and two of our inhibitors are in progress, in a collaboration with Dr. X. Zhu (Kinetix, Pharmaceuticals, Inc.). The results obtained may serve as the basis for the design of Lck and/or Src inhibitors, either peptide or nonpeptide. SL-3111 is a high affinity (IC₅₀ = 8.4 nM) and selective (μ/δ = 2020) δ-opioid receptor peptidomimetic ligand developed in Dr. Hruby's laboratory, as the result of extensive structure-activity relationship (SAR) studies based on peptide leads. However, bioassays (GPI and MVD) and in-vivo antinociception studies on the racemic mixture and both enantiomers of this compound, have shown particular problems such as low potency and toxicity. We have shown the importance of the piperazine ring in this molecule for binding toward the δ-opioid receptor. Thus, maintaining such scaffold we have studied a series of solution and solid-phase approaches toward the synthesis of SL-3111 analogues, which explore wider functional diversity at this heterocyclic ring. Compounds 64-67 were synthesized by solution methods. Analysis of the biological data and molecular modeling studies of these compounds, revealed an interesting trend in terms of the effects of the substituent at position two of the piperazine scaffold. Three different solid-phase protocols were explored toward the development of a combinatorial library of this type of compounds, which may facilitate future SAR studies.This item was digitized from a paper original and/or a microfilm copy. If you need higher-resolution images for any content in this item, please contact us at [email protected] file replaced with corrected file September 2023
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Asymmetric synthesis of stereochemically-defined and conformationally-constrained novel amino acids via direct alkylation of chiral nickel(II)-coordinated Schiff bases of glycine and alanine, and design and synthesis of selective peptide and non-peptide ligands for the delta-opioid receptor
No full textA systematic practical method to prepare highly chi (χ)-constrained amino acids has been developed. It was found that increasing the size of R¹ (see figure) from H to Me to Et to n-Pr led to decreased reactivity of the starting complexes. In the case of R¹ as i-Pr, no alkylation was observed. With an increase of the size of R² from H to Me to Et to i-Bu, the reactivities of the alkyl bromides decreased. The starting Schiff bases had more effective stereocontrol at the α-carbon center than at the β-carbon center. The starting Schiff bases showed differential reactivity toward the racemic electrophile (kinetic resolution). Satisfactory differentiations were obtained at room temperature which makes this method synthetically useful. In the case of R¹ as H (NiGlyBPB), the thermodynamically-controlled stereoselectivity of alkylation was as high as 30:1. [DIAGRAM OMITTED]* A series of dipeptide analogues (TMT-Tic and DMT-Tic) were designed and synthesized to mimic the potent and highly selective delta-opioid receptor pentapeptide ligand-[(2S,3R)TMT¹]-DPDPE and thus to explore the topographical requirements for recognition of ligands at the opioid receptor through bioassays and NMR studies to facilitate the design of non-peptide compounds to be used as therapeutic agents for pain. (2S,3R)-TMT-L-Tic was found to have best binding affinities at the δ-opioid receptor in TMT-Tic series. In preliminary NMR studies, it was found that these designed peptide ligands have their own distinct conformations in the aqueous media. Meanwhile some modified non-peptide analogues of SL-3111 were prepared to continue our efforts to find effective non-peptide ligands for the δ-opioid receptor. More systematic studies are still ongoing using NMR and computational methods. *Please refer to dissertation for diagram
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Asymmetric synthesis of chi-constrained pyroglutamic acids, glutamic acids and prolines for peptides and peptidomimetics
No full textThe recent upsurge of interest in the peptide-based drug molecules has been accompanied by a great deal of attention to the design of stereochemically defined non-proteinogenic amino acids. As a continuous effort to develop efficient syntheses of χ-constrained amino acids in our group, we recently have developed a practical methodology for the asymmetric synthesis of substituted pyroglutamic acid, glutamic acid and proline analogues, which are of important use in examining the relationships between conformation and bioactivities of biologically important peptides (e.g. DPDPE, α-MSH). The key step in this method is an asymmetric Michael addition reaction between a chiral Ni(II)-complex of the glycine Schiff base (S)-NiGlyBPB, and derivatives of α,β-unsaturated carboxylic acids. This new method is the first highly diastereoselective, room temperature, organic base-catalyzed, asymmetric Michael addition reaction. Excellent chemical yields and diastereoselectivity, along with the simplicity of experimental procedure, renders the present method of immediate use for preparation of various novel beta-substituted pyroglutamic acids, glutamic acids and prolines. Decomposing the resulting addition products in acidic medium, followed by neutralizing with ammonia, gave optically pure substituted pyroglutamic; acids in good yields (>80%). The substituted pyroglutamic acids were converted to the corresponding substituted glutamic acids by hydrolysis in 6N HCl, or to substituted proline analogues by selective reduction of amide carbonyl group to a methylene group. Both novel substituted glutamic acids and prolines are being incorporated into biologically important peptide MT-II analogues for structure-activity studies
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Studies of biologically active peptides by NMR and molecular dynamics simulations: From structure and dynamics to design and synthesis
No full textNuclear magnetic resonance spectroscopy and molecular dynamics simulation have been used to study the structure and dynamics of biologically active peptide ligands for glucagon and melanocortin receptors, providing valuable insights into the receptor ligand interactions and useful information for the further design of more potent and selective ligands for these receptors. The NMR structure of the potent glucagon antagonist [desHis¹, desPhe⁶, Glu⁹]glucagon amide consists of an unstructured N-terminal segment (2-5), an irregular helix (7-14), a hinge region (15-18), and a well-defined α-helix (19-29). The two helices form an L-shaped structure with an angle of about 90° 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. This result is the first clear indication of an overall tertiary fold for a glucagon analogue in the micelle-bound state. In addition to the structural difference, molecular dynamics simulations showed both N- and C-terminal residues in the glucagon antagonist are more highly ordered than those in glucagon. The single helix obtained for glucagon in the crystal state was found to unravel in the simulation around the region approximately corresponding to the hinge region in the antagonist. These results may have important implications for the biological activities of both peptides. The conformational study of cyclic alpha-melanocyte stimulating hormone analogues by NMR showed that their overall backbone structures are similar around the message sequence (His⁶-D-Phe⁷/D-Nal(2')⁷-Arg⁸-Trp⁹). beta-Turns spanning His⁶ and D-Phe⁷/D-Nal(2')⁷ were identified in all analogues. However, a stacking between the aromatic rings of His⁶ and D-Phe⁷/D-Nal⁷ was observed for the melanocortin agonists, but not for the antagonists. Based on the NMR structure of MTII, a library of new alpha-MSH analogues was designed and synthesized with a disulfide or lactam bridge used as a conformational constraint and the pharmacophore group in Arg⁸ mimicked by Nᵅ-alkylation via the Mitsunobu reaction. These new analogues exhibited high binding affinity and selectivity for the human melanocortin-4 receptor, thus suggesting the usefulness of the NMR structural model of α-MSH peptides
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