Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His6-d-Nal(2')7-NMe-Arg8-Trp9-Lys]-NH2 (15) and Ac-Nle-c[Asp-His6-d-Nal(2')7-NMe-Arg8-NMe-Trp9-NMe-Lys]-NH2 (17). It is known that the pharmacophore (His6-DNal7-Arg8-Trp9) of the SHU-9119 peptides occupies a ß II-turn-like region with the turn centered about DNal7-Arg8. The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg8 and Trp9 side chains are involved in a majority of the interactions with the receptor. While Arg8 forms polar contacts with D154 and D158 of hMC3R, Trp9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp9-hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

Intestinal permeability of cyclic peptides: common key backbone motifs identified.

Insufficient oral bioavailability is considered as a key limitation for the widespread development of peptides as therapeutics. While the oral bioavailability of small organic compounds is often estimated from simple rules, similar rules do not apply to peptides, and even the high oral bioavailability that is described for a small number of peptides is not well understood. Here we present two highly Caco-2 permeable template structures based on a library of 54 cyclo(-D-Ala-Ala(5)-) peptides with different N-methylation patterns. The first (all-trans) template structure possesses two ß-turns of type II along Ala(6)-D-Ala(1) and Ala(3)-Ala(4) and is only found for one peptide with two N-methyl groups at D-Ala(1) and Ala(6) [(NMe(1,6)]. The second (single-cis) template possesses a characteristic cis peptide bond preceding Ala(5), which results in type VI ß-turn geometry along Ala(4)-Ala(5). Although the second template structure is found in seven peptides carrying N-methyl groups on Ala(5), high Caco-2 permeability is only found for a subgroup of two of them [NMe(1,5) and NMe(1,2,4,5)], suggesting that N-methylation of D-Ala(1) is a prerequisite for high permeability of the second template structure. The structural similarity of the second template structure with the orally bioavailable somatostatin analog cyclo(-Pro-Phe-NMe-D-Trp-NMe-Lys-Thr-NMe-Phe-), and the striking resemblance with both ß-turns of the orally bioavailable peptide cyclosporine A, suggests that the introduction of bioactive sequences on the highly Caco-2 permeable templates may result in potent orally bioavailable drug candidates.

Structural basis of multivalent binding to wheat germ agglutinin.

The inhibition of carbohydrate-protein interactions by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. Crucial for the success of this approach is an understanding of the molecular mechanisms as to how a binding enhancement of a multivalent ligand is achieved. We have synthesized a series of multivalent N-acetylglucosamine (GlcNAc) derivatives and studied their interaction with the plant lectin wheat germ agglutinin (WGA) by an enzyme-linked lectin assay (ELLA) and X-ray crystallography. The solution conformation of one ligand was determined by NMR spectroscopy. Employing a GlcNAc carbamate motif with alpha-configuration and by systematic variation of the spacer length, we were able to identify divalent ligands with unprecedented high WGA binding potency. The best divalent ligand has an IC(50) value of 9.8 microM (ELLA) corresponding to a relative potency of 2350 (1170 on a valency-corrected basis, i.e., per mol sugar contained) compared to free GlcNAc. X-ray crystallography of the complex of WGA and the second best, closely related divalent ligand explains this activity. Four divalent molecules simultaneously bind to WGA with each ligand bridging adjacent binding sites. This shows for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also report a tetravalent neoglycopeptide with an IC(50) value of 0.9 microM being 25,500 times higher than that of GlcNAc (6400 times per contained sugar) and the X-ray structure analysis of its complex with glutaraldehyde-cross-linked WGA. Comparison of the crystal structure and the solution NMR structure of the neoglycopeptide as well as results from the ELLA suggest that the conformation of the glycopeptide in solution is already preorganized in a way supporting multivalent binding to the protein. Our findings show that bridging adjacent protein binding sites by multivalent ligands is a valid strategy to find high-affinity protein ligands and that even subtle changes of the linker structure can have a significant impact on the binding affinity.

Multiple N-methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity: pharmacological and conformational studies.

Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation that resembles other alpha-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains.

Plant sterols in "rafts": a better way to regulate membrane thermal shocks.

Specialized lipid domains (rafts) that are generally enriched in sterols and sphingolipids, are most likely present in cell membranes of animals, plants and fungi. While cholesterol and ergosterol are predominant in vertebrates and fungi, plants possess complex sterol profiles, dominated by sitosterol and stigmasterol in Arabidopsis thaliana. Fully hydrated model membranes of composition approaching those found in rafts of mammals, fungi and plants were investigated by means of solid-state 2H-NMR, using deuterated dipalmitoylphosphatidylcholine (2H(62)-DPPC). The dynamics of such membranes was determined through measuring of membrane ordering or disordering properties. The presence of the liquid-ordered, lo, phase, which may be an indicator of rigid sterol-sphingolipid domains, was detected in all binary or ternary mixtures of all sterols investigated. Of great interest, the dynamics of ternary mixtures mimicking rafts in plants (phytosterol/glucosylcerebroside/DPPC), showed a lesser temperature sensitivity to thermal shocks, on comparing to systems mimicking rafts in mammals and fungi. This effect was particularly marked with sitosterol. The presence of an ethyl group branched on the alkyl chain of sitosterol and stigmasterol is proposed as reinforcing the membrane cohesion by additional attractive van der Waals interactions with the alkyl chains of sphingolipids and phospholipids. As a side result, the elevated resolution of NMR spectra in the presence of sitosterol also suggests domains of smaller size than with other sterols. Finally, the role of phytosterols in maintaining plant membranes in a state of dynamics less sensitive to temperature shocks is discussed.

Systematic Backbone Conformational Constraints on a Cyclic Melanotropin Ligand Leads to Highly Selective Ligands for Multiple Melanocortin Receptors.

Human melanocortin receptors (hMCRs) have been challenging targets to develop ligands that are explicitly selective for each of their subtypes. To modulate the conformational preferences of the melanocortin ligands and improve the biofunctional agonist/antagonist activities and selectivities, we have applied a backbone N-methylation approach on Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH2 (Ac-Nle(4)-c[Asp(5),D-Nal(2')(7),Lys(10)]-NH2), a nonselective cyclic peptide antagonist at hMC3R and hMC4R and an agonist at hMC1R and hMC5R. Systematic N-methylated derivatives of Ac-Nle(4)-c[Asp(5),D-Nal(2')(7),Lys(10)]-NH2, with all possible backbone N-methylation combinations, have been synthesized and examined for their binding and functional activities toward melanocortin receptor subtypes 1, 3, 4, and 5 (hMCRs). Several N-methylated analogues are selective and potent agonists or antagonists for hMC1R or hMC5R or have selective antagonist activity for hMC3R. The selective hMC1R ligands show strong binding for human melanoma cells. We have also discovered the first universal antagonist (compound 19) for all subtypes of hMCRs.

N-Methylated sst2 Selective Somatostatin Cyclic Peptide Analogue as a Potent Candidate for Treating Neurogenic Inflammation.

A focused multiply N-methylated library of a cyclic hexapeptidic somatostatin analogue: MK678 cyclo(-MeAYwKVF-) was generated, which resulted in the unexpected observation of an efficacious tetra-N-methylated analogue, cyclo(-MeAYMewMeKVMeF-) with a potent inhibitory action on sensory neuropeptide release in vitro and on acute neurogenic inflammatory response in vivo. The analogue shows selectivity toward somatostatin receptor subtype 2 (sst2). Extensive 2D NMR spectroscopy and molecular dynamics simulation revealed the solution conformation of the analogue, which can be adopted as a lead for the further structure-activity relationship studies targeting neurogenic inflammation.