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Peptides have recently regained interest as therapeutic candidates, but their development remains confronted with several limitations including low bioavailability. Backbone head-to-tail cyclization, i.e., setting a covalent peptide bond linking the last amino acid with the first one, is one effective strategy of peptide-based drug design to stabilize the conformation of bioactive peptides while preserving peptide properties in terms of low toxicity, binding affinity, target selectivity, and preventing enzymatic degradation. Starting from an active peptide, it usually requires the design of a linker of a few amino acids to make it possible to cyclize the peptide, possibly preserving the conformation of the initial peptide and not affecting its activity. However, very little is known about the sequence-structure relationship requirements of designing linkers for peptide cyclization in a rational manner. Recently, we have shown that large-scale data-mining of available protein structures can lead to the precise identification of protein loop conformations, even from remote structural classes. Here, we transpose this approach to linkers, allowing head-to-tail peptide cyclization. First we show that given a linker sequence and the conformation of the linear peptide, it is possible to accurately predict the cyclized peptide conformation. Second, and more importantly, we show that it seems possible to elaborate on the information inferred from protein structures to propose effective candidate linker sequences constrained by length and amino acid composition, providing the first framework for the rational design of head-to-tail cyclization linkers. Finally, we illustrate this for two peptides using a limited set of amino-acids likely not to interfere with peptide function. For a linear peptide derived from Nrf2, the peptide cyclized starting from the experimental structure showed a 26-fold increase in the binding affinity. For urotensin II, a peptide already cyclized by a disulfide bond that exerts a broad array of biological activities, we were able, starting from models of the structure, to design a head-to-tail cyclized peptide, the first synthesized bicyclic 14-residue long urotensin II analogue, showing a retention of in vitro activity. Although preliminary, our results strongly suggest that such an approach has strong potential for cyclic peptide-based drug design.

Using an integrated transcriptomic and proteomic approach, we characterized the venom peptidome of the European red ant, Manica rubida . We identified 13 "myrmicitoxins" that share sequence similarities with previously identified ant venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O- fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC venom fractions on the blowfly Lucilia caesar , permitting us to identify six myrmicitoxins (i.e., U 3 -, U 10 -, U 13 -, U 20 -MYRTX-Mri1a, U 10 -MYRTX-Mri1b, and U 10 -MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U 10 -MYRTX-Mri1a, -Mri1b, -Mri1c, and U 20 -MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g -1 ). U 13 -MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g -1 ). Finally, U 3 -MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g -1 . Thus, M. rubida employs a paralytic venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.

26RFa, the endogenous QRFPR ligand, is implicated in several physiological and pathological conditions such as the regulation of glucose homeostasis and bone mineralization; hence, QRFPR ligands display therapeutic potential. At the molecular level, functional interaction occurs between residues Arg 25 of 26RFa and Gln 125 of QRFPR. We have designed 26RFa (20-26) analogues incorporating arginine derivatives modified by alkylated substituents. We found that the Arg 25 side chain length was necessary to retain the activity of 26RFa (20-26) and that N-monoalkylation of arginine was accommodated by the QRFPR active site. In particular, [(Me) ω Arg 25 ]26RFa (20-26) (5b, LV-2186) appeared to be 25-fold more potent than 26RFa (20-26) and displayed a position in a QRFPR homology model slightly different to that of the unmodified heptapeptide. Other peptides were less potent than 26RFa (20-26) , exhibited partial agonistic activity, or were totally inactive in accordance to different ligand-bound structures. In vivo, [(Me) ω Arg 25 ]26RFa (20-26) exerted a delayed 26RFa-like hypoglycemic effect. Finally, N-methyl substituted arginine-containing peptides represent lead compounds for further development of QRFPR agonists.

The peptide toxins in the venoms of small invertebrates such as stinging ants have rarely been studied due to the limited amount of venom available per individual. We used a venomics strategy to identify the molecular diversity of the venom peptidome for the myrmicine ant Tetramorium bicarinatum. The methodology included (i) peptidomics, in which the venom peptides are sequenced through a de novo mass spectrometry approach or Edman degradation; (ii) transcriptomics, based on RT-PCR-cloning and DNA sequencing; and (iii) the data mining of the RNA-seq in the available transcriptome. Mass spectrometry analysis revealed about 2800 peptides in the venom. However, the de novo sequencing suggested that most of these peptides arose from processing or the artifactual fragmentations of full-length mature peptides. These peptides, called "myrmicitoxins", are produced by a limited number of genes. Thirty-seven peptide precursors were identified and classified into three superfamilies. These precursors are related to pilosulin, secapin or are new ant venom prepro-peptides. The mature myrmicitoxins display sequence homologies with antimicrobial, cytolytic and neurotoxic peptides. The venomics strategy enabled several post-translational modifications in some peptides such as O-glycosylation to be identified. This study provides novel insights into the molecular diversity and evolution of ant venoms.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of bioactive peptides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of post-translational modifications catalyzed by novel radical S-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a C-to-N directional manner. By combining mutagenesis experiments with labeling studies and investigating the enzyme substrate promiscuity, we deciphered in detail the mechanism of PoyD. We notably identified a critical cysteine residue as a likely key H atom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addition, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with unnatural epimerization patterns.

26RFa, a novel RFamide neuropeptide, is the endogenous ligand of the former orphan receptor GPR103. Intracerebroventricular injection of 26RFa and its C-terminal heptapeptide, 26RFa((20-26)), stimulates food intake in rodents. To develop potent, stable ligands of GPR103 with low molecular weight, we have designed a series of aza-β(3)-containing 26RFa((20-26)) analogues for their propensity to establish intramolecular hydrogen bonds, and we have evaluated their ability to increase [Ca(2+)](i) in GPR103-transfected cells. We have identified a compound, [Cmpi(21),aza-β(3)-Hht(23)]26RFa((21-26)), which was 8-fold more potent than 26RFa((20-26)) in mobilizing [Ca(2+)](i). This pseudopeptide was more stable in serum than 26RFa((20-26)) and exerted a longer lasting orexigenic effect in mice. This study constitutes an important step toward the development of 26RFa analogues that could prove useful for the treatment of feeding disorders.

26RFa is a new member of the RFamide peptide family that has been identified as the endogenous ligand of the orphan GPCR GPR103. As the C-terminal heptapeptide (26RFa((20-26))) mimics the action of the native peptide on food intake and gonadotropin secretion in rodents, we have synthesized a series of analogues of 26RFa((20-26)) and measured their potency to induce [Ca(2+)](i) mobilization in Gα(16)-hGPR103-transfected CHO cells. Systematic replacement of each residue by an alanine (Ala scan) and its D-enantiomer (D scan) showed that the last three C-terminal residues were very sensitive to the substitutions while position 23 tolerated rather well both modifications. Most importantly, replacement of Ser(23) by a norvaline led to an analogue, [Nva(23)]26RFa((20-26)), that was 3-fold more potent than the native heptapeptide. These new pharmacological data, by providing the first information regarding the structure-activity relationships of 26RFa analogues, should prove useful for the rational design of potent GPR103 receptor ligands with potential therapeutic application.