Développement de modulateurs allostériques peptidiques inhibiteurs de l’activité des récepteurs de l’interleukine 1 et de la vasopressine

L’approche Module X a été créée dans le but de concevoir de petits peptides modulateurs ayant des propriétés allostériques. Module X reproduit de petites parties des portions extracellulaires flexibles des récepteurs. Ces petits peptides vont interagir en s’interposant entre deux sous unités ou entre deux régions de la même sous-unité qui interagissent par des liens hydrogènes, des ponts salins ou des liens disulfure. Ces régions sont spécialement choisies à l’extérieur du domaine de liaison du ligand orthostérique et sont situées dans les régions inter domaines, la portion juxta membranaire ou dans les boucles. Étant donné que les boucles sont exposées durant les changements de conformation, une séquence peptidique reproduisant certaines régions de ces boucles pourrait s’insérer à un endroit approprié dans la structure où se lier à son partenaire de signalisation dans le complexe protéique, ce qui aurait comme effet de déplacer l’équilibre de l’ensemble vers un état particulier et modulerait ainsi la signalisation. De cette façon, certaines voies de signalisation pourraient être partiellement inhibées tandis que d’autres voies ne seraient pas touchées puisque le ligand orthostérique pourrait toujours se lier au récepteur. Dans une première étude, nous avons conçu des peptides inhibiteurs du récepteur de l’interleukine 1 (IL-1R/IL-1RAcP) plus précisément en reproduisant des régions flexibles de la protéine accessoire, sous-unité signalisatrice du récepteur. IL-1 est un médiateur majeur de l’inflammation, mais le seul antagoniste disponible est l’analogue naturel de IL-1, IL-1Ra qui compétitionne avec IL-1 pour le site de liaison sur le récepteur. Nous avons conçu plusieurs peptides à partir des boucles de la protéine accessoire. Un de ces peptides, rytvela (101.10) a démontré des propriétés de non-compétitivité et de sélectivité fonctionnelle caractéristiques des modulateurs allostériques. 101.10 bloque la prolifération des thymocytes et la synthèse de PGE2 avec un IC50 de 1 nM mais une efficacité de 100 % et 45 % respectivement et ne déplace pas IL-1 radioactif dans des essais de radioliaisons. De plus, 101.10 n’a qu’un effet minime sur l’affinité de IL-1 pour son récepteur. 101.10 démontre, de plus, une activité inhibitrice in vivo dans des modèles d’inflammation de l’intestin chez le rat (efficacité supérieure aux corticostéroïdes et à IL-1Ra) et de dermatite chez la souris de même que dans un modèle d’hyperthermie induite par IL-1. La deuxième étude démontre que Module X peut être utilisé pour concevoir des inhibiteurs pour une autre grande famille de récepteurs : les récepteurs couplés aux protéines G. La vasopressine joue un rôle important dans l’équilibre hydro-osmotique et un moindre rôle dans la vasomotricité. Six peptides ont été conçus à partir de régions juxta membranaires du récepteur de la vasopressine V2R. Le peptide le plus actif, VRQ397 (IC50 = 0,69 nM dans un modèle de vasorelaxation du crémastère), a démontré de la sélectivité fonctionnelle en inhibant la synthèse de prostacycline mais sans inhiber l’activation de la protéine Gs et la génération d’ AMP cyclique. Le peptide VRQ397 ne pouvait déplacer le ligand naturel AVP marqué radioactivement; de même VRQ397 radioactif ne se liait que sur V2R et non pas sur d’autres récepteurs de la même famille tel que V1R (récepteur de la vasopressine de type I). Ces études décrivent la caractérisation de petits peptides modulateurs de la signalisation de IL-1R et V2R et présentant des propriétés de modulateurs allostériques.The Module X approach was conceived to generate small allosteric peptides that do not (by definition) compete with the natural ligand to inhibit or modulate signalling. Orthosteric inhibition blocks the entire signalling pathways while allosteric modulators will bind to another site on the target and show functional selectivity. By reproducing parts of the flexible regions (loops) of two receptors, the IL-1 and vasopressin receptors, we generated small peptides that showed allosteric properties. To prove our concept we started with a pro-inflammatory target: IL-1 receptor. Interleukin (IL)-1 is a major pro-inflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein (IL-1RacP) subunits. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1RacP, which are putative interactive sites with the IL-1RI subunit. Here we substantiate the merits of one of these peptides, rytvela (we termed, 101.10), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC50 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed non-competitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. The second study involved a representative of the biggest family of membrane proteins: G-protein coupled receptors. Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydro-osmotic equilibrium and to a lesser extent on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, vasopressin (AVP), and hence are competitive ligands. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, CRAVKY (labelled VRQ397), was characterized. VRQ397 was potent (IC50 = 0.69 ± 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function (specifically DDAVP-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events). Dose-response profile revealed non-competitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells, could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). VRQ397 exhibited pharmacological permissiveness on V2R-induced signals as it inhibited DDAVP-induced PGI2 generation, but not that of cAMP or recruitment of — arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. Findings describe the discovery of potent and specific small (peptide) antagonists of IL-1RI and V2R with properties in line with an allosteric negative modulator

Probing Anti-inflammatory Properties Independent of NF-κB Through Conformational Constraint of Peptide-Based Interleukin-1 Receptor Biased Ligands

Interleukin-1β (IL-1β) binds to the IL-1 receptor (IL-1R) and is a key cytokine mediator of inflammasome activation. IL-1β signaling leads to parturition in preterm birth (PTB) and contributes to the retinal vaso-obliteration characteristic of oxygen-induced retinopathy (OIR) of premature infants. Therapeutics targeting IL-1β and IL-1R are approved to treat rheumatoid arthritis; however, all are large proteins with clinical limitations including immunosuppression, due in part to inhibition of NF-κB signaling, which is required for immuno-vigilance and cytoprotection. The all-D-amino acid peptide 1 (101.10, H-d-Arg-d-Tyr-d-Thr-d-Val-d-Glu-d-Leu-d-Ala-NH2) is an allosteric IL-1R modulator, which exhibits functional selectivity and conserves NF-κB signaling while inhibiting other IL-1-activated pathways. Peptide 1 has proven effective in experimental models of PTB and OIR. Seeking understanding of the structural requirements for the activity and biased signaling of 1, a panel of twelve derivatives was synthesized employing the various stereochemical isomers of α-amino-γ-lactam (Agl) and α-amino-β-hydroxy-γ-lactam (Hgl) residues to constrain the D-Thr-D-Val dipeptide residue. Using circular dichroism spectroscopy, the peptide conformation in solution was observed to be contingent on Agl, Hgl, and Val stereochemistry. Moreover, the lactam mimic structure and configuration influenced biased IL-1 signaling in an in vitro panel of cellular assays as well as in vivo activity in murine models of PTB and OIR. Remarkably, all Agl and Hgl analogs of peptide 1 did not inhibit NF-κB signaling but blocked other pathways, such as JNK and ROCK2 phosphorylation contingent on structure and configuration. Efficacy in preventing preterm labor correlated with a capacity to block IL-1β-induced IL-1β synthesis. Furthermore, the importance of inhibition of JNK and ROCK2 phosphorylation for enhanced activity was highlighted for prevention of vaso-obliteration in the OIR model. Taken together, lactam mimic structure and stereochemistry strongly influenced conformation and biased signaling. Selective modulation of IL-1 signaling was proven to be particularly beneficial for curbing inflammation in models of preterm labor and retinopathy of prematurity (ROP). A class of biased ligands has been created with potential to serve as selective probes for studying IL-1 signaling in disease. Moreover, the small peptide mimic prototypes are promising leads for developing immunomodulatory therapies with easier administration and maintenance of beneficial effects of NF-κB signaling

Insertion of multiple α-amino γ-lactam (Agl) residues into a peptide sequence by solid-phase synthesis on synphase lanterns

The insertion of lactams into peptide analogs can enhance potency and improve receptor selectivity. The synthesis of lactam-bridged peptide sequences has been accomplished by a solid-phase approach on SynPhase lanterns using cyclic (R)- and (S)-oxathiazinane ester (2) to annulate the amino lactam residue onto the peptide chain. Parallel synthesis of α-amino γ-lactam analogs of the allosteric modulator of IL-1 receptor 101.10 (D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala: rytvela) was performed by split-mix chemistry on the lanterns. In particular, the double insertion of α-amino γ-lactams in the same peptide sequence has been accomplished by this effective method for the solid-supported combinatorial synthesis of lactam-bridged peptides. Peptides bearing an Agl residue exhibited curve shapes indicative of turn conformations in their circular dichroism spectra

Positional scanning for peptide secondary structure by systematic solid-phase synthesis of amino lactam peptides

Incorporation of amino lactams into biologically active peptides has been commonly used to restrict conformational mobility, enhance selectivity, and increase potency. A solid-phase method using a Fmoc-protection strategy has been developed for the systematic synthesis of peptides containing configurationally defined α- and β-amino γ-lactams. N-Alkylation of N-silyl peptides with five- and six-member cyclic sulfamidates 9 and 8 minimized bis-alkylation and provided N-alkyl peptides, which underwent lactam annulation under microwave heating. Employing this solid-phase protocol on the growth hormone secretagogue GHRP-6, as well as on the allosteric modulator of the IL-1 receptor 101.10, has furnished 16 lactam derivatives and validated the effectiveness of this approach on peptides bearing aliphatic, aromatic, branched, charged, and heteroatomic side chains. The binding affinity IC50 values of the GHRP-6 lactam analogues on both the GHS-R1a and CD36 receptors are reported as well as inhibition of thymocyte proliferation measurements for the 101.10 lactam analogues. In these cases, lactam analogues were prepared exhibiting similar or improved properties compared with the parent peptide. Considering the potential for amino lactams to induce peptide turn conformations, the effective method described herein for their supported construction on growing peptides, and for the systematical amino lactam scan of peptides, has proven useful for the rapid identification of the secondary structure necessary for peptide biological activity

Plateletactivating factor in vasoobliteration of oxygen-induced retinopathy. Invest Ophthalmol Vis Sci 43

PURPOSE. To test whether platelet-activating factor (PAF) directly causes retinovascular endothelial cell (EC) death. METHODS. Retinovascular density was calculated in rat pups exposed to 80% O 2 from postnatal days (P)6 to P14 (to produce oxygen-induced retinopathy [OIR]), using the adenosine diphosphatase (ADPase) technique, in animals treated with distinct PAF receptor blockers (PCA-4248, BN52021, or THG315). PAF levels were then measured in the retinas. Viability of ECs from piglets and humans in response to C-PAF (a stable PAF analogue) was determined by the reduction of the tetrazolium salt 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by viable cells, incorporation of propidium iodide (PI), TUNEL assay, and release of lactate dehydrogenase. Release of thromboxane (TX) was measured in the cell media. RESULTS. PAF levels in retina were markedly increased by exposure of isolated rat retinas to H 2 O 2 (1 M) and of rat pups placed in 80% O 2 . Exposure to 80% O 2 induced retinal vasoobliteration, which was equally significantly inhibited (ϳ60%) by all PAF receptor blockers tested. C-PAF increased incorporation of PI by isolated rat retinal microvasculature. Also, C-PAF caused time-and concentration-dependent death of cultured retinal ECs, which was prevented by the PAF receptor antagonist CV-3988. This effect of C-PAF was selective on retinal and neurovascular ECs, but not on other ECs. DNA fragmentation (TUNEL) was hardly detected, and inhibition of apoptosis-related processes by nicotinamide, cyclosporin A, and Z-DEVD-FMK and Z-VAD-FMK (caspase inhibitors) barely protected against death in EC, whereas C-PAF increased release of lactate dehydrogenase, implying that necrosis is the nature of EC death. Finally, C-PAF-induced cell death was preceded by an increase in TXB 2 levels and was prevented by TXA 2 synthase inhibition (with CGS12970). CONCLUSIONS. The data suggest PAF plays a major role in vasoobliteration in OIR by triggering death of neuroretinal microvascular ECs. The cell death seems to be mediated at least in part by TXA 2 . These effects of PAF may participate in ischemic retinopathies such as diabetes and retinopathy of prematurity. (Invest Ophthalmol Vis Sci. 2002;43:3327-3337