Rational engineering of glycosaminoglycan-based Dickkopf-1 scavengers to improve bone regeneration.

The WNT signaling pathway is a central regulator of bone development and regeneration. Functional alterations of WNT ligands and inhibitors are associated with a variety of bone diseases that affect bone fragility and result in a high medical and socioeconomic burden. Hence, this cellular pathway has emerged as a novel target for bone-protective therapies, e.g. in osteoporosis. Here, we investigated glycosaminoglycan (GAG) recognition by Dickkopf-1 (DKK1), a potent endogenous WNT inhibitor, and the underlying functional implications in order to develop WNT signaling regulators. In a multidisciplinary approach we applied in silico structure-based de novo design strategies and molecular dynamics simulations combined with synthetic chemistry and surface plasmon resonance spectroscopy to Rationally Engineer oligomeric Glycosaminoglycan derivatives (REGAG) with improved neutralizing properties for DKK1. In vitro and in vivo assays show that the GAG modification to obtain REGAG translated into increased WNT pathway activity and improved bone regeneration in a mouse calvaria defect model with critical size bone lesions. Importantly, the developed REGAG outperformed polymeric high-sulfated hyaluronan (sHA3) in enhancing bone healing up to 50% due to their improved DKK1 binding properties. Thus, rationally engineered GAG variants may represent an innovative strategy to develop novel therapeutic approaches for regenerative medicine.

Dipeptide-Derived Alkynes as Potent and Selective Irreversible Inhibitors of Cysteine Cathepsins.

The potential of designing irreversible alkyne-based inhibitors of cysteine cathepsins by isoelectronic replacement in reversibly acting potent peptide nitriles was explored. The synthesis of the dipeptide alkynes was developed with special emphasis on stereochemically homogeneous products obtained in the Gilbert-Seyferth homologation for C≡C bond formation. Twenty-three dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S, and K. Numerous combinations of residues at positions P1 and P2 as well as terminal acyl groups allowed for the derivation of extensive structure-activity relationships, which were rationalized by computational covalent docking for selected examples. The determined inactivation constants of the alkynes at the target enzymes span a range of >3 orders of magnitude (3-10 133 M-1 s-1). Notably, the selectivity profiles of alkynes do not necessarily reflect those of the nitriles. Inhibitory activity at the cellular level was demonstrated for selected compounds.

Descripción del bienestar social y prevalencia del consumo de alcohol en jóvenes de Ciudad del Carmen, México / Description of social welfare and prevalence of alcohol consumption in young people in Ciudad del Carmen, México

Introducción: la juventud ha sido considerada una etapa de mayor riesgo ante conductas saludables o no saludables, asimismo una etapa de menor control parental y un incremento en las actividades sociales que configura el estilo de vida de esta población. Objetivo: analizar la relación del bienestar social y el consumo de alcohol en jóvenes Método: investigación cuantitativa de tipo correlacional realizada en 279 jóvenes de Ciudad del Carmen Campeche, se utilizó un muestreo no probabilístico por conveniencia, como instrumentos de medición se utilizó una cédula de datos personales y de consumo de alcohol y la escala de Bienestar Social de Keyes. Resultados: la prevalencia global (U= 5483.50, p=.026) y en el último año (U= 8432.00, p=.038) muestra diferencias significativas, siendo mayor las puntuaciones en los jóvenes que no han consumido alcohol. Se identificó una relación negativa y significativa del bienestar social y el número de bebidas alcohólicas consumidas (rs=-727, p=.001). Conclusión: los resultados permitieron comprender el grado de bienestar social de los jóvenes, siendo un elemento crucial para su crecimiento y desarrollo para la vida adulta, además se constató la magnitud del consumo de alcohol en esta población, el cual representa un problema de salud pública. (AU)

Introduction: youth has been considered a stage of greater risk for healthy or unhealthy behaviors had a stage of less parental control and an increase in social activities that make up the lifestyle of this population. Objective: to analyze the relationship between social well-being and alcohol consumption in young people Method: correlational-type quantitative research carried out on 279 young people from Ciudad del Carmen Campeche, a non-probabilistic sample was obtained for convenience, as measurement instruments a certificate of personal and alcohol consumption data and the Keyes Social Welfare scale. Results: the global prevalence (U= 5483.50, p=.026) and in the last year (U= 8432.00, p=.038) show significant differences, the proportions being higher in young people who have not consumed alcohol. A negative and significant relationship was identified between social well-being and the number of alcoholic beverages consumed (rs=-727, p=.001). Conclusion: the results allowed us to understand the degree of social well-being of young people, being a crucial element for their growth and development for adult life, in addition, the magnitude of alcohol consumption in this population was verified, which represents a public health problem. (AU)

Sulfated glycosaminoglycans inhibit transglutaminase 2 by stabilizing its closed conformation.

Transglutaminases (TGs) catalyze the covalent crosslinking of proteins via isopeptide bonds. The most prominent isoform, TG2, is associated with physiological processes such as extracellular matrix (ECM) stabilization and plays a crucial role in the pathogenesis of e.g. fibrotic diseases, cancer and celiac disease. Therefore, TG2 represents a pharmacological target of increasing relevance. The glycosaminoglycans (GAG) heparin (HE) and heparan sulfate (HS) constitute high-affinity interaction partners of TG2 in the ECM. Chemically modified GAG are promising molecules for pharmacological applications as their composition and chemical functionalization may be used to tackle the function of ECM molecular systems, which has been recently described for hyaluronan (HA) and chondroitin sulfate (CS). Herein, we investigate the recognition of GAG derivatives by TG2 using an enzyme-crosslinking activity assay in combination with in silico molecular modeling and docking techniques. The study reveals that GAG represent potent inhibitors of TG2 crosslinking activity and offers atom-detailed mechanistic insights.

Conformational dynamics promotes disordered regions from function-dispensable to essential in evolved site-specific DNA recombinases.

Protein intrinsically disordered regions (IDRs) play pivotal roles in molecular recognition and regulatory processes through structural disorder-to-order transitions. To understand and exploit the distinctive functional implications of IDRs and to unravel the underlying molecular mechanisms, structural disorder-to-function relationships need to be deciphered. The DNA site-specific recombinase system Cre/loxP represents an attractive model to investigate functional molecular mechanisms of IDRs. Cre contains a functionally dispensable disordered N-terminal tail, which becomes indispensable in the evolved Tre/loxLTR recombinase system. The difficulty to experimentally obtain structural information about this tail has so far precluded any mechanistic study on its involvement in DNA recombination. Here, we use in vitro and in silico evolution data, conformational dynamics, AI-based folding simulations, thermodynamic stability calculations, mutagenesis and DNA recombination assays to investigate how evolution and the dynamic behavior of this IDR may determine distinct functional properties. Our studies suggest that partial conformational order in the N-terminal tail of Tre recombinase and its packing to a conserved hydrophobic surface on the protein provide thermodynamic stability. Based on our results, we propose a link between protein stability and function, offering new plausible atom-detailed mechanistic insights into disorder-function relationships. Our work highlights the potential of N-terminal tails to be exploited for regulation of the activity of Cre-like tyrosine-type SSRs, which merits future investigations and could be of relevance in future rational engineering for their use in biotechnology and genomic medicine.

Pairing of single mutations yields obligate Cre-type site-specific recombinases.

Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.

Hyaluronan/Collagen Hydrogels with Sulfated Glycosaminoglycans Maintain VEGF165 Activity and Fine-Tune Endothelial Cell Response.

In order to restore the regeneration capacity of large-size vascularized tissue defects, innovative biomaterial concepts are required. Vascular endothelial growth factor (VEGF165) is a key factor of angiogenesis interacting with sulfated glycosaminoglycans (sGAG) within the extracellular matrix. As this interplay mainly controls and directs the biological activity of VEGF165, we used chemically modified sGAG derivatives to evaluate the structural requirements of sGAG for controlling and tuning VEGF165 function and to translate these findings into the design of biomaterials. The in-depth analysis of this interaction by surface plasmon resonance and ELISA studies in combination with molecular modeling stressed the relevance of the substitution position, degree of sulfation, and carbohydrate backbone of GAG. Acrylated hyaluronan (HA-AC)/collagen (coll)-based hydrogels containing cross-linked acrylated, sulfated hyaluronan (sHA-AC) derivatives with different substitution patterns or an acrylated chondroitin sulfate (CS-AC) derivative function as multivalent carbohydrate-based scaffolds for VEGF165 delivery with multiple tuning capacities. Depending on the substitution pattern of sGAG, the release of biologically active VEGF165 was retarded in a defined manner compared to pure HA/coll gels, which further controlled the VEGF165-induced stimulation of endothelial cell proliferation and extended morphology of cells. This indicates that sGAG can act as modulators of protein interaction profiles of HA/coll hydrogels. In addition, sHA-AC-containing gels with and even without VEGF165 strongly stimulate endothelial cell proliferation compared to gels containing only CS-AC or HA-AC. Thus, HA/coll-based hydrogels containing cross-linked sHA-AC are biomimetic materials able to directly influence endothelial cells in vitro, which might translate into an improved healing of injured vascularized tissues.

Dual Action of Sulfated Hyaluronan on Angiogenic Processes in Relation to Vascular Endothelial Growth Factor-A.

Pathological healing characterized by abnormal angiogenesis presents a serious burden to patients' quality of life requiring innovative treatment strategies. Glycosaminoglycans (GAG) are important regulators of angiogenic processes. This experimental and computational study revealed how sulfated GAG derivatives (sGAG) influence the interplay of vascular endothelial growth factor (VEGF)165 and its heparin-binding domain (HBD) with the signaling receptor VEGFR-2 up to atomic detail. There was profound evidence for a HBD-GAG-HBD stacking configuration. Here, the sGAG act as a "molecular glue" leading to recognition modes in which sGAG interact with two VEGF165-HBDs. A 3D angiogenesis model demonstrated the dual regulatory role of high-sulfated derivatives on the biological activity of endothelial cells. While GAG alone promote sprouting, they downregulate VEGF165-mediated signaling and, thereby, elicit VEGF165-independent and -dependent effects. These findings provide novel insights into the modulatory potential of sGAG derivatives on angiogenic processes and point towards their prospective application in treating abnormal angiogenesis.

Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level.

The extracellular matrix (ECM) is a highly dynamic network constantly remodeled by a fine-tuned protein formation and degradation balance. Matrix metalloproteinases (MMPs) constitute key orchestrators of ECM degradation. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAG). Here, we investigated the molecular interplay of MMP2 with different GAG (chondroitin sulfate, hyaluronan (HA), sulfated hyaluronan (SH) and heparin (HE)) and the impact of GAG on MMP2/TIMP3 complex formation using in vitro-experiments with human bone marrow stromal cells, in silico docking and molecular dynamics simulations. SH and HE influenced MMP2 and TIMP3 protein levels and MMP2 activity. Only SH supported the alignment of both proteins in fibrillar-like structures, which, based on our molecular models, would be due to a stabilization of the interactions between MMP2-hemopexin domain and TIMP3-C-terminal tail. Dependent on the temporal sequential order in which the final ternary complex was formed, our models indicated that SH and HA can affect TIMP3-induced MMP2 inhibition through precluding or supporting their interactions, respectively. Our combined experimental and theoretical approach provides valuable new insights on how GAG interfere with MMP2 activity and MMP2/TIMP3 complex formation. The results obtained evidence GAG as promising molecules for fine-balanced intervention of ECM remodeling.

Syntheses of defined sulfated oligohyaluronans reveal structural effects, diversity and thermodynamics of GAG-protein binding.

Binding of sulfated glycosaminoglycans (GAG) to a wide spectrum of extracellular regulatory proteins is crucial for physiological processes such as cell growth, migration, tissue homeostasis and repair. Thus, GAG derivatives exhibit great relevance in the development of innovative biomaterials for tissue regeneration therapies. We present a synthetic strategy for the preparation of libraries of defined sulfated oligohyaluronans as model GAG systematically varied in length, sulfation pattern and anomeric substitution in order to elucidate the effects of these parameters on GAG recognition by regulatory proteins. Through an experimental and computational approach using fluorescence polarization, ITC, docking and molecular dynamics simulations we investigate the binding of these functionalized GAG derivatives to ten representative regulatory proteins including IL-8, IL-10, BMP-2, sclerostin, TIMP-3, CXCL-12, TGF-ß, FGF-1, FGF-2, and AT-III, and we establish structure-activity relationships for GAG recognition. Binding is mainly driven by enthalpy with only minor entropic contributions. In several cases binding is determined by GAG length, and in all cases by the position and number of sulfates. Affinities strongly depend on the anomeric modification of the GAG. Highest binding affinities are effected by anomeric functionalization with large fluorophores and by GAG dimerization. Our experimental and theoretical results suggest that the diversity of GAG binding sites and modes is responsible for the observed high affinities and other binding features. The presented new insights into GAG-protein recognition will be of relevance to guide the design of GAG derivatives with customized functions for the engineering of new biomaterials.

Hyaluronan/collagen hydrogels containing sulfated hyaluronan improve wound healing by sustained release of heparin-binding EGF-like growth factor.

Functional biomaterials that are able to bind, stabilize and release bioactive proteins in a defined manner are required for the controlled delivery of such to the desired place of action, stimulating wound healing in health-compromised patients. Glycosaminoglycans (GAG) represent a very promising group of components since they may be functionally engineered and are well tolerated by the recipient tissues due to their relative immunological inertness. Ligands of the Epidermal Growth Factor (EGF) receptor (EGFR) activate keratinocytes and dermal fibroblasts and, thus, contribute to skin wound healing. Heparin-binding EGF-like growth factor (HB-EGF) bound to GAG in biomaterials (e.g. hydrogels) might serve as a reservoir that induces prolonged activation of the EGF receptor and to recover disturbed wound healing. Based on previous findings, the capacity of hyaluronan (HA) and its sulfated derivatives (sHA) to bind and release HB-EGF from HA/collagen-based hydrogels was investigated. Docking and molecular dynamics analysis of a molecular model of HB-EGF led to the identification of residues in the heparin-binding domain of the protein being essential for the recognition of GAG derivatives. Furthermore, molecular modeling and surface plasmon resonance (SPR) analyses demonstrated that sulfation of HA increases binding strength to HB-EGF thus providing a rationale for the development of sHA-containing hydrogels. In line with computational observations and in agreement with SPR results, gels containing sHA displayed a retarded HB-EGF release in vitro compared to pure HA/collagen gels. Hydrogels containing HA and collagen or a mixture with sHA were shown to bind and release bioactive HB-EGF over at least 72 h, which induced keratinocyte migration, EGFR-signaling and HGF expression in dermal fibroblasts. Importantly, hydrogels containing sHA strongly increased the effectivity of HB-EGF in inducing epithelial tip growth in epithelial wounds shown in a porcine skin organ culture model. These findings suggest that hydrogels containing HA and sHA can be engineered for smart and effective wound dressings. STATEMENT OF SIGNIFICANCE: Immobilization and sustained release of recombinant proteins from functional biomaterials might overcome the limited success of direct application of non-protected solute growth factors during the treatment of impaired wound healing. We developed HA/collagen-based hydrogels supplemented with acrylated sulfated HA for binding and release of HB-EGF. We analyzed the molecular basis of HB-EGF interaction with HA and its chemical derivatives by in silico modeling and surface plasmon resonance. These hydrogels bind HB-EGF reversibly. Using different in vitro assays and organ culture we demonstrate that the introduction of sulfated HA into the hydrogels significantly increases the effectivity of HB-EGF action on target cells. Therefore, sulfated HA-containing hydrogels are promising functional biomaterials for the development of mediator releasing wound dressings.

Twists or turns: stabilising alpha vs. beta turns in tetrapeptides.

Protein-protein interactions involve hotspots as small as 4 sequential amino acids. Corresponding tetrapeptides have no structure in water. Here we report linking side chains of amino acids X and Z to form 24 cyclic tetrapeptides, cyclo-[XAAZ]-NH2, and stabilise 14-18 membered rings that mimic different kinds of non-regular secondary structures found in protein hotspots. 2D NMR spectra allowed determination of 3D structures for 14 cyclic tetrapeptides in water. Five formed two (i, i + 3) hydrogen bonds and a beta/gamma (6, 7) or beta (9, 19, 20) turn; eight formed one (i, i + 4) hydrogen bond and twisted into a non-helical (13, 18, 21, 22, 24) or helical (5, 17, 23) alpha turn; one was less structured (15). A beta or gamma turn was favoured for Z = Dab, Orn or Glu due to a χ1 gauche (+) rotamer, while an alpha turn was favoured for Z = Dap (but not X = Dap) due to a gauche (-) rotamer. Surprisingly, an unstructured peptide ARLARLARL could be twisted into a helix when either a helical or non-helical alpha turn (5, 13, 17, 18, 21-24) with Z = Dap was attached to the N-terminus. These structural models provide insights into stability for different turns and twists corresponding to non-regular folds in protein hotspots.

Nε-Acryloyllysine Piperazides as Irreversible Inhibitors of Transglutaminase 2: Synthesis, Structure-Activity Relationships, and Pharmacokinetic Profiling.

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of Nε-acryloyllysine piperazides as irreversible inhibitors of TGase 2. Systematic structural modifications on 54 new compounds were performed with a major focus on fluorine-bearing substituents due to the potential of such compounds to serve as radiotracer candidates for positron emission tomography. The determined inhibitory activities ranged from 100 to 10 000 M-1 s-1, which resulted in comprehensive structure-activity relationships. Structure-activity correlations using various substituent parameters accompanied by covalent docking studies provide an advanced understanding of the molecular recognition for this inhibitor class within the active site of TGase 2. Selectivity profiling of selected compounds for other transglutaminases demonstrated an excellent selectivity toward transglutaminase 2. Furthermore, an initial pharmacokinetic profiling of selected inhibitors was performed, including the assessment of potential membrane permeability and liver microsomal stability.

Sulfated hyaluronan attenuates inflammatory signaling pathways in macrophages involving induction of antioxidants.

It is well recognized that high molecular weight hyaluronan (H-HA) exerts potent anti-inflammatory effects while its fragmentation into low molecular weight HA (L-HA) is discussed to promote inflammation. Chemical modification of HA with sulfate groups has been shown to foster its anti-inflammatory activity which seems to be maintained in sulfated low molecular weight HA derivatives (sL-HA). However, the molecular mechanisms by which sL-HA produces its anti-inflammatory activity are not understood. In this study, we used global quantitative proteomics combined with targeted analysis of key proteins to characterize the effect of sL-HA on fully differentiated human inflammatory macrophages (iMФ). Culture of iMФ with sL-HA did not affect cell viability but resulted in a reduced pro-inflammatory cytokine response of iMФ after activation indicating a profound counter-regulation of their initial inflammatory phenotype. Rapid internalization of sL-HA involving CD44 and scavenger receptors was observed. Furthermore, an upregulation of the antioxidants SOD2 and SOD3 was found while no oxidative stress was induced. Consequently, activity of transcription factors for inflammatory gene expression was downregulated in iMФ with sL-HA after activation whereas anti-inflammatory proteins were induced. This study proves anti-inflammatory properties of sL-HA and provides information on its regulatory mode of action on iMФ.

Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency.

Bcl2 Homology (BH) proteins can either trigger or prevent programmed cell death or apoptosis. Deregulation of the BH protein family network leads to evasion of apoptosis, uncontrolled proliferation and is a hallmark of cancer. Inhibition of pro-survival BH proteins is a promising chemotherapeutic strategy for certain cancers. We have examined whether helix-constrained peptides based on the BAD BH3 domain (residues 103-127) can be downsized to much smaller more drug-like peptides. We report the preparation, structural characterisation, in vitro Bcl-xL inhibition and leukemic T-cell killing ability of 45 linear, mono-, bi- and tricyclic helical peptidomimetics between 8- and 19-residues in length. We show that the BAD BH3 can be downsized to 8-14 residues and still maintain appreciable affinity for Bcl-xL. In addition, the binding efficiency indices (BEI) of the downsized mimetics are significantly higher than the BAD BH3 and similar stapled BH3 mimetics, approaching drug-like molecules. This suggests that bicyclic and monocyclic mimetics based on BH3 domains are much more efficient binding ligands than the longer peptides which they mimic.

Correction: Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency.

Correction for 'Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency' by Nicholas E. Shepherd et al., Org. Biomol. Chem., 2016, DOI: 10.1039/c6ob02185a.

Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function.

Rational Structure-Based Rescaffolding Approach to De Novo Design of Interleukin 10 (IL-10) Receptor-1 Mimetics.

Tackling protein interfaces with small molecules capable of modulating protein-protein interactions remains a challenge in structure-based ligand design. Particularly arduous are cases in which the epitopes involved in molecular recognition have a non-structured and discontinuous nature. Here, the basic strategy of translating continuous binding epitopes into mimetic scaffolds cannot be applied, and other innovative approaches are therefore required. We present a structure-based rational approach involving the use of a regular expression syntax inspired in the well established PROSITE to define minimal descriptors of geometric and functional constraints signifying relevant functionalities for recognition in protein interfaces of non-continuous and unstructured nature. These descriptors feed a search engine that explores the currently available three-dimensional chemical space of the Protein Data Bank (PDB) in order to identify in a straightforward manner regular architectures containing the desired functionalities, which could be used as templates to guide the rational design of small natural-like scaffolds mimicking the targeted recognition site. The application of this rescaffolding strategy to the discovery of natural scaffolds incorporating a selection of functionalities of interleukin-10 receptor-1 (IL-10R1), which are relevant for its interaction with interleukin-10 (IL-10) has resulted in the de novo design of a new class of potent IL-10 peptidomimetic ligands.

Helix-constrained nociceptin peptides are potent agonists and antagonists of ORL-1 and nociception.

Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i)→Asp(i+4) side chain-side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure-activity relationships leading to the most potent known α-helical ORL-1 agonist (EC50 40 pM, pERK, Neuro-2a cells) and antagonist (IC50 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1-17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED50 70 pmol, IC50 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.