Identification and engineering of potent cyclic peptides with selective or promiscuous binding through biochemical profiling and bioinformatic data analysis.

As our understanding of biological systems grows, so does the need to selectively target individual or multiple members of specific protein families in order to probe their function. Many targets of current biological and pharmaceutical interest are part of a large family of closely related proteins and achieving ligand selectivity often remains either an elusive or time-consuming endeavour. Cyclic peptides (CPs) occupy a key niche in ligand space, able to achieve high affinity and selectivity while retaining synthetic accessibility. De novo cyclic peptide ligands can be rapidly generated against a given target using mRNA display. In this study we harness mRNA display technology and the wealth of next generation sequencing (NGS) data generated to explore both experimental approaches and bioinformatic, statistical data analysis of peptide enrichment in cross-screen selections to rapidly generate high affinity CPs with differing intra-family protein selectivity profiles against fibroblast growth factor receptor (FGF-R) family proteins. Using these methods, CPs with distinct selectivity profiles can be generated which can serve as valuable tool compounds to decipher biological questions.

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

Full-length human GLP-1 receptor structure without orthosteric ligands.

Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.

Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion.

The Munc13 family of exocytosis regulators has multiple Ca(2+)-binding, C2 domains. Here, we probed the mechanism by which Munc13-4 regulates in vitro membrane fusion and platelet exocytosis. We show that Munc13-4 enhances in vitro soluble NSF attachment protein receptor (SNARE)-dependent, proteoliposome fusion in a Ca(2+)- and phosphatidylserine (PS)-dependent manner that was independent of SNARE concentrations. Munc13-4-SNARE interactions, under the conditions used, were minimal in the absence or presence of Ca(2+). However, Munc13-4 was able to bind and cluster liposomes harbouring PS in response to Ca(2+). Interestingly, Ca(2+)-dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca(2+)-liganding aspartate residues of the C2B domain. Analytical ultracentrifugation (AUC) measurements indicated that, in solution, Munc13-4 was a monomeric prolate ellipsoid with dimensions consistent with a molecule that could bridge two fusing membranes. To address the potential role of Munc13-4 as a tethering protein in platelets, we examined mepacrine-stained, dense granule mobility and secretion in platelets from wild-type and Munc13-4 null (Unc13d(Jinx)) mice. In the absence of Munc13-4, dense granules were highly mobile in both resting and stimulated platelets, and stimulation-dependent granule release was absent. These observations suggest that dense granules are stably docked in resting platelets awaiting stimulation and that Munc13-4 plays a vesicle-stabilizing or tethering role in resting platelets and also in activated platelets in response to Ca(2+). In summary, we show that Munc13-4 conveys Ca(2+) sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion.

Munc18b/STXBP2 is required for platelet secretion.

Platelets are vital for hemostasis because they release their granule contents in response to vascular damage. Platelet exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), whose interactions are governed by regulators, eg, Sec/Munc18 proteins. These proteins chaperone syntaxin t-SNAREs and are required for exocytosis. Platelets contain 3 Munc18 isoforms: Munc18a, Munc18b, and Munc18c. We report that Munc18b is the major isoform and is required for platelet secretion. Familial hemophagocytic lymphohistiocytosis type 5 (FHL5) is caused by defects in the Munc18b/STXBP2 gene. We confirm a previous report showing that platelets from FHL5 patients have defective secretion. Serotonin, ADP/ATP, and platelet factor 4 release was profoundly affected in the 2 biallelic patients and partially in a heterozygous patient. Release of lysosomal contents was only affected in the biallelic platelets. Platelets from the FHL5 biallelic patients showed decreased Munc18b and syntaxin-11 levels were significantly reduced; other syntaxins were unaffected. Munc18b formed complexes with syntaxin-11, SNAP-23, and vesicle-associated membrane protein-8 in human platelets. Other potential secretion regulators, Munc13-4 and Rab27, were also found associated. These data demonstrate a key role for Munc18b, perhaps as a limiting factor, in platelet exocytosis and suggest that it regulates syntaxin-11.

Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis.

Activation-dependent platelet granule release is mediated by integral membrane proteins called soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) and their regulators; however, the mechanisms for this process are ill-defined. To further characterize platelet secretion, we analyzed the function of platelets from Unc13d(Jinx) mice. Platelets from these animals lack the putative vesicle priming factor, Munc13-4, and have a severe secretion defect. Release from dense granules was completely ablated and that from alpha-granules and lysosomes was severely compromised. Unc13d(Jinx) platelets showed attenuated aggregation and, consequently, Unc13d(Jinx) mice had prolonged tail-bleeding times. The secretion defect was not due to altered expression of SNAREs or SNARE regulators, defective granule biogenesis, or faulty platelet activation. The defective release could be rescued by adding recombinant Munc13-4 to permeabilized Unc13d(Jinx) platelets. In wild-type mouse platelets, Munc13-4 levels were lower than those of SNAREs suggesting that Munc13-4 could be a limiting component of the platelets' secretory machinery. Consistently, Munc13-4 levels directly correlated with the extent of granule release from permeabilized platelets and from intact, heterozygous Unc13d(Jinx) platelets. These data highlight the importance of Munc13-4 in platelets and indicate that it is a limiting factor required for platelet secretion and hemostasis.

Dissecting the N-ethylmaleimide-sensitive factor: required elements of the N and D1 domains.

N-Ethylmaleimide-sensitive factor (NSF) is a homo-hexameric member of the AAA(+) (ATPases associated with various cellular activities plus) family. It plays an essential role in most intracellular membrane trafficking through its binding to and disassembly of soluble NSF attachment protein (SNAP) receptor (SNARE) complexes. Each NSF protomer contains an N-terminal domain (NSF-N) and two AAA domains, a catalytic NSF-D1 and a structural NSF-D2. This study presents detailed mutagenesis analyses of NSF-N and NSF-D1, dissecting their roles in ATP hydrolysis, SNAP.SNARE binding, and complex disassembly. Our results show that a positively charged surface on NSF-N, bounded by Arg(67) and Lys(105), and the conserved residues in the central pore of NSF-D1 (Tyr(296) and Gly(298)) are involved in SNAP.SNARE binding but not basal ATP hydrolysis. Mutagenesis of Sensor 1 (Thr(373)-Arg(375)), Sensor 2 (Glu(440)-Glu(442)), and Arginine Fingers (Arg(385) and Arg(388)) in NSF-D1 shows that each region plays a discrete role. Sensor 1 is important for basal ATPase activity and nucleotide binding. Sensor 2 plays a role in ATP- and SNAP-dependent SNARE complex binding and disassembly but does so in cis and not through inter-protomer interactions. Arginine Fingers are important for SNAP.SNARE complex-stimulated ATPase activity and complex disassembly. Mutants at these residues have a dominant-negative phenotype in cells, suggesting that Arginine Fingers function in trans via inter-protomer interactions. Taken together, these data establish functional roles for many of the structural elements of the N domain and of the D1 ATP-binding site of NSF.

Endobrevin/VAMP-8-dependent dense granule release mediates thrombus formation in vivo.

Individuals whose platelets lack dense or alpha-granules suffer various degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. In platelets, SNARE proteins mediate the membrane fusion events required for granule cargo release. Endobrevin/VAMP-8 is the primary vesicle-SNARE (v-SNARE) responsible for efficient release of dense and alpha-granule contents; thus, VAMP-8(-/-) mice are a useful model to evaluate the importance of platelet granule secretion in thrombus formation. Thrombus formation, after laser-induced vascular injury, in these mice is delayed and decreased, but not absent. In contrast, thrombus formation is almost completely abolished in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lacks dense granules. Evaluation of aggregation of VAMP-8(-/-) and ruby-eye platelets indicates that defective ADP release is the primary abnormality leading to impaired aggregation. These results demonstrate the importance of dense granule release even in the earliest phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for antiplatelet therapies.

The platelet release reaction: just when you thought platelet secretion was simple.

PURPOSE OF REVIEW: In response to agonists produced at vascular lesions, platelets release a host of components from their three granules: dense core, alpha, and lysosome. This releasate activates other platelets, promotes wound repair, and initiates inflammatory responses. Although widely accepted, the specific mechanisms underlying platelet secretion are only now coming to light. This review focuses on the core machinery required for platelet secretion. RECENT FINDINGS: Proteomic analyses have provided a catalog of the components released from activated platelets. Experiments using a combination of in-vitro secretion assays and knockout mice have led to assignments of both vesicle-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (v-SNARE) and target membrane SNARE to each of the three secretion events. SNARE knockout mice are also proving to be useful models for probing the role of platelet exocytosis in vivo. Other studies are beginning to identify SNARE regulators, which control when and where SNAREs interact during platelet activation. SUMMARY: A complex set of protein-protein interactions control the membrane fusion events required for the platelet release reaction. SNARE proteins are the core elements but the proteins that control SNARE interactions represent key points at which platelet signaling cascades could affect secretion and thrombosis.

Endobrevin/VAMP-8 is the primary v-SNARE for the platelet release reaction.

Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8-/- mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/-, VAMP-3-/-, and VAMP-2+/-/VAMP-3-/- platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3-/- platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8-/- platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8-/- mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.

Platelets from Munc18c heterozygous mice exhibit normal stimulus-induced release.

A critical aspect of hemostasis is the release of clot-forming components from the three intra-platelet stores: dense core granules, alpha-granules and lysosomes. Exocytosis from these granules is mediated by soluble (SNAPs and NSF) and integralmembrane proteins (v- and t-SNAREs). Three SM (Sec1/Munc18) proteins are present in mouse platelets (Munc18a, 18b and 18c) and each potentially regulates exocytosis via modulation of their cognate syntaxin binding partner. To define the molecular machinery required for platelet exocytosis, we analyzed platelets from Munc18c heterozygous knockout mice. These platelets show a decrease in Munc18c but no apparent reduction in other secretory machinery components. No differences in the rates of aggregation or of secretion of [(3)H]-5HT (dense core granules), platelet factor 4 (alpha-granules), or hexosaminidase (lysosomes) were detected between platelets from Munc18c heterozygous knockout or wild-type mice. The platelets also show normal morphology. Contrary to a predicted requirement for Munc18c in platelet secretion, data reported here show that reducing Munc18c levels does not substantially alter platelet function. These data show that despite Munc18c's role in platelet secretion, the lack of a secretion defect may be attributed to compensation by other Munc18 isoforms or that one allele is sufficient to maintain secretion under standard conditions.