On-demand assembly of macromolecules used for the design and application of targeted secretion inhibitors

Neurological and endocrine pathologies such as acromegalie, Cushing’s disease, and neuropathic pain display disregulated exocytosis. Silencing specific cell populations would thus be invaluable to correct these debilitating disorders. To achieve this goal, we reengineered the Botulinum neurotoxin (BoT), a highly potent pharmaceutical compound capable of inhibiting exocytosis, and fused to it a protein “stapling” domain [1,2]. These peptide motifs, that form an irreversible tetrahelical coiled-coil, are able to link a variety of targeting domains onto the enzyme and thus redirect it towards normally unaffected cells. The conformational diversity of this assembly process greatly supersedes traditional protein expression since multiple targeting domains (homo- and hetero-) can be linked onto one scaffold, larger yields can be produced separately, it permits the combination of solid-phase peptide synthesis with recombinant protein expression, and it can avoid the necessity of an N- to C- translational fusion. With only a few dozen building “blocks” it is possible to construct thousands of different complexes specifically tailored for each purpose as every individual component can be linked onto any other cognate stapling moieties

SNARE based peptide linking as an efficient strategy to retarget botulinum neurotoxin’s enzymatic domain to specific neurons using diverse neuropeptides as targeting domains

Many disease states are caused by miss-regulated neurotransmission. A small fraction of these diseases can currently be treated with botulinum neurotoxin type A (BoNT/A). BoNT/A is composed of three functional domains – the light chain (Lc) is a zinc metalloprotease that cleaves intracellular SNAP25 which inhibits exocytosis, the translocation domain (Td) that enables the export of the light chain from the endosome to the cytosol, and the receptor binding domain (Rbd) that binds to extracellular gangliosides and synaptic vesicle glycoproteins while awaiting internalisation [1]. Current endeavours are directed towards retargeting Bont/A as well as finding safer methods of preparation and administration. Recently, our laboratory has developed a SNARE based linking strategy to recombine non-toxic BoNT/A fragments into a functional protein by simple mixing [2]. This SNARE based linking strategy permits the stepwise assembly of highly stable macromolecular complexes [2,3]. Onto these three SNARE peptides, diverse functional groups can be attached to the N- or C- terminus by direct synthesis and/or by genetic design. To enhance the therapeutic potential of BoNT/A, this method enables the rapid assembly of a large array of neuropeptide-SNAREs to their cognate LcTd-SNARE. A substitution of the Rbd with various neuropeptide sequences permits a large throughput combinatorial assay of LcTd to target new cell types. In this study, we have fused LcTd to 3 different Synaptobrevin sequences; we also use a small protein staple, and 26 different Syntaxin-neuropeptide fusions (permitting the assay of 78 new chimeric LcTd proteins with modified targeting domains). These neuropeptides such as, but not exclusively, somatostatin (SS), vasoactive intestinal peptide, substance P, opioid peptide analogues, Gonadotropin releasing hormone, and Arginine Vasopressin, which natively function through G protein coupled receptors (GPCR) can undergo agonist induced internalisation upon activation. The ability of our new constructs, once endocytosed, to inhibit neurotransmitter release was tested on different neuronal cell lines with immunoblotting of endogenous SNAP25. This cleavage by Lc reflects the ultimate readout of the enzyme’s efficacy, which incorporates the cell surface binding, internalisation kinetics, translocation of the Lc to the cytosol, and finally the enzymatic cleavage of SNAP25. Internalisation of the toxins can also be monitored with confocal microscopy and FACS by the substitution of the staple peptide for a fluorescent homologue. Figure 1 shows that whole boNT/A (upper left) can have its Rbd replaced with SNARE peptides, which will fuse together to form highly stable chimeric proteins with an altered targeting domain (right). Figure 1 also shows 4 different neuropeptide synthaxins in complex, resolved on SDS-PAGE gel (bottom left lanes 1-4, boiled 1’-4’). Fig. 1. SNARE-linked botulinum neurotoxins used for the retargeting of Bont/A. 29

Cleaved intracellular SNARE peptides are implicated in a novel cytotoxicity mechanism of botulinum serotype C

Recent advances in intracellular protein delivery have enabled more in-depth analyses of cellular functions. A specialized family of SNARE proteases, known as Botulinum Neurotoxins, blocks neurotransmitter exocytosis, which leads to systemic toxicity caused by flaccid paralysis. These pharmaceutically valuable enzymes have also been helpful in the study of SNARE functions. As can be seen in Figure 1A, SNARE bundle formation causes vesicle docking at the presynapse. Although these toxins are systemically toxic, no known cytotoxic effects have been reported with the curious exception of the Botulinum serotype C [1]. This enzyme cleaves intracellular SNAP25, as does serotype A and E, but also, exceptionally, cleaves Syntaxin 1. Using an array of lipid and polymer transfection reagents we were able to deliver different combinations of Botulinum holoenzymes into the normally unaffected, Neuro2A, SH-SY5Y, PC12, and Min6 cells to analyze the individual contribution of each SNARE protein and their cleaved peptide products

Binary polypeptide system for permanent and oriented protein immobilization.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling. RESULTS: Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. CONCLUSIONS: IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.Published versio

New botulinum neurotoxin constructs for treatment of chronic pain

Chronic pain affects one in five people across human societies, with few therapeutic options available. Botulinum neurotoxin (BoNT) can provide long-lasting pain relief by inhibiting local release of neuropeptides and neurotransmitters, but its highly paralytic nature has limited its analgesic potential. Recent advances in protein engineering have raised the possibility of synthesising non-paralysing botulinum molecules for translation to pain sufferers. However, the synthesis of these molecules, via several synthetic steps, has been challenging. Here, we describe a simple platform for safe production of botulinum molecules for treating nerve injury–induced pain. We produced two versions of isopeptide-bonded BoNT from separate botulinum parts using an isopeptide bonding system. Although both molecules cleaved their natural substrate, SNAP25, in sensory neurons, the structurally elongated iBoNT did not cause motor deficit in rats. We show that the non-paralytic elongated iBoNT targets specific cutaneous nerve fibres and provides sustained pain relief in a rat nerve injury model. Our results demonstrate that novel botulinum molecules can be produced in a simple and safe manner and be useful for treating neuropathic pain

Burkholderia Lethal Factor 1, a Novel Anti-Cancer Toxin, Demonstrates Selective Cytotoxicity in MYCN-Amplified Neuroblastoma Cells

Immunotoxins are being investigated as anti-cancer therapies and consist of a cytotoxic enzyme fused to a cancer targeting antibody. All currently used toxins function via the inhibition of protein synthesis, making them highly potent in both healthy and transformed cells. This non-specific cell killing mechanism causes dose-limiting side effects that can severely limit the potential of immunotoxin therapy. In this study, the recently characterised bacterial toxin Burkholderia lethal factor 1 (BLF1) is investigated as a possible alternative payload for targeted toxin therapy in the treatment of neuroblastoma. BLF1 inhibits translation initiation by inactivation of eukaryotic initiation translation factor 4A (eIF4A), a putative anti-cancer target that has been shown to regulate a number of oncogenic proteins at the translational level. We show that cellular delivery of BLF1 selectively induces apoptosis in neuroblastoma cells that display MYCN amplification but has little effect on non-transformed cells. Future immunotoxins based on this enzyme may therefore have higher specificity towards MYCN-amplified cancer cells than more conventional ribosome-inactivating proteins, leading to an increased therapeutic window and decreased side effects

Selective neuronal silencing using synthetic botulinum molecules alleviates chronic pain in mice

Chronic pain is a widespread debilitating condition affecting millions of people worldwide. Although several pharmacological treatments for relieving chronic pain have been developed, they require frequent chronic administration and are often associated with severe adverse events, including overdose and addiction. Persistent increased sensitization of neuronal subpopulations of the peripheral and central nervous system has been recognized as a central mechanism mediating chronic pain, suggesting that inhibition of specific neuronal subpopulations might produce antinociceptive effects. We leveraged the neurotoxic properties of the botulinum toxin to specifically silence key pain-processing neurons in the spinal cords of mice. We show that a single intrathecal injection of botulinum toxin conjugates produced long-lasting pain relief in mouse models of inflammatory and neuropathic pain without toxic side effects. Our results suggest that this strategy might be a safe and effective approach for relieving chronic pain while avoiding the adverse events associated with repeated chronic drug administration

Nonparalytic botulinum molecules for the control of pain

Local injections of botulinum toxins have been reported to be useful not only for the treatment of peripheral neuropathic pain and migraine but also to cause long-lasting muscle paralysis, a potentially serious side effect. Recently, a botulinum A-based molecule ("BiTox") has been synthesized that retains neuronal silencing capacity without triggering muscle paralysis. In this study, we examined whether BiTox delivered peripherally was able to reduce or prevent the increased nociceptive sensitivity found in animal models of inflammatory, surgical, and neuropathic pain. Plasma extravasation and edema were also measured as well as keratinocyte proliferation. No motor deficits were seen and acute thermal and mechanical nociceptive thresholds were unimpaired by BiTox injections. We found reduced plasma extravasation and inflammatory edema as well as lower levels of keratinocyte proliferation in cutaneous tissue after local BiTox injection. However, we found no evidence that BiTox was transported to the dorsal root ganglia or dorsal horn and no deficits in formalin-elicited behaviors or capsaicin or formalin-induced c-Fos expression within the dorsal horn. In contrast, Bitox treatment strongly reduced A-nociceptor-mediated secondary mechanical hyperalgesia associated with either complete Freund's adjuvant (CFA)-induced joint inflammation or capsaicin injection and the hypersensitivity associated with spared nerve injury. These results imply that although local release of neuromodulators from C-fibers was inhibited by BiTox injection, C-nociceptive signaling function was not impaired. Taken together with recent clinical data the results suggest that BiTox should be considered for treatment of pain conditions in which A-nociceptors are thought to play a significant role

New botulinum neurotoxin constructs for treatment of chronic pain

Chronic pain affects one in five people across human societies, with few therapeutic options available. Botulinum neurotoxin (BoNT) can provide long-lasting pain relief by inhibiting local release of neuropeptides and neurotransmitters, but its highly paralytic nature has limited its analgesic potential. Recent advances in protein engineering have raised the possibility of synthesising non-paralysing botulinum molecules for translation to pain sufferers. However, the synthesis of these molecules, via several synthetic steps, has been challenging. Here, we describe a simple platform for safe production of botulinum molecules for treating nerve injury-induced pain. We produced two versions of isopeptide-bonded BoNT from separate botulinum parts using an isopeptide bonding system. Although both molecules cleaved their natural substrate, SNAP25, in sensory neurons, the structurally elongated iBoNT did not cause motor deficit in rats. We show that the non-paralytic elongated iBoNT targets specific cutaneous nerve fibres and provides sustained pain relief in a rat nerve injury model. Our results demonstrate that novel botulinum molecules can be produced in a simple and safe manner and be useful for treating neuropathic pain

Re-Assembled Botulinum Neurotoxin Inhibits CNS Functions without Systemic Toxicity

The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications