Split luciferase-based assay to detect botulinum neurotoxins using hiPSC-derived motor neurons.

Botulinum neurotoxins (BoNTs) have been widely used clinically as a muscle relaxant. These toxins target motor neurons and cleave proteins essential for neurotransmitter release like Synaptosomal-associated protein of 25 kDa (SNAP-25). In vitro assays for BoNT testing using rodent cells or immortalized cell lines showed limitations in accuracy and physiological relevance. Here, we report a cell-based assay for detecting SNAP-25-cleaving BoNTs by combining human induced Pluripotent Stem Cells (hiPSC)-derived motor neurons and a luminescent detection system based on split NanoLuc luciferase. This assay is convenient, rapid, free-of-specialized antibodies, with a detection sensitivity of femtomolar concentrations of toxin, and can be used to study the different steps of BoNT intoxication.

BoNT/A1 Secondary Failure for the Treatment of Neurogenic Detrusor Overactivity: An Ex Vivo Functional Study.

Management of neurogenic detrusor overactivity (NDO) remains a clinical priority to improve patients' quality of life and prevent dramatic urological complications. Intradetrusor injection of onabotulinumtoxinA (BoNT/A1, botulinum neurotoxin A1) is approved as second therapeutic line in these patients, demonstrating a good efficacy. However, a loss of its efficacy over time has been described, with no clear understanding of the underlying mechanisms. This paper aims at shedding new light on BoNT/A1 secondary failure in NDO through functional and structural analysis. Three groups of patients (either non-NDO, NDO with no toxin history or toxin secondary failure) were investigated using an ex vivo bladder strip assay. Detrusor strips were tensed in organ baths and submitted to electrical field stimulation to generate contractions. Recombinant BoNT/A1 was then added at various concentrations and contractions recorded for 4 h. Histology exploring BoNT/A1 targets, fibrosis and neuronal markers was also used. Detrusor strips from patients with BoNT/A1 secondary failure displayed a smaller sensitivity to toxin ex vivo at 3 nM compared to the other groups. Histological evaluation demonstrated the presence of cleaved Synaptosomal-Associated Protein, 25 kDa (c-SNAP25) in the detrusor from the toxin-secondary failure population, indicating some remaining in vivo sensitivity to BoNT/A1 despite the therapeutic escape. Moreover, residual c-SNAP25 did not affect parasympathetic-driven contractions observed ex vivo. This study confirms the slightly lower efficacy of BoNT/A1 in the BoNT/A1 secondary failure NDO group, suggesting that the escape from BoNT/A1 efficacy in NDO occurs at least at the parasympathetic level and could imply compensatory mechanisms for detrusor contraction.

hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) are zinc metalloproteases that block neurotransmitter release at the neuromuscular junction (NMJ). Their high affinity for motor neurons combined with a high potency have made them extremely effective drugs for the treatment of a variety of neurological diseases as well as for aesthetic applications. Current in vitro assays used for testing and developing BoNT therapeutics include primary rodent cells and immortalized cell lines. Both models have limitations concerning accuracy and physiological relevance. In order to improve the translational value of preclinical data there is a clear need to use more accurate models such as human induced Pluripotent Stem Cells (hiPSC)-derived neuronal models. In this study we have assessed the potential of four different human iPSC-derived neuronal models including Motor Neurons for BoNT testing. We have characterized these models in detail and found that all models express all proteins needed for BoNT intoxication and showed that all four hiPSC-derived neuronal models are sensitive to both serotype A and E BoNT with Motor Neurons being the most sensitive. We showed that hiPSC-derived Motor Neurons expressed authentic markers after only 7 days of culture, are functional and able to form active synapses. When cultivated with myotubes, we demonstrated that they can innervate myotubes and induce contraction, generating an in vitro model of NMJ showing dose-responsive sensitivity BoNT intoxication. Together, these data demonstrate the promise of hiPSC-derived neurons, especially Motor Neurons, for pharmaceutical BoNT testing and development.

Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models.

Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1MY) and E1191Q/S1199W (rBoNT/B1QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease.

Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.

Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT-induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well-tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species-specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

Engineering Botulinum Toxins to Improve and Expand Targeting and SNARE Cleavage Activity.

Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar multi-domain structure at the molecular level. In nature, BoNTs are encoded by DNA in producing clostridial bacteria and, as such, are amenable to recombinant production through insertion of the coding DNA into other bacterial species. This, in turn, creates possibilities for protein engineering. Here, we review the production of BoNTs by the natural host and also recombinant production approaches utilised in the field. Applications of recombinant BoNT-production include the generation of BoNT-derived domain fragments, the creation of novel BoNTs with improved performance and enhanced therapeutic potential, as well as the advancement of BoNT vaccines. In this article, we discuss site directed mutagenesis, used to affect the biological properties of BoNTs, including approaches to alter their binding to neurons and to alter the specificity and kinetics of substrate cleavage. We also discuss the target secretion inhibitor (TSI) platform, in which the neuronal binding domain of BoNTs is substituted with an alternative cellular ligand to re-target the toxins to non-neuronal systems. Understanding and harnessing the potential of the biological diversity of natural BoNTs, together with the ability to engineer novel mutations and further changes to the protein structure, will provide the basis for increasing the scope of future BoNT-based therapeutics.

The Expanding Therapeutic Utility of Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is a major therapeutic agent that is licensed in neurological indications, such as dystonia and spasticity. The BoNT family, which is produced in nature by clostridial bacteria, comprises several pharmacologically distinct proteins with distinct properties. In this review, we present an overview of the current therapeutic landscape and explore the diversity of BoNT proteins as future therapeutics. In recent years, novel indications have emerged in the fields of pain, migraine, overactive bladder, osteoarthritis, and wound healing. The study of biological effects distal to the injection site could provide future opportunities for disease-tailored BoNT therapies. However, there are some challenges in the pharmaceutical development of BoNTs, such as liquid and slow-release BoNT formulations; and, transdermal, transurothelial, and transepithelial delivery. Innovative approaches in the areas of formulation and delivery, together with highly sensitive analytical tools, will be key for the success of next generation BoNT clinical products.

Augmentation of VAMP-catalytic activity of botulinum neurotoxin serotype B does not result in increased potency in physiological systems.

Botulinum neurotoxins (BoNTs) are used extensively as therapeutic agents. Serotypes A and B are available as marketed products. Higher doses of BoNT/B are required to reach an efficacy similar to that of products containing BoNT/A. Advances in our understanding of BoNT/B mechanism of action have afforded the opportunity to make rational modifications to the toxin aimed at increasing its activity. Recently, a mutation in the light chain of BoNT/B (S201P) was described that increases the catalytic activity of the isolated BoNT/B light chain in biochemical assays. In this study, we have produced two full-length recombinant BoNT/B toxins in E.coli-one wild type (rBoNT/B1) and one incorporating the S201P mutation (rBoNT/B1(S201P)). We have compared the activity of these two molecules along with a native BoNT/B1 in biochemical cell-free assays and in several biological systems. In the cell-free assay, which measured light-chain activity alone, rBoNT/B1(S201P) cleaved VAMP-2 and VAMP-1 substrate with an activity 3-4-fold higher than rBoNT/B1. However, despite the enhanced catalytic activity of rBoNT/B1(S201P), there was no significant difference in potency between the two molecules in any of the in vitro cell-based assays, using either rodent spinal cord neurons or cortical neurons. Similarly in ex vivo tissue preparations rBoNT/B1(S201P) was not significantly more potent than rBoNT/B1 at inhibiting either diaphragm or detrusor (bladder) muscle activity in C57BL/6N and CD1 mice. Finally, no differences between rBoNT/B1 and rBoNT/B1(S201P) were observed in an in vivo digit abduction score (DAS) assay in C57BL/6N mice, either in efficacy or safety parameters. The lack of translation from the enhanced BoNT/B1(S201P) catalytic activity to potency in complex biological systems suggests that the catalytic step is not the rate-limiting factor for BoNT/B to reach maximum efficacy. In order to augment the efficacy of BoNT/B in humans, strategies other than enhancing light chain activity may need to be considered.

Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors.

Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.Humans are less sensitive to the therapeutic effects of botulinum neurotoxin B (BoNT/B) than the animal models it is tested on due to differences between the human and the mouse receptors. Here, the authors engineer BoNT/B to improve its affinity to human receptors and enhance its therapeutic efficacy.

Botulinum Neurotoxins Serotypes A and B induce paralysis of mouse striated and smooth muscles with different potencies.

To address the scarcity of direct comparison of botulinum neurotoxin serotypes activity on smooth versus striatal muscle, we have studied the action of BoNT/A1 and BoNT/B1 on ex vivo preparations of both muscle types. We have set up and characterized a model of neurogenic contractions in the isolated mouse bladder, and used this model to explore the effects of the two serotypes on contractions evoked by electrical field stimulation. Both toxins were also tested in the mouse phrenic nerve hemidiaphragm assay, to compare their potency in smooth versus striated muscle. The characterization of the model of neurogenic contractions in the isolated mouse bladder indicates that about half of the activity is driven by purinergic signaling, and about half by cholinergic signaling. Furthermore, we find that BoNT/B1 is more potent than BoNT/A1 in inhibiting activity in the mouse detrusor smooth muscle preparation, but that both toxins have comparable potency on the striated muscle activity of the phrenic nerve hemidiaphragm model. We also show that these findings are mouse strain independent. In conclusion, the established mouse bladder detrusor smooth muscle model is able to discriminate between different botulinum neurotoxin serotypes and could be a useful preclinical tool to explore the pathophysiology of bladder overactivity, as well as the effects of new therapeutic candidates. It is interesting to note that the high proportion of purinergic transmission driving detrusor contractions in this model is similar to that seen in neurodetrusor overactivity disease, making this model relevant with regard to pathophysiological interest.

SCN10A Mutation in a Patient with Erythromelalgia Enhances C-Fiber Activity Dependent Slowing.

Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences.

Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes.

Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1ß1δε, α3ß2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1ß1δε, α3ß2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses.

Reduced efficacy of the intravenous anesthetic agent AZD3043 at GABA(A) receptors with ß2 (N289M) and ß3 (N290M) point-mutations.

AZD3043 (previously named THRX-918661) is a novel short-acting intravenous anesthetic agent in clinical trials. Although AZD3043 is a positive modulator at the γ-aminobutyric acid (GABA)(A)-receptor, its potency and efficacy have not been characterized in detail. Nor is it known whether the point-mutations in the ß-subunit of the GABA(A)-receptor that dramatically reduce the anesthetic effect of propofol (i.e. ß2 (N289M) and ß3 (N290M)), also influence the effect of AZD3043. This study investigated the in vitro pharmacology of AZD3043 at the most common human GABA(A) receptor subtypes. Subunits of four human wild-type (α1ß2, α1ß2γ2, α2ß2γ2 and α2ß3γ2) and two mutant (α1ß2(N289M)γ2 and α2ß3(N290M)γ2) GABA(A) receptor channels were introduced into Xenopus oocytes and studied with two-electrode voltage-clamp. AZD3043 potentiated and directly activated the α1ß2γ2, α2ß2γ2 and α2ß3γ2 GABA(A) receptor subtypes. Moreover, both potency and efficacy of AZD3043 were reduced at the mutant α1ß2(N289M)γ2 and α2ß3(N290M)γ2 subtypes. AZD3043 increased the GABA response also in GABA(A) receptors lacking the γ2-subunit, i.e. α1ß2. In conclusion, AZD3043 is a positive modulator and a direct agonist at human GABA(A) receptors and is not dependent on the γ2-subunit for its effect. Similar to propofol, the effect of AZD3043 is dramatically reduced by point-mutations in the ß2(N289M) and ß3(N290M) subunits, indicating similar molecular mechanisms of action for propofol and AZD3043 at the human GABA(A) receptor.

Linkage analysis and functional evaluation of inherited clinical pain conditions.

Because ion channel function is a fundamental element of any nociceptive signalling, it is not surprising that numerous channelopathies have recently emerged as likely causes of several inherited clinical pain conditions. For example, numerous missense mutations in the Na(v)1.7 gene SCN9A have recently been linked to a congenital inability to sense pain. Establishing the link between a clinical pain phenotype to an inherited molecular dysfunction of a specific protein has its challenges and requires the collaboration between many specialists. However, once established, such a linkage offers the promise of a powerful and elegant way to mechanistically explain the aspects of the disease studied.

Ion channel screening technology.

Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.

Voltage-gated sodium channels in neurological disorders.

Voltage-gated sodium channels play an essential biophysical role in many excitable cells such as neurons. They transmit electrical signals through action potential (AP) generation and propagation in the peripheral (PNS) and central nervous systems (CNS). Each sodium channel is formed by one alpha-subunit and one or more beta-subunits. There is growing evidence indicating that mutations, changes in expression, or inappropriate modulation of these channels can lead to electrical instability of the cell membrane and inappropriate spontaneous activity observed during pathological states. This review describes the biochemical, biophysical and pharmacological properties of neuronal voltage-gated sodium channels (VGSC) and their implication in several neurological disorders.

Biophysical properties of human Na v1.7 splice variants and their regulation by protein kinase A.

The sodium channel Na(v)1.7 is preferentially expressed in nociceptive neurons and is believed to play a crucial role in pain sensation. Four alternative splice variants are expressed in human dorsal root ganglion neurons, two of which differ in exon 5 by two amino acids in the S3 segment of domain I (exons 5A and 5N). Two others differ in exon 11 by the presence (11L) or absence (11S) of an 11 amino acid sequence in the loop between domains I and II, an important region for PKA regulation. In the present study, we used the whole cell configuration of the patch-clamp technique to investigate the biophysical properties and 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) modulation of these splice variants expressed in tsA201 cells in the presence of the beta(1)-subunit. The alternative splicing of Na(v)1.7 had no effect on most of the biophysical properties of this channel, including activation, inactivation, and recovery from inactivation. However, development of inactivation experiments revealed that the isoform containing exon 5A had slower kinetics of inactivation for negative potentials than that of the variant containing exon 5N. This difference was associated with higher ramp current amplitudes for isoforms containing exon 5A. Moreover, 8Br-cAMP-mediated phosphorylation induced a negative shift of the activation curve of variants containing exon 11S, whereas inactivation properties were unchanged. Isoforms with exon 11L were not modulated by 8Br-cAMP-induced phosphorylation. We conclude that alternative splicing of human Na(v)1.7 can specifically modulate the biophysical properties and cAMP-mediated regulation of this channel. Changing the proportions of these variants may thus influence neuronal excitability and pain sensation.

Cellular HTS assays for pharmacological characterization of Na(V)1.7 modulators.

Ion channels are challenging targets in the early phases of the drug discovery process, especially because of the lack of technologies available to screen large numbers of compounds in functionally relevant assays. The electrophysiological patch-clamp technique, which is the gold standard for studying ion channels, has low throughput and is not amenable to screening large numbers of compounds. However, for random high-throughput screening (HTS) of compounds against ion channel targets, a number of functional cellular assays have become available during the last few years. Here we use the sodium channel NaV1.7 stably expressed in human embryonic kidney 293 cells and compare three HTS assays-a Li flux atomic absorption spectroscopy (AAS) assay, a fluorescent imaging plate reader (FLIP, Molecular Devices, Sunnyvale, CA) membrane potential assay, and a fluorescence resonance energy transfer (FRET)-based membrane potential assay-to an automated electrophysiological assay (the Ionworks HT [Molecular Devices] platform) and characterize 11 known NaV inhibitors. Our results show that all three HTS assays are suitable for identification of NaV1.7 inhibitors, but as an HTS assay the Li-AAS assay is more robust with higher Z' values than the FLIPR and FRET-based membrane potential assays. Furthermore, there was a better correlation between the Ionworks assay and the Li-AAS assay regarding the potency of the NaV inhibitors investigated. This paper describes the first comparison between all the HTS assays available today to study voltage-gated NaVs, and the results suggest that the Li-AAS assay is more suited as a first HTS assay when starting an NaV drug discovery campaign.