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Glenn F. King - One of the best experts on this subject based on the ideXlab platform.
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Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of NaV1.7
Biomedicines, 2020Co-Authors: Kathleen Yin, Jennifer R Deuis, Zoltan Dekan, Volker Herzig, Glenn F. King, Ai-hua Jin, Paul F. Alewood, Irina VetterAbstract:Spider Venom is a novel source of disulfide-rich Peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel Peptides from the Venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue Peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue Peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native Peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of Spider Venom-derived Peptides with improved potency and selectivity at NaV channels in the future.
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The antitrypanosomal diarylamidines, diminazene and pentamidine, show anthelmintic activity against Haemonchus contortus in vitro.
Veterinary parasitology, 2019Co-Authors: Samantha A. Nixon, Volker Herzig, Glenn F. King, Natalie J. Saez, Andrew C. KotzeAbstract:Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC50 4.9 μM and 16.1 μM, respectively, in a drug-susceptible isolate) with no existing cross-resistance in two multidrug resistant isolates and a monepantel-resistant isolate. Combinations of pentamidine with commercial anthelmintics showed additive activity, with no significant synergism detected. Pentamidine and diminazene showed different life-stage patterns of activity; both were active against early stage larvae in development assays, but only diminazene was active against the infective L3 stage in migration assays. This suggests some differences in uptake of the two drugs across the nematode cuticle, or differences in the nature and expression patterns of their molecular targets. As pentamidine and diminazene have been reported to be potent inhibitors of mammalian acid-sensing ion channels (ASIC), we tested the activity of known ASIC inhibitors against H. contortus to probe whether these channels may represent potential anthelmintic targets in nematodes. Remarkably, the Spider-Venom Peptide Hi1a, a potent inhibitor of ASIC1a, inhibited H. contortus larval development with an IC50 of 22.9 ± 1.9 μM. This study highlights the potential use of diarylamidines as anthelmintics, although their activity needs to be confirmed in vivo. In addition, our demonstration that ASIC inhibitors have anthelmintic activity raises the possibility that this family of ion channels may represent a novel anthelmintic target.
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the modulation of acid sensing ion channel 1 by pctx1 is ph subtype and species dependent importance of interactions at the channel subunit interface and potential for engineering selective analogues
Biochemical Pharmacology, 2019Co-Authors: Ben Cristoforiarmstrong, Irene R Chassagnon, Glenn F. King, Natalie J. Saez, Lachlan D. RashAbstract:Acid-sensing ion channels (ASICs) are primary acid sensors in the mammalian nervous system that are activated by protons under conditions of local acidosis. They have been implicated in a range of pathologies including ischemic stroke (ASIC1a subtype) and peripheral pain (ASIC1b and ASIC3). Although the Spider Venom Peptide PcTx1 is the best-studied ASIC modulator and is neuroprotective in rodent models of ischemic stroke, little experimental work has been done to examine its molecular interaction with human ASIC1a or the off-target ASIC1b. The complementary face of the acidic pocket binding site of PcTx1 is where these channels differ in sequence. We show here that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. We examined the pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b, and show that inhibitory and stimulatory effects at each ASIC1 variant is driven mostly by a shared set of core Peptide pharmacophore residues that bind to the thumb domain, while Peptide residues that interact with the complementary face of the biding site underlie species and subtype-dependent differences in activity that may allow manipulation of ASIC1 variant selectivity. Finally, the stimulatory effect of PcTx1 on rat ASIC1a when applied under mildly alkaline pH correlates with low receptor occupancy. These new insights into the interactions between PcTx1 with ASIC1 subtypes demonstrates the complexity of its mechanism of action, and highlights important implications to consider when using PcTx1 as a pharmacological tool to study ASIC function.
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Can we resolve the taxonomic bias in Spider Venom research?
Elsevier, 2019Co-Authors: Volker Herzig, Glenn F. King, Eivind A.b. UndheimAbstract:The rate of discovery of new Spider species greatly exceeds the rate of Spider Venom characterisation, leading to an increasing number of species with unstudied Venoms. However, recent advances in proteomics and genomics that enable the study of Venoms from smaller species has expanded the accessible taxonomic range. Thus, although the number of unstudied Spider Venoms is likely to further increase, future research should focus on the characterisation of Venoms and toxins from previously unstudied Spider families. Keywords: Spider, Venom, Peptide, Diversity, Taxonomic, Transcriptomics, Proteomic
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molecular basis of the interaction between gating modifier Spider toxins and the voltage sensor of voltage gated ion channels
Scientific Reports, 2016Co-Authors: Glenn F. King, Mehdi MobliAbstract:Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most Spider-Venom Peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the Spider-Venom Peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.
Lachlan D. Rash - One of the best experts on this subject based on the ideXlab platform.
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the modulation of acid sensing ion channel 1 by pctx1 is ph subtype and species dependent importance of interactions at the channel subunit interface and potential for engineering selective analogues
Biochemical Pharmacology, 2019Co-Authors: Ben Cristoforiarmstrong, Irene R Chassagnon, Glenn F. King, Natalie J. Saez, Lachlan D. RashAbstract:Acid-sensing ion channels (ASICs) are primary acid sensors in the mammalian nervous system that are activated by protons under conditions of local acidosis. They have been implicated in a range of pathologies including ischemic stroke (ASIC1a subtype) and peripheral pain (ASIC1b and ASIC3). Although the Spider Venom Peptide PcTx1 is the best-studied ASIC modulator and is neuroprotective in rodent models of ischemic stroke, little experimental work has been done to examine its molecular interaction with human ASIC1a or the off-target ASIC1b. The complementary face of the acidic pocket binding site of PcTx1 is where these channels differ in sequence. We show here that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. We examined the pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b, and show that inhibitory and stimulatory effects at each ASIC1 variant is driven mostly by a shared set of core Peptide pharmacophore residues that bind to the thumb domain, while Peptide residues that interact with the complementary face of the biding site underlie species and subtype-dependent differences in activity that may allow manipulation of ASIC1 variant selectivity. Finally, the stimulatory effect of PcTx1 on rat ASIC1a when applied under mildly alkaline pH correlates with low receptor occupancy. These new insights into the interactions between PcTx1 with ASIC1 subtypes demonstrates the complexity of its mechanism of action, and highlights important implications to consider when using PcTx1 as a pharmacological tool to study ASIC function.
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discovery and molecular interaction studies of a highly stable tarantula Peptide modulator of acid sensing ion channel 1
Neuropharmacology, 2017Co-Authors: Sing Yan Er, Ben Cristoforiarmstrong, Pierre Escoubas, Lachlan D. RashAbstract:Abstract Acute pharmacological inhibition of acid-sensing ion channel 1a (ASIC1a) is efficacious in rodent models in alleviating symptoms of neurological diseases such as stroke and multiple sclerosis. Thus, ASIC1a is a promising therapeutic target and selective ligands that modulate it are invaluable research tools and potential therapeutic leads. Spider Venoms have provided an abundance of voltage-gated ion channel modulators, however, only one ASIC modulator (PcTx1) has so far been isolated from this source. Here we report the discovery, characterization, and chemical stability of a second Spider Venom Peptide that potently modulates ASIC1a and ASIC1b, and investigate the molecular basis for its subtype selectivity. π-TRTX-Hm3a (Hm3a) is a 37-amino acid Peptide isolated from Togo starburst tarantula (Heteroscodra maculata) Venom with five amino acid substitutions compared to PcTx1, and is also three residues shorter at the C-terminus. Hm3a pH-dependently inhibited ASIC1a with an IC50 of 1–2 nM and potentiated ASIC1b with an EC50 of 46.5 nM, similar to PcTx1. Using ASIC1a to ASIC1b point mutants in rat ASIC1a revealed that Glu177 and Arg175 in the palm region opposite α-helix 5 play an important role in the Hm3a-ASIC1 interaction and contribute to the subtype-dependent effects of the Peptide. Despite its high sequence similarity with PcTx1, Hm3a showed higher levels of stability over 48 h. Overall, Hm3a represents a potent, highly stable tool for the study of ASICs and will be particularly useful when stability in biological fluids is required, for example in long term in vitro cell-based assays and in vivo experiments. This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.’
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Selective inhibition of ASIC1a confers functional and morphological neuroprotection following traumatic spinal cord injury [version 1; referees: 1 approved, 1 approved with reservations]
F1000Research, 2016Co-Authors: Liam M. Koehn, Glenn F. King, Lachlan D. Rash, Qing Dong, Katarzyna M. Dziegielewska, Norman R. Saunders, Mark D. HabgoodAbstract:Tissue loss after spinal trauma is biphasic, with initial mechanical/haemorrhagic damage at the time of impact being followed by gradual secondary expansion into adjacent, previously unaffected tissue. Limiting the extent of this secondary expansion of tissue damage has the potential to preserve greater residual spinal cord function in patients. The acute tissue hypoxia resulting from spinal cord injury (SCI) activates acid-sensing ion channel 1a (ASIC1a). We surmised that antagonism of this channel should provide neuroprotection and functional preservation after SCI. We show that systemic administration of the Spider-Venom Peptide PcTx1, a selective inhibitor of ASIC1a, improves locomotor function in adult Sprague Dawley rats after thoracic SCI. The degree of functional improvement correlated with the degree of tissue preservation in descending white matter tracts involved in hind limb locomotor function. Transcriptomic analysis suggests that PcTx1-induced preservation of spinal cord tissue does not result from a reduction in apoptosis, with no evidence of down-regulation of key genes involved in either the intrinsic or extrinsic apoptotic pathways. We also demonstrate that trauma-induced disruption of blood-spinal cord barrier function persists for at least 4 days post-injury for compounds up to 10 kDa in size, whereas barrier function is restored for larger molecules within a few hours. This temporary loss of barrier function provides a "treatment window" through which systemically administered drugs have unrestricted access to spinal tissue in and around the sites of trauma. Taken together, our data provide evidence to support the use of ASIC1a inhibitors as a therapeutic treatment for SCI. This study also emphasizes the importance of objectively grading the functional severity of initial injuries (even when using standardized impacts) and we describe a simple scoring system based on hind limb function that could be adopted in future studies.
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Molecular dynamics and functional studies define a hot spot of crystal contacts essential for PcTx1 inhibition of acid-sensing ion channel 1a.
British journal of pharmacology, 2015Co-Authors: Natalie J. Saez, Irene R Chassagnon, Mehdi Mobli, Evelyne Deplazes, Ben Cristofori-armstrong, Xiaozhen Lin, Alan E. Mark, Lachlan D. Rash, Glenn F. KingAbstract:Background and Purpose The Spider‐Venom Peptide PcTx1 is the most potent and selective inhibitor of acid‐sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic β‐hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous Peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important.
Mehdi Mobli - One of the best experts on this subject based on the ideXlab platform.
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Recombinant production, bioconjugation and membrane binding studies of Pn3a, a selective NaV1.7 inhibitor
Biochemical pharmacology, 2020Co-Authors: Gagan Sharma, Jennifer R Deuis, Irina Vetter, Xinying Jia, Alexander Mueller, Mehdi MobliAbstract:Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (NaVs) play a critical role in this process. In particular, the NaV subtype 1.7, has emerged as a promising universal target for the treatment of pain. Recently, a Spider Venom Peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of NaV1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development. We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the Peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the Peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.
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Evaluation of Chemical Strategies for Improving the Stability and Oral Toxicity of Insecticidal Peptides
MDPI AG, 2018Co-Authors: Volker Herzig, Yanni K Y Chin, Mehdi Mobli, Monique J Windley, Aline Dantas De Araujo, Kathryn P. Greenwood, Youmie Chong, Markus Muttenthaler, Neil Audsley, Graham M. NicholsonAbstract:Spider Venoms are a rich source of insecticidal Peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal Spider-Venom Peptide ω-HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the Peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability
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potent neuroprotection after stroke afforded by a double knot Spider Venom Peptide that inhibits acid sensing ion channel 1a
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Irene R Chassagnon, Claudia A Mccarthy, Yanni K Y Chin, Sandy S Pineda, Angelo Keramidas, Mehdi Mobli, Vi Pham, Michael T De Silva, Joseph W Lynch, Robert E WiddopAbstract:Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich Spider Venom Peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury.
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molecular basis of the interaction between gating modifier Spider toxins and the voltage sensor of voltage gated ion channels
Scientific Reports, 2016Co-Authors: Glenn F. King, Mehdi MobliAbstract:Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most Spider-Venom Peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the Spider-Venom Peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.
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Molecular dynamics and functional studies define a hot spot of crystal contacts essential for PcTx1 inhibition of acid-sensing ion channel 1a.
British journal of pharmacology, 2015Co-Authors: Natalie J. Saez, Irene R Chassagnon, Mehdi Mobli, Evelyne Deplazes, Ben Cristofori-armstrong, Xiaozhen Lin, Alan E. Mark, Lachlan D. Rash, Glenn F. KingAbstract:Background and Purpose The Spider‐Venom Peptide PcTx1 is the most potent and selective inhibitor of acid‐sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic β‐hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous Peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important.
Maya Topf - One of the best experts on this subject based on the ideXlab platform.
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The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Nav1.7
Journal of the American Chemical Society, 2017Co-Authors: Zoë V. F. Wright, Stephen Mccarthy, Rachael Dickman, Francis E. Reyes, Silvia Sanchez-martinez, Adam Cryar, Ian R. Kilford, Adrian Hall, Andrew K. Takle, Maya TopfAbstract:Spider Venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid Peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such Peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear Peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK Spider Venom Peptide not bound to a substrate.
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The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Nav1.7
2017Co-Authors: Zoë V. F. Wright, Stephen Mccarthy, Rachael Dickman, Francis E. Reyes, Silvia Sanchez-martinez, Adam Cryar, Ian R. Kilford, Adrian Hall, Andrew K. Takle, Maya TopfAbstract:Spider Venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid Peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such Peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear Peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK Spider Venom Peptide not bound to a substrate
Zhonghua Liu - One of the best experts on this subject based on the ideXlab platform.
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engineering gain of function analogues of the Spider Venom Peptide hntx i a potent blocker of the hnav1 7 sodium channel
Toxins, 2018Co-Authors: Yunxiao Zhang, Song-ping Liang, Qiuchu Yang, Qingfeng Zhang, Dezheng Peng, Minzhi Chen, Xi Zhou, Zhonghua LiuAbstract:Pain is a medical condition that interferes with normal human life and work and reduces human well-being worldwide. The voltage-gated sodium channel (VGSC) human NaV1.7 (hNaV1.7) is a compelling target that plays a key role in human pain signaling. The 33-residue Peptide µ-TRTX-Hhn2b (HNTX-I), a member of NaV-targeting Spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian VGSCs, including the hNaV1.7 channel. We engineered analogues of HNTX-I based on sequence conservation in NaSpTx family 1. Substitution of Asn for Ser at position 23 or Asp for His at position 26 conferred potent activity against hNaV1.7. Moreover, multiple site mutations combined together afforded improvements in potency. Ultimately, we generated an analogue E1G⁻N23S⁻D26H⁻L32W with >300-fold improved potency compared with wild-type HNTX-1 on hNaV1.7 (IC50 0.036 ± 0.007 µM). Structural simulation suggested that the charged surface and the hydrophobic surface of the modified Peptide are responsible for binding affinity to the hNaV1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNaV1.7 channel.
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Engineering Gain-of-Function Analogues of the Spider Venom Peptide HNTX-I, A Potent Blocker of the hNaV1.7 Sodium Channel
MDPI AG, 2018Co-Authors: Yunxiao Zhang, Song-ping Liang, Qiuchu Yang, Qingfeng Zhang, Dezheng Peng, Minzhi Chen, Xi Zhou, Zhonghua LiuAbstract:Pain is a medical condition that interferes with normal human life and work and reduces human well-being worldwide. Human voltage-gated sodium channel NaV1.7 (hNaV1.7) is a compelling target that plays a key role in human pain signaling. The 33-residue Peptide µ-TRTX-Hhn2b (HNTX-I), a member of NaV-targeting Spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian voltage-gated sodium channels (VGSCs), including the hNaV1.7 channel. We engineered analogues of HNTX-I based on sequence conservation in NaSpTx family 1. Substitution of Asn for Ser at position 23 or Asp for His at position 26 conferred potent activity against hNaV1.7. Moreover, multiple site mutations combined together afforded improvements in potency. Ultimately, we generated an analogue E1G–N23S–D26H–L32W with >300-fold improved potency compared with wild-type HNTX-I on hNaV1.7 (IC50 0.036 ± 0.007 µM). Structural simulation suggested that the charged surface and the hydrophobic surface of the modified Peptide are responsible for binding affinity to the hNaV1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNaV1.7 channel
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Spider toxin Peptide lycosin i functionalized gold nanoparticles for in vivo tumor targeting and therapy
Theranostics, 2017Co-Authors: Huaxin Tan, Song-ping Liang, Yazhou Huang, Bo Chen, Peng Zhang, Lehui Xiao, Zhonghua LiuAbstract:Cell penetrating Peptides (CPPs) are commonly utilized for intracellular delivery of functional materials to circumvent biomembrane barrier. However, further application of CPPs is hindered by lacking selectivity toward targeted cells. The Spider Venom Peptide, lycosin-I, is a CPP with potent cytotoxicity to cancer cells, which might enable lycosin-I to deliver functional materials into cancer cells selectively. In this study, we demonstrated that the lycosin-I-conjugated spherical gold nanoparticles (LGNPs) not only exhibited efficient cellular internalization efficiency toward cancer cells but also displayed unprecedented selectivity over noncancerous cells. Although LGNPs were removed from the living circulatory system via reticuloendothelial system-dominant clearance modes without noticeable adverse effects to animals, they actually displayed active tumor-targeting effects and efficient accumulation in tumors in vivo. Furthermore, the potential application of this platform for cancer therapy was explored by lycosin-I-conjugated gold nanorods (LGNRs). LGNRs exhibited selective intracellular translocation towards cancer cells and efficient photothermal effect under near infrared (NIR, 808 nm) irradiation, which consequently killed cancer cells in vitro and in vivo effectively. Therefore, the established LGNPs and LGNRs possessed great potential in cancer-targeting delivery and photothermal therapy.
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The Spider Venom Peptide Lycosin-II Has Potent Antimicrobial Activity against Clinically Isolated Bacteria
Toxins, 2016Co-Authors: Yong-jun Wang, Zhonghua Liu, Ling Wang, Huali Yang, Haoliang Xiao, Athar Farooq, Xiaoliu ShiAbstract:Antimicrobial Peptides have been accepted as excellent candidates for developing novel antibiotics against drug-resistant bacteria. Recent studies indicate that Spider Venoms are the source for the identification of novel antimicrobial Peptides. In the present study, we isolated and characterized an antibacterial Peptide named lycosin-II from the Venom of the Spider Lycosa singoriensis. It contains 21 amino acid residue lacking cysteine residues and forms a typical linear amphipathic and cationic α-helical conformation. Lycosin-II displays potent bacteriostatic effect on the tested drug-resistant bacterial strains isolated from hospital patients, including multidrug-resistant A. baumannii, which has presented a huge challenge for the infection therapy. The inhibitory ability of lycosin-II might derive from its binding to cell membrane, because Mg2+ could compete with the binding sites to reduce the bacteriostatic potency of lycosin-II. Our data suggest that lycosin-II might be a lead in the development of novel antibiotics for curing drug-resistant bacterial infections.
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Beneficial effects of trypsin inhibitors derived from a Spider Venom Peptide in L-arginine-induced severe acute pancreatitis in mice.
PloS one, 2013Co-Authors: Weiwen Ning, Song-ping Liang, Yong-jun Wang, Xiaoliu Shi, Fan Zhang, Hengyun Wang, Fan Wang, Xiaojuan Wang, Huaxin Tang, Zhonghua LiuAbstract:HWTI is a 55-residue protein isolated from the Venom of the Spider Ornithoctonus huwena. It is a potent trypsin inhibitor and a moderate voltage-gated potassium channel blocker. Here, we designed and expressed two HWTI mutants, HWTI-mut1 and HWTI-mut2, in which the potassium channel inhibitory activity was reduced while the trypsin inhibitory activity of the wild type form (approximately 5 EPU/mg) was retained. Animal studies showed that these mutants were less toxic than HWTI. The effects of HWTI and HWTI-mut1 were examined in a mouse model of acute pancreatitis induced by intraperitoneal injection of a large dose of L-arginine (4 mg/kg, twice). Serum amylase and serum lipase activities were assessed, and pathological sections of the pancreas were examined. Treatment with HWTI and HWTI-mut1 significantly reduced serum amylase and lipase levels in a dose dependent manner. Compared with the control group, at 4 mg/kg, HWTI significantly reduced serum amylase level by 47% and serum lipase level by 73%, while HWTI-mut1 significantly reduced serum amylase level by 59% and serum lipase level by 72%. Moreover, HWTI and HWTI-mut1 effectively protected the pancreas from acinar cell damage and inflammatory cell infiltration. The trypsin inhibitory potency and lower neurotoxicity of HWTI-mut1 suggest that it could potentially be developed as a drug for the treatment of acute pancreatitis with few side effects.