NaV1.7 targeted fluorescence imaging agents for nerve identification during intraoperative procedures.

Surgeries and trauma result in traumatic and iatrogenic nerve damage that can result in a debilitating condition that approximately affects 189 million individuals worldwide. The risk of nerve injury during oncologic surgery is increased due to tumors displacing normal nerve location, blood turbidity, and past surgical procedures, which complicate even an experienced surgeon's ability to precisely locate vital nerves. Unfortunately, there is a glaring absence of contrast agents to assist surgeons in safeguarding vital nerves. To address this unmet clinical need, we leveraged the abundant expression of the voltage-gated sodium channel 1.7 (NaV1.7) as an intraoperative marker to access peripheral nerves in vivo, and visualized nerves for surgical guidance using a fluorescently-tagged version of a potent NaV1.7-targeted peptide, Tsp1a, derived from a Peruvian tarantula. We characterized the expression of NaV1.7 in sensory and motor peripheral nerves across mouse, primate, and human specimens and demonstrated universal expression. We synthesized and characterized a total of 10 fluorescently labeled Tsp1a-peptide conjugates to delineate nerves. We tested the ability of these peptide-conjugates to specifically accumulate in mouse nerves with a high signal-to-noise ratio in vivo. Using the best-performing candidate, Tsp1a-IR800, we performed thyroidectomies in non-human primates and demonstrated successful demarcation of the recurrent laryngeal and vagus nerves, which are commonly subjected to irreversible damage. The ability of Tsp1a to enhance nerve contrast during surgery provides opportunities to minimize nerve damage and revolutionize standards of care across various surgical specialties.

Pulmonary sclerosing pneumocytoma - approaching a solitary pulmonary nodule and the limitations of risk prediction models.

Our case is an asymptomatic, non-smoking, East Asian woman in her 40s presenting with a solitary pulmonary nodule (SPN). On imaging, the 1.7 cm solid SPN located in the left upper lobe, was rounded in morphology and moderately fluorodeoxyglucose avid. The clinical pretest probability of malignancy assessed by risk prediction models such as Brock (19.1%), Mayo Clinic (56.2%) and Herder (51.4%) was discordant. She underwent a percutaneous CT-guided needle biopsy, establishing a diagnosis of pulmonary sclerosing pneumocytoma (PSP). PSP is a rare benign lung neoplasm with indolent growth characteristics that has been described predominantly in non-smoking women. Our case illustrates the limitations of applying existing risk prediction models in Asia where the epidemiology and biology of lung cancer differ significantly from the Caucasian derivation cohorts. Additionally, the risk models do not account for tuberculosis, which is endemic in Asia and can mimic malignancy. Non-surgical lung biopsy remains useful in minimising unnecessary thoracotomy.

Three shades of an unusual mediastinal tumour.

Thoracic SMARCA4-deficient undifferentiated tumour (SMARCA4-UT) is an unusual and aggressive tumour. While there are approximately 100 cases of this tumour reported in the literature, there are very few detailed descriptions of its cytomorphologic characteristics, and only rare cases in which primary diagnosis was made on cytologic material. Herein we present a case with a detailed description of the appearance on three specimen types: transbronchial needle aspiration (TBNA) cytology, transbronchial needle biopsy (TBNB) and effusion cytology. Thoracic SMARCA4-UT is an important diagnosis to clinch in modern pathology because of its prognostic and therapeutic implications. We discuss an integrated approach to clinching the diagnosis with reference to clinical, radiographic, morphologic and immunohistochemical features. We also discuss possible differential diagnoses, and how they can be excluded. Cytologic and/or small biopsy diagnosis is valuable in these cases as these tumours are typically not amenable to surgical resection. With the correct diagnosis, the patient may instead be a candidate for immune checkpoint inhibitors or experimental therapy targeting SWI/SNF deficiency.

Cysteine-Rich α-Conotoxin SII Displays Novel Interactions at the Muscle Nicotinic Acetylcholine Receptor.

α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 310-helix frequently employed by framework I α-conotoxins; α-SII acted at the muscle nAChR with half-maximal inhibitory concentrations (IC50) of 120 nM (adult) and 370 nM (fetal) though weakly at neuronal nAChRs. Truncation of α-SII to a two disulfide bond amidated peptide with framework I disulfide connectivity led to similar activity. Surprisingly, the more constrained α-SII was less stable under mild reducing conditions and displayed a unique docking mode at the nAChR.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

Venom-derived modulators of epilepsy-related ion channels.

Epilepsy is characterised by spontaneous recurrent seizures that are caused by an imbalance between neuronal excitability and inhibition. Since ion channels play fundamental roles in the generation and propagation of action potentials as well as neurotransmitter release at a subset of excitatory and inhibitory synapses, their dysfunction has been linked to a wide variety of epilepsies. Indeed, these unique proteins are the major biological targets for antiepileptic drugs. Selective targeting of a specific ion channel subtype remains challenging for small molecules, due to the high level of homology among members of the same channel family. As a consequence, there is a growing trend to target ion channels with biologics. Venoms are the best known natural source of ion channel modulators, and venom peptides are increasingly recognised as potential therapeutics due to their high selectivity and potency gained through millions of years of evolutionary selection pressure. Here we describe the major ion channel families involved in the pathogenesis of various types of epilepsy, including voltage-gated Na+, K+, Ca2+ channels, Cys-loop receptors, ionotropic glutamate receptors and P2X receptors, and currently available venom-derived peptides that target these channel proteins. Although only a small number of venom peptides have successfully progressed to the clinic, there is reason to be optimistic about their development as antiepileptic drugs, notwithstanding the challenges associated with development of any class of peptide drug.

Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization.

BACKGROUND: Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theater with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider, Haplopelma schmidti, and conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence. RESULTS: Hs1a-FL showed specific and stable binding to the sodium channel NaV1.7, present on the surface of human and mouse nerves. Hs1a-FL allowed epifluorescence visualization of sciatic mouse nerves with favorable nerve-to-muscle contrast. CONCLUSIONS: Fluorescent NaV1.7-targeted tracers have the potential to be adopted clinically for the intraoperative visualization of peripheral nerves during surgery, providing guidance for the surgeon and potentially improving the standard of care.

Venom Peptides with Dual Modulatory Activity on the Voltage-Gated Sodium Channel NaV1.1 Provide Novel Leads for Development of Antiepileptic Drugs.

Voltage-gated sodium (NaV) channels play a fundamental role in normal neurological function, especially via the initiation and propagation of action potentials. The NaV1.1 subtype is found in inhibitory interneurons of the brain and it is essential for maintaining a balance between excitation and inhibition in neuronal networks. Heterozygous loss-of-function mutations of SCN1A, the gene encoding NaV1.1, underlie Dravet syndrome (DS), a severe pediatric epilepsy. We recently demonstrated that selective inhibition of NaV1.1 inactivation prevents seizures and premature death in a mouse model of DS. Thus, selective modulators of NaV1.1 might be useful therapeutics for treatment of DS as they target the underlying molecular deficit. Numerous scorpion-venom peptides have been shown to modulate the activity of NaV channels, but little is known about their activity at NaV1.1. Here we report the isolation, sequence, three-dimensional structure, recombinant production, and functional characterization of two peptidic modulators of NaV1.1 from venom of the buthid scorpion Hottentotta jayakari. These peptides, Hj1a and Hj2a, are potent agonists of NaV1.1 (EC50 of 17 and 32 nM, respectively), and they present dual α/ß activity by modifying both the activation and inactivation properties of the channel. NMR studies of rHj1a indicate that it adopts a cystine-stabilized αß fold similar to known scorpion toxins. Although Hj1a and Hj2a have only limited selectivity for NaV1.1, their unusual dual mode of action provides an alternative approach to the development of selective NaV1.1 modulators for the treatment of DS.

A selective NaV1.1 activator with potential for treatment of Dravet syndrome epilepsy.

Dravet syndrome (DS) is a catastrophic epileptic encephalopathy characterised by childhood-onset polymorphic seizures, multiple neuropsychiatric comorbidities, and increased risk of sudden death. Heterozygous loss-of-function mutations in one allele of SCN1A, the gene encoding the voltage-gated sodium channel 1.1 (NaV1.1), lead to DS. NaV1.1 is primarily found in the axon initial segment of fast-spiking GABAergic inhibitory interneurons in the brain, and the principle mechanism proposed to underlie seizure genesis in DS is loss of inhibitory input due to dysfunctional firing of GABAergic interneurons. We hypothesised that DS symptoms could be ameliorated by a drug that activates the reduced population of functional NaV1.1 channels in DS interneurons. We recently identified two homologous disulfide-rich spider-venom peptides (Hm1a and Hm1b) that selectively potentiate NaV1.1, and showed that selective activation of NaV1.1 by Hm1a restores the function of inhibitory interneurons in a mouse model of DS. Here we produced recombinant Hm1b (rHm1b) using an E. coli periplasmic expression system, and examined its selectivity against a panel of human NaV subtypes using whole-cell patch-clamp recordings. rHm1b is a potent and highly selective agonist of NaV1.1 and NaV1.3 (EC50 ~12 nM for both). rHm1b is a gating modifier that shifts the voltage dependence of channel activation and inactivation to hyperpolarised and depolarised potentials respectively, presumably by interacting with the channel's voltage-sensor domains. Like Hm1a, the structure of rHm1b determined by using NMR revealed a classical inhibitor cystine knot (ICK) motif. However, we show that rHm1b is an order of magnitude more stable than Hm1a in human cerebrospinal fluid. Overall, our data suggest that rHm1b is an exciting lead for a precision therapeutic targeted against DS.

Novel venom-derived inhibitors of the human EAG channel, a putative antiepileptic drug target.

Recently, we and other groups revealed that gain-of-function mutations in the human ether à go-go voltage-gated potassium channel hEAG1 (Kv10.1) lead to developmental disorders with associated infantile-onset epilepsy. However, the physiological role of hEAG1 in the central nervous system remains elusive. Potent and selective antagonists of hEAG1 are therefore much sought after, both as pharmacological tools for studying the (patho)physiological functions of this enigmatic channel and as potential leads for development of anti-epileptic drugs. Since animal venoms are a rich source of potent ion channel modifiers that have been finely tuned by millions of year of evolution, we screened 108 arachnid venoms for hEAG1 inhibitors using electrophysiology. Two hit peptides (Aa1a and Ap1a) were isolated, sequenced, and chemically synthesised for structure-function studies. Both of these hEAG1 inhibitors are C-terminally amidated peptides containing an inhibitor cystine knot motif, which provides them with exceptional stability in both plasma and cerebrospinal fluid. Aa1a and Ap1a are the most potent peptidic inhibitors of hEAG1 reported to date, and they present a novel mode of action by targeting both the activation and inactivation gating of the channel. These peptides should be useful pharmacological tools for probing hEAG1 function as well as informative leads for the development of novel anti-epileptic drugs.