Neuropeptide signalling systems – an underexplored target for venom drug discovery

Abstract

Neuropeptides are signalling molecules mainly secreted from neurons that act as neurotransmitters or peptide hormones to affect physiological processes and modulate behaviours. In humans, neuropeptides are implicated in numerous diseases and understanding their role in physiological processes and pathologies is important for therapeutic development. Teasing apart the (patho)physiology of neuropeptides remains difficult due to ligand and receptor promiscuity and the complexity of the signalling pathways. The current approach relies on a pharmacological toolbox of agonists and antagonists displaying high selectivity for independent receptor subtypes, with the caveat that only few selective ligands have been discovered or developed. Animal venoms represent an underexplored source for novel receptor subtype-selective ligands that could aid in dissecting human neuropeptide signalling systems. Multiple endogenous-like neuropeptides as well as peptides acting on neuropeptide receptors are present in venoms. In this review, we summarise current knowledge on neuropeptides and discuss venoms as a source for ligands targeting neuropeptide signalling systems.

1. Neuropeptides – ancient signalling peptides widely distributed across the animal kingdom

Neuropeptides are the largest and most diverse class of cell-to-cell signalling molecules with significant roles in animal physiology, behaviour and homeostasis [1]. To date, over 6,000 neuropeptides have been identified belonging to more than 60 neuropeptide families found across nearly 500 different species (source: NeuroPep [2]). The widespread distribution of neuropeptides across Bilateria is due to whole genome as well as local gene duplication events [3].

Neuropeptide signalling systems are evolutionarily ancient and seem to have evolved in the earliest of the metazoans, predating bilaterians, as they are found in the pre-bilaterian metazoans Ctenophora (comb jellies), Cnidaria, Porifera (sponges) and Placozoa (Figure 1A) [3–5]. Porifera and Placozoa do not technically possess a nervous system, meaning that they do not possess neurons, yet studies have found neuropeptide-like sequences in both [4–8]. Neuropeptides mediate their effects mainly through G protein-coupled receptors (GPCRs) [1]. There is ample evidence for the presence of GPCRs in Porifera, Placozoa, Cnidaria and Ctenophora [9, 10], and the study of GPCRs in early pre-bilaterian metazoans has provided insights into the evolution of neuropeptide signalling systems [4, 5]. For instance, genomic evidence was found for bilaterian neuropeptide receptor orthologues of several glycoprotein hormones (follicle-stimulating hormone, luteinizing hormone, and thyroid stimulating hormone) as well as relaxin, and bursicon in Cnidaria and Placozoans [11]. In addition, several genes encoding proteins with characteristics of neuropeptides were identified in the placozoan Trichoplax adhaerens [12] and cnidarian Nematostella vectensis [13]. However, in most cases neuropeptide receptor families are considered evolutionarily ancient based on the occurrence of orthologous neuropeptide-type receptors in one or more deuterostome species and one or more protostome species [3]. Within this definition, at least 31 different neuropeptide families are of pre-bilaterian origin and considered evolutionarily ancient [3].

Figure 1. Overview of animal evolution, neuropeptide biosynthesis, release and signalling.

(A) Simplified phylogenetic tree illustrating selected animal phyla where neuropeptides are found, including non-bilaterian phyla. The deuterostome and protostome phyla diverged ~600 million years ago (mya). *, phyla with no evidence of a nervous system. Adapted from [7]. (B) Neuropeptide precursors are synthesised in the soma of the neuron. The precursor neuropeptide is produced in the endoplasmic reticulum (ER) from mRNA that is translated on the cytosolic ER membrane. After folding of the protein and addition of N-linked carbohydrate (if the appropriate sites are present in the precursor), the neuropeptide is transported to the Golgi via small vesicles that bud from the ER and fuse with the Golgi. The precursor protein is packaged into secretory vesicles, where they are further processed. Upon stimuli, the vesicle fuses with the cell membrane and releases the mature neuropeptides. Mature neuropeptides can be released from nerve endings, dendrites or the cell soma. Neuropeptides can either act at short range on the secreting neuron or through diffusion on neighbouring cells (1), or reach long range target receptors through secretion into the blood stream (2) or through non-synaptic dispersion (3). Once released, the neuropeptides are subject to processing by extracellular peptidases. This generally results in degradation and inactivation of the neuropeptides, except for some cases (e.g., bradykinin and angiotensin II) where this extracellular processing is required to produce the active mature neuropeptide. (C) Schematic of a model neuropeptide precursor made up of a signal peptide (red), propeptide (green) and mature peptides (blue and yellow). Precursors are cleaved by endopeptidases and carboxypeptidases at dibasic cleavage sites to produce the neuropeptides, which can further mature through additional post-translational modifications once in the Golgi. Image was created with Biorender.com.

High conservation of evolutionarily ancient neuropeptide families is generally associated with important physiological roles. Although the function of many conserved neuropeptide families has diverged across animal lineages, emerging evidence demonstrates evolutionary conservation of related neuropeptide-receptor pairs in the regulation of similar aspects of animal physiology and behaviour across species [14]. For example, the neurohypophyseal neuropeptide family (oxytocin and vasopressin) has a conserved role in reproduction, water homeostasis and feeding [15–18], which are essential functions for survival. Other examples include insulin in regulating growth and metabolism [19], thyrotropin-releasing hormone (TRH) in growth [20], prolactin water and salt in metabolism [21], adrenocorticotropic hormone (ACTH) in the immune system [22], and gonadotropin in reproduction [23].

Many neuropeptide families have evolved more recently, yet still regulate important physiological processes. For example, the opioid family, which is a very diverse family and therapeutically important for pain management [24]. Current evidence suggests that opioids are a vertebrate novelty as there is no evidence of opioid signalling system in invertebrates [3]. Similarly, bradykinin is only found in vertebrates and plays a crucial role in inflammation and is also involved in fluid homeostasis, pain and blood pressure [25].

2. Neuropeptide biosynthesis and signalling

Neuropeptides are produced as precursor proteins which consist of an N-terminal signal peptide, a propeptide and one or more mature neuropeptides (Figure 1C) [26]. The precursor protein requires further proteolytic processing to generate the smaller, bioactive neuropeptides. After removal of the signal peptide in the endoplasmic reticulum by signal peptidase, the peptide precursor is transported to the Golgi, where most neuropeptides are released from the precursor protein through cleavage at dibasic residues (K/R-K/R) and, in some cases, single basic residues (K/R) by proprotein convertases 1/3 and 2 (Figure 1B, C) [26]. The neuropeptides are then transported in large dense core vesicles to the nerve terminals. Neuropeptides can undergo post-translational modifications (PTMs) including C-terminal amidation, N-terminal pyroglutamylation, acetylation, phosphorylation, sulfation, and glycosylation to generate the final mature neuropeptides [1, 26–29]. Mature neuropeptide release is mediated by prolonged increases in calcium levels at the axon terminals, leading to the fusion of the secretory vesicles to the cell membrane. In some cases, such as bradykinin and angiotensin II, further peptide processing by enzymes occurs after release to produce the mature peptide [30, 31]. Once released, peptides can either act at short range (within the synapse or local diffusion) [32] or act at a distance (‘peptide hormones’) [33], typically after being released into the bloodstream (Figure 1B) [14].

A neuropeptide precursor can contain one or more of the same or different mature peptides and proteolytic processing plays a key regulatory function [1, 34]. In addition, mature peptides within one neuropeptide precursor can belong to different neuropeptide families; for instance, the proopiomelanocortin (POMC) precursor contains the melanocortin neuropeptides ACTH and melanocyte-stimulating hormone (MSH) as well as the opioid peptide b-endorphin [26, 35]. Further neuropeptide diversity results from alternative splicing of neuropeptide genes, resulting in neuropeptide precursor isoforms as is the case for the calcitonin/calcitonin gene-related peptide (CGRP) family which is alternatively spliced to generate either calcitonin precursor mRNA or CGRP precursor mRNA [26, 36]. In this instance, alternative splicing is tissue-dependent and results in tissue-specific expression of calcitonin and CGRP [26, 36]. The nature of PTMs can also be tissue- and state-dependent as a result of differential expression of processing enzymes. For example, during fasting, a-MSH from POMC accumulates due to an increased rate of processing of POMC, likely as a result of altered expression of proprotein convertases [37]. In summary, neuropeptide precursors can contain more than one mature peptide, and gene splicing and precursor processing contribute to neuropeptide diversity.

A single neuropeptide may bind to a variety of receptor subtypes and thereby mediate a wide range of effects [38]. Vasopressin is a clear example for this since vasopressin activates all three vasopressin receptors (V1aR, V1bR, V2R) as well as the oxytocin receptor (OTR) with varying potencies [39, 40]. In blood vessels, for example, vasopressin acts on the V1aR to mediate pressor effects via smooth muscle contraction, whereas in the kidney vasopressin mediates antidiuretic effects through the V2R. In addition, different ligands binding at the same receptor subtype can activate different signalling pathways; binding of morphine or endogenous enkephalin to the µ-opiate receptor results in recruitment of distinct b-arrestin isoforms, which differentially affect desensitisation [14, 41]. Thus, neuropeptide signalling systems can be very complex as they involve several GPCR subtypes linked to multiple signalling cascades and are expressed in various tissues with different functions [42].

3. Neuropeptides in humans

So far, over 200 different neuropeptides from 47 different neuropeptide families have been identified in humans (source: NeuroPep [2]). They are distributed in both the central nervous system (CNS) and the peripheral nervous system (PNS), where they regulate numerous physiological processes including reproduction, fluid homeostasis, cardiovascular function, energy homeostasis, pain, memory and learning, and circadian rhythm (Figure 2) [1]. In some cases, neuropeptides play essential physiological roles, for example, pituitary adenylate cyclase-activating peptide (PACAP) is necessary for nerve growth, nerve differentiation, and neuroprotection, and PACAP gene knockout in mice leads to early death after birth [43]. In addition, neuropeptides modulate behaviour including social behaviour (oxytocin and vasopressin), feeding behaviour (leptin, gastrin, ghrelin, neuropeptide Y, oxytocin, and melanin-concentrating hormone), and stress and anxiety (neuromedins, galanin, cholecystokinin, melanin-concentrating hormone, neuropeptide S, neuropeptide Y, somatostatin, tachykinins, urotensin-2, oxytocin, vasopressin). A single neuropeptide can contribute to and regulate many different physiological processes and/or behaviours. This is mostly mediated through tissue-dependent release and receptor subtype expression. For example, V1bR activation in the paraventricular nucleus of the hypothalamus initiates ACTH secretion signalling whereas V1bR activation in the colon mediates proinflammatory properties [44].

Figure 2. Selected neuropeptides and the physiological processes they are involved in.

MCH, melanin-concentrating hormone; CRH, corticotropin-releasing hormone; NPY, neuropeptide Y; GnRH, gonadotropin-releasing hormone. This figure has been designed using resources from Freepik.com.

Although some neuropeptides are crucial for survival and development, many are modulatory in nature or play a minor role [41]. Subsequently, neuropeptide gene knockout mice often show little change in phenotype [41]. For example, vasoactive intestinal peptide is a immunoregulatory neuropeptide, yet vasoactive intestinal peptide knock out mice survived weaning and have normal basal immune characteristics [45]. In addition, there is evidence of functional compensation of neuropeptide signalling systems; oxytocin knockout mice are unable to release milk but have otherwise largely normal parturition and maternal behaviour, possibly due to compensation from the closely-related vasopressin signalling system [46].

Under pathological conditions neuropeptide signalling can play a more important role and become therapeutically relevant [41]. For example, the vasoconstriction of blood vessels caused by vasopressin binding to V1aR in the vascular system plays a minor role in the regulation of blood pressure in a healthy individual. However, under pathophysiological conditions such as severe extracellular fluid volume depletion, hypotension, general anaesthesia, or haemorrhage, high levels of vasopressin in the blood can significantly exacerbate vasopressor effects [44].

4. Clinical application of neuropeptides

Neuropeptides are implicated in multiple physiological processes and, consequently, numerous disease processes [47]. Disease areas where neuropeptides play a key role and where neuropeptide signalling systems are a validated therapeutic target or are actively being investigated as a therapeutic target include obesity, heart failure, epilepsy, sleep disorders, autism and depression [47–55].

Both neuropeptide analogues and small molecules targeting neuropeptide receptors have been developed for therapeutic treatments ranging from cardiovascular disorders, diabetes, pain, and labour to gastrointestinal disorders. Table 1 provides an overview of approved drugs targeting neuropeptide signalling systems. A range of other neuropeptide targets are also under investigation [47].

Table 1. Approved drugs targeting neuropeptide receptors and neuropeptides used as biomarkers or diagnostic tools.^a .

DRUGS
Receptor	Receptor subtypes	Drug and indication
Angiotensin (AT) receptor	AT1 and AT2 receptors	Small molecules: Valsartan, Losartan, Irbesartan, Olmesartan, Eprosartan, Telmisartan, AT1 antagonists for hypertension; Candesartan, AT1 antagonist for chronic heart failure.
Bradykinin receptors	B1, B2 receptors	Peptide: Icatibant, selective B2 receptor antagonist, for hereditary angioedema.
Calcitonin (CT) receptor	CT receptor	Peptides: Synthetic salmon calcitonin or synthetic human calcitonin for the treatment of hypercalcemia (postmenopausal osteoporosis, Paget’s disease of bone, Sudeck’s atrophy, and malignancy-associated hypercalcemia)[174].
Endothelin (ET) receptors	ETA and ETB receptors	Small molecules: Bosentan and Macitentan, dual receptor antagonists; Ambrisentan, a selective ETA receptor antagonist; All used for the treatment of pulmonary hypertension [175].
Glucagon receptors	Glucagon receptor, GLP-1 receptor b, GLP-2 receptor	Peptides: Recombinant glucagon, for severe hypoglycaemia; Exenatide, liraglutide, albiglutide, lixisenatide, dulaglutide, semaglutide, GLP-1 receptor agonists, used for diabetes treatment; Teduglutide, human GLP-2 analogue, used for short bowel syndrome, malabsorption.
Gonadotropin-releasing hormone (GnRH) receptor	GnRH receptor	Peptides: Nafarelin, GnRH receptor agonist used for treatment of central precocious puberty and endometriosis; Cetrorelix, GnRH receptor antagonist used to inhibit premature luteinizing hormone surges in controlled ovarian stimulation; Elagolix, GnRH receptor antagonist, used for management of moderate to severe pain associated with endometriosis; Degarelix, GnRH receptor antagonist, used for management of advanced prostate cancer; Leuprolide, GnRH agonist, used to treat prostate cancer, endometriosis, uterine fibroids and premature puberty.
Growth hormone receptor	Growth hormone receptor	Peptide / protein: Somatotropin, recombinant human growth hormone, used for growth hormone deficiency, Turner syndrome, Noonan syndrome, Prader-Willi syndrome, short stature homeobox-containing gene deficiency, chronic renal insufficiency, idiopathic short stature and children small for gestational age; Pegvisomant, growth hormone receptor antagonist, used for acromegaly.
Growth hormone releasing hormone (GHRH) receptor	GHRH receptor	Peptides: Sermorelin, GHRH1–29 peptide fragment, for treatment of growth failure in children; Tesamorelin, GHRH1–44 peptide fragment, for lipodystrophy in HIV patients.
Insulin receptor	Insulin receptor, IGF-1 c receptor	Peptides: Human insulin, used for glycaemic control in patients with diabetes mellitus; Mecasermin, recombinant human IGF-1, approved for growth failure in children with primary insulin-like growth factor-1 deficiency or growth hormone gene deletion.
Leptin receptor	Leptin receptor	Peptide: Metreleptin, a leptin analogue used for diabetes and/or hypertriglyceridemia.
Melanocortin (MC) receptors	MC1-5 receptors	Peptide: Bremelanotide, MC1, MC3-5 agonist, used for premenopausal women with hypoactive sexual desire disorder.
Natriuretic peptide receptors (NPR)	NPR-A, -B, and -C receptors	Small molecules: Nitroprusside, Nitroglycerin, Isosorbide dinitrate, Erythrityl tetranitrate, and Amyl nitrite, act as nitric oxide source, vasodilators used for chest pain and high blood pressure. Peptide: Nesiritide, recombinant human BNP d for congestive heart failure.
Opioid receptors	µ-, δ-, κ-opioid receptors	Small molecules: Butorphanol, Tramadol, Sufentanil, Alfentanil, Hydrocodone, Fentanyl, Dextropropoxyphene, Morphine, Codeine, Hydromorphone, Meperidine, µ-, δ-, and/or κ-opioid receptor agonists for pain management, anaesthetic, analgesia; Naltrexone, Nalbuphine, Buprenorphine, Dezocine, Pentazocine, Naloxone, Levallorphan, µ-, δ-, and/or κ-opioid receptor antagonists for pain and/or addiction management, treatment of narcotic depression.
Orexin (OX) receptors	OX1, OX2 receptors	Small molecule: Suvorexant, OX1R and OX2R antagonist used for insomnia.
Parathyroid hormone (PTH) type 1 receptor	PTH type 1 receptor	Peptides: Teriparatide, recombinant human PTH used in treatment of osteoporosis; Abaloparatide, PTH-related protein analogue, used for post-menopausal women with osteoporosis.
Somatostatin (SST) receptors	SST1-5 receptors	Peptides: Octreotide, lanreotide, SST2 / SST5 receptor agonists used for acromegaly and to reduce cancer chemotherapy side effects; Pasireotide, pan agonist used for Cushing’s Disease;
Tachykinin receptors	NK1, NK2, NK3, Neuromedin-K receptors	Small molecules: Fosaprepitant, Aprepitant, Netupitant, Rolapitant, NK1 antagonist used for chemotherapy-induced emesis.
Oxytocin (OT) and vasopressin receptors	OT, V1a, V1b, V2 receptors	Peptides: Atosiban, OTR antagonist used as labour suppressant and IVF treatment; Pitocin and Carbetocin, OTR agonists used for labour induction, post-partum bleeding, and haemorrhage; Desmopressin, V2R agonist for diabetes insipidus and nocturnal enuresis; Terlipressin, V2R agonist used to stop bleeding in the oesophagus. Small molecules: Conivaptan, V1aR and V2R antagonist used for hyponatremia; Tolvaptan, V2R antagonist used for hyponatremia.
BIOMARKERS OR DIAGNOSTIC TOOLS
Neuropeptide	Receptor	Compound and application
ATCH	MC2 receptor	Diagnostic agent used to screen for adrenocortical insufficiency.
BNP	n.a.e	Diagnostic marker for heart failure [176].
Chromogranin A	n.a.	An indicator for pancreas and prostate cancer in carcinoid syndrome [177].
Corticotropin-releasing hormone (CRH)	CRH receptor 1	Corticorelin, synthetic human CRH, is used to test the HPA axis and ATCH secretion.
Gastrin	n.a.	Diagnostic marker for gastrin-secreting tumours, atrophic gastritis, gastric ulcers, and pernicious anaemia [178].
Ghrelin	Ghrelin receptor	Ghrelin mimetic Macimorelin is used to diagnose adult GH deficiency.
GnRH	GnRH receptor	Gonadorelin, synthetic GnRH, is used to evaluate anterior pituitary performance.
Insulin	Insulin receptor	Biomarker for diabetes (complementary); used for identification of insulinomas; insulin-induced hypoglycaemia test used to diagnose suspected ACTH or growth hormone deficiency [179].
Pancreatic polypeptide	n.a.	Diagnostic test of vagal nerve function [180].
Procalcitonin Prolactin	n.a. n.a.	A biomarker of infection and sepsis [181, 182]. Diagnostic marker in sex hormone check-up; epilepsy type diagnosis [21].
Thyrotropin-releasing hormone (TRH)	TRH receptor	Protirelin, a TRH analogue, is used to test anterior pituitary gland function to aid thyroid disorder diagnosis [183].
Somatostatin	SST1–5 receptors	Various radiolabelled somatostatin analogues used for cancer treatment (e.g. Edotreotide gallium Ga-68 and Lutetium Lu 177 dotatate).

^aInformation from DrugBank database (www.drugbank.ca) [184] where an ‘approved’ drug has been approved in at least one jurisdiction, anywhere;

^bGLP, Glucagon-like peptide;

^cIGF, insulin-like growth factor;

^dBNP, B-type natriuretic peptide;

^en.a. Not applicable.

There are clear examples of the clinical importance of neuropeptides including opioid analgesics, insulin treatment for type 2 diabetes mellitus, oxytocin to induce labour, and parathyroid hormone to treat osteoporosis (Table 1). However, many trials targeting neuropeptide signalling systems have been disappointing in part due to lack of efficacy, receptor dimerization, unexpected side effects, and dosage-dependent effects [41, 47, 56, 57]. One potential cause of lack of efficacy is the complexity of neuropeptide signalling, which displays temporal variations and signalling redundancy, but also a lack of knowledge on signalling mechanisms [41, 47, 58]. Consequently, it is no longer the expectation that a single neuropeptide will cure a single disease and there is much scope for targeting neuropeptide signalling systems for therapeutic treatment in a wide range of diseases [41].

Neuropeptides, and in some cases neuropeptide precursors, are also valuable biomarkers and diagnostic probes to aid disease diagnosis and prognosis (Table 1). As the various roles of neuropeptides in (patho)physiological processes are elucidated, their value as biomarkers and diagnostic markers is often assessed.

Neuropeptide signalling systems represent a great opportunity for the development of therapeutics, in particular for peptide therapeutics. An in-depth understanding of the mechanistic roles of neuropeptides in health and disease is essential to translate their therapeutic potential into drug targets, therapeutic leads, diagnostics and biomarkers. Although technical advances within next-generation sequencing and mass spectrometry have greatly facilitated neuropeptide discovery and characterisation of their signalling systems [59–62], most neuropeptide signalling systems remain poorly understood [14]. In addition to their complex signalling pathways (multiple G protein-dependent and -independent pathways), neuropeptides have numerous characteristics that make them difficult to identify using mass spectrometry and sequencing technology, especially because of the high occurrence of PTMs, and the existence of isoforms [62]. Identification based on mass is further complicated by (i) varying and typically low in vivo concentrations (up to 1,000-fold lower than classical neurotransmitters); (ii) lack of enzymatic fragments, requiring highly-sensitive proteomic detection methods; and (iii) rapid in vivo degradation [62]. Consequently, the therapeutic potential of many neuropeptide systems remain unrealised [41]. Subtype selective ligands that can selectively inhibit or stimulate neuropeptide receptors are key pharmacological tools to dissect their functions in health and disease [58]. These ligands also constitute therapeutic leads. Such pharmacological toolboxes already exist for some neuropeptide signalling systems. For instance, bradykinin acts via two receptors, B1, B2, and receptor-selective agonists and antagonists have been developed for both receptors [63, 64].

5. Neuropeptides in venoms – an underexplored field

Endogenous neuropeptides from animals throughout the animal kingdom represent a solid starting point for discovery. Neuropeptide receptor sequence conservation means that there are numerous occurrences of neuropeptides from one phylum activating the corresponding receptors in another phylum. For example, vertebrate substance P is able to activate a Drosophila tachykinin receptor expressed in Xenopus oocytes [65], and mammalian neuropeptide Y peptides can activate the Drosophila neuropeptide F receptor, the invertebrate neuropeptide Y receptor orthologue [66]. Small sequence variations in ligands and receptors across species result in diverse pharmacological profiles including differences in selectivity, affinity and function (e.g. agonism, antagonism, and biased signalling) [67, 68]. This diversity can be exploited to benefit human health as neuropeptides identified from different organisms represent a rich source for molecular probes and therapeutic leads to dissect neuropeptide signalling in health and disease. This hypothesis has been underpinned by multiple studies, including new oxytocin and vasopressin ligands identified from insects [69, 70], salmon calcitonin used to treat hypercalcemia [71]; and pramlintide (based on rat amylin) to reduce blood glucose levels in type 1 and type 2 diabetes mellitus patients [72].

Another, and maybe even richer, source for ligands targeting neuropeptide signalling systems is animal venom. Venoms are highly complex mixtures mostly made up of proteins and peptides (~90%) that disrupt normal biochemical and physiological processes in the prey/predator for the benefit of the venomous animal [73–76]. Venom peptides have an array of targets including receptors and enzymes affecting biological functions in various locations including the CNS, PNS, blood and muscle tissue [77]. Importantly, venom components have undergone strong natural selection for in vivo stability, potency, selectivity and delivery to engage in precise molecular interactions exogenously to warrant their survival through defence and predation [73–76, 78, 79]. Many of these features are also highly desirable in drug development, thus venoms represent an advantageous drug discovery starting point compared to combinatorial or DNA/RNA display libraries [78, 80–83]. Indeed, venoms are a highly successful source for drug leads with five venom-derived peptide drugs on the market, including Exenatide (Byetta^®) and Lixisentide (Adlyxin, Lyxumia) from the Gila monster to treat type 2 diabetes [84, 85]; Eptifibatide (Integrilin^®), an antiplatelet agent from the rattlesnake Sistrurus miliarius barbourin [86]; and Bivalirudin (Angiomax, Angiox) from the leech Hirudo medicinalis, which is used as an anticoagulant agent [87]. Furthermore, conotoxins from the marine predatory cone snail have transformed neuropathic pain research with one conotoxin drug approved to treat severe chronic pain (Ziconatide, Prialt^®) [88]. Spider toxins have also provided outstanding therapeutic leads for stroke, pain, and epilepsy through their actions on ion channels Nav1.1 [89, 90], Nav1.7 [91, 92], and acid-sensing ion channels 1a (ASIC1a) [93], respectively. Chlorotoxin, isolated from the venom of the scorpion Leiurus quinquestriatus and initially characterised as a voltage-gated chloride channel ligand [94], was subsequently identified as a potent and selective matrix metalloproteinase-2 receptor ligand and is currently undergoing clinical trials to visualise glioblastomas [95]. Finally, venom peptides and their use as receptor-subtype selective pharmacological probes have revolutionised our understanding of many human receptors and links to disease [96–102]. For instance, bradykinin-potentiating peptides, originally identified in the venom of the snake Bothrops jararaca, led to the development of a new class of hypotensive drugs, angiotensin converting enzyme inhibitors, including captopril, for the treatment of high blood pressure and heart failure [103].

Venom proteins and peptides are a result of gene duplication and selective expression of an endogenous protein/peptide in the venom gland, a process called neofunctionalization [73, 75, 76, 104–107]. Neuropeptides are no exception, and at least 176 different neuropeptides from 16 different neuropeptide families have been found in the venom gland transcriptomes or venoms of 107 different species, including spiders, scorpions, insects, centipedes, cone snails, octopus, monotremes, snakes, lizards and fish (Table 2).

Table 2. Neuropeptides in animal venoms (proteomic evidence and venom organ transcriptome evidence).

Neuropeptide family	Neuropeptide	Species	Taxonomic group	UniProt/ Genbank/ PMID ID
Bradykinin	Megascoliakinin	Megascolia flavifrons	Insect	P12797
Bradykinin	Thr6-Bradykinin	Polybia occidentalis	Insect	P0DM70
CCAP a	ConoCAP-a	Conus villepinii	Mollusc	E3PQQ8
CCAP	ConoCAP-Gm	Conus gloriamus	Mollusc	28531118
Elevenin	Elevenin-Vc1	Conus victoriae	Mollusc	A0A0F7YZQ7
Endothelin	Bibrotoxin	Atractaspis bibroni	Lepidosauromorpha (Lamprophiidae)	P80163
Endothelin	Sarafotoxin-A, Serisoform, S6A	Atractaspis engaddensis	Lepidosauromorpha (Lamprophiidae)	P13209
Endothelin	Sarafotoxin-A, Thrisoform	Atractaspis engaddensis	Lepidosauromorpha (Lamprophiidae)	P13208
Endothelin	Sarafotoxin-B, S6B	Atractaspis engaddensis	Lepidosauromorpha (Lamprophiidae)	P13208
Endothelin	Sarafotoxin-C, S6C	Atractaspis engaddensis	Lepidosauromorpha (Lamprophiidae)	P13208
Endothelin	Sarafotoxin-E, S6E	Atractaspis engaddensis	Lepidosauromorpha (Lamprophiidae)	P13208
Endothelin	Sarafotoxin-i1	Atractaspis irregularis	Lepidosauromorpha (Lamprophiidae)	P0DJK0
Endothelin	Sarafotoxin-i2	Atractaspis irregularis	Lepidosauromorpha (Lamprophiidae)	P0DJK1
Endothelin	Sarafotoxin-m (SRTX-m)	Atractaspis microlepidota microlepidota	Lepidosauromorpha (Lamprophiidae)	Q6RY99
Endothelin	Sarafotoxin-m1 (SRTX-m1)	Atractaspis microlepidota microlepidota	Lepidosauromorpha (Lamprophiidae)	Q6RY100
Endothelin	Sarafotoxin-m2 (SRTX-m2)	Atractaspis microlepidota microlepidota	Lepidosauromorpha (Lamprophiidae)	Q6RY101
Endothelin	Sarafotoxin-m3 (SRTX-m3)	Atractaspis microlepidota microlepidota	Lepidosauromorpha (Lamprophiidae)	Q6RY102
Endothelin	Sarafotoxin-m4 (SRTX-m4)	Atractaspis microlepidota microlepidota	Lepidosauromorpha (Lamprophiidae)	Q6RY103
Enkephalin	BmK-YA	Buthus martensii	Arachnid (scorpion)	22792309
Enkephalin	BmK-YA	Mesobuthus martensii	Arachnid (scorpion)	Q9Y0X6
Enkephalin	His4-BmK-YA	Buthus martensii	Arachnid (scorpion)	22792309
Enkephalin	Met-enkephalin	Meiacanthus atrodorsalis	Actinopterygii	P0DP56
Enkephalin	Met-enkephalin-His-Asp	Meiacanthus atrodorsalis	Actinopterygii	P0DP56
Enkephalin	Met-enkephalin-Se	Meiacanthus r atrodorsalis	Actinopterygii	P0DP56
Enkephalin	Synenkephalin	Meiacanthus atrodorsalis	Actinopterygii	P0DP56
FARP b	Conorfamide Tx1.3	Conus textile	Mollusc	P0DL71
FARP	Conorfamide-As1	Conus cancellatus	Mollusc	P0DQH7
FARP	Conorfamide-As2	Conus cancellatus	Mollusc	P0DQH8
FARP	Conorfamide-Gm	Conus gloriamus	Mollusc	28531118
FARP	Conorfamide-Sr1	Conus spurius	Mollusc	P58805
FARP	Conorfamide-Sr2	Conus spurius	Mollusc	P85871
FARP	Conorfamide-Sr3	Conus spurius	Mollusc	P0DM28
FARP	Conorfamide-Tx1	Conus textile	Mollusc	P0DM26
FARP	Conorfamide-Tx2	Conus textile	Mollusc	P0DM27
FARP	Conorfamide-Vc1	Conus victoriae	Mollusc	P0DOZ7
GLP-1 c	Exendin-4	Heloderma suspectum	Lepidosauromorpha (lizard; Helodermatidae)	8396143
GLP-1	GLP-1 like	Ornithorhynchus anatinus	Monotreme	A0A1L3MY50
Insulin	Cono-insulin Pl1	Conus planorbis	Mollusc	KX034591
Insulin	Cono-insulin Tx1	Conus textile	Mollusc	A0A0B5A7N8
Insulin	Cono-insulin Tx2	Conus textile	Mollusc	KX034587
Insulin	Cono-insulin Bn1	Conus bandanus	Mollusc	KX034581.1
Insulin	Cono-insulin Eb1	Conus eburneus	Mollusc	KX034589
Insulin	Cono-insulin Eb2	Conus eburneus	Mollusc	KX034590
Insulin	Cono-insulin F1	Conus floridulus	Mollusc	A0A0B5AC98
Insulin	Cono-insulin F2	Conus floridulus	Mollusc	A0A0B5ADT9
Insulin	Cono-insulin F2b	Conus floridulus	Mollusc	A0A0B5A7N1
Insulin	Cono-insulin F2c	Conus floridulus	Mollusc	A0A0B5A7N5
Insulin	Cono-insulin G1	Conus geographus	Mollusc	A0A0B5AC95
Insulin	Cono-insulin G121	Conus geographus	Mollusc	BAO65654.1
Insulin	Cono-insulin G1b	Conus geographus	Mollusc	A0A0B5A8Q2
Insulin	Cono-insulin G1c	Conus geographus	Mollusc	A0A0B5A7P2
Insulin	Cono-insulin G2	Conus geographus	Mollusc	A0A0B5ABD9
Insulin	Cono-insulin G2b	Conus geographus	Mollusc	A0A0B5ADT3
Insulin	Cono-insulin G3	Conus geographus	Mollusc	A0A0B5A8P4
Insulin	Cono-insulin G3b	Conus geographus	Mollusc	A0A0B5AC86
Insulin	Cono-insulin Im1	Conus imperialis	Mollusc	A0A0B5A7M7
Insulin	Cono-insulin Im2	Conus imperialis	Mollusc	A0A0B5A8P8
Insulin	Cono-insulin K1	Conus kinoshitai	Mollusc	AZS18883.1
Insulin	Cono-insulin K2	Conus kinoshitai	Mollusc	AZS18884.1
Insulin	Cono-insulin M1	Conus marmoreus	Mollusc	A0A0B5A8Q6
Insulin	Cono-insulin M2	Conus marmoreus	Mollusc	KX034588
Insulin	Cono-insulin Me1	Conus memiae	Mollusc	A0A0B5ADV0
Insulin	Cono-insulin Pu1	Conus pulicarius	Mollusc	KX034592
Insulin	Cono-insulin Q1	Conus quercinus	Mollusc	A0A0B5ABE6
Insulin	Cono-insulin Q1b	Conus quercinus	Mollusc	A0A0B5ABE4
Insulin	Cono-insulin T1	Conus tulipa	Mollusc	A0A0B5ADU4
Insulin	Cono-insulin T2	Conus tulipa	Mollusc	A0A0B5AC90
Insulin	Cono-insulin T2	Conus tulipa	Mollusc	MH879035.1
Insulin	Cono-insulin T3	Conus tulipa	Mollusc	A0A0B5ABD5
Insulin	Cono-insulin Tr1	Conus tribblei	Mollusc	KX034595
Insulin	Cono-insulin Ts1	Conus tessulatus	Mollusc	KX034593
Insulin	Cono-insulin Ts2	Conus tessulatus	Mollusc	KX034594
Insulin	Cono-insulin Va1	Conus varius	Mollusc	KX034596
Insulin	Cono-insulin Va2	Conus varius	Mollusc	KX034597
Insulin	Cono-insulin Vc1	Conus victoriae	Mollusc	A0A0F7YYV0
Insulin	Cono-insulin Vi1	Conus virgo	Mollusc	AOF40168.1
Insulin	Insulin	Conus lividus	Mollusc	ATG85034.1
Insulin	Insulin partial	Conus araneosus	Mollusc	AQM52451.1
Insulin	Insulin-like protein	Conus amadis	Mollusc	AKZ17800.1
Insulin	Insulin-like protein partial	Conus buxeus loroisii	Mollusc	AMB57283.1
Insulin	Insulin-like protein partial	Conus frigidus	Mollusc	ARU12135.1
Insulin	Insulin-like protein partial	Conus litteratus	Mollusc	ARS01446.1
Insulin	Insulin-related protein	Conus ermineus	Mollusc	AXL95338.1
Insulin	Insulin-related protein	Conus ermineus	Mollusc	AXL95359.1
Insulin	Insulin-related protein	Conus ermineus	Mollusc	AXL95374.1
Insulin	Insulin-related protein	Conus ermineus	Mollusc	AXL95395.1
Insulin	Insulin-related protein	Conus ermineus	Mollusc	AXL95461.1
Insulin	Insulin-related protein	Conus ermineus	Mollusc	AXL95708.1
Insulin	Tu304	Conus tulipa	Mollusc	30669642
Insulin	Tu478	Conus tulipa	Mollusc	30669642
Insulin	Tu479	Conus tulipa	Mollusc	30669642
ITP/CHH d	α-latrotoxin associated LMWPe	Latrodectus geometricus	Arachnid (spider)	V9QF69
ITP/CHH	α-latrotoxin associated LMWP	Latrodectus hesperus	Arachnid (spider)	V9QFH5
ITP/CHH	α-latrotoxin associated LMWP	Latrodectus tredecimguttatus	Arachnid (spider)	P49125
ITP/CHH	α-latrotoxin associated LMWP 2	Latrodectus geometricus	Arachnid (spider)	V9QFG7
ITP/CHH	α-latrotoxin associated LMWP 2	Latrodectus hesperus	Arachnid (spider)	V9QEI7
ITP/CHH	α-latrotoxin associated LMWP 2	Latrodectus tredecimguttatus	Arachnid (spider)	Q4U4N3
ITP/CHH	α-latrotoxin associated LMWP 2	Steatoda grossa	Arachnid (spider)	V9QFH8
ITP/CHH	α-latrotoxin associated LMWP SGV150-311	Steatoda grossa	Arachnid (spider)	V9QFG9
ITP/CHH	α-latrotoxin associated LMWP SGV242-280	Steatoda grossa	Arachnid (spider)	V9QER4
ITP/CHH	BLTX280	Nephila pilipes	Arachnid (spider)	A0A076KUK2
ITP/CHH	k-scoloptoxin(03)-Ssd1a	Scolopendra subspinipes dehaani	Myriapod	A0A0R4I951
ITP/CHH	k-scoloptoxin(03)-Ssm1a	Scolopendra mutilans	Myriapod	I6RU32
ITP/CHH	k-scoloptoxin(03)-Ssm1d	Scolopendra mutilan	Myriapod	I6RU46
ITP/CHH	k-scoloptoxin(03)-Ssm1e	Scolopendra mutilan	Myriapod	I6RA73
ITP/CHH	µ-scoloptoxin(03)-Ssm2a	Scolopendra mutilan	Myriapod	P0DL36
ITP/CHH	U-scoloptoxin(03)-Sa1b	Phoneutria nigriventer	Myriapod	P0DPW8
ITP/CHH	U-scoloptoxin(03)-Ssd1b	Polistes lanio	Insect	P0DPV3
ITP/CHH	U1-agatoxin-Ta1a	Scolopendra mutilan	Myriapod	O46166
ITP/CHH	U1-agatoxin-Ta1b	Scolopendra subspinipes dehaani	Myriapod	O46167
ITP/CHH	U1-agatoxin-Ta1c	Scolopendra alternans	Myriapod	O46168
ITP/CHH	Venom protein 10	Microctonus hyperodae	Insect	A9YME6
Myoactive tetradeca-peptide	Conomap-Vt	Conus vitulinius	Mollusc	P0C260
Natriuretic peptide	CNP f	Philodryas olfersii	Lepidosauromorpha (colubrid)	Q09GK2
Natriuretic peptide	CNP	Bothrops insularis	Lepidosauromorpha (viper)	P68515
Natriuretic peptide	CNP	Cerastes cerastes	Lepidosauromorpha (viper)	A8YPR9
Natriuretic peptide	CNP	Crotalus atrox	Lepidosauromorpha (viper)	P0CV87
Natriuretic peptide	CNP	Crotalus durissus collilineatus	Lepidosauromorpha (viper)	Q2PE51
Natriuretic peptide	CNP	Crotalus durissus terrificus	Lepidosauromorpha (viper)	Q90Y12
Natriuretic peptide	CNP	Echis ocellatus	Lepidosauromorpha (viper)	A8YPR6
Natriuretic peptide	CNP	Gloydius blomhoffii	Lepidosauromorpha (viper)	P01021
Natriuretic peptide	CNP	Lachesis muta muta	Lepidosauromorpha (viper)	Q27J49
Natriuretic peptide	CNP	Sistrurus catenatus edwardsii	Lepidosauromorpha (viper)	B0VXV8
Natriuretic peptide	CNP	Trimeresurus gramineus	Lepidosauromorpha (viper)	P0C7P6
Natriuretic peptide	CNP 2	Takifugu rubripes	Actinopterygii	Q805D5
Natriuretic peptide	CNP 39	Ornithorhynchus anatinus	Monotreme	P84715
Natriuretic peptide	Tf-CNP	Protobothrops flavoviridis	Lepidosauromorpha (viper)	P0C7P5
Natriuretic peptide	Natriuretic peptide	Rhabdophis tigrinus tigrinus	Lepidosauromorpha (colubrid)	D1MZV3
Natriuretic peptide	Natriuretic peptide	Heloderma horridum	Lepidosauromorpha (lizard; Helodermatidae)	E8ZCG5
Natriuretic peptide	Natriuretic peptide	Heloderma suspectum cinctum	Lepidosauromorpha (lizard; Helodermatidae)	C6EVG7
Natriuretic peptide	Natriuretic peptide	Micrurus altirostris	Lepidosauromorpha (elapid)	F5CPE8
Natriuretic peptide	AsNP-a (Fragment)	Austrelaps superbus	Lepidosauromorpha (elapid)	A8S6B3
Natriuretic peptide	BF131	Bungarus flaviceps flaviceps	Lepidosauromorpha (elapid)	D5J9S0
Natriuretic peptide	BM026	Bungarus multicinctus	Lepidosauromorpha (elapid)	P0DMD5
Natriuretic peptide	CnNP-a (Fragment)	Cryptophis nigrescens	Lepidosauromorpha (elapid)	Q1ZYW1
Natriuretic peptide	CnNP-b (Fragment)	Cryptophis nigrescens	Lepidosauromorpha (elapid)	Q1ZYW0
Natriuretic peptide	Coa_NP1	Crotalus oreganus abyssus	Lepidosauromorpha (viper)	B3EWY3
Natriuretic peptide	Coa_NP2	Crotalus oreganus abyssus	Lepidosauromorpha (viper)	B3EWY2
Natriuretic peptide	DNP	Dendroaspis angusticeps	Lepidosauromorpha (elapid)	P28374
Natriuretic peptide	DNP-2	Dendroaspis angusticeps	Lepidosauromorpha (elapid)	Q8QGP7
Natriuretic peptide	GNP1	Varanus varius	Lepidosauromorpha (lizard; Anguidae)	Q2XXL8
Natriuretic peptide	HsNP-a (Fragment)	Hoplocephalus stephensii	Lepidosauromorpha (elapid)	Q3SAE6
Natriuretic peptide	HsNP-b (Fragment)	Hoplocephalus stephensii	Lepidosauromorpha (elapid)	Q3SAE5
Natriuretic peptide	Mc-NP	Micrurus corallinus	Lepidosauromorpha (elapid)	P79799
Natriuretic peptide	Mf-NP	Micrurus fulvius	Lepidosauromorpha (elapid)	B8K1V9
Natriuretic peptide	Na-NP	Naja atra	Lepidosauromorpha (elapid)	D9IX97
Natriuretic peptide	NP2	Crotalus durissus cascavella	Lepidosauromorpha (viper)	P0DKY6
Natriuretic peptide	NsNP-a (Fragment)	Notechis scutatus scutatus	Lepidosauromorpha (elapid)	Q3SAE8
Natriuretic peptide	NsNP-b (Fragment)	Notechis scutatus scutatus	Lepidosauromorpha (elapid)	Q3SAE7
Natriuretic peptide	Oh-NP	Ophiophagus hannah	Lepidosauromorpha (elapid)	D9IX98
Natriuretic peptide	OmNP-d (Fragment)	Oxyuranus microlepidotus	Lepidosauromorpha (elapid)	Q3SAF8
Natriuretic peptide	OmNP-e (Fragment)	Oxyuranus microlepidotus	Lepidosauromorpha (elapid)	Q3SAF7
Natriuretic peptide	OsNP-d (Fragment)	Oxyuranus scutellatus scutellatus	Lepidosauromorpha (elapid)	Q3SAX8
Natriuretic peptide	PaNP-a (Fragment)	Pseudechis australis	Lepidosauromorpha (elapid)	Q3SAF5
Natriuretic peptide	PaNP-b (Fragment)	Pseudechis australis	Lepidosauromorpha (elapid)	Q3SAF4
Natriuretic peptide	PaNP-c (Fragment)	Pseudechis australis	Lepidosauromorpha (elapid)	Q3SAF3
Natriuretic peptide	PaNP-d (Fragment)	Pseudechis australis	Lepidosauromorpha (elapid)	Q3SAF2
Natriuretic peptide	PNP	Pseudocerastes persicus	Lepidosauromorpha (viper)	P82972
Natriuretic peptide	PpNP-a (Fragment)	Pseudechis porphyriacus	Lepidosauromorpha (elapid)	Q3SAF1
Natriuretic peptide	PpNP-b (Fragment)	Pseudechis porphyriacus	Lepidosauromorpha (elapid)	Q3SAF0
Natriuretic peptide	PtNP-a (Fragment)	Pseudonaja textilis	Lepidosauromorpha (elapid)	Q3SAF6
Natriuretic peptide	TcNP-a (Fragment)	Tropidechis carinatus	Lepidosauromorpha (elapid)	Q3SAE9
Natriuretic peptide	TNP-a	Oxyuranus microlepidotus	Lepidosauromorpha (elapid)	P83224
Natriuretic peptide	TNP-a	Oxyuranus scutellatus canni	Lepidosauromorpha (elapid)	P83226
Natriuretic peptide	TNP-a	Oxyuranus scutellatus scutellatus	Lepidosauromorpha (elapid)	P83225
Natriuretic peptide	TNP-b	Oxyuranus microlepidotus	Lepidosauromorpha (elapid)	P83227
Natriuretic peptide	TNP-b	Oxyuranus scutellatus canni	Lepidosauromorpha (elapid)	P83229
Natriuretic peptide	TNP-b	Oxyuranus scutellatus scutellatus	Lepidosauromorpha (elapid)	P83228
Natriuretic peptide	TNP-c	Oxyuranus scutellatus canni	Lepidosauromorpha (elapid)	P83231
Natriuretic peptide	TsNP	Tityus serrulatus	Arachnid	P0DMD6
Natriuretic peptide	natriuretic/ helokinestatin-Cwar1	Celestus warreni	Lepidosauromorpha (lizard; Anguidae)	E2E4J4
Natriuretic peptide	natriuretic/ helokinestatin-Ginf1	Gerrhonotus infernalis	Lepidosauromorpha (lizard; Anguidae)	E2E4J3
Natriuretic peptide	Peptide TNP-c	Oxyuranus microlepidotus	Lepidosauromorpha (elapid)	P83230
Neuropeptide Y	Cono-NPY	Conus praecellens	Mollusc	28922871
Neuropeptide Y	Cono-NPY-Gm	Conus gloriamus	Mollusc	28531118
Neuropeptide Y	Neuropeptide Y	Meiacanthus atrodorsalis	Actinopterygii	P0DP55
Neuropeptide Y	Neuropeptide Y1	Conus betulinus	Mollusc	P0CJ22
Neuropeptide Y	Neuropeptide Y2	Conus betulinus	Mollusc	P0CJ23
Neurotensin	Contulakin G	Conus geographus	Mollusc	Q9XYR5
Neuro-hypophyseal	Conopressin Cn	Conus consors	Mollusc	22079299
Neuro-hypophyseal	Conopressin G	Conus andremenezi	Mollusc	ATF27388.1; ATF27387.1
Neuro-hypophyseal	Conopressin G	Conus araneosus	Mollusc	ATJ04126.1
Neuro-hypophyseal	Conopressin G	Conus buxeus loroisii	Mollusc	ATJ04131.1
Neuro-hypophyseal	Conopressin G	Conus ebraeus	Mollusc	ATJ04127.1
Neuro-hypophyseal	Conopressin G	Conus ermineus	Mollusc	AXL95508.1
Neuro-hypophyseal	Conopressin G	Conus geographus	Mollusc	P05486
Neuro-hypophyseal	Conopressin G	Conus gloriamus	Mollusc	28531118
Neuro-hypophyseal	Conopressin G	Conus imperialis	Mollusc	7940591
Neuro-hypophyseal	Conopressin G	Conus lividus	Mollusc	ATJ04129.1
Neuro-hypophyseal	Conopressin G	Conus monile	Mollusc	QDE14046.1
Neuro-hypophyseal	Conopressin G	Conus praecellens	Mollusc	ATF27585.1
Neuro-hypophyseal	Conopressin S	Conus striatus	Mollusc	P05487
Neuro-hypophyseal	Conopressin T	Conus tulipa	Mollusc	P0DL76
Neuro-hypophyseal	Conopressin Tx	Conus textile	Mollusc	P86255.1
Neuro-hypophyseal	Conopressin Tx1	Conus textile	Mollusc	P02716
Neuro-hypophyseal	Conopressin Tx2	Conus ermineus	Mollusc	A0A346CJ19
Neuro-hypophyseal	Conopressin Tx2	Conus lenavati	Mollusc	A0A0K8TTS0
Neuro-hypophyseal	Conopressin Tx2	Conus lividus	Mollusc	A0A291NVW3
Neuro-hypophyseal	Conopressin Tx2	Conus miles	Mollusc	A0A291NVU9
Neuro-hypophyseal	Conopressin Tx2	Conus monile	Mollusc	A0A4Y5X186
Neuro-hypophyseal	Conopressin Tx2	Conus textile	Mollusc	Unpublished
Neuro-hypophyseal	Conopressin Tx2	Conus tribblei	Mollusc	A0A0C9RYL1
Neuro-hypophyseal	Conopressin Vil	Conus villepinii	Mollusc	P85141
Prohormone-4	PH4	Conus andremenezi	Mollusc	28922871
Prohormone-4	PH4	Conus praecellens	Mollusc	28922871
Prohormone-4	PH4-Gm1	Conus gloriamus	Mollusc	28531118
Prohormone-4	PH4-Gm2	Conus gloriamus	Mollusc	28531118
Prohormone-4	PH4-Vc1	Conus victoriae	Mollusc	A0A0F7YYX3
Prohormone-4	Prohormone-4 like	Octopus vulgaris	Mollusc	XP 029651323.1
Tachykinin	Eledoisin	Eledone cirrhosa	Mollusc	P62933
Tachykinin	Eledoisin	Eledone moschata	Mollusc	P62934
Tachykinin	Oct-TK-I	Octopus vulgaris	Mollusc	Q8I6S3
Tachykinin	Oct-TK-II	Octopus vulgaris	Mollusc	Q8I6S2
Tachykinin	PhM1	Phoneutria nigriventer	Arachnid (spider)	15573368
Tachykinin	Sialokinin I	Aedes aegypti	Insect	P42634
Tachykinin	Sialokinin II Tachykinin-like	Aedes aegypti	Insect	P42634
Tachykinin	peptide-I (PllTkP-I) Tachykinin-like	Polistes lanio	Insect	P85879
Tachykinin	peptide-II (PllTkP-II)	Polistes lanio	Insect	P85880
Tachykinin	Tachykinin-like peptide-IX (PnTkP-IX)	Phoneutria nigriventer	Arachnid (spider)	P86306
Tachykinin	Tachykinin-like peptide-VI (PnTkP-VI)	Phoneutria nigriventer	Arachnid (spider)	P86303
Tachykinin	Tachykinin-like peptide-X (PnTkP-X)	Phoneutria nigriventer	Arachnid (spider)	P86307
Tachykinin	Tachykinin-like peptide-XI (PnTkP-XI)	Phoneutria nigriventer	Arachnid (spider)	P86308
Tachykinin	Tachykinin-like peptide-XIII	Phoneutria nigriventer	Arachnid (spider)	P86310

^aCCAP, Crustacean cardioactive peptide;

^bFARP, FMRFamide related peptide family;

^cGLP-1, Glucagon-like peptide 1;

^dITP/CHH, Ion transport peptide/crustacean hyperglycaemic hormone;

^eLMWP, low molecular weight protein;

^fCNP, C-type natriuretic peptide; Genbank accession number are underlined, in bold.

There exist several examples where venom neuropeptides have demonstrated their therapeutic potential. Exendin-4, a GLP-1 receptor agonist isolated from the venom of the Gila monster (Heloderma suspectum), was used to develop Exenatide as well as Lixisenatide for the treatment of type 2 diabetes mellitus [85, 108]; contulakin-G, a neurotensin homologue isolated from Conus geographus venom is under investigation for the treatment of acute and chronic pain [109]; natriuretic peptides found in snake venoms are used to understand human natriuretic peptide receptor signalling [110]; sarafotoxin, identified in the venom of the snake Atractaspis engaddensis, acts on endothelin receptors, has been used to understand endothelin receptor signalling and aided development of endothelin receptor antagonists for the treatment of cardiovascular disorders [111]; conopressin-T, a vasopressin-like peptide identified in the cone snail Conus tulipa venom, contributed towards the development of novel vasopressin receptor antagonists that are used for the treatment of hyponatremia [112].

Although venom neuropeptides have been utilised for their therapeutic potential, very little is known on the physiological role venom neuropeptides play in predation and defence. Venom natriuretic peptides and sarafotoxins likely contribute to prey capture by targeting the cardiovascular system and affecting vasodilation or vasoconstriction and heart contractility [110, 113, 114]. Cono-insulins reduce blood glucose levels in fish which results in sluggish behaviour facilitating capture [115]. Pain and inflammation are common responses to envenomation, often for defensive purposes [116] and for envenomation of mice by the wasp Polistes lanio lanio these effects were attributed to tachykinin-like peptides acting on the neurokinin-1 (NK1) receptor [117, 118]. Conorfamide-Sr2, an FMRF-amide related peptide isolated from worm-eating Conus spurius venom, was hypothesized to have a defensive function as it had mild paralytic effects in the limpet Patella opea [119]. In addition, based on the neuropeptide families they belong to, venom neuropeptides are implicated in various physiological processes including cardiovascular functions (natriuretic peptide [120], crustacean cardioactive peptide [121], RFamide [122], neurotensin [123]), metabolism (insulin [124], glucagon-like peptide-1 [GLP-1] [125], RFamide [122]), reproduction (oxytocin [16]), fluid homeostasis (natriuretic peptide [126], vasopressin [44], bradykinin [127]) and pain (tachykinin [128], bradykinin [25], enkephalin [129], RFamide [122]).

In addition to venom neuropeptides with high homology to the human neuropeptides, venoms also contain peptides with very little homology to neuropeptides that potently bind to neuropeptide receptors. Venom peptides are generally characterised by well-conserved disulfide bond frameworks with loops that are highly mutable, thereby providing an evolutionary easily amendable scaffold able to adapt to a wide range of targets [76, 130]. A good example of this is mambaquaretin-1 isolated from the green mamba (Dendroaspis angusticeps), which binds allosterically to the vasopressin V2R (GPCR) and protects against renal cyst development, making it an interesting therapeutic lead for the treatment of polycystic kidney disease [131]. This was unexpected, since Mambaquaretin-1 has a Kunitz-fold, which is a well-characterised scaffold with three disulfide bonds, two antiparallel beta strands and a short alpha helix [131, 132] that is commonly found in venoms and known to target ion channels and proteases [110, 131, 133, 134]. Other examples of venom peptides that have no homology to neuropeptides but target neuropeptide receptors include hypotensin-I isolated from the scorpion Tityus serrulatus and which is a B2 bradykinin receptor agonist [135]; d-ctenitoxin-Pn1a from the spider Phoneutria nigriventer that targets opioid receptors [136]; t-CnVA and conorphins from cone snail venom that target the somatostatin and opioid receptors, respectively [137, 138]; and Crotalphine isolated from the venom of the snake Crotalus durissus terrificus that targets the k-opioid receptor and causes analgesia in rats [139]. The overall chemical and functional diversity of venom toxins is what renders venoms such interesting sources for the discovery of novel drug leads, diagnostic probes and pharmacological tools, which could accelerate our understanding of neuropeptide signalling in humans, if explored properly.

The number of venom neuropeptides listed in Table 2 is likely to represent only a small fraction of the total number of neuropeptides recruited into animal venoms as a result of limited characterisation. The broad phylogenetic range of venomous animals and number of known venom compounds (~6,000) versus the number of known venom neuropeptides suggests there are many more venom neuropeptides to be discovered [76, 140]. One explanation for the low number of neuropeptides is that only a limited number of venoms have been characterised to date and there is a bias towards certain animal lineages at the expense of others [140]. For example, snakes and cone snails have been heavily studied in contrast to invertebrates such as centipedes, cephalopods (octopus, squid and cuttlefish) and cnidarians [140, 141]. In fact, it is hypothesised that less than 1% of the total number of venom proteins and peptides have been characterised [77].

Another reason for the low number of known neuropeptides in animal venoms may have to do with the methods employed to characterise them. The majority of neuropeptides listed in Table 2 were identified by transcriptomics. The transcriptome is the sum of all mRNA molecules in one cell or a population of cells [142]; it provides information on transcript expression level, transcriptome dynamics across different tissues or conditions, and is not dependent on an existing genomic sequence. Transcriptomics, often combined with proteomics, is the most common venom characterisation method and allows a non-biased and non-targeted approach to characterise venom glands [140, 143–145]. However, it has also revealed high intraspecific variation due to diet, geographical location, age, and gender which necessitates care when interpreting results [146–148]. The correct and efficient use of bioinformatics is essential to be able to process and integrate the large amount of data generated in a transcriptomic study [149]. Annotation of transcriptome sequences relies on sequence comparison to known, annotated proteins and peptides in databases such as UniProt and the National Center for Biotechnology Information (NCBI) database [150]. The short length of neuropeptides and high sequence diversity of neuropeptide precursors makes them difficult to identify using sequence homology methods such as the NCBI Basic Local Alignment Search Tool (BLAST) [61, 151]. Consequently, it is likely that a large number of neuropeptide homologues in venoms have not been detected in ‘omics’ data. Therefore, it seems reasonable to expect identification of a higher number of neuropeptides in animal venoms using a targeted approach. This seems to have been the case in cone snails where neuropeptide-specific search in the venom gland transcriptome of Conus victoriae led to the identification of five different neuropeptides [152], in contrast to the two neuropeptides previously identified in C. victoriae [153].

6. Neuropeptide identification

Venom and neuropeptide research employ comparable strategies and technologies to identify peptides and face similar challenges including low peptide concentrations, PTMs, variable size, high degree of structural diversity and a large number of isoforms [60, 62]. A combined approach of mass spectrometry, sequencing technologies (i.e. transcriptomics) and bioinformatics (termed ‘Integrated Venomics’) has been very successful for both venom characterisation [77, 102, 140, 145] and neuropeptide identification [29, 59, 154]. Mass spectrometry is very sensitive, fast, allows de novo sequencing and enables identification of PTMs, although more material is required compared to transcriptomics [60, 62]. For animals with no or only a partial reference genome, transcriptomics in particular has greatly accelerated characterisation of animal venoms and identification of neuropeptides [140, 155]. Advances in sequencing technology and mass spectrometry combined with in silico prediction and bioinformatic tools has facilitated research in both areas [59, 60, 150, 156].

PTMs include the precursor protein cleavage sites that produce the mature peptides as well as residue modifications and can only be partially predicted from the nucleotide sequence [151]. Although advancements in mass spectrometry have increased selectivity and sensitivity, PTM determination of low abundance peptides remains challenging and requires sample preparation and separation methods such as liquid chromatography or capillary electrophoresis to maximise sensitivity [59, 62]. This is particularly relevant for venom characterisation of animals that produce very small amounts of venom. D-amino acids, a PTM identified in spider, mollusc and mammalian venom peptides, as well as mollusc neuroexcitatory peptides and crustacean neurohormones, are notoriously difficult to detect due to a lack of a sequence change or mass defect [157]. New mass spectrometry methods have been developed that measure the distinct molecular fragmentation patterns among peptide diastereomers with tandem mass spectrometry (MS/MS) [59].

Computational methods are necessary to filter, process, store and integrate the multifaceted information generated in mass spectrometry and sequencing studies. In addition, bioinformatic tools have been developed to facilitate neuropeptide and venom peptide identification including prohormone prediction tools (NeuroPID [158]), precursor protein cleavage site prediction tools (NeuroPred [159, 160], ProP [161], SignalP [162]), and peptide databases (neuropeptide database NeuroPep [2] and venom peptide databases [163–166]). The advancement and development of novel bioinformatic tools is critical to meet the computational demands of high-throughput mass spectrometry and sequencing technology which produce ever larger and more complex datasets [60].

Identification of a peptide, be that a neuropeptide or venom peptide, using mass spectrometry or polynucleotide sequencing technology does not provide information on the peptide target. After being identified with an ‘omics’ approach, peptides are typically synthesised and their activity characterised. Fortunately, synthesis of peptides (under 50 residues) is relatively straightforward using solid-phase peptide synthesis and the use of selective amino acid protecting groups allows directed disulfide-bond formation, which is important for characterising disulfide-rich peptides displaying potentially several disulfide bond isomers [167, 168]. In addition, recombinant expression, native chemical ligation, semisynthetic methods, and ligase/enzymatic ligations enable synthesis of longer and more complex peptides [100, 169, 170]. Subsequent testing on high-throughput assays such as fluorescent plate readers which measure Ca²⁺, Na⁺, K⁺ signalling (such as Fluorescence Imaging Plate Reader, FLIPR), electrophysiology, or single cell assays enables assessing of activity at various receptors [171, 172]. However, in vitro characterization of venom peptides provides limited insight into their function, and often unexpected effects are observed once administered in vivo [173].

7. Summary and future perspectives

Neuropeptide signalling systems are ubiquitous throughout the animal kingdom and regulate many fundamental physiological processes. They typically contain multiple receptor subtypes with complex signalling cascades that remain poorly understood due to a lack of receptor subtype-selective ligands. Because of their high level of conservation, endogenous neuropeptides, and in particular venom neuropeptides, represent a source for novel neuropeptide receptor ligands with varying pharmacological properties. Known venom neuropeptides are likely to be the tip of the iceberg and technological advances in mass spectrometry and sequencing technology combined with the development of novel bioinformatics tools promise to facilitate the identification of venom neuropeptides and characterisation of neuropeptide signalling systems. However, many challenges remain including a limited venom species diversity and identification of PTMs. Nevertheless, neuropeptides recruited into animal venoms offer a viable source for novel pharmacological tools and therapeutic leads to study the role of neuropeptides in health and disease.

List of abbreviations

ACTH

Adrenocorticotropic hormone

BLAST

Basic Local Alignment Search Tool

CGRP

Calcitonin/calcitonin gene-related peptide

CNS

Central nervous system

CRH

Corticotropin-releasing hormone

ER

Endoplasmic reticulum

FLIPR

Fluorescence Imaging Plate Reader

GLP-1

Glucagon-like peptide-1

GnRH

Gonadotropin-releasing hormone

GPCR

G protein-coupled receptors

MCH

Melanin-concentrating hormone

MS/MS

Tandem mass spectrometry

MSH

Melanocyte-stimulating hormone

NCBI

National Center for Biotechnology Information

NPY

Neuropeptide Y

OTR

Oxytocin receptor

PACAP

Pituitary adenylate cyclase-activating peptide

PNS

Peripheral nervous system

POMC

Proopiomelanocortin

PTM

Post-translational modifications

TRH

Thyrotropin-releasing hormone