Venom peptides as pharmacological tools and therapeutics for diabetes

Accepted Manuscript Venom peptides as pharmacological tools and therapeutics for diabetes Samuel D. Robinson, Helena Safavi-Hemami PII:

S0028-3908(17)30308-8

DOI:

10.1016/j.neuropharm.2017.07.001

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Neuropharmacology

Received Date: 3 February 2017 Revised Date:

24 June 2017

Accepted Date: 4 July 2017

Please cite this article as: Robinson, S.D., Safavi-Hemami, H., Venom peptides as pharmacological tools and therapeutics for diabetes, Neuropharmacology (2017), doi: 10.1016/ j.neuropharm.2017.07.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Review ACCEPTED MANUSCRIPT Venom peptides as pharmacological tools and therapeutics for diabetes Samuel D. Robinson1 and Helena Safavi-Hemami1* Department of Biology, University of Utah, Salt Lake City, UT 84112, USA *To whom correspondence should be addressed: [email protected]

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Abstract

Diabetes mellitus is a chronic disease caused by a deficiency in production of insulin by the beta cells

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of the pancreas (type 1 diabetes, T1D), or by partial deficiency of insulin production and the ineffectiveness of the insulin produced (type 2 diabetes, T2D). Animal venoms are a unique source of compounds targeting ion channels and receptors in the nervous and cardiovascular systems. In recent

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years, several venom peptides have also emerged as pharmacological tools and therapeutics for T1D and T2D. Some of these peptides act directly as mimics of endogenous metabolic hormones while others act on ion channels expressed in pancreatic beta cells. Here, we provide an overview of the discovery of these venom peptides, their mechanisms of action in the context of diabetes, and their

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therapeutic potential for the treatment of this disease.

Contents

1. The role of pancreatic hormones in glucose homeostasis 2. The role of ion channels in insulin secretion

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3. Diabetes mellitus – pathophysiology and treatment 4. Venom peptides as pharmacological tools and therapeutics for diabetes

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4.1 GLP-1 analogues from venoms 4.1.1 Exendin-4 - a first-in-class treatment for diabetes from lizard venom

4.1.2 Exendin-4 analogues as diagnostic tools for diabetes 4.1.3 A new GLP-1 analogue from the platypus and echidna

4.2 Cone snail venom insulins 4.3 Potassium channel blockers from venoms 4.3.1 KV2.1 blockers from tarantula venom enhance insulin secretion 4.3.2 Conkunitzin-S1 from cone snail venom reveals KV1.7 as a molecular target for T2D 4.3.3 Blockade of BK channels by iberiotoxin enhances insulin secretion in human beta cells 1

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5. Perspectives Acknowledgments References

1. The role of pancreatic hormones in glucose homeostasis In mammals glucose homeostasis is regulated by a finely tuned interplay between the two pancreatic

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hormones, insulin and glucagon. Insulin is produced in the beta cells of the pancreas and released into the bloodstream in response to elevated blood glucose levels following food intake. Insulin induces glucose uptake from the bloodstream into adipose, skeletal muscle and the liver cells (Figure 1). Glucagon, which is released by the alpha cells of the pancreas when blood glucose levels are too low, has the opposite effect. The insulin-glucagon system maintains steady blood glucose levels and

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prevents episodes of low or high blood glucose, known as hypo- or hyperglycemia, respectively. In addition to insulin and glucagon, several other hormones have been identified which also contribute to

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glucose homeostasis (Aronoff et al., 2004). One of these, the incretin hormone glucagon-like peptide-1 (GLP-1), which was not discovered until the early 1980s (Lund et al., 1982), is now known to play a major role in maintaining healthy blood glucose levels (Nadkarni et al., 2014). Following food intake, GLP-1 is released from intestinal L-cells whereby, among other actions, it stimulates insulin secretion from pancreatic beta cells and suppresses glucagon secretion from pancreatic alpha cells (Komatsu et

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al., 1989; Mojsov et al., 1987; Schmidt et al., 1985).

2. The role of ion channels in insulin secretion

Pancreatic beta cells share many similarities with neuronal cells (Atouf et al., 1997; Eberhard, 2013). In

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pancreatic beta cells, ion channels play a critical role in regulating the glucose-stimulated secretion of insulin (Cherki et al., 2006). Similar to the release of neurotransmitters by neuronal cells, exocytosis of insulin granules involves membrane depolarization and action potential firing. A simplified overview

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of this process is provided in Figure 1. Uptake and metabolism of glucose in beta cells generates an increase in the ratio of ATP over ADP. This causes closure of ATP-sensitive potassium (KATP) channels leading to cell depolarization. As a response, voltage-gated calcium (CaV) channels open and the resulting increase in intracellular Ca2+ ions triggers action potential firing and the release of insulin granules from the pancreas (Ashcroft and Rorsman, 1989; Rajan et al., 1990). Following exocytosis of insulin-containing granules, the cell repolarizes through the activation of large-conductance calciumactivated potassium (BK) channels and voltage-gated potassium (KV) channels, including KV2.1 and KV1.7. Because of their critical role in insulin secretion, beta cell ion channels have become important targets for pharmacological interventions in the treatment of diabetes (Cherki et al., 2006; Herrington, 2007). 2

3. Diabetes mellitus – pathophysiology and treatment ACCEPTED MANUSCRIPT Disruption of glucose homeostasis can lead to diabetes mellitus. Two major types of diabetes exist. Type 1 diabetes (T1D) is an autoimmune disorder that is characterized by the destruction of pancreatic islets leading to insulin deficiency. Daily insulin injections are the only effective treatment for T1D. The ultimate goal of insulin therapy is to maintain healthy blood glucose levels. However, owing to the physicochemical properties of insulin, application of the natural hormone can result in slow absorption

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of mealtime insulin and insufficient levels of basal insulin, often resulting in episodes of hyper- and hypoglycemia. This has initiated the design of both rapid-acting (e.g. lispro and aspart) and long-acting (e.g. detemir and glargine) insulin analogues (Owens, 2002). Injection of a long-acting, basal analogue in combination with a rapid-acting mealtime analogue provides better control of blood glucose. Type 2 diabetes (T2D) is caused by a partial deficiency in pancreatic insulin secretion coupled with the

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inability of cells to adequately respond to insulin (insulin resistance). T2D is characterized by reduced uptake of glucose by muscle, liver and adipose cells and subsequent hyperglycemia. Oral drugs used to

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treat T2D include compounds that enhance insulin secretion from the pancreas (Sulfonylureas, such as glyburide and glipizide), decrease the production of glucose in the liver (Biguanides, such as metformin) or increase the sensitivity of adipose and muscle cells to insulin (Thiazolidinediones, such as rosiglitazone and pioglitazone) (Blonde, 2009). Insulin therapy is prescribed if blood glucose cannot be effectively controlled using oral medication alone.

A third type of diabetes, gestational diabetes mellitus (GDM), is a condition where women without

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previous diagnosis of diabetes exhibit abnormal blood glucose levels during pregnancy. In normal pregnancy, secretion of several hormones by the placenta (growth hormone, corticotropin-releasing hormone, placental lactogen and progesterone) leads to an increase in insulin resistance. This is

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compensated by elevated insulin secretion due to pancreatic beta cell hyperplasia (Mack and Tomich, 2017). GDM is characterized by the inability of the body to overcome insulin resistance despite beta cell hyperplasia. Exercise and a healthy diet are effective in most GDM patients. However, insulin

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injections may be prescribed if blood glucose is not effectively controlled by lifestyle changes alone.

4. Venom peptides as pharmacological tools and therapeutics for diabetes Animal venoms are a unique natural source for drug discovery (King, 2011; Lewis and Garcia, 2003; Zambelli et al., 2016). The majority of venom peptides and proteins characterized so far target the cardiovascular or nervous system of prey (or predators). The discovery and characterization of the GLP-1 analogue exendin-4 (Eng et al., 1990) provided the first example for the therapeutic application of a venom component that targeted metabolic function. In addition to exendin-4, several other venom peptides have now been discovered which have potential utility in the development of new diabetes therapeutics (Table 1). These include a GLP-1 analogue from the platypus and echidna, specialized 3

insulin from the venom of a cone snail and potassium channel blockers from the venoms of tarantulas, ACCEPTED MANUSCRIPT a cone snail and a scorpion.

4.1 GLP-1 analogues from venoms

4.1.1 Exendin-4 - a first-in-class treatment for diabetes from lizard venom

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The incretin hormone GLP-1 has a number of attributes that made it a promising potential treatment for diabetes. In addition to stimulation of insulin secretion, GLP-1 inhibits glucagon secretion, delays gastric emptying and suppresses appetite (Flint et al., 1998; Komatsu et al., 1989; Wettergren et al., 1993; Willms et al., 1996). Furthermore, the effects of GLP-1 on insulin secretion are glucosedependent, thus reducing the risk of hypoglycaemia, one major unwanted action of many anti-diabetic

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agents. Finally, GLP-1 also stimulates pancreatic beta cell proliferation and prevents beta cell death

complications associated with T2D.

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(Farilla et al., 2002), suggesting it can not only prevent, but also perhaps partly reverse some of the

Early studies investigating the potential of GLP-1 as a treatment for diabetes confirmed the antidiabetic actions of the peptide (Nauck et al., 1993; Rachman et al., 1997). However, a major drawback halted the development of the peptide as a drug. GLP-1 has a very short in vivo half-life (2-3 minutes). It is rapidly degraded by the peptidase dipeptidyl-peptidase-IV (DPP-IV) and other ectopeptidases (Mentlein et al., 1993). GLP-1 was only effective with repeated injections or continuous infusion.

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Thus, despite its promising anti-diabetic effects, the obvious impracticalities associated with administration rendered the peptide unviable as an anti-diabetic agent. It was clear that a more stable analogue of GLP-1 would be required. Such a peptide came from a series of academic studies of the

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venoms of helodermatid lizards (reviewed in (Furman, 2012)). Helodermatidae is a family of venomous lizards, which are found in the South Western United States, Mexico and as far south as Guatemala. In helodermatid lizards, venom is produced in premandibular glands and secreted onto

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specialized grooved teeth of the lower jaw. Venom is used in defence and also for subduing prey (rodents, other small mammals and reptiles) (Russell and Bogert, 1981). The dominant clinical phenotype associated with envenomation by these lizards is a rapid drop in blood pressure that can lead to hypotensive shock (Patterson, 1967). However, the venoms are complex and, additional, less clinically obvious mechanisms of action exist, including disruption of metabolic function. Two peptides, designated exendin-3 and exendin-4 were isolated from the venoms of the Mexican beaded lizard Heloderma horridum (Eng et al., 1990) and the Gila monster Heloderma suspectum (Eng et al., 1992), respectively. Both peptides produced an increase in cAMP signalling in pancreatic acinar cells with exendin-4 having superior selectivity (Raufman et al., 1991; Raufman et al., 1992). Sequence similarity to mammalian GLP-1 was recognized, and it was soon confirmed that the actions of exendin4 were mediated through the GLP-1 receptor (Göke et al., 1993). In fact, in essentially every aspect 4

tested, including anti-diabetic activity, exendin-4 appeared to mimic the actions of mammalian GLP-1 ACCEPTED MANUSCRIPT (Raufman et al., 1992; Young et al., 1999). However, key differences in the primary structure of exendin-4 resulted in in vivo stability that was several orders of magnitude greater than that of GLP-1 (Thum et al., 2002) (Figure 2A). Exendin-4 has an in vivo half-life of around 2.4 h (20- to 30-fold longer than GLP-1) and accordingly, a 5,500-fold greater potency in lowering plasma glucose (Parkes et al., 2001; Young et al., 1999). The greater stability of the venom peptide exendin-4 sparked its rapid development as an anti-diabetic

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agent. Following successful clinical trials, synthetic exendin-4 (Exenatide; ByettaTM) entered the market in 2005 as a first-in-class incretin-mimetic for the treatment of T2D. In 2008, US sales peaked at $678 million. In 2016, Intarcia Therapeutics completed Phase 3 clinical trials for the continuous subcutaneous

delivery

of

exenatide

using

a

match-stick

sized

mini-pump

(ITCA650,

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injection-free GLP-1 receptor agonist therapy for T2D.

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http://www.intarcia.com/pipeline-technology/itca-650.html). If approved, this will represent the first

4.1.2 Exendin-4 analogues as diagnostic tools for diabetes

GLP-1 analogues, including exendin-4, are now also being pursued as potential clinical tools for the early diagnosis of diabetes through non-invasive in vivo assessment of pancreatic beta cell mass (BCM). Reduction in BCM is a key factor in the development in both T1D and T2D, and usually begins years before the clinically obvious disease is evident. Current tests, such as evaluation of fasting

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plasma glucose or the oral glucose tolerance test detect the disease only once the majority of the beta cells are destroyed and the disease is too advanced for any preventive measures to be taken. Non-invasive in vivo imaging of BCM has the potential to provide an earlier diagnosis facilitating

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therapeutic intervention. Because of its specific expression in beta cells and strong interaction with its ligands, the GLP-1 receptor is a promising beta-cell biomarker (Tornehave et al., 2008). Several radio- and fluorescent-labeled exendin-4 analogues have now been developed (Connolly et al.,

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2012; Gotthardt et al., 2006; Reiner et al., 2011; Selvaraju et al., 2013; Wild et al., 2010; Wu et al., 2011) and appear to be promising tools for the evaluation of BCM in vivo. However, several issues such as non-specific beta cell uptake and high accumulation in nearby organs need to be addressed before these compounds can become clinically relevant (Manandhar and Ahn, 2015).

4.1.3 A new GLP-1 analogue from the platypus and echidna New venom gland-derived GLP-1 analogues were recently reported from monotremes (Tsend-Ayush et al., 2016). Monotremata is an order of mammals, found in Australia and New Guinea, which include the platypus (Ornithorhynchus anatinus) and four species of echidna. Male platypus possess a venom gland on the hind limb. They use their venom in defence, particularly against other male platypus 5

during the breeding season (Wong ACCEPTED et al., 2012). Echidna species also possess an equivalent gland but MANUSCRIPT its biological role is unclear (Wong et al., 2013). Analysis of Gcg expression (the gene encoding GLP-1) in the platypus and echidna indicated mRNA expression in venom gland tissue (Tsend-Ayush et al., 2016). Accordingly, it was proposed that the predicted GLP-1 peptides may serve as venom components in monotremes. The monotreme peptides showed interesting biophysical characteristics that may offer valuable insight in the development of

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new GLP-1-based anti-diabetic agents. They are resistant to degradation in human serum in a similar way to exendin-4, and while both monotreme peptides showed reduced potency at GLP-1 receptors, platypus GLP-1 and echidna GLP-1 (at a concentration of 100 nanomolar) showed equivalent stimulation of insulin secretion from mouse islet cells to human GLP-1 and exendin-4. Significantly, the monotreme GLP-1 peptides produced distinct GLP-1 receptor signal bias relative to human GLP-1

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and exendin-4. Human GLP-1 and exendin-4 show relative bias for cAMP and intracellular Ca2+ mobilization (involved in promotion of insulin release) (Koole et al., 2013) whereas the monotreme

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peptides show relative bias toward pERK1/2 (involved in activation of mitogenic signaling pathways). Exploration of the consequences of the distinct signaling differences engendered by these peptides may offer valuable insight into the development of new GLP-1-based anti-diabetic agents.

4.2 Cone snail venom insulins

Insulin therapy is essential for the treatment of patients with T1D and for many late-stage patients with

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T2D. As a standard administration method, these diabetic patients must inject insulin subcutaneously once or multiple times per day. One of the major limitations of insulin therapy is that the native hormone oligomerizes into a stable hexamer consisting of three insulin dimers held together by a

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central zinc ion (Adams et al., 1969). Following subcutaneous injection the hexamer has to dissociate into the dimer then monomer to be able to bind to the insulin receptor. Hexamer-to-monomer conversion is a slow process that leads to a significant delay in glucose control. This limitation drove

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the search for insulin analogues with reduced dimerization (and thus oligomerization) rates (Owens, 2002). However, despite decades of research the best fast-acting insulin formulations are not truly monomeric and still require between 15-90 min to effectively lower blood glucose levels (Elleri et al., 2011). This is because the region involved in dimerization of the insulin molecule, the C-terminus of the insulin B chain, is also of critical importance for receptor activation (Menting et al., 2013). Removal of this region to abolish dimerization could not be achieved without compromising biological activity (Bao et al., 1997; De Meyts et al., 1978). Novel insights for solving this long-standing problem recently came from the discovery of an insulin peptide found in the venom of the fish-hunting cone snail, Conus geographus (Safavi-Hemami et al., 2015). C. geographus belongs to a large genus of predatory marine snails (Conus) that use venom for prey capture, defense and competitive interactions (Olivera, 1997). It is famous for having caused the 6

greatest number of human fatalitiesACCEPTED of any cone snail species; in the absence of medical intervention, MANUSCRIPT 70% of human stinging cases are fatal (Kohn, 2016). C. geographus insulin (Con-Ins G1) was shown to be an abundant component of the venom of this species and is used by the snail to rapidly induce insulin shock (dangerously low blood sugar) in its fish prey (Robinson and Safavi-Hemami, 2016; Safavi-Hemami et al., 2015). Con-Ins G1 represented the smallest insulin identified from a natural source, and remarkably, lacked the region of the B chain that

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is critical for both receptor engagement and dimerization in human insulin (Menting et al., 2016; Safavi-Hemami et al., 2015). Furthermore, the sequence of Con-Ins G1 was more similar to fish insulin than to the endogenous snail insulin (Safavi-Hemami et al., 2016), strongly suggesting that it would be active at the fish insulin receptor. Indeed, injection of Con-Ins G1 into zebrafish produced a rapid drop in blood glucose with a potency comparable to that of human insulin (Safavi-Hemami et al., 2015).

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Additionally, simply adding Con-Ins G1 to the water column resulted in an immediate impairment of the fish’s swimming behavior suggesting that this peptide was taken up into the blood stream via the

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gills. Together these findings demonstrated that the snail insulin Con-Ins G1 was active at the fish insulin receptor and capable of inducing hypoglycemia. Given the high sequence similarity of insulin and its receptor between fish and human it was anticipated that Con-Ins G1 would also be active against the human insulin receptor. Indeed, it was subsequently demonstrated that Con-Ins G1 was an agonist of the human insulin receptor and induced downstream insulin signaling, albeit at lower potency than that of human insulin (Menting et al., 2016). To identify the structural motifs rendering

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Con-Ins G1 active despite the absence of the C-terminus of the B chain, the structure of this peptide was solved and modeled against regions of the human insulin receptor that form the primary binding site of insulin (Menting et al., 2016). These studies revealed that certain residues within Con-Ins G1

2B).

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(TyrB15 and TyrB20) could act as surrogates of the C-terminus of the B chain in human insulin (Figure

Con-Ins G1 represents the first truly monomeric insulin analogue with significant activity at the human

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receptor but its lower activity compared with human insulin and potential immunogenicity effects may hinder the development of Con-Ins G1 as an effective drug for T1D. However, current and future findings on the interaction between Con-Ins G1 and the human insulin receptor may inform the design of a novel class of fast-acting insulin analogues for the treatment of diabetes. Furthermore, several other fish–hunting cone snail species also express venom insulins that share basic characteristics of Con-Ins G1 (i.e. are predicted to be monomeric) but have diverse primary structures (Safavi-Hemami et al., 2015). Structure-function studies on these venom peptides may reveal further insights into the molecular mechanisms of insulin receptor binding and signaling. 4.3 Potassium channel blockers from venoms

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As outlined in section 2 and illustrated in FigureMANUSCRIPT 1, insulin secretion is triggered by closure of KATP ACCEPTED channels upon an increase in intracellular glucose (and therefore ATP) levels. Because of their critical role in insulin secretion, KATP channels have been a target of drug development, and several small molecule drugs inhibiting these channels (e.g. Sulfonylureas) are now widely used for treating T2D (Bonfanti et al., 2015; Bryan et al., 2005; Sturgess et al., 1985). However, a problem commonly associated with these drugs is that inhibition of KATP channels is independent of basal glucose levels

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and hypoglycemia is a potential side-effect (Stahl and Berger, 1999). This limitation has led to efforts to identify alternative ion channel targets for enhancing pancreatic insulin secretion. As outlined in section 2, pancreatic beta cells possess both BK channels and delayed rectifier KV channels that are important for repolarization of the action potential following insulin secretion (Smith et al., 1990). Because they only open when beta cells are depolarized in the presence of elevated glucose, blocking

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these channels was expected to enhance secretion in a strictly glucose-dependent manner. However, testing this hypothesis was not trivial because small molecule inhibitors of specific potassium channel

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subtypes did not exist. Several venom peptides have since led the way in identifying specific potassium channels in pancreatic beta cells and confirming that blocking these channels can produce a surge in glucose-dependent insulin secretion.

4.3.1 KV2.1 blockers from tarantula venom enhance insulin secretion Of the various KV channels known to be expressed in pancreatic beta cells (Yan et al., 2004), KV2.1

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subunits were thought to be a major contributor of delayed rectifier currents. The first insight into the role of KV2.1 channels in insulin secretion came from studies of the venom of the Chilean rose tarantula Grammostola rosea (Swartz and MacKinnon, 1995), a species from the desert regions

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of Northern Chile, Bolivia, and Argentina. The peptide hanatoxin (κ-theraphotoxin-Gr1a), a KV2.1 channel blocker, was discovered in its venom (Swartz and MacKinnon, 1995). Hanatoxin was able to inhibit delayed rectifier currents in mouse and human beta cells and augmented glucose-dependent

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insulin secretion (Herrington et al., 2005; Tamarina et al., 2005). This finding indicated that the KV2.1 channel had potential to serve as a new target for the treatment of T2D. Further functional testing was performed using another KV2.1 channel blocker, guangxitoxin-1 (κ-theraphotoxin-Pg1a) from the Chinese earth tiger tarantula (Chilobrachys guangxiensis), a species from the Hainan Province of Southern China. It was confirmed that in beta cells, KV2.1 inhibition by guangxitoxin-1, led to an increase in intracellular Ca2+ and stimulated insulin secretion in a glucose-dependent fashion (Herrington et al., 2006). However, neither peptide was pursued further as a drug lead due to their broad pharmacological profile; In addition to blocking KV2.1, both peptides also potently inhibited KV2.2 and KV4 channels (Herrington, 2007), which although absent from pancreatic beta cells (Roe et al., 1996; Yan et al., 2004), are widely expressed in other human tissues (Isbrandt et al., 2000; Johnston et al., 2008). Nevertheless these venom peptides were instrumental in discovering and characterizing 8

the role of KV2.1 channels in insulinACCEPTED secretion. MANUSCRIPT In addition to the role of KV2.1 channels in glucose-stimulated insulin secretion, Kv2.1−/− mice also display reduced fasting blood glucose and elevated serum insulin levels (Jacobson et al., 2007). This may result from longer action potential durations during the small bursts of beta cell electrical activity between meals. Further studies will be necessary to establish whether inhibition of KV2.1 represents a

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viable avenue to tackle T2D.

4.3.2 Conkunitzin-S1 from cone snail venom reveals KV1.7 as a molecular target for T2D Further insights into the molecular identity of KV channel subtypes contributing to delayed rectifier currents in pancreatic beta cells came from the discovery of Conkunitzin-S1 (Conk-S1), a peptide isolated from the venom of the cone snail Conus striatus (Bayrhuber et al., 2005), a fish-hunting

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species found in the Indo-Pacific. Functional studies of Conk-S1 demonstrated that this peptide, in contrast to hanatoxin and guangxitoxin-1, was selective for its molecular target, KV1.7, not blocking, in

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the sub-micromolar range, any other murine potassium channels from the sub-families KV1 - KV4. Importantly, it was also revealed that inhibition of KV1.7 produced an increase in glucose-stimulated insulin secretion from rat islets (Finol-Urdaneta et al., 2012). Although mRNA encoding KV1.7 had previously been detected in mouse pancreatic islet cells by in situ hybridization (Kalman et al., 1998), prior to this finding, KV1.7 channels had not been implicated in insulin secretion. Administration of Conk-S1 blocked ~18% of delayed rectifier currents demonstrating that reducing a comparably small

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proportion of rectifier currents could have a significant impact on insulin secretion (Finol-Urdaneta et al., 2012). In vivo testing of Conk-S1 in healthy rats showed promising results. Injection of Conk-S1 resulted in a temporary increase in insulin release and a decrease of the transient increase in blood

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glucose concentration upon subsequent glucose challenge (Finol-Urdaneta et al., 2012). Injection of the KATP channel blocker glibenclamide (used for treating T2D) also resulted in a reduction of blood glucose following glucose challenge. However, an important difference between the effect of

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glibenclamide was that Conk-S1 only transiently reduced glucose levels without affecting basal glucose. Thus, in contrast to blockers of KATP channels, Conk-S1 does not produce hypoglycemia (Finol-Urdaneta et al., 2012). Conk-S1 is an important tool for dissecting the role of KV1.7 in the regulation of insulin secretion and a valuable potential drug lead for the treatment of T2D.

4.3.3 Blockade of BK channels by iberiotoxin enhances insulin secretion in human beta cells Electrophysiological characterisation of human pancreatic beta cells indicated that additional potassium channels contribute to delayed rectifier currents (Braun et al., 2008). These studies identified a transient component that activates rapidly upon membrane depolarization, depends on Ca2+-influx and was blocked by the venom peptide iberiotoxin (also known as IbTx or α-KTx 1.3). IbTx, isolated from the venom of the Eastern Indian red scorpion Buthus tamulus, is a selective blocker of BK 9

channels (Galvez et al., 1990), thus BK channels likely contribute to delayed rectifier currents. In ACCEPTED MANUSCRIPT human pancreatic beta cells, IbTx was able to increase action potential amplitude and enhance insulin secretion by 70% (Braun et al., 2008). While IbTx may not represent a viable drug candidate for the treatment of diabetes (BK channels play important roles in many cellular processes outside of the pancreas), this venom peptide was a useful tool in identifying BK channels as important in insulin

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secretion.

5. Perspectives

Animal venoms continue to reveal creative strategies for manipulating vital functions. Exendin-4 and Con-Ins G1 are two examples of venom peptides that mimic endogenous hormones and are used specifically by the animal to target the metabolic function of their prey (or predator). The unique

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requirements of use in venom has resulted in attributes that make these peptides superior to their endogenous counterparts as therapeutic agents. For exendin-4 this is remarkably stability, while for

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Con-Ins G1, it is rapid activity. We anticipate that these peptides represent only a small fraction of the potentially useful metabolic agents harbored in animal venoms.

Each ion channel that is discovered as a new player in the pathophysiology of diabetes will potentially open new pharmacological possibilities for interventions in the future. Unlike many small molecule drugs, venom peptides often selectively modulate their molecular targets. As is exemplified by the KV channel blockers from tarantula and cone snail venoms, unique selectivity profiles have greatly

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contributed to uncovering new ways of manipulating beta cell function to augment insulin secretion. Investigating the potential of KV channel blockers for the treatment of diabetes, including addressing beta cell selectivity, may become an exciting field of research. Venoms have already proven a rich

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source of selective KV channel blockers (Chen and Chung, 2015; Mouhat et al., 2008; Olivera et al., 2015) and it will be of considerable interest to see what new agents emerge over the coming years.

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Acknowledgments

We would like to thank Prof. Heinrich Terlau and Dr. Briony Forbes for their comments and suggestions for this review. H. Safavi-Hemami acknowledges research funding from the Margolis Foundation, the Utah Diabetes and Metabolism Center and Juvenile Diabetes Research Foundation (JDRF).

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Table 1: Venom peptides as pharmacological tools and therapeutics for diabetes

Peptide

Sequence

Gila monster (Heloderma suspectum)

Exendin-4 (Exenatide; Byetta ™)

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

Platypus (Ornithorhynchus anatinus) Echidna (Tachyglossus aculeatus)

Platypus GLP-1 (pGLP-1)

HSEGTFTNDVTRLLEEKATSEFIAWLLKGLE

Echidna GLP-1 (eGLP-1)

HFDGVYTDYFSRYLEEKATNEFIDWLLKGQE

Geography cone (Conus geographus)

Conus geographus insulin 1 (Con-Ins G1)

GVVγHCCHRPCSNAEFKKYC* (A chain) TFDTOKHRCSGSγITNSYMDLCYR (B chain)

Chilean rose tarantula (Grammostola rosea)

Hanatoxin

ECRYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS

Chinese earth tiger tarantula (Chilobrachys guangxiensis)

Guangxitoxin-1

EGECGGFWWKCGSGKPACCPKYVCSPKWGLCNFPMP

Eastern Indian red scorpion (Buthus tamulus)

Iberiotoxin (IbTx)

ZFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ

Striated cone (Conus striatus)

Conkunitzin-S1 (Conk-S1)

KDRPSLCDLPADSGSGTKAEKRIYYNSARKQCLRFDYTG QGGNENNFRRTYDCQRTCLYT

Mode of action GLP-1 receptor agonist

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Species

GLP-1 receptor agonist GLP-1 receptor agonist Insulin receptor agonist

Summary Highly stable GLP-1 analogue, promotes glucose-dependent insulin secretion, inhibits glucagon secretion and suppresses appetite. FDAapproved for T2D Stable GLP-1 analogue with distinct signal bias Stable GLP-1 analogue with distinct signal bias Monomeric, fast-acting insulin analogue

KV2.1 channel blocker

Established the role of this KV subtype in insulin secretion

KV2.1 channel blocker

Established the role of this KV subtype in insulin secretion

BK channel blocker

Established the role of BK channels in insulin secretion

KV1.7 channel blocker

Promotes glucose-dependent insulin secretion. Selective MOA

*; C-terminal amidation, γ; gamma carboxy-glutamate, O; hydroxyproline, Z; pyroglutamic acid.

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Figure 1. Schematic of pancreatic beta cell insulin secretion and mechanism of action of venom peptides with therapeutic potential for the treatment of diabetes. Following food intake, glucose is taken up into pancreatic beta cells via the glucose transporter (GLUT) leading to mitochondrial conversion of ADP to ATP. An increase in the ATP/ADP ratio leads to closure of KATP channels and depolarization of the cell, triggering an action potential. CaV channels open producing an increase in intracellular Ca2+, which leads to the release of insulin-containing granules. Following insulin release, beta cells repolarize through the action of KV channels, including KV2.1 and KV1.7. Food intake also stimulates GLP-1 release from intestinal L-cells. GLP-1 action on the beta cell GLP-1 receptor (GLP1R) results in an increase in cAMP, subsequent release of calcium from intracellular stores and insulin secretion. Released insulin undergoes hexamer-to-monomer conversion and acts at insulin receptors (IRs) on muscle, liver and adipose tissue cells, promoting glucose uptake. The venom peptides exendin-4 and pGLP-1 act as stable GLP-1 mimetics, Con-Ins G1 as a fast-acting insulin mimetic, while iberiotoxin, guangxitoxin, hanatoxin and conkunitzin-S1 block different potassium channels involved in beta cell repolarization. Insulin secretion involves numerous other ion channels and receptors not shown in this schematic, but which may become future targets for diabetes treatments. BK channel; large-conductance calcium-activated potassium channel, CaV; voltage-gated calcium channel, GLP-1; glucagon-like peptide-1, GLP1R, GLP-1 receptor, GLUT; glucose transporter, IR; insulin receptor, KATP; ATP-sensitive potassium channel, KV; voltage-gated potassium channel.

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Figure 2. A) Comparison of the primary structure of exendin-4 with that of human GLP-1. Exendin4 and human GLP-1 share similar potency at the GLP-1 receptor, but because exendin-4 lacks the DPP-IV cleavage site (highlighted in blue) and several of the neutral endopeptidase cleavage sites of human GLP-1, exendin-4 is several orders of magnitude more stable. B) Comparison of the primary structure and disulfide connectivity of the venom insulin Con-Ins G1 with that of human insulin. Regions of the hormone known to be associated with oligomerization in human insulin are highlighted in blue. The aromatic triplet FFY in the B chain of human insulin (highlighted in blue) is important for receptor activation. Residues in Con-Ins G1 suggested to compensate for the loss of the aromatic triplet are highlighted in orange. Post-translational modifications are shown in red (γ: γcarboxyglutamate, O: hydroxyproline, *: C-terminal amidation).

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Animal venoms are a unique source of peptides targeting various ion channels and receptors Several venom peptides have emerged as pharmacological tools and therapeutics for diabetes These include GLP-1 and insulin analogues as well as KV channel blockers An overview of their mechanisms of action and their therapeutic potential is provided

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