Venoms have evolved over hundreds of millions of years as a way of defense from predation and securing sources of food. They are highly effective in targeting receptors, enzymes and ion channels to cause such disastrous effects as paralysis, coagulating blood to cause clotting, the breakdown of muscle and tissue and affecting the cardiovascular and nervous system (Patrick Walker et al., 2013). However, according to the World Toxin Bank in 2014, animal toxins are a source of 20 medications to treat the likes of diabetes, chronic pain and heart attacks (World Toxin Bank, 2014). Further research has unfolded to show many more promising properties of venoms and potential discoveries for new treatments.
Current Pain Medications
The treatment of chronic pain through pharmaceuticals is difficult to achieve with the currently available analgesic drugs. There are currently a number of treatments, including both oral and topical, that are available over the counter or via a prescription. Oral medications include anti-inflammatory drugs, paracetemol and opioids. However, chronic pain is an individualized experience and can be caused by a multitude of ailments; including cancer pain, neuralgia, regional pain syndrome, HIV/AIDS neuropathy and as a result of injury. Chronic pain is defined as “pain that extends beyond the expected period of healing” (Loeser & Bonica, 2001) with common markers being between 3 months and 12 months since onset (David A. Scott *, Christine E. Wright, 2002).
Pain, or nociception, is a somatic sensation in response to a stimulus causing our bodies nociceptors to be activated and send action potentials through nerve fibers in the peripheral nerve system to the dorsal horn of the spinal cord. The result of these action potentials reaching the central terminals of sensory afferents is membrane depolarization, activation of voltage-gated calcium channels (VGCCs) and a calcium influx, which triggers synaptic vesicle exocytosis (Magee & Johnston, 1995). This triggers the release of neurotransmitters glutamate, substance P and calcitonin in the dorsal horn (Park & Luo, 2010) into the synaptic cleft, with nociceptive information being transmitted to the brain, which can activate the descending pathways, leading to the spinal modulation of sensory signals (Chahl, 1996; Park & Luo, 2010). The signals are sent to the thalamus, which then relays the messages to the somatosensory cortex, the frontal cortex and the limbic system, which are responsible for physical sensation, thinking and emotions respectively. These descending neurons, which originate in the somatosensory cortex and the hypothalamus, can inhibit the ascending nerve signals and stimulate natural opiate neurotransmitters for pain relief (analgesia), which mean VGCCs may contribute to both the ascending and descending modulation of sensory signals. Therefore the expression and function on these channels mean VGCCs can prove as potential targets for pain management; specifically chronic pain (Mao, 2017).
Opioids work by preventing the depolarization of the nerve terminal by blocking calcium entry through the N-type voltage-sensitive Ca2+ channels by means of the G-protein mechanism. They also increase the outward movement of K+ through opening of the K+ channels, thus shortening the repolarization time (Chahl, 1996). However, tolerance and dependence for opioids is higher than any other drug (Chahl, 1996) and as a consequence, higher doses of opioids are required to produce an effect when there is a tolerance for them (Mattick, Breen, Kimber, & Davoli, 2009).
Voltage-Sensitive Calcium Channels
Nervous systems exist in order for animals to make appropriate responses to changes in their environment in ways that promote their survival. They rely on rapid changes in electrical potential and passage through ion channels (primarily calcium channels), caused by depolarization, which causes the release of neurotransmitters to eventually pass information to the brain (Layer & McIntosh, 2006). There are a number of Cav channels that have their own distinct subunits, physiological and pharmacological properties. There are currently ten identified calcium channel families within mammals that all serve as cellular signal transducers. Calcium channels mediate the influx of calcium to regulate the physiological events induced by electrical signals within cells (Catterall, 2000).
Those calcium channels that have been well characterized consist architecturally of four to five subunits; with a1 , the largest subunit, governing pore conduction, the voltage sensor, gating apparatus and managing the known sites of channel regulation by intracellular signaling molecules, drugs and toxins (Takahashi, Nakajima, Hirosawa, Nakajima, & Onodera, 1987). The a1 subunit is comprised of four homologous domains with six trans-membrane segments (S1-S6) within each (Tang et al., 2014). The S4 segment detects the changes in the electrical field and, as it contains the gating charges, opens the pore (Lacinová, Klugbauer, & Hofmann, 1999). The α1B subunit encodes the pore-forming unit of N-type calcium channels (CaV2.2) (Westenbroek et al., 1992).
Due to the varying physiological and pharmacological properties of calcium currents in different cell types, distinguishing alphabetical nomenclature evolved (Snutch, Tomlinson, Leonard, & Gilbert, 1991). Both N-type and P/Q-type calcium currents require strong depolarization for activation (Nowycky, Fox, & Tsien, 1985).
Calcium channels are controlled by brief interactions with G protein βγ subunits, vesicle mediating proteins, calcium-modulated and sensor proteins and phosphorylation of protein kinases (Catterall & Few, 2008). Interactions with Rem and RIM proteins regulate expression and localization. N-type calcium channels localize vesicles at the synaptic cleft and mediate key events including synaptogenesis and neurotransmission (Su et al., 2012). The synprint region bind syntaxin and synaptotagmin; components of the SNARE complex (Sheng, Westenbroek, & Catterall, 1998). Rem proteins bid to the CaVβ subunit and reduce expression, whilst RIM proteins bind to the C-terminal, specifically the PDX domain and tethers presynaptic calcium channels to the active zone for both N and P/Q-type (Han, Kaeser, Südhof, & Schneggenburger, 2011). This phosphorylation of a1 subunit of the N-type calcium channel causes a calcium influx due to the open probability increasing. It has been concluded that N-type calcium channels are substrates for cyclin-dependent kinase 5 (Cdk5) and this is what affects the presynaptic functions (Su et al., 2012). Whilst RIM interacts with the β auxiliary subunit of P/Q-type channels to suppress inactivation, allowing calcium influx (Su et al., 2012).
N-type channels are of particular interest in the study of treating chronic pain due to their ability to lesson neurotransmission in the spinal cord. They are located in high amounts at the pre-synaptic terminals of neurons and their subunits are unregulated when tissue damage occurs. N-type voltage-gated calcium channels enriched in the neurons play a prominent role in neurotransmitter release as they are localized at the synaptic terminal (Weber et al., 2010). When action potentials reach the desired threshold, calcium flows into the N-type channels and triggers vesicles containing the SNARE proteins to be ‘zipped’ with the plasma membrane of the presynaptic terminal (Jahn, Lang, & Südhof, 2003). N-type channels are associated with the release of neurotransmitters such as glutamate, GABA, acetylcholine, dopamine, and norepinephrine. These neurotransmitters can then not bind to the sensory neurons where pain is felt. Glutamate and glutamate receptors are also located in areas of the brain, spinal cord and periphery that are involved in pain sensation and transmission. However, their widespread distribution and array of function has often resulted in drugs targeting these sites having undesirable side-effects (Wozniak, Rojas, Wu, & Slusher, 2012). Glutamate works as an excitatory and encourages the firing of nerve impulses, whilst GABA has the opposite effect and is an inhibitory neurotransmitter, though is synthesized from glutamate. Acetylcholine is used at the neuromuscular junction and activates muscles in the peripheral nervous system as well as influencing cognitive functions in the central nervous system. Studies on the cardiovascular system show that only N-type channels are involved in the release of norepinephrine through introducing ω-Conotoxin (Molderings, Likungu, & Göthert, 2000).
P-type calcium channels are similar to N-type in the way the α1 subunit determines the channels properties and are involved in neurotransmitter release at the presynaptic terminal. They were discovered initially within the Purkinje neurons by Llinás and Sugimori in 1980 (Llinás & Sugimori, 1980). P-type and Q-type are similar due to them being produced from the same gene via alternative splicing, though they may have different subunit compositions (Matsuyama et al., 1999). Strong depolarization’s activating the P-channel cause ω-agatoxin IVA to have a very low affinity and it can no longer block the channel. ω-agatoxin IVB binds much slower than IVA but still cannot block the channel upon strong depolarization (Nimmrich & Gross, 2012). ω-Conotoxin MVIIC blocks the P-channel at the hippocampal CA3-CA1 synapse to stop synaptic transmission. However, it’s effects are slow (Su et al., 2012). As well as this, mutations in P-type calcium channels result in a decrease of intracellular free calcium. Without maintaining calcium homeostasis and fluctuating concentrations can trigger multiple diseases, which can also result in neuronal death (Matsuyama et al., 1999). An example of this is a study of mice with mutations in the α1 subunit of their P/Q-type channels. These mutations caused a delayed onset of seizures and ataxia. Mutation of the P-regoin, which is responsible for forming the pore of the channel, can lead to ataxia, severely altered respiration and symptoms associated with atelectasis (Wakamori et al., 1998).
Conotoxins
Conotoxins derive from the venomous marine molluscs known as cone snails, genus Conus and are a group of neurotoxic peptides. There are currently an estimated 500-700 species identified, each containing a large number of active peptides toxins; between 50-200 (Layer & McIntosh, 2006). Conotoxins typically consists of between 12-30 amino acids, with their small size and structural stability giving them a good basis for pharmaceutical study. These neurotoxins produce an adverse effect on the central and peripheral nervous system and causes damage to the nervous tissue through altering the regular activity between the neurons (Cunha-Oliveira, Rego, & Oliveira, 2008). However, their ability to modulate the activity of ion channels (Terlau & Olivera, 2004) makes them of particular interest as a study into pain therapeutics. Conus is split into families based upon sequence, homology, cysteine bond structure and function (Layer & McIntosh, 2006).
Omega Conotoxins As Therapeutics
MVIIA is a ω-Conotoxin that derives from Conus magus and has been synthesized into the drug Ziconotide, and most recently Prialt®. Ziconotide; as a synthetic peptide has proven refractory to other treatment modalities and has been approved by the US Food and Drug Administration and the European Medicines Agency (Schmidtko, Lötsch, Freynhagen, & Geisslinger, 2010). It is a new drug in a class of N-type voltage-sensitive calcium channel blockers and is a synthetic equivalent to a component of the venom of the marine snail (Miljanich, 2004). Ziconotides’ therapeutic profile derives from its selective blocking of neuronal N-type voltage-sensitive calcium channels. The direct blockage then inhibits the activity of neurons, which include the pain-sensing primary nociceptors (Schmidtko et al., 2010).
Ziconotide is administered by intrathecal drug injection into the spinal cord, utilizing implantable pumps that continuously deliver drugs through catheters placed into intrathecal space. Though protected by the blood brain barrier, making delivery to the CNS difficult, intrathecal injection has proven to be safe but only used for chronic pain that is unresponsive to oral or intravenous medications as it is an invasive procedure. Drugs van then be concentrated within the spinal cord where the analgesic actions take place, minimizing concentrations at distant sites where side effects are often mediated (Benedetti & Stein, 2007). This also regulates the drug concentration and minimizes fluctuations, which is typical of oral dosing (Layer & McIntosh, 2006).
Through Nuclear Magnetic Resonance spectroscopy, Hα shifts relative to random coil values can be measured to give backbone conformation. This can then be used to visualize conformational differences between otherwise similar peptides (Adams, Smith, Schroeder, Yasuda, & Lewis, 2003). Omega-conotoxins MVIIA, MVIIC and CVID are almost identical in secondary structure, though there is much differentiation between primary structures. Cone snails have used this framework to readily evolve ligands to target structurally related but selectively different ion channels (Katherine J. Nielsen, Schroeder, & Lewis, 2000). MVIIC is harvested from Conus magus and is also an ω-conotoxin but is selective for the P/Q-type calcium channels. These P/Q-type channel blockers, such as MVIIC are not considered useful leads as therapeutics due to them likely being lethal to mammals (Zamponi, 1997).
It is peculiar that MVIIA and MVIIC would have such similar primary structures but select differently for N-type and P/Q-type respectively. This is why both conotoxins have proved beneficial in assessing structure-activity relationships.
CVID was first isolated from Conus catus and is another ω-conotoxin that is highly selective for N-type over P/Q-type VSCCs. Studies have also found that CVID also inhibits neurotransmitter release in the preganglionic parasympathetic neurons, which is otherwise resistant to inhibition by other calcium channel antagonists (Adams et al., 2003). CVID was found to have the larger ratio to behavioral toxicity than MVIIA and was effective in managing chronic inflammatory pain (Scott, Wright, & Angus, 2002). Its side-effect producing doses were also 10-fold higher than MVIIA in producing antinociception (Smith, Cabot, Ross, Robertson, & Lewis, 2002).Therefore, based on its current clinical profile, it may produce significantly less side effects than MVIIA and has a greater therapeutic ratio relative to MVIIA due to its selectivity for N-type calcium channel variant within the preganglionic nerve terminals (Adams, Smith, Schroeder, Yasuda, & Lewis, 2002).
Objectives
Methods (1000)
I will use pre-discovered and tested protein sequences of omega-conotoxins and pharmaceuticals used in chronic pain management to download and compare their protein alignments and 3D structures. I will use these for further bioinformatics alignments from the database, National Centre of Biotechnology Information (NCBI).
I will highlight the main motifs and residues in the pre-discovered proteins that make them applicable in the use of therapeutics for chronic pain. I will then input the alignments into software such as Ensembl to compare the genomics to other alignments that may also prove useful as a way to block nerve activity and therefore act as a therapeutic for chronic pain.
Results and discussion
1 5 10 15 20
Toxin Sequence Target Net Charge
CVID CKSKGAKCSKLMYDCCSGS CSGTVGRC N-type +5
MVIIA CKGKGAKCSRLMYDCCTGSC -R- SGKC N-type +6
MVIIC CKGKGAPCRKTMYDCCSGSCGR-RGKC
P/Q-type +7
Loop 1 Loop 2 Loops 3 Loop 4
Conserved Cys residues
Conserved Tyr residue (Tyr13)
Conserved Gly residues
Other conserved residues
MVIIA and MVIIC Similarity: 76.92%
E-value: 2.6e-9
MVIIA and CVID Similarity:
E-value:
MVIIC and CVID Similarity:
E-value:
Structure
Omega-conotoxins have been shown to be relatively rigid peptides, with a structure comprising of a cysteine scaffold that stabilizes the four-loop framework, making it unlikely that they will be altered on binding to voltage sensitive calcium channels (K.J. Nielsen et al., 1999). Their configuration ascertains the canonical ω-conotoxin fold, made up of a triple-stranded β sheet and a β bridge (Pallaghy, Nielsen, Craik, & Norton, 1994). It has recently been observed that the β bridge is formed by residues 1-2 and 14-16 (Lewis et al., 2000) with a possibility that it is stabilized by a salt-bridge between the side chains of Lys2 and Asp14 in MVIIA, MVIIC and CVID (K.J. Nielsen et al., 1999; Skalicky, Ciesla, Pardi, Metzler, & Galdes, 1993). This arrangement makes for a stable base from which the side chains, which are critical for selectivity, are attached (Schroeder & Lewis, 2006).
Loops 2 and 4
Loops 2 and 4 have been attributed to be the most important sites for VSCC selectivity and binding through harboring the distinct residues needed to bind to the varying subtypes. By comparing the 3D structures of CVID, MVIIA and MVIIC, an analysis of reactions between the loops can be established with the influence conformational changes has upon binding. A study conducted by Nielsen et al (1999) found that there are little major conformational differences between MVIIA and MVIIC’s cysteine framework and folding. However, loop 4 was fond to be shorter in MVIIA (K.J. Nielsen et al., 1999). There may also be subtle differences between MVIIC and MVIIA in loop 2, the region that is integral to VSCC subtype selectivity (Basus, Nadasdi, Ramachandran, & Miljanich, 1995). The differences in loop 2 have been found to affected by the constituents of loop 4 and so, loop 4 may also influence binding (K.J. Nielsen et al., 1999).
Many of the differences within loops 2 and 4 are as a result from differences in primary structure. There are three apparent conformational changes between MVIIC at Pro7, Gly21 and Arg22 compared to Lys7, Arg21 and Ser22 at MVIIA. Arg21 of MVIIA corresponds more closely to Arg23 rather than Arg22 of MVIIC, which is not clear from the primary structure. This extra charged residue (Arg22) in MVIIC is the biggest conformational difference in loop 4, with it not being present in MVIIA and is replaced by Ser22 at the same position, a non-charged residue. Although it may have no effect on binding or selectivity, Arg22’s side chain may augment P/Q-type potency as well as result in conformational changes in loop 2. There are also possibly two more allosteric changes in loop 2 resulting from long-range effects made by substitutions of loop 4 at Thr11 and Tyr13 of MVIIC and Leu11 of MVIIA (K.J. Nielsen et al., 1999).
Further to this, an examination of the side chains in both MVIIA and MVIIC show identical orientation with Lys2, Lys4, Ala6, Met12, Tyr13, Asp14, Ser19 and Lys24 of MVIIA positioned identically in both peptides, forming a localized patch. There are a number of side chains located at these positions that are involved in binding of MVIIA to N-type VSCCs. Therefore, it can be assumed that MVIIC binds to its P/Q-type VSCC subtype in a similar manner. However, varying surface exposure of these side chains and differences in position or type is likely to be what gives the peptides their selectivity. This also further authenticates loop 2 and 4 as the important sites for selectivity as it is primarily within these that differing amino acids and varying extents of surface exposure is present (K.J. Nielsen et al., 1999).
NMR studies have shown that loop 4 of CVID is curved towards loop 2, creating a globular surface, making it structurally different to both MVIIA and MVIIC. Whereas In MVIIA and MVIIC, loop 4 is parallel to loop 2 (David A. Scott *, Christine E. Wright, 2002). This is due to hydrogen bonds between Lys10 in loop 2 and Gly22 in loop 4 (Lewis et al., 2000) that have not been previously reported in MVIIA or MVIIC (Adams et al., 2002) and studies have shown this may have lead to instability in loop 2 of MVIIA compared to CVID; instead with the isomerization of Cys8 and Cys20 attributing to the motion of loop 2 (Atkinson, Kieffer, Dejaegere, Sirockin, & Lefèvre, 2000).
Within loop 2, the only amino acid variant between MVIIA and CVID is Lys10 present in CVID. This residue has been studied to find that its presence enhances CVIDs ability to bind to the preganglionic VSCC and block neurotransmission. The study replaced the lysine residue with that of arginine as found in MVIIA by manual stepwise synthesis using in situ neutralization. This limited its access to the binding site (Adams et al., 2002; Zamponi, 1997) and for this reason, it can be assumed that the preganglionic ion channel is a N-type Cav2.2 variant.
Nielsen’s study of hybrids of MVIIA and MVIIC took loops 1, 2, 3 and 4 from each and interchanged them to create 14 variants. With ‘A ‘representing MVIIA and ‘C’ representing MVIIC in order of loops (AAAC is an example where loops 1-3 are taken from MVIIA and loop 4 is utilized from MVIIC). The study discovered that the upper and lower bounds for potency at the P/Q-type VSCC were not by MVIIC and MVIIA, but by the hybrids ACAC and CACA respectively (K.J. Nielsen et al., 1999). This insinuates that hybrids where loops 1 and 3 of each peptide are interchanged for the alternate VSCC-binding peptide (N-type and P/Q-type) will increase its potency for its own proposed VSCC. This increased potency may cause a hybrid of Loop 1 and 3 of MVIIC combined with loops 2 and 4 of MVIIA to increase neurotransmission inhibition at N-type VSCCs in order to create a more effective therapeutic.
Surface Profile
It is clear that there are four residues that are integral to specific binding of the N-type VSCC and for their specificity in MVIIA. These are all located in loops 2 and 4 and comprise of Tyr13, Arg10, Leu11 and Arg21. MVIICs important residues for binding to P/Q-type comprise of Lys10, Arg23 along with Ty13. Tyr13 is conserved amongst all 3 peptides and is the most integral residue for binding to both the N-type and P/Q-type VSCCs, with the other residues being secondary binders (K.J. Nielsen et al., 1999). An analogue of CVID, where the tyrosine was replaced with phenylalanine, proved that Tyr13 is integral to binding as the analogue produced no effects in the excitatory postsynaptic potential (Livett et al., 2006). The inability of analogues of Tyr13 to act as a binding site appears to be from either (i) the loss of the Tyr13 hydrogen bond, (ii) Leu11 causing a hydrophobic reaction and/or (iii) Arg10 crating an apparent electrostatic interaction (Katherine J. Nielsen et al., 1999). Ty13 effects may also be limited in MVIIA and MVIIC through Arg22’s (not present in CVID) effect on stabilizing the a1 conformation over other backbone conformations; giving reason to CVIDs higher affinity binding at N-type VSCCs and increased selectivity against P/Q-type over MVIIA.
Another residue outside of loops 2 and 4 but considered still important for binding is Lys2 in loop 1. For this to be directly involved in interaction with the binding would requite encapsulation of the entire conotoxin, which is unlikely but still possible. For this to occur the VSCC would need to make a significant conformational change and as this is conserved across MVIIA, MVIIC and CVID, this would mean conformational changes occur within N-type, P/Q-type and the N-type variant. There is no further study into this as, through observation of a salt bridge between the side chains of Lys2 and Asp14 (K.J. Nielsen et al., 1999), it is assumed to contribute to the structural scaffolding of loop 2’s anchoring. Previous studies have indicated that replacement of Lys2-Ala2 may only induce little disruption. However, further studies revealed that analogues in the position of Lys2 critically affected th structure of loop 2 (K.J. Nielsen et al., 1999).