α-Conotoxins PnIA and [A10L]PnIA Stabilize Different States of the α7-L247T Nicotinic Acetylcholine Receptor

Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the ␣-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.conotoxins ͉ drug delivery ͉ molecular engineering V enoms from marine snails of the Conus genus comprise a myriad of peptides called conotoxins (CTXs) for the rapid immobilization of prey (1, 2). These 12-to 30-aa peptides target membrane receptors with exquisite selectivity and potency and have become invaluable neurophysiological probes and drug leads. Recently, the CTX ziconotide (MVIIA) was approved for use in the treatment of severe chronic pain by the FDA, and other CTXs have entered clinical trials as treatments for pain (3,4). In addition, CTXs have played a critical role in dissecting the molecular mechanisms of ion channel and transporter functions in the nervous system (2). One family of CTXs, the ␣-CTXs, consists of members that antagonize the nicotinic acetylcholine receptors (nAChRs). Ranging in size from 12 to 19 residues, ␣-CTXs are the smallest of all of the CTXs, yet this family is the most widely distributed among Conus venoms (5).Despite their exciting applications, many peptide toxins are susceptible to enzymatic degradation by proteases. This characteristic may limit the therapeutic applications of CTXs, and, hence, methods that provide improvements in biological half-life are valuable. Cyclization has been used in the past as a strategy in the pharmaceutical industry for stabilizing and locking the conformation of small peptides (6). Similarly, microorganisms are known to produce cyclized peptides, such as cyclosporin A, which is now in widespread use as an immunosuppressant. Such a strategy has not been applied in the past to disulfide-rich proteins, but with the recent discovery of the cyclotide family of macrocyclic miniproteins (7), it is clear that the approach can be applied to disulfide-rich toxins to produce additional stabilization with the potential to dramatically increase the therapeutic potential of these molecules when limited by poor in vivo stability.This stu...

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“…22. cDNA encoding the rat ␣2-7 and ␤2-4 nAChR subunits were provided by J. Patrick (Baylor College of Medicine, Houston).…”

Section: Methodsmentioning

confidence: 99%

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Clark

Fischer

Dempster

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…-Ctx discovery in conjunction with chemical peptide synthesis has provided an increasing array of biologically active compounds with varying subtype selectivity [13,14,[16][17][18][19][20][21][22][23]56]. Based on the observation that the residue at position 10 of the 4/7 class of -Ctxs can influence nAChR M a n u s c r i p t…”

Section: Discussionmentioning

confidence: 99%

“…[A10L]PnIA analogues were applied directly into the bath and incubated for at least 5 min, before a further application of agonist [23]. Directly adding peptides to the bath conserved were determined and t-tests were used to determine significance.…”

Section: Electrophysiological Recordings From Exogenously Expressed Nmentioning

confidence: 99%

“…In further studies, -Ctx [A10L]PnIA, acted as an antagonist of chick A c c e p t e d M a n u s c r i p t 5 wild-type 7 nAChRs expressed in Xenopus oocytes, as well as being an 7-L247T receptor agonist [23]. The 7-L247T receptor renders the closed state of the receptor conductive, and can be used to probe ligand interactions with otherwise electropharmacologically silent states.…”

Section: Introductionmentioning

confidence: 99%

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Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors

Hopping

Wang

Hogg³

et al. 2014

Biochemical Pharmacology

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Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease.-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian 7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptorligand interactions that determine the role of position 10 in determining potency and selectivity for the 7 and 3 2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished 7 binding.Molecular docking revealed dominant hydrophobic interactions with several 7 and 3 2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the 7 subtype (Ki = 44 nM) though selectivity reverted to 3 2 (IC 50 = 0.7 nM). It appears that that placement of a single methyl group determines selectivity between 7 and 3 2 nAChRs via different molecular determinants.

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“…Electrophysiology-Expression and electrophysiological recording from nAChRs was carried out as described previously (34). Briefly, oocytes were injected intranuclearly with 2 ng of cDNA that coded for either the human ␣7 or ␣3␤2 receptors (cDNAs were a gift from J. P. Changeux).…”

Section: Comparison Of Ntii Spatial Structure With Structures Ofmentioning

confidence: 99%

Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

Lyukmanova

Шенкарев

Schulga

et al. 2007

Journal of Biological Chemistry

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Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain ␣-neurotoxins preferentially inhibit muscle-type nAChRs, whereas longchain ␣-neurotoxins block both muscle-type and ␣7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of ␣-and -neurotoxins is essential for their action on the ␣7 and ␣3␤2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfidecontaining loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain ␣-neurotoxins and -neurotoxins. The correct folding and structural stability for both chimeras were shown by 1 H and 1 H-15 N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I block ␣7 nAChRs with similar potency (IC 50 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain ␣-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for ␣7 nAChR (IC 50 126 nM) but did not convey activity at ␣3␤2 nAChRs. Docking of both chimeras to ␣7 and ␣3␤2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of -neurotoxins underlie their selectivity for ␣3␤2 nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 belong to the class of ligand-gated ion channels (1-3). All nAChRs are composed of five homologous subunits, which span the plasma membrane to form a transmembrane ion-conducting pore. The muscle-type receptors are composed of four types of subunits, ␣2␤␥␦ in the fetal and ␣2␤⑀␦ in the mature receptor. The best studied nAChR of this type is the receptor from electric organ of ray Torpedo marmorata (4). Neuronal receptors consist of two types of subunits, ␣ (␣2-␣10) and ␤ (␤2-␤4), and can have homooligomeric (for example, ␣7-type nAChR) or heterooligomeric (for example, ␣3␤2-type receptor) composition. Neuronal nAChRs are widely expressed both pre-and postsynaptically, and presynaptic receptors modulate synaptic activity and the release of different neurotransmitters. Neuronal nAChRs are found also in non-neuronal tissues, such as keratinocytes and pulmonary and other tissues (5-8).Because of their role in cellular communication, nAChRs play important roles in the normal functioning of the organism. Dysfunctions of these receptors have been proposed to contribute to a number of disorders of the central and peripheral nervous systems such as schizophrenia, epilepsy, depression, nicotine and alcohol dependence, Alzheimer disease, Parkinson disease, and autis...

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α-Conotoxins PnIA and [A10L]PnIA Stabilize Different States of the α7-L247T Nicotinic Acetylcholine Receptor

Cited by 59 publications

References 23 publications

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors

Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

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