Synthetic peptides as tools to investigate the structure and pharmacology of potassium channel-acting short-chain scorpion toxins

Bhn'himie (1998) 81), 151 -.154 0 Socid|6 |'run~'aisetie biochimie el bioh~gie moldctdahc : F.Ise~ieL Pari~,

Synthetic peptides as tools to investigate the structure and pharmaco|ogy of potassium channe|-acfing short-chain scorpion toxins C L e c o m t e , JM Sabatier, J Van R i e t s c h o t e n , H R o c h a t I.aboratoire de Biochimie. ingt;ni6rie des Protdines, (', 'RS UMR 6560. IFR .lean Roche. f'acultd tie M&tecine :v,.'d. Bd Picrre-Dramard. 13916 Mm'seille ceth'x 20, France

(Received I 8 November 1997; accepted 6 January ! 998) ~|ll|l|nal'~ ~ ~ Ii~ Ihe lasl decade, numerous polypeptide toxins acling on ion channels have been isolaled arid characterized from diverse scorpiot~ velloms, These toxins are useful pharmacological probes to study ion-specific channel proteins because they hllcracl s,..icctively with these channels and modulate their activities. Since low illllOilil|s of natural toxins can be isolated from ,;corpion venoms, the chemical synthesis approach is extremely useful Io produce larger quantities of toxins and toxin analogs. This report is a succinct overview of the possibilitie,; offered by the chernical synthesis to investigate pharmacoh)gicai and slrtlClUl'al i,roperties of these conlpounds. (G SociallY' I'ranqaise dc biochimie el biologic moldculaire 1 Elsevier, Paris). scorpion toxin I synthetic peptides / ion channel I structure-activity Introduction

Polypeplide scorpion toxins are potent and selective compounds that act on ion channels. Because of their P+l+ecificity and high affinity, they have been used ;is tools h) characterize various receptor proteins involved in ion channel functioning. Structural and pharmacolqgical studies allowed to distinguish two nlahl families of scorpion neurotoxins: the short-chain toxins (29 Io 39 amir|o acid residues: three disulfide bridges) which ;ire mainly acting on potassium cllannels; a11d the hmg-chain toxitts (60 to 70 amino acid residues, four disull'ide bridges} 11-41 which act on voltage-gated sodium channels, However, two short-chain scorpion toxins crossqinked by hmr disulfide bridges (it, maurotoxin and Pandimts imperator toxin I ) have been re° cently reported to act on potassium channels 15, 61. Despite their variable length and specificity, scorpion toxins share a common structural motif, the (x/I] scaflbid, consisting in an ~-helix cross-linked to a double stranded ~-sheet by two disulfide bridges 17 I. So I'ar only short-chain neurotoxins (see fig I)and their structural analogs have been chemically synthesized by Ihe solid-phase. Long-chain synthetic scorpion toxins are still difficult to obtain mainly because of the insolubility of the reduced form in current oxidative systems or inadequate folding leading to inactive oxidized form(s) 181. In contrast to the puril'ication fi'om natural source, the chemical synthesis allows to obtain the large amounts of well-characterized synthetic products which are necessary to extensive structural studies such as the deter,nination of distlll'ide bridge pairing, and of the 3-D structure ot" toxins/analogs. Further, pharmacological data obtained

with a synthetic toxin may often prove to be more reliable than those obtained using a natqral toxin isolated from the scorpion venom since the risk of contamination of the toxin preparation by another active compound is avoided. For vaccination purposes, chemical synthesis could be used to produce non-lethal and immunogenic anatoxins, in the case of the scorpion Amh'o~'tom~s australis he,'mr (AaHk injection in mice of a toxin I!~derived anatoxin (in which the eight hall'-cystines of the natural toxin II were replaced by the tx+amim~ butyrate derivatives) was reported to protect aullllals against a cllallengc of several lethal doses of either natural toxin II or crude AaH venom I~i. l:;'I'[!~IllelilSOf toxins, oi' (~I 1 the prolcinn fOl'llliilglhe ion challllels, can also be synthesized and used ;,is imnltlnogens

to elicit site-directed anti-peptide antibodies. These specific antibodies, which can be raised against immuno,~ilent reo gions, are useful probes to delineate the region of the toxin involved in its pharmacological activity Ipharmacophorc region), or the domain(s) of the ion channel protein(s) ino wflved in toxin binding, by testing their ability to inhibit the interaction of the toxin with the ion channel and to neuo tralize the toxin-induced lethal effects in vivo (the neutrao lizing a n t i - p e p t i d e antibodies can be used for anti-scorpionic serolherapy). These synthetic peplide flagnlenls could be also tested directly in binding assays as potential competitors of either the toxin or the ion channel protein{s} to map the domain{s} involved in the toxin-ion channel interaction. Agonist or antagonist compounds of great pharmacological interest could be found from such studies. The synthetic toxin fragments used as antigens could also serve to address the immunological properties of a p:lrlicu°

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FIR I. Primary structures in the one-letter code (IUPAC convention} of short-chain scorpion toxins which were obtained by solid phase chemical synthesis. From top to bottom: ieiurotoxin I (LTx) 1121; I)(_)5ll31; BmP05 1271; Pill 1281; tityus kapa {TsK) 1291; charybdotoxin (ChTx) 1301. i~riotoxin (IbTx) I31]. noxiustoxin {NTx) i321: margatoxin (MgTx)1331: kaliotoxin (KTx) 1181; and maun)toxin (MTx) I341. Recently. the chemical synthesis of [pTxa. a short-chain toxin (33 amino acid residues, lhree disulfide bridges} from the Pandim~s imperator scorpion venom, has been reported. It is acling on Ca -~+ release chanuelstryanodine receptors 1351.

lar toxin by mapping the toxin epitopes with anti-toxin polycional sera. For example, synthetic fragments of toxin !1 from AaH allowed to delineate four major antigenic regions within the molecule [ 10. l l I. one of which is able to elicit neutralizing antibodies. The main application of the chemical synthesis is the structureoactivity relatiollship study of toxins using structuo ral analogs. These analogs could be truncated or pointomuo tated 'toxins', chimeric compounds, biotinylatedt fluo~scentlphotoreactive toxin derivatives, pseudopeptides or products containing unnatural ~sidues or D-amino acids. It is noteworthy that some of these analogs could not be obtained by other techniques, eg genetic engineering. Obviously, the chemical synthesis approach has highly contributed to our current knowledge of the toxin to receptor interaction at a molecular level.

C h e m i c a l synthesis in the study o f short-chain scorpion toxins

TIo~'e-dimettsimt(d slrl~ctro~, dele~'l.i.ation ~.I"xy¢llh('lic I¢~Xita,~' Until now about ten structu~s have bt~en determined it) solution by IH-NMR spectroscopy using chemically-synthesized scorpion t~t~xins. This represents 30--35% of the known scorpion toxin structures, the chemical synthesis

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Fig 2. Comparison of the structures of different short-chain scorpion toxins which were obtained from synthetic peptides: leiurot,sin 1 {LTx) 1151:P05 1171: POI 1281; tityus kapa (TsK) 1291; kaliotoxin (KTx) 1361; and maumtoxin (MTx ) 137].

h¢iug thus an imporlant source n!' toxin.~ available for strut. rural determination. The cnmparison ()f these 3-D structures (fig 2) 171 show.~ that the main differences between !he tnxil)s are 1he length of the Noterminns which, when extended, is able to fonu the third strand t)l"the [~-sheet. and the size of the o~-helix, in addition, several structures of toxin analogs have been reported which helped to assess first, the role of particular residues or domains in the folding process in vimJ. and second, the correlation between peptide conforms/ion and its bioactivity.

St,'at'tare-activity reh~tio,ship study ,sing synthetic toxin analogs Since the last decade, most of the progress realized in the study of the scorpion toxins mode of action and selectivity. as well as in the functioning of their targets, the ion channels. as also in the identification of their various families and subtyl~s, could be undoubtedly attributed to the extensive use of chemically-synthesized toxins or toxin-derived pn~ucts. The peptide-based studies highlighted the importance of the I~sitively-charged arginine residues located within the or-helical structure, as well as .he nature, free carboxylate very'us amidated of the C-ten'ritual extremity, for the phar-

153 macological activity of short-chain scorpion toxins acting on small conductance Ca~+-activated K ÷ ~SK) channels, eg ieiurotoxin ! and P05 112-141. In the case of |he study on P05, it is noteworthy that the synthetic ~-amidated limn of the toxin behaves as an irreversible iigand of SK channels ill vittv. The 3-D structures of these toxins indicate that the 'critical" residues in positions 6, 7, and 31, are located on the same side of the molecules [15-17]. Since it has been demonstrated thai the three-dimensional structure is not affected by these mutations, the observed variations of activity relied on the chemical function carried by the side chain. For toxins acting on voltage-gated (Kv) and/or big-conductance Ca2+-activated K+ (BK) channels (eg kaliotoxin, iberiotoxin, noxiustoxin, and charybdotoxin), the involvement of specific residues within the ~-sheet structure in the toxin bioactivity has been also evidenced using synlhetic peptides. In the case of KTx, a study targeting the C-ternlinal region, which is highly conserved arnong BK and/or Kv acting scorpion toxins, has shown that the peptidu mimicking domain 26-32 may ~ n i a i n a binding subsite essential for K + channel recognition 1181. in 'he case of toxin, exhibiting dual activity, the use of poi~,,~-tm,tatedltruncated toxins has allowed to define the structural trite:i:, of toxin selectivity towards a channel tinnily tog niamotoxm, unpublished results). The use of chimeric peptldc:-;poss,.~ssing part of distinct toxins could also lead to the determinatl,.m of such criteria. For example, a study using synthetic iberiotoxin/charybdotoxin mutants has shown that the C-terminus of charybdotoxin supports activity on voltage-gated K + channels 1191. The chemical synthesis of peptides also allows to study the relationslfips between prinmry structure and the backbone folding, as well as the infhtence of conformational changes on the peptide phalmacological activity. Two main studies have been reported concerning leiurotoxin ! and charybdotoxin, ht both cases, N oterminal truncaled analogs were obtained which possessed parent toxin-like conlbrmation, demonstrating that the lack of N-terminus did not a f l'ect significantly the folding process and formation of the ot/[] scaffold which is conserved among the scorpion toxins 120, 211. The influence of each of the three disulfide bridges on the conformation and bioactivity was also investigated on these toxins. The conformational and functional characterization of synthetic leiurotoxin I analogs lacking one disulfide bridge suggests that incorporation of a pair of ~-amino butyrate residues (Abu) in the positions of halfcystines 3 and 21 did not affect the structure and activity of this Ibld, whereas this substitution in positions 8 and 26, or 12 and 28, had large effects on folding, altering both peptidc structure (eg 0~-helix) and activity 122, 231. in the case of charybdotoxin, the most active analog (Chab 11) lacks disulfide bridge Cys13-Cys33 (ie the intermediate disulfide), which corresponds by analogy to disulfide Cys8-Cys26 of leiurotoxin I according to the consensus disulfide organization of short-chain scorpion toxins. The circular dichroism analysis of Chab 11 indicates that it retained a grossly cha-

rybdotoxm-like conformation '~vhereas other analogs exo hibiled significantly different slrtlclures 124, 25 I. Therefore. it appears that the activity of toxin analogs lacking one disulfide bridge could solely rely on the relative contribution of that particular disulfide to the maintenance of specific structural domain conformation(s). These data show that the use of synthetic peptides could be also of general interest in the field of protein folding. The elucidation of the structure-activity relationships of the K+ channel toxins from scorpion venoms may shed new light on the structures of various K + channels. Clearly, the chemical synthesis proves to be a powerful method to investigate the pharmacology, immunology and structure of scorpion toxins, allowing to dissect the molecular mechanistns of toxin to ion channel interaction. The study of K + charmel-acting toxins is particularly attractive because lowmass toxins could be, hi most cases, synthesized in good yields+ and there are a number of different types of K + channels which could be either selectively blocked or altered in their gating properties by these molecules. Apart from the synthetic peptide-based studies, it is noteworthy that recom+ binant products obtained by genetic engineering have also largely contributed to improve our knowledge in the field of scorpion toxins and ion channels [261. The two methods. ie chemical synthesis and genetic e-,,; .... rin,, _..~,.,,,,, ~ , are c o m p l e m e n t a r y . However. the main limitation of the latter tech.,.tique remains the low amount of product recovered as compared to the peptide synthesis. Of course, the chemical syrlthesi~ approach is not limited to the field of scorpion toxins and lheir targets but could be applied to most of the polypeptide compounds.

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