Conotoxins Univ 2006-comp

Конотоксины – природные комбинаторные библиотеки и лекарства из ядовитых морских улиток Conus.

Shells of some Conus species C.geographus (the geography cone)

C.quercinus (the oak cone)

C.radiatus C. striatus (the striated cone)

(the radial cone)

C.gloriamaris (the glory-of-the –sea)

C.textile (the cloth-of-gold cone)

C.magus (the magus cone)

C.purpurascens

C.marmoreus

(the purple cone)

(the marble cone)

Myers R.A., Cruz L.J., Rivier J.E. and Olivera B.M. (1993) Chem. Rev. 93, 1923-1936

Конотоксины и другие пептиды из морских ракушек семейства Conidae 1. Классификация и химические структуры • аминокислотные последовательности

• расположение дисульфидов

• нуклеотидные последовательности

• препропептиды и зрелые пептиды

• посттрансляционные модификации

• природные комбинаторные библиотеки

2. Пространственные структуры в кристалле и растворе 3. Биологические мишени: рецепторы и ионные каналы 4. Токсин-рецепторные взаимодействия: • существенные остатки и поверхности взаимодействия • роль гидрофобных и электростатических взаимодействий • роль конформационной подвижности ? • идентификация парных взаимодействий в циклах «двойных мутаций» • сравнение с другими токсинами, действующими на те же рецепторы

5. Медицинское использование конотоксинов 6. Более подробно: взаимодействие с никотиновыми ацетилхолиновыми рецепторами • разное сродство к двум центрам связывания рецептора Torpedo • фотоактивируемые α-конотоксины • синтез аналогов α-конотоксинов для детекции различных типов холинорецепторов

Peptide and polypeptide toxins Primary structure Toxins α-neurotoxins receptors

number of a.a.residues

Target

disulfide bridges

Snakes

(nAChR) muscarinic toxins (mAChR) fasciculins calciseptins dendrotoxins dendroaspin adhesion cardiotoxins

60-75

4-5

nicotinic acetylcholine

65

4

muscarinic acetylcholine receptors

61

4

acetylcholinesterase (AChE) Ca2+-channels K+-channels cell

60 57-60 58

4 3 4

60

4

47

3

65 31 37

4 3 3

36

3

18

2

membranes

Sea anemona Toxin II Anemonia sulcata channels

Na+-

Scorpions

α -scorpion neurotoxins leirutoxin I charybdotoxin

Na+-channels K+-channels K+-channels

Spiders ω-atrachotoxin insects)

Ca2+-channels (from

Bees apamin

K+-channels

Conus peptides and their targets Ligand-gated ion channels • • • •

α -Conotoxins αA-Conotoxins ψ-Conotoxins σ -Conotoxin

• Conantokins

Nicotinic acetylcholine receptors Serotonin 5-HT3 receptor NMDA receptor

Voltage-gated ion channels • • • •

ω-Conotoxins µ-Conotoxins δ-Conotoxins µO-Conotoxins

• κ-Conotoxins • κA-Conotoxins

Ca2+ channels Na+ channels

K+-channels

G-protein coupled receptors • Conopressin • Conutalakin-G

• • • • •

Contryphans mr10a (T-superfamily) λ-Conotoxins Gla-containing conopeptide Conodipine -M

Vasopressin receptor Neurotensin receptors Different activities behavioral effects antinociceptive seizures, paralysis spasmodic phospholipase

Новые конотоксины и конопептиды Conorfamide-Sr1 GPMGWVPVFYRF-NH2 (семейство RF амидов) синдром гиперактивности

Mailto et al. (2002) Toxicon 40, 401-408

Conophysin-R принадлежит к семейству нейротензинов 48 аминокислотных остатков, 7 дисульфидов

Lizarn et al (2002) Toxicon 40, 901-908

ρ-Conopeptide ρ-TIA FNWRCCLIPACRRNHKKFC неконкурентный блокатор α1-адренорецепторов

Sharpe et al (2001) Nat.Neurosci. 4, 902-907

χ-Conopeptides MrIA NGVCCGYKLCHOC неконкурентный блокатор норадреналиновых транспортеров,

Sharpe et al (2001) Nat.Neurosci. 4, 902-907

анальгетическая активность

McIntosch et al (2000)

Structural features of Cys-containing Conus peptides Peptide

Cysteine and disulfide postions

Number of residues

α -Conotoxins

CC- -C- - -C

12-19

λ -Conotoxins

CC- -C- - -C

11-13

αΑ -Conotoxins

CC- -C- - -C - - - C- - -C

25-30

ψ -Conotoxins

CC- -C- - -C - - - CC

24

µ -Conotoxins

CC- -C- - -C - - - CC

22

µΟ -Conotoxius

C- - - C- - -CC - - C - - C

31

δ -Conotoxins

C- - - C- - -CC - - C- - -C

21-31

κ -Conotoxius

C- - - C- - -CC - - C- - -C

27

ω-Conotoxins σ − Conotoxins Contryphans

C - - C - - CC - - C - - C C - -C - - C - - CC - - CC- - - - CC C---C

24-29 41 7-8

Posttranslational modifications of Conus peptides Modification reaction

Modified group or residue

Enzyme

1. Disulfide bond formation

-S-S-

Disulfide isomerase

2. Amidation of C-terminal carboxylate

-NH2

Protein amidating monooxygenase

3. Cyclization of N-terminal Gln

pGlu

Glutaminyl cyclase

O

Proline hydroxylase

4. Hydroxylation of Pro 5. γ− Carboxylation of Glu 6. Glycosylation of Thr

Gla (β l → 3)GalNAc(α1→ ) Thr

7. Isomerization of Trp or Leu

D-Trp or D-Leu

γ -Glutamate carboxylase Polypeptide HexNAc transferase Tryptophan or leucine epimerase

8. Bromination of Trp

Trp(Br)

Bromo peroxidase

9. Sulfation of Tyr

Tyr(SO4)

Tyrosyl sulfotransferase

Bromocontryphan

GCOw EPW(Br)C-NH2

(+)-Tubocurarine and α-conotoxin G1 differential protection of specified Torpedo AChR subunits from labeling by photoactivatable α-neurotoxins. ___________________________________________________________ displacer, labeled subunits, α-neurotoxin IC50, µM ___________________________________________________________

αH

γ

αL

δ

α-conotoxin G1, AzBz-Lys26 NT-II

0.76

5.01

(+)-tubocurarine, AzBz-Lys46 NT-II

1.25

AzBz-Lys26 NT-II Diaz-Lys23 CTX

562 0.64

3.0

6.3

412 200

160

___________________________________________________________ Yu.N.Utkin, Y.Kobayashi, F.Hucho, V.I.Tsetlin. Relationship between the binding sites for an α-conotoxin

and snake venom neurotoxins in the nicotinic acetylcholine receptor from Torpedo californica. Toxicon 32, 1153-1157 (1994). Yu.N.Utkin et al. Eur. J.Biochem. 253, 229-235 (1998)

Affinity of α-conotoxins for the two sites in different receptors

_____________________________________________________________________________________ Toxin AChR source High-affinity Low affinity References IC50, nM IC50, µM _________________________________________________________________________________________________ ___

G1

Torpedo

(α/γ) 1.9 (α/δ)

760

5.0 (α/δ)

Utkin et al (1994) Toxicon 32, 1153-7.

15

Kreienkamp et al. (1994) J.Biol. Chem. 269, 8108-14.

(α/γ)

M1

mouse muscle

M1

Torpedo

2.6

(α/γ)

2.3 (α/δ)

Groebe et al. (1995) Mol.Pharmacol. 48,105-111

M1

Torpedo

4.5

(α/γ)

0.5 (α/δ)

Martinez et al. (1995) Biochemistry 34,14519-26.

M1

BC3H1

1.5 (α/δ)

22

(α/γ)

Groebe et al. (1995) Mol.Pharmacol. 48,105-111

SIA

BC3H1

7.7 (α/δ)

200

SI

BC3H1

680 (α/δ)

(α/γ) (α/γ)

“------------------” “------------------”

E1

Torpedo

0.4 (α/δ)

(α/γ)

Martinez et al. (1995) Biochemistry 34,14519-14526

E1

BC3H1

9.4 (α/δ)

(α/γ)

“-----------------”

220 0.2 0.3

Свойства α-конотоксинов

Избирательное блокирование конотоксинами определенных типов мышечных и нейрональных холинорецепторов

H-NMR solution structures of α-conotoxins G1 and IMI acting on muscle-type and α7 AChRs, respectively

1

α-conotoxin G1

α-conotoxin IMI

a: Major form superimposed on the X-ray structure shown in red b: Minor conformation

Maslennikov IV, SobolAG, Gladky KV, Lugovskoy AA, Ostrovsky AG, Tsetlin VI, Ivanov VT, Arseniev AS Eur J Biochem. 254, 238-47(1998).

Maslennikov IV, Shenkarev ZO, Zhmak MN, Ivanov VT, Methfessel C, Tsetlin VI, Arseniev AS. FEBS Lett. 444, 275-80 (1999). NMR structures of α-conotoxin ImI and its mutants published by other groups: Rogers J.P. el al. (1999) Biochemistry 38, 3874-82. Couda H. and Hirono S. (1999) Biochim. Biophys. Acta 1431,384-394 Gehrmann J. et al. (1999). J. Med. Chem. 42, 2364-72. Lamthanh et al. (1999) FEES Lett. 454, 293-298. Rogers J.P. et al. (2000). J. Mol. Biol. 304, 911-926.

α-Conotoxins acting on neuronal acetylcholine receptors α-Conotoxin IMI PnIB PnIA MII AuIB EpI

Receptor α7 α7 α3β2 (α7) α3β2 and α6 α3β4 α3β2, α3β4

Cho J.-H., Mok K.H., Olivera B.M. et al (2000) J.Biol. Chem.275, 8680 Superimposed structures of α-conotoxins AuIB (green) and MII (blue).

Comparison of solvent-accessible surfaces of α-conotoxins acting on different neuronal nicotinic receptors.

Surfaces are colored according to electrostatic potential. Negative and positive charges are represented by red and blue colors, respectively. Different orientations are shown in (a) and (b). Rogers J.P. et al. (1999). Biochemistry 38, 3874-3882.

Different binding sites on the neuronal α7 nicotinic receptor for α conotoxin PnIB and α-conotoxin ImI  

GCCSLPPCALSNPDYC

α7(5HT-3)

α-conotoxin PnIB    S4          Y93, W149        L5  S34, R186, Y188, Y195      P6-----------W149, Y93        P7----     --Y93          A9           L10--------W149  GCCSLPPCAANNPDYC         α3β2 α-conotoxin PnIA

GCCSDPRCAWRC

α7(5HT-3)

α-conotoxin IMI W55, T77,S34, D64,  Q117, Y195 D5--------------- W149, Y151, G153           P6            R7---------------Y195 W10---------------T77, N111

Изомеры α-конотоксинов по дисульфидным связям

Схематическое представление структур ацетилхолинового рецептора и ацетилхолинсвязывающего белка

Модели связывания α -конотоксинов c нейрональными ацетилхолиновыми рецепторами

ImI и PnIB и

α7 AChR

PnIA и MII

α3β2 AChR

Mode of κ -Conotoxin PVIIA blocking the Shaker K+ channel

Spatial structure: Savarin et al. (1988.) Biochemistry 37, 5407-5416. Ala walk mutagenesis: Jacobsen et al. (2000). J. Biol. Chem. 275, 24639-24644 (red color: about 1000-fold drop in affinity, yellow::1000-100-fold, green: 10-fold, white : no substitution or no proper folding Earlier,using chimeras of channels sensitive and insensitive to PVIIA, the binding determinant was found to be the pore region of the channel . Two groups published the very smilar spatial structures, but proposed different binding mechanisms. One group (from Australia) suggested that His11, Arg18, Lys 19 and Arg 22 are essential, because similar residues are present in charybdotoxin (Lys11, Arg25, Lys27 and Arg31). Menez et al (France) believed that it should be a dyad motif of lysine and aromatic residues (Lys 7 and Phe 9 or Phe 23) that are also found in unrelated toxins acting on voltage-gated potassium channels (including charybdotoxin and sea anemone toxins). Alanine walk mutagenesis proved that the French authors are right: considerable loss of activity upon mutating Arg 2, Lys 7, and Phe 9. (additional role for Lys 25 which is in Van der Waals contact with Lys 7). By mutating the channel residues (double mutant cycles),

for those residues of κ-conotoxin PVIIA partners were not identified

Spatial structure of ω-conotoxins

Kobayashi K .et al. (2000). Three-dimensional solution structure of ω-conotoxin TxVII, an L-type calcium channel blocker . Biochemistry 39, 14761-14767

Kasheverov I., Rozhkova A., Zhmak M., Utkin Yu., Ivanov V., Tsetlin V. Photoactivatable α-conotoxins reveal contacts with all subunits as well as antagonist-induced rearrangements in the Torpedo californica acetylcholine receptor. Eur.J.Biochem. 268, 3664-3673 (2001)

Участки связывания α-конотоксинов и αбунгаротоксина на ацетилхолиновом рецепторе Torpedo californica перекрываются, но не идентичны.

Выводы о структурно-функциональных взаимоотношениях α-конотоксинов , основанные только на конкуренции с радиоактивным αбунгаротоксином, могут быть некорректными

α -Conotoxins ImI and ImII: Similar α 7 nicotinic receptor antagonists act at different sites. Ellison MA, McIntosh JM, Olivera BM (2002). J Biol Chem .

Aromatic substitutions in α-conotoxin IMI. Synthesis of iodinated photoactivatable derivatives. Y.N.Utkin, M.N.Zhmak, C.Methfessel, V.I.Tsetlin. Toxicon 37, 16831695. (1999)

Role of natural or artificially introduced charges in α-conotoxins targeting muscletype or neuronal nicotinic acetylcholine receptors

Competition of native α-conotoxins and their analogs with radioiodinated α-bungarotoxin for binding to L.stagnalis (a) and A.californica (b) AChBP

Celie P.H.N., Kasheverov I.E., Mordvintsev D.Y., Hogg R.C., van Nierop P., van Elk R., van Rossum-Fikkert S.E., Zhmak M.N., Bertrand D., Tsetlin V., Sixma T.K., Smit A.B.

Nature Str.&Mol.Biol. 12, 582-588 (2005)

Figure 4 Conformational changes in AChBP upon toxin binding. (a), Superposition of one subunit (yellow) of AcAChBP-PnIA[A10L,D14K] on Ac-AChBP-HEPES (grey), displaying relative rigid body movement in complementary subunit (blue) indicated by the arrow. (b), Zoom-in of the ligand binding-site of above superposition, with a-Ctx side chains (orange). Movement of E-loop upon toxin binding is indicated by the arrow. AChBP Tyr53 makes novel H-bond with Tyr91 main chain. c, Opening of C-loop upon toxin binding. Ac-AChBPHEPES in grey, Ac-AChBP-PnIA[A10L,D14K] in yellow, toxin in red, Cys residues in green. (d), C-loop closes upon HEPES binding (grey) and nicotinic agonist binding13 (magenta) and opens up upon a-Ctx binding (yellow) and in the absence of HEPES (green) (see also Figure 2a, green subunit)..

Figure 5 Models of α-conotoxin binding to other ligand-binding domains. (a), Comparison of PnIA[A10L,D14K (violet) bound to a Ls-AChBP model, showing C-loop (orange) and complementary side (green) to PnIA[A10L,D14K] (red) bound to Ac-AChBP in the crystal structure, showing only E-loop (blue, with cyan residues) (C-loops are similar). The predicted salt bridge between Ctx-Lys14 and Glu110 in LsAChBP model is shown (red dotted line). (b), Comparison of PnIA[A10L,D14K] (red) and PnIA[A10L] (violet) built into a model of the a7-nAChR extracellular domain, showing C-loop (in yellowish colors) and complementary side (blue). Predicted hydrogen bond between Lys75 and Ctx-Asp14 of PnIA[A10L] is indicated with a red dotted line.

Structural determinants of selective α–conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP Chris Ulens, Ronald C. Hogg, Patrick H. Celie, Daniel Bertrand, Victor Tsetlin, August B. Smit, and Titia K. Sixma¶

Proc. Natl. Acad. Sci. USA 103, 3615-3620 (2006)

Модель связывания конотоксина SIA[D12K] c AChR Torpedo californica

Kasheverov IE, Zhmak MN, Vulfius CA, Gorbacheva EV, Mordvintsev DY, Utkin YN, van Elk R, Smit AB, Tsetlin VI. FEBS J. 273, 4470-81 (2006)

Identification of diverse nicotinic acetylcholine receptors with snake and snail toxins Toxin

Number of aa residues

S-S bonds

Target acetylcholine receptor

Snake venoms

Short-chain α-neurotoxins

60-62

4

Torpedo (α2βγδ) and muscle (α2βγδ or α2βεδ)

Long-chain α-neurotoxins

66-74

5

Torpedo, muscle; neuronal α7, α7-like Cl-channels on Aplysia and L.stagnalis

Long-chain κ-neurotoxins

66

5

Neuronal α3β2, (α4β2)

63-66

5

Torpedo and muscle-type, neuronal α7; α7-like Cl-channels of L.stagnalis

13-18 12 16 16 16 15

2 2 2 2 2 2

Torpedo and muscle

“Weak” toxins

Conus snail venoms

α-conotoxins G1, M1, SI, EI α-conotoxins ImI and ImII PnIB , [A10L] PnIA α-conotoxins PnIA , GIC α-conotoxin MII α-conotoxin AuIB

α7 α7 α3β2 α3β2, α6 α3β4

Конотоксины и конопептиды – потенциальные медицинские средства ___________________________________________________________________________________________________

Пептид

Мишень

Применение

Примечания

___________________________________________________________________________________________________ ω-конотоксин МVIIA Ca-каналы Антиболевое действие Одобрен FDA и в Евросоюзе (Ziconotide, Prialt) N-типа (раковые больные), Хроническая боль ω-конотоксин CVID

Ca-каналы N-типа

Контулакин -G

Рецептор нейротензина

Конантокин G

NMDA рецептор

α-конотоксин ACV1

нейрональные никотиновые рецепторы

‘-------------------------“ нейропатическая боль послеоперационная боль антиноцицептивная активность противоэпилептическое действие периферийная нейропатическая боль

Фаза II Фаза II Фаза II

Фаза I

___________________________________________________________________________________________________________________