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Конотоксины –

природные комбинаторные
библиотеки и лекарства из
ядовитых морских улиток
Conus.
Shells of some Conus species

C.geographus C.quercinus
(the geography cone) (the oak cone)

C.radiatus
(the radial cone)
C. striatus
(the striated cone)
C.gloriamaris
(the glory-of-the –sea)
C.textile
C.magus
(the cloth-of-gold
cone) (the magus cone)

C.purpurascens C.marmoreus
(the marble cone)
(the purple 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 number of a.a.residues disulfide bridges Target
Snakes
α-neurotoxins 60-75 4-5 nicotinic acetylcholine
receptors

(nAChR)
muscarinic toxins 65 4 muscarinic acetylcholine receptors

(mAChR)
fasciculins 61 4 acetylcholinesterase (AChE)
calciseptins 60 4 Ca2+-channels
dendrotoxins 57-60 3 K+-channels
dendroaspin 58 4 cell
adhesion
cardiotoxins 60 4 membranes
Sea anemona
Toxin II Anemonia sulcata 47 3 Na+-
channels
Scorpions
α -scorpion neurotoxins 65 4 Na+-channels
leirutoxin I 31 3 K+-channels
charybdotoxin 37 3 K+-channels
Spiders
ω-atrachotoxin 36 3 Ca2+-channels (from
insects)
Bees
apamin 18 2 K+-channels
Conus peptides and their targets
Ligand-gated ion channels
• α -Conotoxins Nicotinic acetylcholine receptors
• αA-Conotoxins
• ψ-Conotoxins
• σ -Conotoxin Serotonin 5-HT3 receptor
• Conantokins NMDA receptor
Voltage-gated ion channels
• ω-Conotoxins Ca2+ channels
• µ-Conotoxins Na+ channels
• δ-Conotoxins
• µO-Conotoxins
• κ-Conotoxins K+-channels
• κA-Conotoxins
G-protein coupled receptors
• Conopressin Vasopressin receptor
• Conutalakin-G Neurotensin receptors
Different activities
• Contryphans behavioral effects
• mr10a (T-superfamily) antinociceptive
• λ-Conotoxins seizures, paralysis
• Gla-containing conopeptide spasmodic
• Conodipine -M phospholipase
Новые конотоксины и конопептиды
Conorfamide-Sr1 Mailto et al. (2002)
GPMGWVPVFYRF-NH2 Toxicon 40, 401-408
(семейство RF амидов)
синдром гиперактивности

Conophysin-R Lizarn et al (2002)


принадлежит к семейству Toxicon 40, 901-908
нейротензинов
48 аминокислотных остатков,
7 дисульфидов

ρ-Conopeptide ρ-TIA Sharpe et al (2001)


FNWRCCLIPACRRNHKKFC Nat.Neurosci. 4, 902-907
неконкурентный блокатор
α1-адренорецепторов

χ-Conopeptides Sharpe et al (2001)


MrIA NGVCCGYKLCHOC Nat.Neurosci. 4, 902-907
неконкурентный блокатор
норадреналиновых транспортеров,

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 C - - C - - CC - - C - - C 24-29

σ − Conotoxins C - -C - - C - - CC - - CC- - - - CC 41
Contryphans C---C 7-8
Posttranslational modifications of Conus peptides
Modification reaction Modified group or Enzyme
residue
1. Disulfide bond formation -S-S- Disulfide isomerase
2. Amidation of C-terminal -NH2 Protein amidating
carboxylate monooxygenase
3. Cyclization of N-terminal Gln pGlu Glutaminyl cyclase

4. Hydroxylation of Pro O Proline hydroxylase


5. γ− Carboxylation of Glu Gla γ -Glutamate carboxylase

6. Glycosylation of Thr (β l → 3)GalNAc(α1→ ) Polypeptide HexNAc


Thr transferase
7. Isomerization of Trp or Leu D-Trp or D-Leu 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 1.25 562
AzBz-Lys26 NT-II 0.64 412
Diaz-Lys23 CTX 3.0 6.3 200 160
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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
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___
G1 Torpedo 760 (α/γ) 5.0 (α/δ) Utkin et al (1994) Toxicon 32, 1153-7.
M1 mouse muscle 1.9 (α/δ) 15 (α/γ) Kreienkamp et al. (1994)
J.Biol. Chem. 269, 8108-14.
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 (α/γ) “------------------”
SI BC3H1 680 (α/δ) 220 (α/γ) “------------------”

E1 Torpedo 0.4 (α/δ) 0.2 (α/γ) Martinez et al. (1995)


Biochemistry 34,14519-14526
E1 BC3H1 9.4 (α/δ) 0.3 (α/γ) “-----------------”
Свойства α-конотоксинов
Избирательное блокирование конотоксинами определенных типов
мышечных и нейрональных холинорецепторов
1
H-NMR solution structures of α-conotoxins G1 and IMI
acting on muscle-type and α7 AChRs, respectively
α-conotoxin G1 α-conotoxin IMI

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

b: Minor
conformation
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
Maslennikov IV, SobolAG, Gladky KV,
1431,384-394
Lugovskoy AA, Ostrovsky AG, Tsetlin VI,
Gehrmann J. et al. (1999). J. Med. Chem. 42, 2364-72.
Ivanov VT, Arseniev AS Eur J Biochem.
Lamthanh et al. (1999) FEES Lett. 454, 293-298.
254, 238-47(1998).
Rogers J.P. et al. (2000). J. Mol. Biol. 304, 911-926.
α-Conotoxins acting on neuronal acetylcholine receptors
α-Conotoxin Receptor
IMI α7
PnIB α7
PnIA α3β2 (α7)
MII α3β2 and α6
AuIB α3β4
EpI 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) GCCSDPRCAWRC α7(5HT-3)


α-conotoxin PnIB α-conotoxin IMI
   S4          Y93, W149    W55, T77,S34, D64, 
    L5  S34, R186, Y188, Y195 Q117, Y195
     P6-----------W149, Y93 D5--------------- W149, Y151, G153
       P7----     --Y93           P6
         A9            R7---------------Y195
          L10--------W149 W10---------------T77, N111

 GCCSLPPCAANNPDYC         α3β2
α-conotoxin PnIA
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 α-
бунгаротоксина на ацетилхолиновом рецепторе
Torpedo californica перекрываются, но не идентичны.

Выводы о структурно-функциональных
взаимоотношениях α-конотоксинов , основанные
только на конкуренции с радиоактивным α-
бунгаротоксином, 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, 1683-
1695. (1999)
Role of natural or artificially introduced
charges in α-conotoxins targeting muscle-
type 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 Ac-
AChBP-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-AChBP-
HEPES 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 Ls-
AChBP 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
Number of S-S Target acetylcholine receptor
Toxin aa residues bonds
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)

“Weak” toxins 63-66 5 Torpedo and muscle-type, neuronal α7;


α7-like Cl-channels of L.stagnalis
Conus snail venoms
α-conotoxins G1, M1, SI, EI 13-18 2 Torpedo and muscle
α-conotoxins ImI and ImII 12 2 α7
PnIB , [A10L] PnIA 16 2 α7
α-conotoxins PnIA , GIC 16 2 α3β2
α-conotoxin MII 16 2 α3β2, α6
α-conotoxin AuIB 15 2 α3β4
Конотоксины и конопептиды – потенциальные медицинские средства
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Пептид Мишень Применение Примечания

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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 ‘-------------------------“ Фаза II


N-типа нейропатическая боль

Контулакин -G Рецептор послеоперационная боль Фаза II


нейротензина

Конантокин G NMDA рецептор антиноцицептивная активность Фаза II


противоэпилептическое
действие

α-конотоксин ACV1 нейрональные периферийная нейропатическая боль Фаза I


никотиновые
рецепторы

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