justin du bois department of chemistry stanford university · t u r n i n g t o x i n s i n t o t o...
TRANSCRIPT
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Voltage-Gated Ion Channels: Remarkable Molecular Machines �
• transmembrane proteins that function to transport charged ions across lipid bilayer
• found in all electrically excitable cells; respond to changes in membrane voltage
• selective for K+, Na+, Ca2+, Cl– ; ions pass at rates on the order of 107 per second! • crystallographic analysis of bacterial and mammalian K+ ion channels�
The Voltage-Gated Sodium Channel
19Å cryo-EM image Sato Nature 2001�
• ~260 kDa α-subunit; functional expression in heterologous vectors
• α-subunit comprised of four repeat domains, each with 6 TM helices
• 10 gene loci encoding α-subunit identified in mammalian cells
taken from King Channels 2008, 2, 100�
Tools of the Channelologist
electrophysiology immuno -cytology & histology fluorescent protein labeling
fast and dynamic nature of neuronal processes necessitates methods that afford rapid temporal response
Sodium Ion Channel Modulators
H
Me
O
OH
MeN
O O
Me O
NHMe
Me
O
H
O
OH
OH
OHNHN
H2N
HO
HO
OHO
H
N NH
HN NH
NH2HO
HO
H2N
O NH2
O
O
MeO
O
H
MeMe
HMe
Me
AcOOH
OMe
OH
OHOMeO
Me
O
MeN
MeO O
Ph
O
O
O
O
O
O
O
OO
O O
Me
O
Me
Me
Me Me
Me
Me
H
H
H
H
H
HH H
H
H
H
HO
HH
H
H
CHO
tetrodotoxin
saxitoxin
zetekitoxin AB
pyrethrin I
batrachotoxin
aconitine
brevetoxin A
N
HN N
H2N
OHN
O
OH
NO
HO
HO
O
N H–O3SO
NH2
HO
A Total Synthesis of (+)-Saxitoxin – The Cliffs Notes Version�
N
NHBoc
OSitBuPh2
PMB O
H
3 steps from N-Boc L-serine ester�
Mbs = OMeS
O
O
NH
NH
NMbs
O NH2
ONH
MbsN
H2N
NH
NH
NMbs
O NH2
ONH
MbsN
H2N
N NH
NHHN
NH2
H2N
HO
HO
O
O
NH2
(+)-saxitoxin
N
NHBoc
OSitBuPh2
PMB OH
NH
MbsN SMe
1 step� 7 steps�
N NH
NHHN
NH2
H2N
O
O
NH2HO
β-saxitoxinol�
B(O2CCF3)3��
84%�
[O]��
>70%�
• desired target assembled in 14 linear steps from commercial material
• de novo construction provides access to a unique chemical probe�
HN
NHN
NMbs
HO HOO
NH2
MbsN
NH2
O
10 mol% OsCl3�oxone, Na2CO3�
57%�
Mbs = OMeS
O
O
Toxin Conjugates as Molecular Probes of NaV Function
H2N
N NH
HN NH
NH2HO
HO
O
O
NH
HN
OH2N
N NH
HN NH
NH2HO
HO
O
O
NH
NH3+
O
O
N
O
O
H2N
N NH
HN NH
NH2HO
HO
O
O
NH
+HN
O
N N
MeMeMeMe
Me
STX-Cy5
Fluorescent probes
pH = 9.5
H2O/CH3CN
STX-18F
PET imaging agents
STX-maleimide
Affinity labels STX-maleimide
Affinity labels
A ‘Library’ of Bis-Guanidinium Toxins
saxitoxin (STX) neosaxitoxin gonyautoxin 2
dc-gonyautoxin 1 hydroxybenzoate STX gonyautoxin 5
Lyngbya wollei toxin 3
STX ethanoic acid
An Asymmetric Synthesis of (+)-Gonyautoxin – Abridged
61% isolated yield single diastereomer
770%�N NH
NH
O
OTBDPSBF3•OEt2
CH2Cl2
N NH
NHHN
NH2
H2N
HO
HO–O3SO
O
O
NH2
gonyautoxin 2/3
7 steps�
N NH
N
O
OSitBuPh2
from L-serine ester in 4 steps
N NH
NH
NSO2OCH2CCl3
H2N
NC(O)CCl3
OTBDPS
4 steps�
N NH
NHHN
NC(O)CCl3
TcesN
H
OTBDPS
BF3•OEt2
Et3SiH
75%�
2 mol%Rh2(esp)2PhI(OAc)2
N NH
NHHN
NH2
H2N
O
O
NH2HO
HO
Second Generation Improvements to Streamline Synthesis
5 steps
N NH
NHHN
NC(O)OCH2CCl3
Cl3CCH2OSO2N
HO
HOO
O
NH2
N NH
NH
TcesN
H2N
N
O
CCl3
OSitBuPh2
N N
NH
TcesN
H2N
NH2
OSitBuPh2
N NH
NH
TcesN
H2N
N
O
OCH2CCl3
OSitBuPh2
N N
NH
TcesN
H2N
NH O
OCH2CCl3
OSitBuPh2
+ TrocCl
iPrNEt, CH2Cl2
[O]N NH
NHHN
NH2
H2N
O
O
NH2HO
HOHO
HO
11,11-dihydroxy-STX
recently isolated PSP from Alexandrium tamerense – Quilliam J. Nat. Prod. 2008, 71, 1518
H2cat. Pd/C
CF3CO2HMeOH
76%
10 : 1�1 : 6�
DMSO
DCCC5H5NH+ –TFA
88%
CH2Cl2
–OTf
NMeTrocN
Ph
885%�65%�
‘Unnatural’ Toxins: C10-Substitution
55%�
74%�
C10-modified GTX 3
A Highly Evolved Cork Stoppers Ion Flux
extracellular Domain I Domain II Domain III Domain IV
outer vestibule: E403 E758 M1240 D1532 selectivity filter: D400 E755 K1237 A1529
IV III I
N NH
HN NH
NH2HO
HO
H2N
O
O
NH2
D400K1237
D1532
E403
E758
E755
M1240
NaV1.1 NaV1.2 NaV1.3 NaV1.4 NaV1.5 NaV1.6 NaV1.7 NaV1.8 NaV1.9 NaX
SCN1A SCN2A SCN3A SCN4A SCN5A SCN8A SCN9A SCN10A SCN11A SCN7A
CNS CNS, PNS Brain, DRG, PNS Skeletal muscle Cardiac CNS, DRG, PNS DRG, SN, spinal cord DRG DRG Glial cells, DRG
���������������?�
TTX/STX sensitivity localization gene locus name
Novakovic Trends Neurosci 2001, 24, 473 Llewellyn Nat. Prod. Rep. 2006, 23, 200
A Comparison of Toxin Volume and Surface Potentials�
N NH
HN NH
NH2HO
HO
H2N
O NH2
O
(+)-saxitoxin
N NH
HN NH
NH2HO
HO
H2N
–O3SO O NH2
O
(+)-gonyautoxin III
O
OH
OH
OHNHN
H2N
HO
HO
OHO
H
(–)-tetrodotoxin
305 Å3 242 Å3 252 Å3
electrostatic surface potential –4 kT to +4 kT
Double Mutant Cycle Analysis to Study Protein Structure
WTNaV � STX
ΔΔGint = ΔG1 – ΔG2 – (ΔG3 – ΔG4)
ΔG1
WTNaV � MutSTX
ΔG2
MutNaV1.4 � STX
ΔG3
MutNaV � MutSTX
ΔG4
� Magnitude of ΔΔGint varies with distance between two groups
� Quantitative assessment of localized interactions
� Previous studies limited by structural diversity of natural toxins used
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
70686664626058
ob
serv
ed
cur
rent
(nA
)
time (ms)
–100�
0�
–50�a
pp
lied
po
tentia
l (mV
)
Evaluating STX Block: Whole-cell Voltage-Clamp Recordings�
1.0 nM STX
5.0 nM STX
STX washed off
no STX
N NH
HN NH
NH2HO
HO
H2N
O NH2
O
(+)-saxitoxin
IC50 = 2.9 ± 0.1 nM against NaV1.4 (CHO)
Na+�
pipette
electrode
cell membrane
ion channel
N NH
HN NH
NH2HO
HO
H2N
O NH2
O
(+)-saxitoxin
IC50 = 2.9 ± 0.1 nM against NaV1.4 (CHO)
Na+��
pipette
electrode
cell membrane
ion channel
β-saxitoxinol
IC50 = 32 μM
N NH
HN NH
NH2HO
H
H2N
O
NH2
O
αα-saxitoxinol
IC50 = 559 nM gonyautoxin III
IC50 = 15 nM
N NH
HN NH
NH2HO
HO
H2N
–O3SO
O
NH2
ON NH
HN NH
NH2H
HO
H2N
O
NH2
O
11,11-dihydroxysaxitoxin
IC50 = 2.25 μM
structure-activity data for C11- and C12-derivatives challenge binding model �
Evaluating STX Block: Whole-cell Voltage-Clamp Recordings�
N NH
HN NH
NH2HO
HO
H2N
O
NH2
O
IIIIIV
D400K1237
M1240
D1532
E403
D758
E755
N NH
HN NH
NH2HO
HO
H2N
O
NH2
O
IIIIIV
D400K1237
M1240
D1532
E403
E758
E755
E758D
A Single Point Mutation Gives a Dramatic Result
IC50 = 2.9 nM for wild type NaV1.4
IC50 = 3.2 μM for E758D mutant
STX resistance in Mya arenaria ascribed to E→D mutation
Catterall & Trainer, Nature 2005
A Highly Evolved Cork Stoppers Ion Flux
extracellular Domain I Domain II Domain III Domain IV
outer vestibule: E403 E758 M1240 D1532 selectivity filter: D400 E755 K1237 A1529
IV III I
N NH
HN NH
NH2HO
HO
H2N
O
O
NH2
D400K1237
D1532
E403
E758
E755
M1240
NaV1.1 NaV1.2 NaV1.3 NaV1.4 NaV1.5 NaV1.6 NaV1.7 NaV1.8 NaV1.9 NaX
SCN1A SCN2A SCN3A SCN4A SCN5A SCN8A SCN9A SCN10A SCN11A SCN7A
CNS CNS, PNS Brain, DRG, PNS Skeletal muscle Cardiac CNS, DRG, PNS DRG, SN, spinal cord DRG DRG Glial cells, DRG
���������������?�
TTX/STX sensitivity localization gene locus name
Novakovic Trends Neurosci 2001, 24, 473 Llewellyn Nat. Prod. Rep. 2006, 23, 200
Human Pain Conditions linked to Sodium Channel Gene Mutations
Two autosomal dominant mutations in the NaV1.7 gene which cause a hyperpolarizing shift in activation resulting in redness, hyperalgesia, and burning pain�
Erythermalgia
Congenital Pain Insensitivity
NaV1.7 gene functional deletion - inability�to sense pain. �
Heckert, J. “The Hazards of Growing Up Painlessly,”
The New York Times Magazine Nov. 15, 2012
Human NaV1.7 is TTX ‘Sensitive’ and STX ‘Resistant’�
Time (s)
I/I 0
NaV1.4 control
+ 100 nM STX
Time (s)
NaV1.7 control
+ 100 nM STX
Time (s)
I/I 0
NaV1.4 control
+ 100 nM TTX
Time (s)
NaV1.7 control
+ 100 nM TTX
L L L L L L
M M M M M M
T T T T T T
Q Q Q Q Q Q
D D D D D D
F F Y Y Y Y
W W W W W W
E E E E E E
N N N N N N
T T T T T T
V V V I V V
L L L L L L
C C C C C C
G G G G G G
E E E E E E
W W W W W W
I I I I I I
E E E E E E
1.1 1.2 1.3 1.4 1.6 1.7
NaV Domain I Domain II Domain III Domain IV
A A A A A A
I I I I I I
T T T T T T
F F F F F F
K K K K K K
G G G G G G
W W W W W W
M M M M M T
D D D D D I
L L L L L L
T T T T T T
T T T T T T
S S S S S S
A A A A A A
G G G G G G
W W W W W W
D D D D D D
G G G G G G
N NH
HN NH
NH2HO
HO
H2N
O NH2
O
(+)-saxitoxin
N NH
HN NH
NH2HO
HO
H2N
–O3SO O NH2
O
(+)-gonyautoxin III
O
OH
OH
OHNHN
H2N
HO
HO
OHO
H
(–)-tetrodotoxin
toxin NaV1.4 Na
V1.4
M1240T Na
V1.4
D1241I Na
V1.4
M1240T
D1241I
NaV1.7 Na
V1.7
T1398M
I1399D
TTX 17.1 nM 466 8.7 90 18.6 5.0
STX 2.8 73 53 1153 702 2.3
GTX-III 14.9 228 38 1084 1513 22
A Possible Explanation For Differences in Toxin Affinity?�
Domain III Amino Acids Alter Pore Dimensions & Electrostatic Surface�
NaV1.4 NaV1.7
Site II – The Inner Pore
out
in
1
2
STX, GTX, TTX
V = 582 Å3 V = 502 Å3 V ~ 633 Å3
A Common NaV Receptor Site for all three Lipid-Soluble Neurotoxins?�
batrachotoxin�
H
Me
O
OH
MeN
O O
Me O
NHMe
Me
O
HAcO
OHOMe
OH
OHOMeO
Me
O
Me N
MeO O
OHO
OH
OHN
HO
Me
OH
HO
O
OMe
OMe
O
aconitine� veratridine�
Musutani, et al J. Pharm. Exp. Therap. 1981, 217, 812
Kosower FEBS Lett. 1983, 163, 161
Codding J. Am. Chem. Soc. 1983, 105, 3172
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VVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVV ================================================================================================================================================================ 55555555555555555555555555555555555555555555555555555555888888888888888888888888888888888888888888888222222222222222222222222222222222222222222222222222222222222222222 ÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅÅ33333333333333333333333333333333333333333333333333333333333333
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CoCoCoCoCoCoCoCCoCCoCCoCoCCCCCCCoCoCoCoCoCoCoCoCCoCoCoCoCCCCCCCCCoCCoCCoCoCoCoCoCoCCoCCoCCoCoCCCCCCCCooCoCCoCCCCoCCCCCoCCCoCoCoCCoCCoCoCoCoCoCCCCCoCCoCoCoCCoCoCoCoCoooCoCoCoCCCoooCoCoCooooooCooCoCCCCoCooCCCCCCooCoooCCCCCooCoooCCCCCCCCCCCCCoCoCooCoCCCCCooCooCoCoCoCooooCCoCCCCCCCCCCCooCoCoooCoCoCCCCCCCCCoCCoCooooCCoCoCoCCCCCoCoooCooooCoCoCoCoCoCCCCoCoCoCCoCCCCoCoCoCCCoCooCooCoCCCoCooooCooCCCoooCCoCCCoooooooooooooCCCooooooddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddd iniiininininnininininniniiininiiiinnininnniniiininnnnininnnnnninnnininnnnnnnininininnnnnninnnnninnnnnnninnnnnnnnnnnnnnnnggggggggggggggggggggggggggggggggggggggg
A Common NaV Receptor Site for all three Lipid-Soluble Neurotoxins?�
batrachotoxin�
H
Me
O
OH
MeN
O O
Me O
NHMe
Me
O
HAcO
OHOMe
OH
OHOMeO
Me
O
Me N
MeO O
OHO
OH
OHN
HO
Me
OH
HO
O
OMe
OMe
O
aconitine� veratridine�
O��O� O�
2.4 �
4.5 �
2.8 Å*�
Musutani, et al J. Pharm. Exp. Therap. 1981, 217, 812
Kosower FEBS Lett. 1983, 163, 161
Codding J. Am. Chem. Soc. 1983, 105, 3172
avg. distance to N ~6.0 �
holding open a lysine gate?�
NH
HH
+O
OO
H3N+
A Common NaV Receptor Site for all three Lipid-Soluble Neurotoxins?�
batrachotoxin�
H
Me
O
OH
MeN
O O
Me O
NHMe
Me
O
HAcO
OHOMe
OH
OHOMeO
Me
O
Me N
MeO O
OHO
OH
OHN
HO
Me
OH
HO
O
OMe
OMe
O
aconitine� veratridine�
electrophysiology recordings (current vs time) reflect differences in mode of action
The Venom of the Columbian Arrow Poison Frog�
batrachotoxin (BTX)�
Phyllobates terribilis
• first isolated from phyllobates aurotaenia (1965) (family Dendrobatidae) • 5000 frogs delivered 27 mg of pure toxin • structural elucidation/pharmacological evaluation by Daly and Witkop (NIH) • mouse LD50 (subcutaneous) = 2 μg/kg
Pitohui dichrous (Papua New Guinea) Daly & Witkop Science 1971, 172, 995
homobatrachotoxin�H
Me
O
OH
MeN
O O
Me O
NH
Me
O
H
Me
batrachotoxin�
NMe
O
O
NH
Me
Me
O
HO
Me
HOH
Me
O
Designing A Synthesis of Batrachotoxin – Prior Art �
Kishi J. Am. Chem. Soc. 1998, 120, 6627�Wehrli Helv. Chim. Acta 1973, 56, 139�
O
Me
Me
H H
H
OMe
H
HO
Me
H
O
OH
Wehrli (1973) Kishi (1998)
NMe
OH
O
HO
Me
HOH
Me
O
batrachotoxinin A�
NMe
OR
HO
HO
Me
OH
Me
O
HNMe
OR
HO
HO
Me
OH
Me
OHA
C D
A Truncated Form of Batrachotoxin
batrachotoxin
R =
final step esterification affords C/D/E ring analogue structures
H
Me
O
OH
MeN
O O
Me O
NHMe
Me
O
H
Electrophysiological Analysis of BTX C/D/E Analogues
IC50(μμM)
81.4 ± 4.8 41.4 ± 1.0 38.8 ± 1.0 17.4 ± 0.4
R =
19.2 ± 2.4 11.1 ± 0.3
–100
0
–50
0.0
–0.2
–0.4
–0.6
–0.8
–1.0
–1.2
58 60 62 64 66 68
ob
serv
ed
cur
rent
(nA
)
time (ms)
ap
plie
d p
ote
ntial (m
V)
70
control 200 μM CDE-naph
0.0
–0–0–0–0–00–0000000–0000000000–000–000–00––0000.222222222222222222
–0.4
–0.6
–0.8
–1.0
–1.2
58 60
ob
ob
ob
obb
obb
obb
ob
ob
ob
ob
obb
ob
obb
ob
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dde
de
dde
de
de
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ccccccccccccccccccccccccccccccccccccururururrururururrurrururururrururururururrrrururururrurrururururururrrrururrurrururrrrrruuuuuuuuuuuuuuuuuu
rerererereerererererererereeerereeererereeeeererereererereeeereeereerererereeeeererereeereeereeerrrrrrrrntntntnttntntntntntntntntntntntntttntntntntttntntntntnttntnttntntnttntntntnttttnttnnnnnnnnnnnnnnnnnnnnnnn
(((((((((((((((((((((((((((((((((((((((((((((((nAnAnAnAnAAnAnAnAAnAnAnAAAAAAnAAAAnAAAAAnAnAnnnnnnnnnnnnnnnnnnnnnn
25 μM CDE-naph 5 μM BTX
Relative Potency of CDE-naph Against NaV Subtypes�
CDE-naph
NaV isoform IC50 (µM) Stdev
rNaV1.2 30.1 ± 6.9
rNaV1.4 17.4 ± 0.4
hNaV1.5 19.3 ± 3.5
hNaV1.7 7.8 ± 1.7
Minimal differences in IC50 between subtypes
S6 Residues that line inner pore > 96% conserved
CDE-naph (M)
Rela
tive
cur
rent
(%
)
CDE naph
IC50(μM) 13.8 ± 0.7 15.4 ± 0.6
Mirror image isomers display nearly identical potency
Through the ‘Looking Glass’�
Single Point Mutagenesis Experiments with NaV1.4
CDE-naph
300
200
100
0
IC50
(μμ
M)
D1 D2 D3 D4
D1 D2 D3 D4
Homology Model of the Inner Pore (NaVMs)�
Catterall et al. Nature 2011 Clapham, Yan Nature 2012 Wallace et al. Nature 2013
out
in
Minimal differences in IC50 between subtypes
S6 Residues that line inner pore > 96% conserved
N434
F1579
N1584
Rationalizing the Lack of Enantiospecificity�� binding is state-dependent to open channel
� hyperpolarizes threshold activation by 30–50 mV
� eliminates inactivation gating
� reduces single channel conductance by ~60%
� modifies Na+ ion selectivity
H
Me
O
OH
MeN
O O
Me O
NHMe
Me
O
H
Many Outstanding Questions Remain�
batrachotoxin
Acknowledgements
Collaborators
Dr. Sandip Biswal, Aileen Hoehne, Deepak Behera — Radiology
Prof. Merritt Maduke — Mol. & Cell. Physiology
Prof. W. E. Moerner, Hsiao-Lu Lee, Shigeki Iwanaga — Chemistry
Prof. Vijay Pande, Paul Novick — Chemistry
Prof. David Yeomans — Anesthesiology
Dr. J. Zablocki, Dr. M. McGregor — CVT/Gilead
Support
National Institutes of Health
Pfizer
Center for Molecular Analysis and Design
SiteOne Therapeutics, Inc.
Brian Andresen
Matt Axtman
Rosemary Conrad
Sloan Devlin
Darren Finkelstein
James Fleming
Andrew Hinman
Arun Thottumkara
James Walker
Dr. Matt McReynolds
Dr. Alan Whitehead
Dr. Alison Ondrus
Dr. Fred Menard
Dr. Tatsuya Toma
Matt Logan
Jeff Merit
John Mulcahy
William Parsons
Heather Roberts
Rhiannon Thomas
Zahra Taji
Bio-X Stanford University
Stanford Dean’s Fellowship
NSERC Canada
Japan Society for the Promotion of Science