Protoxin-II variants and methods of use
First Claim
Patent Images
1. An isolated Protoxin-II variant comprising the sequence X1X2X3CX4X5WX6QX7CX8X9X10X11X12CCX13X14FX15CX16LWCX17KKLW (SEQ ID NO:
- 403), whereinX1 is G, P, A or deleted;
X2 is P, A or deleted;
X3 is S, Q, A, R or Y;
X4 is Q, R, K, A or S;
X5 is K, S, Q or R;
X6 is M or F;
X7 is T, S, R, K or Q;
X8 is D or T;
X9 is S, A or R;
X10 is E, R, N, K, T or Q;
X11 is R or K;
X12 is K, Q, S or A;
X13 is E, Q or D;
X14 is G or Q;
X15 is V or S;
X16 is R or T; and
X17 is K or R;
optionally having an N-terminal extension or a C-terminal extension,wherein the polypeptide inhibits human Nav1.7 activity with an IC50 value of about 1×
10−
7 M or less,wherein the IC50 value is measured using a membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×
10−
6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7 and using bis-(1,3-diethylthiobarbituric acid)trimethine oxonol as an electron acceptor and Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm.
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Abstract
The present invention relates to Protoxin-II variants, polynucleotides encoding them, and methods of making and using the foregoing.
22 Citations
28 Claims
-
1. An isolated Protoxin-II variant comprising the sequence X1X2X3CX4X5WX6QX7CX8X9X10X11X12CCX13X14FX15CX16LWCX17KKLW (SEQ ID NO:
- 403), wherein
X1 is G, P, A or deleted; X2 is P, A or deleted; X3 is S, Q, A, R or Y; X4 is Q, R, K, A or S; X5 is K, S, Q or R; X6 is M or F; X7 is T, S, R, K or Q; X8 is D or T; X9 is S, A or R; X10 is E, R, N, K, T or Q; X11 is R or K; X12 is K, Q, S or A; X13 is E, Q or D; X14 is G or Q; X15 is V or S; X16 is R or T; and X17 is K or R; optionally having an N-terminal extension or a C-terminal extension, wherein the polypeptide inhibits human Nav1.7 activity with an IC50 value of about 1×
10−
7 M or less,wherein the IC50 value is measured using a membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×
10−
6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7 and using bis-(1,3-diethylthiobarbituric acid)trimethine oxonol as an electron acceptor and Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
- 403), wherein
-
15. An isolated Protoxin-II variant comprising an amino acid sequence that is at least 90%, identical to the amino acid sequence of SEQ ID NO:
- 78 (GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH), wherein a) the amino acid sequence has Q at position 1, Q at position 7 and F at position 19, when residue numbering is according to SEQ ID NO;
1;
b) the polypeptide inhibits human Nav1.7 activity with an IC50 value of about 30×
10−
9 M or less, wherein the IC50 value is measured using a membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×
10−
6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7 and using bis-(1,3-diethylthiobarbituric acid)trimethine oxonol as an electron acceptor and Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm; and
c) the polypeptide selectively inhibits Nav1.7.
- 78 (GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH), wherein a) the amino acid sequence has Q at position 1, Q at position 7 and F at position 19, when residue numbering is according to SEQ ID NO;
Specification