Inhibitors of oplophorus luciferase-derived bioluminescent complexes

1. A compound of formula (I), or a salt thereof:

wherein:

R 1 is

optionally substituted with one or more R W , wherein each R W is independently selected from the group consisting of C 1-10 alkyl, C 1-10 haloalkyl, halogen, —CN, —OR A , —C 1-10 alkylene-OR A , —CO—R A , —C 1-10 alkylene-CO—R A , —CO—OR A , —C 1-10 alkylene-CO—OR A , —CO—NHR A , —C 1-10 alkylene-CO—NHR A , —NR B R C , —C 1-10 alkylene-NR B R C , —NH—CO—C 1-4 alkyl, —C 1-10 alkylene-NH—CO—C 1-4 alkyl, phenyl, and phenyl substituted with 1, 2, 3, or 4 R D groups;

R 2 is C 1-10 alkyl, C 1-10 haloalkyl, halogen, —CN, —OR A , —C 1-4 alkylene-OR A , —CO—R A , —C 1-4 alkylene-CO—R A , —CO—OR A , —C 1-4 alkylene-CO—OR A , —CO—NHR A , —C 1-4 alkylene-CO—NHR A , —NR B R C , —C 1-4 alkylene-NR B R C , —NH—CO—C 1-4 alkyl, —C 1-4 alkylene-NH—CO—C 1-4 alkyl, phenyl, phenyl substituted with 1, 2, 3, or 4 R D groups, —C≡C—R A , or —C≡C—C 1-4 alkylene-OR A , or two R 2 together with the carbon atoms of the

moiety to which they are attached form a 5- or 6-membered fused ring;

R 3 is C 1-10 alkyl, C 1-10 haloalkyl, halogen, —CN, —OR A , —C 1-4 alkylene-OR A , —CO—R A , —CO—OR A , or —CO—NHR A , or two R 3 together with the carbon atoms of the

moiety to which they are attached form a 5- or 6-membered fused ring;

R 4 is H or C 1-4 alkyl;

p is 0, 1, 2, 3, or 4;

q is 0, 1, 2, 3, or 4;

R A at each occurrence is independently H, C 1-4 alkyl, or C 1-4 haloalkyl;

R B and R C at each occurrence are independently H or C 1-4 alkyl, or R B and R C together with the N atom to which they are attached form a 5- or 6-membered heterocycle; and

R D at each occurrence is independently C 1-4 alkyl, —OC 1-4 alkyl, —CN, or halogen.

2. The compound of claim 1 , or a salt thereof, wherein p is 1, and R 2 is C 1-4 alkyl, halogen, C 1-4 haloalkyl, —OH, —C 1-4 alkylene-OH, —OC 1-4 alkyl, or —NH 2 .

3. The compound of claim 1 , or a salt thereof, wherein q is 1, and R 3 is C 1-4 alkyl, halogen, —CN, —OH, or —OC 1-4 alkyl.

4. The compound of claim 1 , or a salt thereof, wherein the compound has formula (I-a):

wherein:

p is 0, 1, or 2;

q is 0 or 1;

R 3 is C 1-4 alkyl, halogen, —CN or —OR A ;

R A is H or C 1-4 alkyl.

5. The compound of claim 4 , or a salt thereof, wherein R 1 is unsubstituted or R 1 is substituted with 1, 2, or 3 R W , each R W being independently halogen, C 1-4 alkyl, C 1-4 haloalkyl, or phenyl.

6. The compound of claim 4 , or a salt thereof, wherein p is 1 or 2, and each R 2 is independently C 1-4 alkyl, halogen, C 1-4 haloalkyl, —OH, —C 1-4 alkylene-OH, —OC 1-4 alkyl, —NH 2 , phenyl, or —CO—OC 1-4 alkyl, or two R 2 together with the

moiety to which they are attached form

7. The compound of claim 4 , or a salt thereof, wherein the compound has formula (I-a-1):

wherein:

R 2 is halogen, C 1-4 alkyl, C 1-4 haloalkyl, —OH, —C 1-4 alkylene-OH, —OC 1-4 alkyl, or —NH 2 .

8. The compound of claim 1 , selected from the group consisting of:

N-(benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamido)benzamide;

methyl 3-(4-(N-(2-(benzo[b]thiophen-2-ylcarbamoyl)phenyl)sulfamoyl)phenyl)propanoate;

N-(benzo[b]thiophen-2-yl)-2-((3-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-2-((4-(3-hydroxypropyl)phenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-3-((4-methylphenyl)sulfonamido)-2-naphthamide;

N-(benzo[b]thiophen-2-yl)-5-methyl-2-((4-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-5-cyano-2-((4-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-2-((4-pentylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-5-butyl-2-((4-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-5-methoxy-2-((4-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-4-methoxy-2-((4-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-2-((3-methoxyphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-4-fluoro-2-((4-methylphenyl)sulfonamido)benzamide;

N-(benzo[b]thiophen-2-yl)-5-fluoro-2-((4-methylphenyl)sulfonamido)benzamide; and

N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzamide,

or a salt thereof.

9. A kit comprising:

(a) the compound of claim 1 ; and

(b) a first polynucleotide encoding a non-luminescent peptide of an Oplophorus -derived luciferase; and

(c) a second polynucleotide encoding a non-luminescent polypeptide of an Oplophorus -derived luciferase, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide.

10. A bioluminescence resonance energy transfer (BRET) system comprising:

(a) a first fusion comprising a first molecule and a non-luminescent peptide or polypeptide of an Oplophorus -derived luciferase;

(b) a second fusion comprising a second molecule and a fluorescent acceptor molecule;

(c) a non-luminescent complementary polypeptide or peptide of Oplophorus -derived luciferase capable of forming a bioluminescent complex with the non-luminescent peptide or polypeptide of an Oplophorus -derived luciferase;

(d) a coelenterazine substrate; and

(e) the compound of claim 1 .

11. A method of inhibiting luciferase activity of an Oplophorus luciferase-derived bioluminescent complex, the method comprising contacting the bioluminescent complex with the compound of claim 1 .

12. A method for modulating luminescence of an Oplophorus luciferase-derived bioluminescent complex in a sample, the method comprising:

(a) contacting the sample with a coelenterazine substrate and the compound of claim 1 ; and

(b) detecting luminescence in the sample,

wherein the compound of claim 1 causes a decrease in the luminescence from the bioluminescent complex.

13. A method to detect an interaction or co-localization between a first molecule and a second molecule in a sample, the method comprising:

(a) contacting a sample with a coelenterazine substrate and the compound of claim 1 , wherein the sample comprises:

(i) a first fusion, wherein the first fusion comprises a non-luminescent peptide or polypeptide of an Oplophorus -derived luciferase and a first molecule; and

(ii) a second fusion, wherein the second fusion comprises a non-luminescent complementary polypeptide or peptide of an Oplophorus -derived luciferase and a second molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; and

(b) detecting luminescence in the sample,

wherein the detection of luminescence indicates an interaction or co-localization between the first molecule and the second molecule.

14. A method to detect an interaction or co-localization of a first molecule and a second molecule in a sample, the method comprising:

(a) contacting a sample with a non-luminescent polypeptide or peptide of an Oplophorus -derived luciferase, a coelenterazine substrate, and the compound of claim 1 , wherein the sample comprises:

(i) a first polynucleotide encoding a first fusion, wherein the first fusion comprises an non-luminescent complementary peptide or polypeptide of an Oplophorus -derived luciferase and a first molecule, wherein the non-luminescent complementary peptide or polypeptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide or peptide; and

(ii) a second polynucleotide encoding a second fusion, wherein the second fusion comprises a fluorescent acceptor molecule and a second molecule; and

(b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicating an interaction or co-localization of the first molecule and the second molecule.

15. A method to detect interaction or co-localization of molecules in a sample, the method comprising:

(a) contacting a sample with a coelenterazine substrate and the compound of claim 1 , wherein the sample comprises:

(i) a first fusion comprising a non-luminescent polypeptide of an Oplophorus -derived luciferase and a first molecule;

(ii) a second fusion comprising a non-luminescent peptide of an Oplophorus -derived luciferase and a second molecule, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; and

(iii) a third fusion comprising a fluorescent acceptor molecule and a third molecule; and

(b) detecting bioluminescence resonance energy transfer (BRET) in the sample, indicating an interaction or co-localization between the first molecule, second molecule, and third molecule in the sample.

16. A method to detect a molecule of interest in a sample, the method comprising:

(a) contacting a sample comprising the molecule of interest fused to a non-luminescent peptide or polypeptide of an Oplophorus -derived luciferase with

(i) a coelenterazine substrate;

(ii) the compound of claim 1 ; and

(iii) a non-luminescent complementary polypeptide or peptide of an Oplophorus -derived luciferase, wherein the non-luminescent complementary polypeptide or peptide is capable of forming a bioluminescent complex with the non-luminescent peptide or polypeptide; and

(b) detecting luminescence in the sample,

wherein detection of luminescence indicates formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

17. A method to detect a molecule of interest in a sample, the method comprising:

(a) contacting a sample comprising the molecule of interest fused to a non-luminescent peptide or polypeptide of an Oplophorus -derived luciferase with

(i) a coelenterazine substrate;

(ii) a compound of claim 1 ; and

(iii) a fusion comprising a non-luminescent complementary polypeptide or peptide of an Oplophorus -derived luciferase and a fluorescent moiety, wherein the non-luminescent complementary polypeptide or peptide is capable of forming a bioluminescent complex with the non-luminescent peptide or polypeptide; and

(b) detecting bioluminescence resonance energy transfer (BRET) in the sample, indicating detection of the molecule.

18. A bioluminescence resonance energy transfer (BRET) system comprising:

(a) a first fusion comprising a non-luminescent peptide or polypeptide of an Oplophorus -derived luciferase and a first molecule;

(b) a second fusion comprising a non-luminescent complementary polypeptide or peptide of an Oplophorus -derived luciferase and a fluorescent moiety, wherein the non-luminescent complementary polypeptide or peptide is capable of forming a bioluminescent complex with the non-luminescent peptide or polypeptide;

(c) a coelenterazine substrate; and

(d) the compound of claim 1 .