Diagnostic marker for pancreatic cancer
The invention provides a compound characterized by formula (1): X1-Thr-Thr-Ala-Arg-X2, wherein cleavage of the compound into a fragment 1 comprising X1 and a fragment 2 comprising X2 generates a detectable signal. The invention further provides an in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with the compound of the invention and detecting a signal, wherein the body fluid may comprise a hydrolytic enzyme derived from pancreatic cancer cells. Furthermore, the invention provides a kit comprising the compound of the invention and a measurement buffer. In addition, the invention provides the use of the compound, the in vitro method or the kit of the invention for the detection of pancreatic cancer, or for monitoring a subject that is suspected of having pancreatic cancer, has an increased risk of developing pancreatic cancer, or has had pancreatic cancer. The invention also provides the use of the compound of the invention in a method of treating pancreatic cancer, the method comprising carrying out the in vitro method for detecting protease activity in a subject's body fluid, and treating pancreatic cancer in a subject for which protease activity, has been detected.
1 . A compound characterized by formula 1:
X1-Thr-Thr-Ala-Arg-X2 (formula),
wherein cleavage of the compound into a fragment 1 comprising X1 and a fragment 2 comprising X2 generates a detectable signal,
wherein X1 comprises or consists of a component C1 and X2 comprises or consists of a component C2 and components C1 and C2 are separated by no more than 10 amino acids,
wherein the detectable signal is generated upon spatial separation of C1 and C2, by hydrolytic cleaving of the compound, and
wherein C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor.
2 . The compound according to claim 1 , wherein the sequence Thr-Thr-Ala-Arg is accessible for a hydrolytic enzyme.
3 . The compound according to claim 2 , wherein the sequence Thr-Thr-Ala-Arg is accessible for a hydrolytic enzyme cleaving the compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH 2 -X2 (fragment 2).
4 . The compound according to claim 1 , wherein one of C1 and C2 is a chromophore having an absorption maximum 1 (AMI) at a wavelength 1, and the compound has an absorption maximum 2 (AM2) at a wavelength 2 that is different from wavelength 1.
5 . The compound according to claim 4 , wherein C2 is a chromophore having an absorption maximum 1 (AMI) at a wavelength 1, and the compound has an absorption maximum 2 (AM2) at a wavelength 2 that is different from wavelength 1.
6 . The compound according to claim 1 , wherein the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/para-nitroaniline (pNA), ABZ/5-amino-2-nitrobenzoic acid (ANB-NH 2 ), ABZ/2,4-dinitrophenyl (DNP), ABZ/ethylene diamine 2,4-dinitrophenyl (EDDNP), 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid (EDANS)/2-(N, N-dimethyl-4-aminophenyl) azobenzenecarboxylic acid (DABCYL), arboxytetramethylrhodamine (TAM)/5-(dimethylamino) naphthalene-1-sulfonyl (DANSYL), ABZ/Tyr (3-NO 2 ).
7 . The compound according to claim 6 , wherein the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH 2 .
8 . The compound according to claim 1 , wherein X2 consists essentially of component C2.
9 . The compound according to claim 1 , wherein components C1 and C2 are separated by 4 amino acids.
10 . A kit comprising a compound according to claim 1 and a measurement buffer.
11 . An in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with a compound and detecting a signal and
wherein the compound is characterized by formula 1:
X1-Thr-Thr-Ala-Arg-X2 (formula),
wherein X1 comprises or consists of a component C1 and X2 comprises or consists of a component C2 and components C1 and C2 are separated by no more than 10 amino acids,
wherein C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor, and
wherein cleavage of the compound into a fragment 1 comprising X1 and a fragment 2 comprising X2 generates a detectable signal if the body fluid comprises a hydrolytic enzyme derived from pancreatic cancer cells.
12 . The in vitro method according to claim 11 for detecting the presence or absence of pancreatic cancer in a subject, wherein the presence of protease activity in the body fluid indicates the presence of pancreatic cancer and the absence of protease activity in the body fluid indicates the absence of pancreatic cancer.
13 . The in vitro method according to claim 11 wherein the subject is suspected of having pancreatic cancer, has been assessed to have an increased risk of developing pancreatic cancer, or has had pancreatic cancer.
14 . The in vitro method according to claim 11 , wherein the body fluid is urine.
15 . The method according to claim 11 , wherein the compound is provided at a concentration of 0.1-10 mg/mL, in a measurement buffer having neutral or alkaline pH.
16 . The in vitro method according to claim 15 , wherein the compound is provided at a concentration 0.125-10 mg/ml.
17 . The in vitro method according to claim 16 , wherein the compound is provided at a concentration 0.25-7.5 mg/ml.
18 . The in vitro method according to claim 17 , wherein the compound is provided at a concentration 0.5-5 mg/ml.
19 . The in vitro method according to claim 18 , wherein the compound is provided at a concentration 0.75-2 mg/ml.
20 . The in vitro method according to claim 19 , wherein the compound is provided at a concentration about 1 mg/ml.
21 . The in vitro method according to claim 11 , wherein the hydrolytic enzyme is a protease.
22 . The method according to 11 , wherein detecting the signal comprises measuring absorbance or fluorescence.
23 . The method according to claim 22 , wherein detecting the signal comprises measuring absorbance intensity at 300-500 nm.
24 . The method according to claim 23 , wherein detecting the signal comprises measuring absorbance at 380-430 nm.
25 . The method according to claim 24 , wherein detecting the signal comprises measuring absorbance or fluorescence for 40-60 min at 25-40° C.
26 . The method according to claim 25 , wherein detecting the signal comprises measuring absorbance or fluorescence at 36-38° C.
27 . A method of treating pancreatic cancer, the method comprising the steps of:
(a) carrying out the method according to claim 11 , and
(b) treating pancreatic cancer in a subject for which protease activity has been detected in step a.
28 . A method of treating pancreatic cancer, the method according to claim 27 , wherein in step a the protease activity that has been detected is increased protease activity.