Novel activity-based probes for neutrophil elastase and their use
AssigneeTakeda Pharmaceutical Company Limited
InventorsLaura EDGINGTON-MITCHELL, Bethany M. ANDERSON, Daniel P. POOLE, Luigi AURELIO, Paulina KASPERKIEWICZ, Marcin DRAG, Nigel BUNNETT
Life of this patent
Estimating the term…
Abstract
The present invention relates to compounds of formula I, wherein D is a detectable moiety, or salts thereof, which can be used as activity-based probes for neutrophil elastase, as well as to methods for detecting neutrophil elastase (NE) activity in a tissue sample lysate, and related diagnostic methods using compounds of formula I.
Claims40
A method of detecting neutrophil elastase (NE) activity in a tissue sample lysate, comprising
- (1) preparing the lysate from a tissue sample obtained from a subject,
- (2) contacting the lysate with a compound of formula I
- or a salt thereof,
- wherein D is a detectable element,
- (3) subsequently subjecting at least an aliquot of the lysate of step (2) to gel electrophoresis;
- and thereafter
- (4) measuring a detectable signal.
The method of any one of the preceding claims, wherein the subject is a human subject.
The method of any one of the preceding claims, wherein an activated form of NE that is a trimmed form of mature NE is detected, optionally wherein the tissue sample is selected from the group consisting of an oral biopsy, an esophagus sample, a stomach sample, a small intestine sample, a colon sample, a proximal colon sample, a distal colon sample, a rectal sample, a fecal sample, and a mucosal biopsy, optionally wherein the mucosal biopsy is selected from the group consisting of an oral mucosal biopsy, an esophagus mucosal biopsy, a small intestine mucosal biopsy, a colon mucosal biopsy, and a rectal mucosal biopsy.
A method of diagnosing a disease associated with NE activity in a subject comprising
- (1) preparing a lysate from a tissue sample obtained from the subject,
- (2) contacting the lysate with a compound of formula I
- or a salt thereof,
- wherein D is a detectable element,
- (3) subsequently subjecting the lysate to gel electrophoresis;
- and thereafter
- (4) measuring a detectable signal.
The method of any one of the preceding claims, wherein preparing the lysate comprises a clearing step.
The method of any one of the preceding claims, wherein the gel electrophoresis is a one-dimensional or a two-dimensional gel electrophoresis, and/or wherein the gel electrophoresis is an SDS-PAGE or a native PAGE, preferably an SDS-PAGE.
The method of any one of the preceding claims, wherein the detectable element is selected from the group consisting of a fluorescent label, a biotin label, a radiolabel, a chelator, and a bioorthogonal ligation handle;
- and/or
- wherein the detectable signal is measured by fluorescence measurement or radiography,
- optionally wherein the fluorescence measurement is in-gel fluorescence;
- or
- optionally wherein the fluorescence measurement is preceded by secondary labeling, optionally wherein the secondary labeling is selected from the group consisting of secondary labeling with tagged streptavidin, secondary labeling with a fluorophore, and secondary labeling with a tagged antibody.
The method of any one of the preceding claims, wherein in step (2), the lysate is contacted with a compound having the formula IA:
- or a salt thereof,
- wherein D is a detectable element.
An in vitro method of diagnosing an inflammatory bowel disease in a subject, comprising detecting an activated form of NE that is a trimmed form of mature NE, optionally wherein the subject is a human subject.
A compound of formula I
- or a salt thereof,
- wherein D is a detectable element,
- with the proviso that compounds wherein D corresponds to one of the following formulas are excluded:
- wherein in each of the above formulas, the curled line represents the point of connection to the remainder of the molecule.
A compound of formula II
- or a salt thereof.
A compound of formula IIA
- or a salt thereof.
Dependencies are inferred from the claim text.