Bioprobes for lysyl oxidases and uses thereof
AssigneePharmaxis Ltd.
InventorsAlison Dorothy Findlay, Craig Ivan Turner, Mandar Deodhar, Jonathan Stuart Foot, Wolfgang Jarolimek, Heidi Schilter Sambade, Wenbin Zhou, Lara Anne Perryman
Life of this patent
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Abstract
The present invention relates to novel bioprobes which are capable of binding to certain amine oxidase enzymes. These bioprobes are useful in methods of detecting and determining the concentration of certain amine oxidase enzymes in a sample as well as in methods for the quantitative assessment of inhibition of certain amine oxidases.
Claims35
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A method for determining a measure of the concentration of a protein selected from a lysyl oxidase (LOX) protein and a lysyl oxidase-like (LOXL) protein in a sample containing or suspected of containing the protein, comprising:
- (a) obtaining a sample containing or suspected of containing the protein from a subject;
- (b) contacting the sample with:
- (i) a capture antibody specific for the protein under conditions sufficient to form a protein/capture antibody complex;
- and
- (ii) a bioprobe of Formula W-L-Z, wherein W represents an affinity tag;
- L represents a linker;
- and Z represents a moiety that binds to the protein;
- wherein Z binds to an enzymatic pocket of the protein wherein Z is a moiety of Formula Ib:
- wherein:
- R1a is selected from the group consisting of hydrogen, C1-6alkyl, —O—C1-6alkyl and —C(O)NR2R3 or represents an attachment point for L;
- and
- R2 and R3 are independently selected from the group consisting of hydrogen and C1-6alkyl;
- and
- (c) performing an immunoassay to measure the total concentration of the protein in the sample, wherein the immunoassay is a digital ELISA.
A method for determining the extent of enzyme inhibition of a protein selected from a lysyl oxidase (LOX) protein and a lysyl oxidase-like (LOXL) protein by a LOX or LOXL inhibitor, the method comprising:
- (a) obtaining a sample containing or suspected of containing the protein from a subject;
- (b) contacting the sample with a first amount of the LOX or LOXL inhibitor;
- (c) contacting the sample with:
- (i) a capture antibody specific for the protein under conditions sufficient to form a protein/capture antibody complex;
- and
- (ii) a bioprobe of Formula W-L-Z to the sample, wherein W represents an affinity tag;
- L represents a linker;
- and Z represents a moiety that binds to the protein;
- wherein Z binds to the same enzymatic pocket of the protein as the LOX or LOXL inhibitor;
- wherein Z is a moiety of Formula Ib:
- wherein:
- R1a is selected from the group consisting of hydrogen, C1-6alkyl, —O—C1-6alkyl and —C(O)NR2R3 or represents an attachment point for L;
- and
- R2 and R3 are independently selected from the group consisting of hydrogen and C1-6alkyl;
- and
- (d) performing an immunoassay to measure the concentration of the protein with the enzymatic pocket occupied by the bioprobe, wherein the immunoassay is a digital ELISA.
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A kit for determining a measure of the concentration of a protein selected from a lysyl oxidase (LOX) protein and a lysyl oxidase-like (LOXL) protein in a sample, the kit comprising
- a capture antibody specific for the protein, and
- a bioprobe of Formula W-L-Z, wherein W represents an affinity tag;
- L represents a linker;
- and Z represents a moiety that binds to the protein;
- wherein Z binds to an enzymatic pocket of the protein;
- wherein Z is a moiety of Formula Ib:
- wherein:
- R1a is selected from the group consisting of hydrogen, C1-6alkyl, —O—C1-6alkyl and —C(O)NR2R3 or represents an attachment point for L;
- and
- R2 and R are independently selected from the group consisting of hydrogen and C1-6alkyl.
A kit for determining the extent of enzyme inhibition of a protein selected from a lysyl oxidase (LOX) protein and a lysyl oxidase-like (LOXL) protein by a LOX or LOXL inhibitor, the kit comprising:
- a capture antibody specific for the protein,
- a bioprobe of Formula W-L-Z, wherein W represents an affinity tag;
- L represents a linker;
- and Z represents a moiety that binds to the protein;
- wherein Z binds to an enzymatic pocket of the protein;
- wherein Z is a moiety of Formula Ib:
- wherein:
- R1a is selected from the group consisting of hydrogen, C1-6alkyl, —O—C1-6alkyl and —C(O)NR2R3 or represents an attachment point for L;
- and
- R2 and R3 are independently selected from the group consisting of hydrogen and C1-6alkyl, and
- a detection antibody specific for the protein.
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A bioprobe of formula W-L-Z, wherein W represents an affinity tag;
- L represents a linker;
- and Z represents a moiety that binds to a protein selected from the group consisting of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 protein, wherein Z is a moiety of Formula Ib:
- wherein:
- R1a is selected from the group consisting of hydrogen, C1-6alkyl, —O—C1-6alkyl and —C(O)NR2R3 or represents an attachment point for L;
- and
- R2 and R are independently selected from the group consisting of hydrogen and C1-6alkyl.
Dependencies are inferred from the claim text.