Compounds and methods of use thereof for assaying lysophospholipase D activity
AssigneeEchelon Biosciences Incorporated
InventorsColin Ferguson, Glenn Prestwich
Life of this patent
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Abstract
Fluorogenic lysophosphatidic acid derivatives which can be used as substrates in a continuous, fluorogenic assay that can be performed in microtiter plates. The assays permit measuring LysoPLD activity levels in normal events such as pregnancy or disease states such as cancer. In addition, the present invention can be adopted to high throughout screening(HTS) for identification of potential inhibitors of lysoPLD activity.
Claims16
A method for assaying the LysoPLD activity of ATX comprising the steps of:
- exposing a LysoPLD substrate comprising a lysophospholipid derivative labeled with a fluorescent group and a quencher group to a solution to be tested and determining whether LysoPLD activity is present in said solution by measuring the fluorescent signal, wherein said lysophospholipid derivative is represented by the formula:
- 2
- wherein A represents O or S, B represents H, Na+, K+, Li+, or an alkyl group with 1-20 carbons, E represents O, CH2, CHF or CF2, G represents H, OH, F, Oacyl or Oalkyl with 1-20 carbons, K, and K2 represent H, F, an alkyl with 1-20 carbons, OH, or CO2H, W represents C(O), NH(CH2)m, [NHC(O)(CH2)n,]pC(O) or NH(CH2CH2O)qC(O) wherein m is an integer from 1 to 10, n is an integer from 1 to 12, p is an integer from 1 to 20, q is an integer from 1 to 33, X represents NH, N(CH3)2 or CH2;
- Y represents O, S or NH;
- Z represents C(O), (CH2)mNH, [C(O)(CH2)n]pC(O)NH or (CH2CH2O)qC(O)NH;
- R1 and R2 is a fluorescent group or a quencher group, respectively.
A kit for assaying the LysoPLD activity of ATX comprising a lysophospholipid derivative labeled with a fluorescent group and a quencher group wherein said lysophosphatidic acid derivative is represented by the formula:
- 4
- wherein A represents O or S, B represents H, Na+, K+, Li+, or an alkyl group with 1-20 carbons), E represents O, CH2, CHF or CF2, G represents H, OH, F, Oacyl or Oalkyl with 1-20 carbons, K1 and K2 represent H, F, an alkyl with 1-20 carbons, OH or CO2H, W represents C(O), NH(CH2)m, [NHC(O)(CH2)n]pC(O) or NH(CH2CH2O)qC(O) wherein m is an integer from 1 to 10, n is an integer from 1 to 12, p is an integer from 1 to 20, q is an integer from 1 to 33, X represents NH, N(CH3)2 or CH2;
- Y represents O, S or NH;
- Z represents C(O), (CH2)mNH, [C(O)(CH2)n]pC(O)NH or (CH2CH2O)qC(O)NH;
- R1 and R2 is a fluorescent group or a quencher group, respectively.
A LysoPLD substrate comprising a lysophosphatidic acid derivative labeled with a fluorescent group and a quencher group which is represented by the formula:
- 6
- wherein A represents O or S, B represents H, Na+, K+, Li+, or an alkyl group with 1-20 carbons, E represents O, CH2, CHF or CF2, G represents H, OH, F, Oacyl or Oalkyl with 1-20 carbons, K1 and K2 represent H, F, an alkyl with 1-20 carbons, OH, or CO2H, W represents C(O), NH(CH2)m[NHC(O)(CH2)n]pC(O) or NH(CH2CH2O)qC(O) wherein m is an integer from 1 to 10, n is an integer from 1 to 12, p is an integer from 1 to 20, q is an integer from 1 to 33, X represents NH, N(CH3)2 or CH2;
- Y represents O, S or NH;
- Z represents C(O), (CH2)mNH, [C(O)(CH2)n]pC(O)NH or (CH2CH2O)qC(O)NH;
- R1 and R2 is a fluorescent group or a quencher group, respectively.
A lysophospholipid derivative selected from the group consisting of:
- 7
Dependencies are inferred from the claim text.