Reaction #420759

ord-f8457f81ae4e465baaf3137ffd382180

Reaction equation

NC(CO)(CO)CO
Tris
O=c1ccc2[n+]([O-])c3ccc(O)cc3oc-2c1
resazurin
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
[Cl-].[Na+]
NaCl
OCCS
beta-mercaptoethanol
[Cl-].[Cl-].[Mg+2]
MgCl2
→
O=c1ccc2[n+]([O-])c3ccc(O)cc3oc-2c1
resazurin
O=c1ccc2nc3ccc(O)cc3oc-2c1
resorufin

Reactants

NC(CO)(CO)CO
Tris
O=c1ccc2[n+]([O-])c3ccc(O)cc3oc-2c1
resazurin
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
[Cl-].[Na+]
NaCl
OCCS
beta-mercaptoethanol
[Cl-].[Cl-].[Mg+2]
MgCl2

Reagents

None

Solvents

CS(C)=O
DMSO

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    workup.ADDITIONwas added
  2. 2
    workup.ADDITION25 ul of enzyme mix
  3. 3
    workup.ADDITIONwas added (0.1 ug/mL ICDH1 G97D)
  4. 4
    workup.ADDITIONwas added
  5. 5
    workup.WAITthe mixture further incubated for 5 minutes at room temperature

Procedure

Assays were conducted in a standard 384-well plate in a reaction volume of 76 uL assay buffer (150 mM NaCl, 10 mM MgCl2, 20 mM Tris pH 7.5, 0.05% bovine serum albumin, 2 mM beta-mercaptoethanol) as follows: To 25 uL of substrate mix (4 uM NADPH, 1 mM aKG), 1 uL of test compound in DMSO was added. The plate was centrifuged briefly, and then 25 ul of enzyme mix was added (0.1 ug/mL ICDH1 G97D) followed by a brief centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at room temperature, then 25 ul of detection mix (30 uM resazurin, 36 ug/ml diaphorase) was added, and the mixture further incubated for 5 minutes at room temperature. The activity readout as a result of the conversion of resazurin to resorufin was detected by fluorescent spectroscopy at excitation 544 nm and emission 590 nm (c/o 590 nm).

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US08883438B2uspto-grants-2014_11