Reaction #659877

ord-7f6e04c2710d448b81751a7866d5507f

Reaction equation

Cl
HCl
Cl.NC(CO)(CO)CO
Tris-HCl
[Cl-].[Cl-].[Mg+2]
MgCl2
O=C(O)CN(CCN(CC(=O)O)CC(=O)O)CC(=O)O
Ethylenediaminetetraacetic acid
O[C@H](CS)[C@H](O)CS
dithiothreitol
CC(C)(COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc32)[C@H](O)[C@@H]1OP(=O)(O)O)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C[C@@](C)(O)CC(=O)O
HMG-CoA
CC(=O)CC(=O)SCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc32)[C@H](O)[C@@H]1OP(=O)(O)O
acetoacetyl-CoA
Cl
HCl
→
CC(C)(COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc32)[C@H](O)[C@@H]1OP(=O)(O)O)[C@@H](O)C(=O)NCCC(=O)NCCS
CoA

Reactants

Cl
HCl
Cl.NC(CO)(CO)CO
Tris-HCl
[Cl-].[Cl-].[Mg+2]
MgCl2
O=C(O)CN(CCN(CC(=O)O)CC(=O)O)CC(=O)O
Ethylenediaminetetraacetic acid
O[C@H](CS)[C@H](O)CS
dithiothreitol
CC(C)(COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc32)[C@H](O)[C@@H]1OP(=O)(O)O)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C[C@@](C)(O)CC(=O)O
HMG-CoA
—
solution
—
1[14C]-acetyl-CoA
CC(=O)CC(=O)SCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc32)[C@H](O)[C@@H]1OP(=O)(O)O
acetoacetyl-CoA
—
assay mixture
Cl
HCl

Reagents

None

Solvents

CS(C)=O
dimethylsulfoxide

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    workup.ADDITIONEnzyme protein (ca. 12.2 μg) is added to a solution
  2. 2
    workup.ADDITIONin the presence or absence of the test compound (added as, e.g.
  3. 3
    OtherThe incubation is in a volume of 0.085 ml at 30° C. in a shaking water bath
  4. 4
    workup.ADDITIONis added
  5. 5
    Otherto give final concentrations of 0.1 and 0.4 mM
  6. 6
    workup.ADDITIONis again added to each vial and the heating
  7. 7
    workup.WAITcontinued for another hour
  8. 8
    workup.ADDITIONis added to each vial and finally 10 ml of scintillation liquid

Procedure

Intrinsic activity of HMG-CoA synthase is measured in the following in vitro assay. Enzyme protein (ca. 12.2 μg) is added to a solution containing 117 mM Tris-HCl (pH 8.0), 11.7 mM MgCl2, 1.17 mM Ethylenediaminetetraacetic acid (EDTA), 0.58 mM dithiothreitol, in the presence or absence of the test compound (added as, e.g., a 2 μg/ml solution in dimethylsulfoxide). The incubation is in a volume of 0.085 ml at 30° C. in a shaking water bath. After 5 minutes, 15 μl of a solution containing acetoacetyl-CoA and 0.1 μCi of 1[14C]-acetyl-CoA is added to give final concentrations of 0.1 and 0.4 mM, respectively. The incubation is continued for 2 more minutes and the reaction stopped by the addition of 50 μl of the assay mixture to 0.2 ml of 6N HCl in a glass scintillation vial. The vial is heated for 1 hour at 110° C. after which time 0.2 ml more of 6N HCl is again added to each vial and the heating continued for another hour. Following this, 1.0 ml of 0.9% saline is added to each vial and finally 10 ml of scintillation liquid. Radioactivity is determined in a Packard Tri-Carb liquid scintillation counter. Percent inhibition is calculated a standard formula. IC50 values are determined by plotting the log of the concentration of the test compound verses the percentage inhibition and fitting a straight line to the resulting data by using the least squares method.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US09029413B2uspto-grants-2015_05