Journal: mBio

Article Title: The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase

doi: 10.1128/mbio.00633-24

Figure Lengend Snippet: A malachite-green-based nucleotidase-coupled assay measures the activity of purified Cho1 protein. ( A ) Blue native PAGE gel of the purified hexameric tag-free Cho1 protein. Purified Cho1 and protein ladder with known MW are indicated. The gel was stained with Coomassie Blue R-250. ( B ) Schematic representation of the malachite-green-based nucleotidase-couple assay. Cho1 synthesizes PS from CDP-DAG (cytidyldiphosphate-diacylglycerol) and serine. This releases PS and CMP (cytidylmonophosphate). The phosphate from CMP is cleaved by the nucleotidase CD73 to release inorganic phosphate, which can be bound by the malachite green reagent and measured colorimetrically at OD 620 . AB-680 is a potent inhibitor of CD73 and can, thus, inhibit the reaction. ( C ) OD 620 signal from the malachite green reagent that was added to the reaction (shown in B ) at different time points after the reaction started. Reactions were set up with the same conditions and stopped by adding malachite green at the time indicated. The dots represent the mean of four biological replicates, and the error bars are ±standard deviation (S.D.) values. ( D ) Inhibition of the nucleotidase-coupled assay by AB-680 is shown for a series of replicates in 384-well format and ( E ) is quantified for a total of 21 replicates. Statistics were conducted using one-way ANOVA using Tukey’s multiple comparisons test (ns, not significant; **** P < 0.0001).

Article Snippet: A total of 30–35 ng of purified Cho1 protein was used in each reaction in 50 mM Tris-HCl (pH 8.0) for primary screening, combined with 100 μM CDP-DAG (Avanti, cat# 870510), 5 mM serine, 0.4 ng CD73 nucleotidase (R&D systems, cat# EA002), 1 mM MnCl 2 , 0.1% APX-100, and 0.1% digitonin, in a total volume of 10 μL.

Techniques: Activity Assay, Purification, Blue Native PAGE, Staining, Standard Deviation, Inhibition