Nonessential amino acid additives

HEK-293 cells in DMEM (Capricorn) were supplemented
with fetal bovine serum (10%, Gibco), l-glutamine (2 mM,
Lonza), and nonessential amino acid additives (Gibco). Cells were
maintained at 37 °C under a humidified atmosphere of 5% CO₂. Compound 23 was tested on the HEK-293 cell
line on cell viability using CellTox Green (G8741, Promega) and the
CellTiter-Glo 2.0 kit (G9241, Promega), respectively, to establish
their cytotoxic effect. Using a Tecan Spark 10 M instrument, the measurements
were performed as end point methods in the multiplex. For multiplex
measurement, 7500 cells were seeded per well in 50 μL of culture
media and cultured for 24 h before the addition of selected compounds.
Compound 23 was used at concentrations of 1 and 10 μM
(1% (v/v) total concentration of DMSO in the reaction). As a vehicle
control, cells were treated with 1% DMSO. As a positive control, 100
μM valinomycin treatment was used. White solid bottom 96 well
microplates were used for the measurement. First, the fluorescent
CellTox Green Cytotoxicity Assay (Ex/Em 485/530 nm) was performed.
Next, the CellTiter-Glo Luminescent Cell Viability Assay with an integration
time of 500 ms was determined.

A previously published cell
line with overexpressed 17β-HSD10
(HEK-293-HSD10)^{15 (link)} was used to measure 17β-HSD10
inhibition in the cellular environment. For this purpose, the (−)–CHANA
fluorogenic probe was used. The cells (density 1 × 10⁴ cells per well) were seeded in DMEM (200 μL) without phenol
red (Gibco). The cells were supplemented with fetal bovine serum (10%,
Gibco), l-glutamine (2 mM), nonessential amino acid additives
(Gibco), and 4.5 g/L glucose (Sigma) into black clear bottom 96 well
plates (Brand, 781971). The cells were incubated for 20 h following
the treatment with compound 23 in DMSO/DMSO only (vehicle
control). After 2 h of compound treatment, the (-)-CHANA probe was
added at the final concentration of 20 μM. The changes in fluorescent
intensities were measured immediately after (-)-CHANA addition and
2 h later. The fluorescence intensities of the CHANK product were
taken by using the TECAN SPARK 10 M instrument (Ex/Em = 380/525 nm).
The residual 17β-HSD10 activity was calculated as ΔF between
2 and 0 h after (-)-CHANA treatment, and the data were normalized
between nontreated HEK-293-HSD10 and native, nontransfected HEK-293
controls (using relative response ratio). Compound 23 was used at 1, 5, 10, 15, and 25 μM concentrations to detect
the ability to penetrate the cells and to influence the 17β-HSD10
enzyme activity inside the cells.