Black clear bottom 384 well plates

To identify GSK1820795A, 2256 compounds were dispensed into black clear‐bottom 384‐well plates (Greiner) as solutions in 100% DMSO (40 nL/well; final concentration 1 µmol/L), using an Echo accoustic dispenser (Labcyte). GeneBLAzer™ T‐REx™‐G2A(GPR132)‐NFAT‐bla FreeStyle™ 293F cells were thawed, resuspended in 90% DMEM; 10% dialyzed FBS; 0.1 mmol/L NEAA; 25 mmol/L HEPES (pH 7.3); 100 U/mL Penicilin; 100 µg/mL Streptomycin, and added to wells (10 000 cells/well in 40 µL). Media was supplemented with 1 ng/mL doxycycline to induce expression of GPR132, resulting in high GPR132 constitutive activity. After incubtion (18 hours at 37°C; 5% CO₂), LiveBLAzer™‐FRET β‐galactosidase substrate (CCF‐AM) prepared according to manufacturer's instructions (ThermoFisher Scientific, Loughborough, UK) was added (8 µL/well; room temperature for 2 hours). Inverse agonists including GSK1820795A were identified from the 460/530 nm emission ratio (Envision, Perkin Elmer) by reference to doxycycline‐treated and untreated controls. Half‐maximal inhibition (pIC₅₀) values were determined in similar experiments, except that cells were supplemented with SKF‐95667 (1 µmol/L).

Black clear bottom 384-well
plates, white TC 384-well plates and μClear Chimney 96-well
black clear-bottom plates TC sterile were from Greiner Bio-One. Microtiter
MicroAmp Optical Adhesive films were bought from Applied Biosystems.
All other materials were purchased from Sigma-Aldrich.
Antisera
against BamA and PhoE (cross-reactive to OmpC and OmpF) were kind
gifts from J. Tommassen (Utrecht University, The Netherlands), against
BamB from D. Kahne (Harvard University, USA), and against DegP from
J. Beckwith (Harvard Medical School, USA. Antisera against LepB and
LpoB were from our own collections. HRP-conjugated affinity purified
antirabbit IgG from Rockland was used as secondary antibody.
Synthesis of compound 14 (MW: 346.19 g/mol, 2-(2-aminopyridin-4-yl)-N4-benzyl-5,6,7,8-tetrahydroquinazoline-4,6-diamine) is
described in the Supporting Information. Compound 2 (MW: 348.96 g/mol, N-(2-(1-(4-chlorobenzyl)cyclohexyl)ethyl)-1-methylpiperidin-4-amine)
was obtained from Vitas M-lab (catalog number: STK661348).