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nhrDUO℠ - Mode of Action Analysis

The LeadHunterSM Discovery Services team is proud to offer you complete compound characterization by providing multiple functional readouts for the same NHR receptor. Nuclear translocation assays monitor the movement of a cytoplasmic NHR to the nuclear compartment. Protein interaction assays detect NHR interaction with a co-activator fusion protein. This direct, non-imaging approach results in fewer off-target effects and the ability to select novel chemotypes based on the disruption of novel protein: protein interactions.

nhrDUO            
Ideal tool for studying compound action by selecting a single target in both protein interaction and nuclear translocation readouts.

Multi-mode Screening and Profiling

With nhrDUO, you can screen or profile compounds using the nuclear translocation and protein interaction readouts in parallel. Quantitatively examining these pathways in parallel can uncover potency and efficacy differences between compounds and aid in defining compound function to better predict compound activity in vivo.

GR NHRTRANS Assay

GR NHRPRO Assay
CHO GR Nuclear Translocation Assay CHO GR NHR PRO Assay
The PathHunter GR Nuclear translocation (NHRTRANS) assay shows no inhibition after addition of Mifepristone (Left)  while the GR protein interaction (NHRPRO) assay shows almost complete inhibition of the Dexamethasone response (Right), indicating that Mifepristone acts on the target of interest by blocking co-activator recruitment.

nhrDUO Assay Panel

Listed below are the assays currently available for screening and profiling.

 
Target Gene ▲Common NameNuclear TranslocationProtein Interaction
ARAndrogen receptor (AR)XX
GRNuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) (NR3C1)XX
LXRαNuclear receptor subfamily 1, group H, member 3 (Liver X receptor α) (NR1H3)XX
MRNuclear receptor subfamily 3, group C, member 2 (Mineralcorticoid receptor) (NR3C2)XX
PRβProgesterone receptor β (PGR)XX

Data Analysis & Interpretation 

Definitions

Percent Activity/Inhibition

The results for single concentration (primary screen) for tested compound(s) are reported in your study report and spreadsheets as '% Activity' or '% Inhibition' and are calculated in the following manner:
 

% Activity
(Agonist Mode) =
100% x
 [
Mean RLU (test sample)- Mean RLU(vehicle control)
Mean MAX RLU (control ligand)- Mean RLU (vehicle control)
 ]
 

test sample = client supplied compound
vehicle control = DMSO (0% activity)
control ligand = control compound (100% activity)
 

% Inhibition
(Antagonist mode) =
100% x
 [ 1- 
Mean RLU (test sample)- Mean RLU(vehicle control)
Mean MAX RLU (control ligand)- Mean RLU (vehicle control)
 ]
 

test sample = client supplied compound
vehicle control = DMSO (0% activity)
EC80 control = control compound (80% activity) 
 

Dose Response Curve (EC50/IC50)

The results for 10-point dose response curve compound/target interactions are reported in the study report and spreadsheets as EC50/IC50, which are values derived using the Hill equation:
hill equation