Amino Alcohol Acrylonitriles as Activators of the Aryl Hydrocarbon Receptor Pathway: An Unexpected MTT Phenotypic Screening Outcome. (28th February 2020)

• Record Type: Journal Article
• Authors: Baker, Jennifer R.; Russell, Cecilia C.; Gilbert, Jayne; Sakoff, Jennette A.; McCluskey, Adam
• Is Part Of: ChemMedChem. Volume 15:Number 6(2020)
• Page Start: 490
• Page End: 505
• Publication Date: 2020-02-28
• Subjects: dichlorophenylacrylonitriles -- aryl hydrocarbon receptor 2 -- breast cancer -- MTT assay 4 -- SRB assay; Pharmaceutical chemistry -- Periodicals; 615.19005
• DOI: 10.1002/cmdc.201900643
• Languages: English
• ISSNs: 1860-7179

Abstract

Abstract: Lead ( Z )‐ N ‐(4‐(2‐cyano‐2‐(3, 4‐dichlorophenyl)vinyl)phenyl)acetamide, 1 showed MCF‐7 GI50 =30 nM and 400‐fold selective c.f. MCF10A (normal breast tissue). Acetamide moiety modification (13 a ‐g ) to introduce additional hydrophobicity was favoured with MCF‐7 breast cancer cell activity enhanced at 1.3 nM. Other analogues were potent against the HT29 colon cancer cell line at 23 nM. Textbook SAR data was observed in the MCF‐7 cell line, in an MTT assay, via the ortho (17 a ), meta (17 b ) and para (13 f ). The amino alcohol ‐OH moiety was pivotal, but no stereochemical preference noted. But, these data did not fit our homology modelling expectations. Aberrant MTT ((3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyl‐tetrazolium bromide) screening results and metabolic interference confirmed by sulforhodamine B (SRB) screening. Interfering analogues resulted in 120 and 80‐fold CYP1A1 and CYP1A2 amplification, with no upregulation of SULT1A1. This is consistent with activation of the AhR pathway. Piperidine per‐deuteration reduced metabolic inactivation. 3‐OH / 4‐OH piperidine analogues showed differential MTT and SRB activity supporting MTT assay metabolic inactivation. Data supports piperidine 3‐OH, but not the 4‐OH, as a CYP substrate. This family of β‐amino alcohol substituted 3, 4‐dichlorophenylacetonitriles show broad activity modulated via the AhR pathway. By SRB analysis the most potent analogue was 23 b, ( Z )‐3‐(4‐(3‐(4‐phenylpiperidin‐1‐yl)‐2‐hydroxypropoxy)phenyl)‐2‐(3, 4‐dichlorophenyl)‐acrylonitrile. Abstract : Microscopy examination reveals an unexpected interference in the MTT assay screening of a range of dichlorophenylacrylonitrile amino alcohols. SRB assays reveal significantly reduced cell viability. Critically, in this study the application of the SRB assay revealed that no true SAR data could be extracted, and this adds a significant note of caution as the MTT assay with this series of compounds has clearly shown a bias towards significant overestimation of compound potency in the MCF‐7 cell line.