Collecting data through high throughput in vitro early toxicity and off-target liability assays to rapidly identify limitations of novel thyromimetics

Abstract

In order to rapidly identify the phenotypic profile and possible off-target liability effects of novel synthesized thyromimetics for selection of lead compounds for further optimization studies, we performed in vitro screening on a new small library of synthetic thyromimetics. A comprehensive panel of early toxicity assays comprising cytotoxicity on 4 different cell lines (osteosarcoma, U2OS; lung fibroblast, hTERT; human breast adenocarcinoma, MCF7; human embryonic kidney, HEK293), hERG liability, cytochrome P450 inhibition (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 isoforms), and off-target liability against selected proteins (Aurora B kinase and phosphodiesterase PDE4C1) and epigenetic enzymes (HDAC4, HDAC6, HDAC8, HDAC9 & SIRT7). All the compounds were screened at 10 μM in at least triplicate using well-established in vitro assays with readouts in luminescence or fluorescence polarization mode. The raw data were processed using Microsoft Excel and the Z' for each assay was calculated (acceptable Z' >0.40). The processed and normalized data were organized in tables and visualized using spider plots. The results which are reported in the present manuscript can be used in prediction studies of early toxicity and off-target liabilities of other thyromimetics using in silico methods. The data reported herein support our research article entitled "Design, synthesis and biological evaluation of novel TRβ selective agonists sustained by ADME-Toxicity analysis" by Runfola M., Sestito S., et al. [1].

Keywords: ADME-Tox profile; Fatty-liver disorder; Liver regeneration; Off-target liability; Screening; TRβ selective agonist; Thyronamine; Triiodothyronine.

Fig. 1

Cytotoxicity of new thyromimetics. The % Cytotoxicity determinations were made in four different cell-lines (HEK293, hTERT, MCF7, and U2OS) at 24 h (red) and 48 h (yellow). See Table 1 for further details.

Fig. 2

CYP450 liability of new thyromimetics. % Inhibition of five CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4). See Table 1 for further details.

Fig. 3

Epigenetic off-target enzyme liability of new thyromimetics. % Inhibition of epigenetic off-targets (HDAC4, HDAC6, HDAC8, HDAC9 and SIRT7). See Table 1 for further details.

Fig. 4

Aurora B kinase and PDE4C1 off-target enzyme liability of new thyromimetics. % Inhibition of Aurora B kinase and PDE4C1. See Table 1 for further details.

Fig. 5

hERG liability of new thyromimetics. % Inhibition of hERG. See Table 1 for further details.