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, 60 (1), 180-201

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand From a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen


Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand From a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen

Matthew D Cheeseman et al. J Med Chem.


Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.

Conflict of interest statement

The Institute of Cancer Research has a potential financial interest in inhibitors of the HSF1 pathway and operates a Rewards to Discoverers.


Figure 1
Figure 1
Inhibitors of HSF1-mediated HSP72 induction.
Figure 2
Figure 2
HSF1 pathway inhibitor, bisamide 1.
Scheme 1
Scheme 1. Synthesis of the Benzodioxane Bisamide Replacements
Reagents and conditions: (i) oxalyl chloride, DMF, DCM, then, pyridine, DCM; (ii) Fe, NH4Cl, EtOH/H2O; (iii) RPhCO2H, HATU, DIPEA, DMF.
Scheme 2
Scheme 2. Synthesis of Solubilizing Group Analogues
Reagents and conditions: (i) oxalyl chloride, DMF, 1,4-benzodioxane-6-carboxylic acid, DCM, then, 4-methyl-3-nitroaniline, pyridine, DCM, RT, then Pd/C (10%), H2 (1 atm), EtOH; (ii) NaH, RCH2OH, THF, 0 °C to reflux; (iii) n-BuLi, CO2(s), THF, −78 °C; (iv) HATU, DIPEA, DMF, RT.
Figure 3
Figure 3
Efficacy of bisamide 26 against SK-OV-3 human ovarian carcinoma xenograft model. Blue, vehicle control, n = 8; red, 26, 20 mg/kg po qd, n = 8 (vehicle = 10% DMSO, 90% of a 25% (2-hydroxypropyl)-β-cyclodextrin in 50 mM citrate buffer pH 5). Error bars: arithmetic mean ± SEM. Dosing breaks were carried out on days 5–12, 14, 16, 18, 20, 22, 24, 26, 29, 31.
Figure 4
Figure 4
Tool compounds for target identification. All quoted cellular activities are in the SK-OV-3 cell line, the numbers of repeats are in parentheses.
Figure 5
Figure 5
Representative SPR sensorgram and binding isotherm of bisamide 26 bound to recombinant pirin. The dotted-line represents the time-point the equilibrium response was measured. The binding isotherm was fitted to a one-site specific binding model using Graphpad Prism 6.07.
Figure 6
Figure 6
X-ray crystal structure of bisamide 26. PDB 5JCT, Pymol image of pirin (light-blue cartoon representation) in complex with bisamide 26 (cyan stick representation), 2FoFc map contoured at 1.0 σ (blue mesh) and Pymol image of pirin (magenta, blue stick representation) in complex with bisamide 26 (cyan stick representation), distances shown are in Å, 2FoFc contoured at 1.0 σ (blue mesh). Crystals belonged to the space group P212121 and diffracted to a resolution of 1.73 Å.
Figure 7
Figure 7
Antimigratory activity of chemical probe bisamide 26. (A) Wound healing images of WM266.4 cells; after 30 h, the wound has almost completely healed in the control in contrast to 100 nM 26. (B) Quantification of the relative wound density reveals the maximum antimigratory activity is achieved at 100 nM 26. Each point represents the arithmetic mean ±SEM of the study carried out in triplicate.

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