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

Cheeseman, Matthew D.; Chessum, N.; Rye, Carl S.; Pasqua, Adele E.; Tucker, Michael; Wilding, Birgit; Evans, Lindsey; Lepri, Susan; Richards, Meirion; Sharp, Swee Y.; Ali, Salyha; Rowlands, Martin; O’Fee, Lisa; Miah, Asadh; Hayes, Angela; Henley, Alan T.; Powers, Marissa; Poele, Robert te; Billy, Emmanuel de; Pellegrino, Loredana; Raynaud, Florence I.; Burke, Rosemary; Montfort, R.L.M. van; Eccles, Suzanne A.; Workman, Paul; Jones, Keith

doi:10.1021/acs.jmedchem.6b01055

Cited by 55 publications

(72 citation statements)

References 102 publications

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“…Therefore, we initially designed a synthetically tractable 15-atom linker that we predicted would not affect the affinity of the PDP for the isolated target proteins. 26 Analysis of the crystal structure of the chemical probe 1 bound to pirin ( Figure 1 , PDB 5JCT) 11 suggested that the solvent-exposed solubilizing group vector should be amenable to linker attachment. We selected a CRBN-targeting thalidomide ligand as the basis of our E3 ligase binding motif due to its low molecular weight.…”

Section: Results and Discussionmentioning

confidence: 99%

“…Trapping the carbonylation intermediate with 2-(trimethylsilyl)ethan-1-ol gave the (trimethylsilyl)ethyl ester 7 , 29 which facilitated TBAF-selective hydrolysis. Amide coupling to the previously described bis-aniline derivative 8 ( 11 ) was followed by hydrolysis of the aliphatic linker ester to give acid 9 . Final amide coupling to the CRBN-targeting derivative 4 gave the first-generation pirin-targeting PDP 3 in 10 steps and 0.4% overall yield.…”

Section: Results and Discussionmentioning

confidence: 99%

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Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

et al. 2018

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Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets.

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Section: Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

et al. 2018

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“…13,16,[30][31] However, to pharmacologically verify that pirin is involved in TGF- dependent gene expression, we used CCT251236, the previously published pirin inhibitor. 28 Human primary dermal fibroblasts were activated with TGF- for 24 hours with or without CCG-222740 or CCG-257081 as well as CCT251236. All three compounds significantly reduced TGF induced ACTA2 expression ( Figure 4A).…”

Section: Inhibition or Ablation Of Pirin Reduces Tgf- Induced Profibmentioning

confidence: 99%

“…The SRE.L Luciferase reporter contains several SRF binding sites that were modified to be dependent on MRTF, but lack responsiveness to ETS factors. 1 CCT251236 has nM potency in a cell-based assay to identify inhibitors of Heat Shock Factor 1 transcription and binds to pirin in vitro with a KD of 44 nM as measured by SPR 28. CCT251236 had a remarkable effect on SRE.L luciferase expression, with an IC50 of 3.3 nM, without affecting luciferase catalytic activity directly (Figure 3Aand SupplementalFigure 3).…”

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confidence: 99%

Identification of Pirin as a Molecular Target of the CCG-1423/CCG- 203971 Series of Anti-Fibrotic and Anti-Metastatic Compounds

Lisabeth¹,

Kahl²,

Gopallawa³

et al. 2018

Preprint

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A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF dependent luciferase screen has shown remarkable efficacy in a variety of in vitro and in vivo models, including melanoma metastasis and bleomycin-induced fibrosis. Although these compounds are efficacious, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent co-transcription factor, as a target of this series of compounds. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds in vitro. We also show with genetic approaches that pirin modulates MRTF-dependent SRE.L Luciferase activation. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF- induced gene expression in primary dermal fibroblasts. A recently developed analog, CCG-257081, which co-crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.

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“…The fully autonomous beamline MASSIF-1 at the ESRF not only automates the process of sample handling but also runs complex crystal location, characterisation and decision making routines for every sample processed Svensson et al, 2015). This level of automation allows a wide range of projects to use the beamline, from those that require extensive screening to find the best diffracting crystal Na et al, 2017;Naschberger et al;Sorigué et al, 2017;Xu et al, 2018) to small molecule fragment screening (Cheeseman et al, 2017;Hiruma et al, 2017) and experimental phasing at high and low resolutions (Kharde et al, 2015;Schulze et al, 2018). The routines optimise data collection by centring crystals using X-ray diffraction quality to determine the location of the best volumes to centre , measuring crystal volumes to dynamically adapt the beam dimeter to match the crystal and also to determine the dose the sample can receive before sustaining significant radiation damage Svensson et al, 2015;Zeldin et al, 2013;Bourenkov & Popov, 2010).…”

Section: Introductionmentioning

confidence: 99%

A comparative anatomy of protein crystals: lessons from the automatic processing of 56,000 samples

Svensson

Gilski

Nurizzo

et al. 2019

Preprint

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SynopsisThe automatic processing of over 56,000 crystals by the autonomous ESRF beamline MASSIF-1 has provided a data set of crystal characteristics and properties that allows many theoretical proposals and assumptions to be evaluated experimentally. AbstractThe fully automatic processing of crystals of macromolecules has presented a unique opportunity to gather information on the samples that is not usually recorded. This has proved invaluable in improving the sample location, characterisation and data collection algorithms. After operating for four years, MASSIF-1 has now processed over 56,000 samples, gathering information at each stage, from the volume of the crystal to the unit cell dimensions, space group, quality of the data collected and the reasoning behind the decisions made in data collection. This provides an unprecedented opportunity to analyse these data together, providing a detailed landscape of macromolecular crystals and intimate details of their contents and, importantly, how the two are related. The data show that mosaic spread is unrelated to the size or shape of crystals and demonstrate experimentally that diffraction intensities scale in proportion to crystal volume and molecular weight. It is also shown that crystal volume scales inversely with molecular weight.The results set the scene for the development of X-ray crystallography in a changing environment for structural biology.

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

Cited by 55 publications

References 102 publications

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

Identification of Pirin as a Molecular Target of the CCG-1423/CCG- 203971 Series of Anti-Fibrotic and Anti-Metastatic Compounds

A comparative anatomy of protein crystals: lessons from the automatic processing of 56,000 samples

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