CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Chen, Dan; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, P.

doi:10.1038/ncomms11040

Cited by 133 publications

(153 citation statements)

References 36 publications

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“…However, compound-induced quenching of protein target recognition was observed by both our ELISA assay using several combinations of RIPK1 antibodies and a commercially available ELISA kit. This quenching effect has already been reported 4,24 and the effects on RIPK1 protein might be dependent on RIPK1 associated with a variety of proteins 14,15,25 . Therefore, we focused on establishing a widely used Western blotting method in a semi-automated procedure.…”

Section: Resultssupporting

confidence: 74%

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CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens

Ishii

Okai

Iwatani-Yoshihara

et al. 2017

Sci Rep

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The proof of target engagement (TE) is a key element for evaluating potential investment in drug development. The cellular thermal shift assay (CETSA) is expected to facilitate direct measurement of intracellular TE at all stages of drug development. However, there have been no reports of applying this technology to comprehensive animal and clinical studies. This report demonstrates that CETSA can not only quantitatively evaluate the drug-TE in mouse peripheral blood, but also confirm TE in animal tissues exemplified by using the receptor interacting protein 1 kinase (RIPK1) lead compound we have developed. Our established semi-automated system allows evaluation of the structure-activity relationship using native RIPK1 in culture cell lines, and also enables estimation of drug occupancy ratio in mouse peripheral blood mononuclear cells. Moreover, optimized tissue homogenisation enables monitoring of the in vivo drug-TE in spleen and brain. Our results indicate that CETSA methodology will provide an efficient tool for preclinical and clinical drug development.

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Section: Resultssupporting

confidence: 74%

“…All of the data are shown as mean ± SEM. For the T agg shift and the ITDR experiments, data were analysed in GraphPad Prism using the Boltzmann sigmoid equation and the four-parameter logistic curve 4,24 . Student’s t -test was used to calculate P values with Microsoft Excel.…”

Section: Methodsmentioning

confidence: 99%

CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens

Ishii

Okai

Iwatani-Yoshihara

et al. 2017

Sci Rep

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“…Second, for compounds that need to be activated in the cell (prodrugs), the F ic of the dosed parent molecule might not be relevant. However, metabolically activated species can easily be monitored as well, such as we previously did for compounds targeting thymidylate synthase (31). Third, the method is designed to give information on exposure in the whole cell (i.e., an average of cytosolic and organelle exposure), which limits the resolution for targets located in specific subcellular compartments.…”

Section: Discussionmentioning

confidence: 99%

“…In a preliminary study with a single cytosolic target (thymidylate synthase), we showed that intracellular concentrations of active hits and reference drugs correlate with intracellular target engagement (31). We, therefore, hypothesized that F ic might be a practical metric for predicting compound access to intracellular targets.…”

mentioning

confidence: 99%

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery

Mateus

Gordon

Wayne

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (F ic ) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined F ic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. F ic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery. intracellular drug bioavailability | drug exposure | target engagement | published kinase inhibitor set | MAPK14

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“…TS catalyzes the reductive methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) . As the TS pathway provides the sole de novo synthesis of dTMP, disrupting the pathway can significantly affect the cell viability . Accordingly, TS has been regarded as an important target against infectious diseases.…”

Section: Introductionmentioning

confidence: 99%

Proteochemometric modeling of the origin of thymidylate synthase inhibition

Rasti

Shahangian

2018

Chem Biol Drug Des

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Due to its crucial role in DNA synthesis, thymidylate synthase (TS) has been considered as a potential therapeutic target. Inhibition of the enzyme is a promising strategy for the treatment of some hyperproliferative diseases, including infections. As TS species-specific inhibitors would be able to distinguish between the host and the pathogens, developing highly selective inhibitors is of great clinical importance. TS is among the most highly conserved enzymes over evolutionary history, making the design of its species-selective inhibitor significantly challenging. The chemical interaction space, governed by a set of non-selective TS inhibitors, has been explored for human TS and its homologous proteins in both Toxoplasma gondii and Escherichia coli using proteochemometrics modeling (PCM). Validity, robustness, and prediction power of the PCM model have been assessed applying a diverse set of internal/external validation approaches. Our PCM model has provided major structural information, which is indeed of great help to design new TS species-specific inhibitors with the simultaneous inhibition ability toward both T. gondii and E. coli. To show applicability of the PCM model, new compounds have been designed based on structural information provided by the constructed model. Final results have been very promising with regard to selectivity ratios of the designed compounds for different TS isoforms, confirming the applicability of the PCM model.

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CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

Cited by 133 publications

References 36 publications

CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens

CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery

Proteochemometric modeling of the origin of thymidylate synthase inhibition

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