Chemical Biology Approaches for Characterization of Epigenetic Regulators

Baršytė-Lovejoy, Dalia; Szewczyk, Magda; Prinos, Panagiotis; Lima‐Fernandes, Evelyne; Ackloo, Suzanne; Arrowsmith, C.H.

doi:10.1016/bs.mie.2016.01.011

Cited by 5 publications

(5 citation statements)

References 50 publications

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“…Treatment of SCs with Noggin (Prepotech) or DKK1 (Prepotech) was done at 100 ng/ml concentration. SCs and SC-derived primary myoblasts were treated with 1 μM of chemical probes provided by the Structural Genomics Consortium (SGC, http://www.thesgc.org/chemical-probes/epigenetics) 29,30 . OICR-9429 and Bromodomain Inhibitors were originally described in 19,31–39.…”

Section: Methodsmentioning

confidence: 99%

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals

Schwörer

Feller

et al. 2016

Nature

112

110

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SUMMARY The functionality of stem cells declines during aging thereby contributing to aging-associated impairments in tissue regeneration and function1. Alterations in developmental pathways have been associated with declines in stem cell function during aging2–6 but the nature of this process remains poorly defined. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in aging7,8. This study identifies an altered epigenetic stress response in muscle stem cells (also known as satellite cells = SCs) of aged compared to young mice. This includes aberrant global and site-specific induction of active chromatin marks in activated SCs from aged mice resulting in the specific induction of Hoxa9 among all Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor separating gene expression of SCs from aged compared to young mice. This includes most of the currently known inhibitors of SC function in aging muscle such as Wnt-, TGFß-, JAK/STAT- and senescence signaling2–4,6. Inhibition of aberrant chromatin activation or deletion of Hoxa9 suffices to improve SC function and muscle regeneration in aged mice, while overexpression of Hoxa9 mimics aging-associated defects in SCs from young mice, which can be rescued by inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated SCs from aged mice, which limits SC function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

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Section: Methodsmentioning

confidence: 99%

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals

Schwörer

Feller

et al. 2016

Nature

112

110

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“…Indeed, it is extremely unlikely that two compounds with distinct chemical scaffolds would both bind not only the same target but also the same off-target. For instance, treatment with the SMYD2 inhibitor LLY-507 (Table ) results in loss of cell viability and apoptosis of glioblastoma primary cells; however, this phenotype is not reproduced with Bay-598, a chemically distinct SMYD2 inhibitor, indicating that the cytotoxicity of LLY-507 is an off-target effect . Developing two unrelated chemical probes for the same target is demanding from a medicinal chemistry perspective, but our data indicates that this additional effort is justified.…”

Section: Negative Controls Have a High Chance To Not Bind Off-targetsmentioning

confidence: 84%

“…For instance, treatment with the SMYD2 inhibitor LLY-507 (Table 1) results in loss of cell viability and apoptosis of glioblastoma primary cells; however, this phenotype is not reproduced with Bay-598, a chemically distinct SMYD2 inhibitor, indicating that the cytotoxicity of LLY-507 is an off-target effect. 23 Developing two unrelated chemical probes for the same target is demanding from a medicinal chemistry perspective, but our data indicates that this additional effort is justified. When available, medicinal chemists should consider progressing multiple hits from a primary screening campaign toward the development of more than one chemical probe.…”

Section: ■ Negative Controls Have a High Chance To Not Bind Off-targetsmentioning

confidence: 88%

The Promise and Peril of Chemical Probe Negative Controls

Lee

Schapira

2021

ACS Chem. Biol.

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Chemical probes are selective modulators that are used in cell assays to link a phenotype to a gene and have become indispensable tools to explore gene function and discover therapeutic targets. Chemical probe off-targets are a confounding factor as the observed phenotype may be driven by inhibition of an unknown off-target instead of the targeted protein. A negative control, a close chemical analog of the chemical probe that is inactive against the intended target, is typically used to verify that the phenotype is indeed driven by the targeted protein. Here, we compare the selectivity profiles of four unrelated chemical probes and their respective negative controls. We find that controls that chemically deviate from the probe by a single heavy atom can be inactive against up to 80% of known off-targets if the chemical modification has a charge-neutralizing effect. In such cases, a loss in phenotype upon treatment with the negative control may be driven by loss of inhibition of an off-target. To expand this analysis, we inspect the crystal structures of 90 pairs of unrelated proteins, where both proteins within each pair is in complex with the same drug-like ligand. We computationally estimate that in 50% of cases, methylation of the ligand (a simple chemical modification often used to generate negative controls) at a position that will preclude binding to one protein (the intended target) will also preclude binding to the other (the off-target). These results emphasize the need to select negative controls with care and profile both chemical probes and negative controls against diverse protein arrays to verify that off-targets of probes are also hit by negative controls. When available, a best practice should be to verify that two unrelated chemical probes targeting the same protein elicit the same phenotype.

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“…Utilising a previously established serum-free, growth factor-enriched spheroid culture that enriches for CC-ICs in patient-derived CRC samples 17 , we screened a collection of 22 selective chemical probes that inhibit epigenetic regulatory proteins using viable cell count as a readout (Supplementary Figure 1a). Each probe was used at a single concentration aiming for an estimated cellular IC 90 concentration 18 (Fig. 1a, Supplementary Table 1).…”

Section: Resultsmentioning

confidence: 99%

Targeting bivalency de-represses Indian Hedgehog and inhibits self-renewal of colorectal cancer-initiating cells

et al. 2019

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In embryonic stem cells, promoters of key lineage-specific differentiation genes are found in a bivalent state, having both activating H3K4me3 and repressive H3K27me3 histone marks, making them poised for transcription upon loss of H3K27me3. Whether cancer-initiating cells (C-ICs) have similar epigenetic mechanisms that prevent lineage commitment is unknown. Here we show that colorectal C-ICs (CC-ICs) are maintained in a stem-like state through a bivalent epigenetic mechanism. Disruption of the bivalent state through inhibition of the H3K27 methyltransferase EZH2, resulted in decreased self-renewal of patient-derived C-ICs. Epigenomic analyses revealed that the promoter of Indian Hedgehog ( IHH ), a canonical driver of normal colonocyte differentiation, exists in a bivalent chromatin state. Inhibition of EZH2 resulted in de-repression of IHH, decreased self-renewal, and increased sensitivity to chemotherapy in vivo. Our results reveal an epigenetic block to differentiation in CC-ICs and demonstrate the potential for epigenetic differentiation therapy of a solid tumour through EZH2 inhibition.

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Chemical Biology Approaches for Characterization of Epigenetic Regulators

Cited by 5 publications

References 50 publications

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals

The Promise and Peril of Chemical Probe Negative Controls

Targeting bivalency de-represses Indian Hedgehog and inhibits self-renewal of colorectal cancer-initiating cells

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