Communication
ChemComm
Intact protein mass spectrometry indicated that histones isolated PHD2 and FIH by CCT1 in vitro does not correlate with
H3.1 and H3.2 from HEK293T cells treated with CCT1 have an the strong stabilisation of HIF and inhibition of HIF hydroxylation
overall positive shift in mass, indicative of increased levels of observed on treatment of cells by CCT1, raising the possibility of
post-translational modification compared to untreated cells (an) unidentified iron-availability mechanism(s) for regulating HIF
(Fig. 5D); CCT1 manifested no effect on modifications to H2A, levels and activity. Given the importance of HIF in the hypoxic
H2B, and H4 by this technique (SF4). Since histone methylation response and regulation of proteins such as erythropoietin, such
principally occurs on H3 proteins, the results support the mechanisms are of biomedicinal interest and subject of ongoing
proposal that CCT1 is a JmjC KDM inhibitor in cells. CCT1 investigations.
(Fig. 5A) and some analogues (Fig. 4B and D) demonstrated
selectivity for KDM4 against other isolated recombinant KDM Lejeune for chiral resolution, and the SGC biotechnology team
subfamilies. for KDM protein production; Cancer Research UK (CCT), a
We thank Richard Hopkinson for helpful advice, Clarisse
The results with isolated enzymes indicate that CCT1 is Royal Society Dorothy Hodgkin fellowship (AK), the SGC, the
selective for the JmjC KDMs over, at least some, other human NIH (EDM), the Friends of the Cancer Center, the Welch
2OG oxygenases (Fig. 5A); in particular CCT1 was inactive Foundation (EDM), and CPRIT for funding.
against isolated recombinant hypoxia-inducible transcription factor
(HIF) prolyl hydroxylase PHD2 (MS-based assay: IC50 4 100 mM;
References
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antibody-based AlphaScreens assay: IC50 = 96 mM (Fig. 5A, SF5A))
under standard conditions, and non-denaturing mass spectrometry
of PHD2 did not indicate binding of CCT1 (SF5B/C); similarly, CCT1
was inactive against isolated recombinant factor inhibiting HIF
(FIH) (MS-based assay: IC50 4 100 mM). HIF-a levels are regulated
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Treatment of HeLa cells with CCT1, but not CCT2, caused
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˜
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Chem. Commun.
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