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M.K.P. Jayatunga et al. / European Journal of Medicinal Chemistry xxx (2014) 1e8
1e3, and 2,2-disubstituted indandiones 4e6 (Fig. 2). The obser-
vation that a structurally diverse set of quinone derivatives dis-
played similar levels of activity suggested that the core quinone
may be the active component. Indeed, we found that simple
commercially available quinones were also active (Fig. 3). The re-
sults showed that the benzoquinone core is sufficient for activity,
with potency correlating well with reported oxidative potentials
[42,43]. Thus, anthraquinone
7
(half wave potential (E1/
2) ¼ ꢀ1.26 V) was much less active than naphthoquinone 8 (E1/
¼ ꢀ1.03 V) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
2
9 (E1/2 ¼ ꢀ0.34 V) [44,45].
Dipyridyldisulfide 13, which contains a disulfide, as do the ETP
inhibitors, was also weakly active (IC50 z 100
mM). Analogous
aromatic compounds, which did not contain quinone functionality,
displayed no activity (e.g. 8 compared to 11) although hydroqui-
nones 10 and 12 showed modest activity. Hydroquinones may also
be involved in a redox process, generating reactive quinones in
situ. Spontaneous oxidation of hydroquinone and catechol by
molecular oxygen has been observed to covalently modify DNA,
suggesting that such redox cycles may be responsible for activity in
our assay [46,47]. A common feature of the active compounds is
the presence of electrophilic groups able to react with cysteines/
thiols [48,49].
Fig. 1. View from an NMR structure of a fragment of the HIF-1
a C-terminal trans-
activation domain (C-TAD) (785e826) (red) complexed with the CH1 domain of p300
(323e423) (green) with structurally important p300 zinc ions shown in magenta (PDB:
IL3E) [14]. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
We then investigated the nature of the indandione-mediated
inhibition. A range of 2-amino-, 2-imino- and 2-amidoindandiones
5, 14e20 was synthesized to investigate SAR (Fig. 4a). The amino-
and imino-derivatives 18e20 were less active than amido-
derivatives 5 and 14e17 (Fig. 4b). Notably, ninhydrin 23 the
parent compound of the indandione derivatives, displayed similar
potency to the amido-derivatives (Fig. 4b, Supp. Info e Table S1),
suggesting that ninhydrin may be the active component of the
indandione compound class. Indeed, mass spectrometric and NMR
analysis indicated that an aqueous solution of amido-compound 5
generates ninhydrin 23 (Fig. 5a). The decay of 5 to picolinamide and
23 was monitored by 1H NMR at pH 8 (D2O, 10 mM phosphate
buffer), indicating that 80% hydrolysis occurs after one hour
(Fig. 5b). Compounds that are structurally similar, but lack the
reactive C-2 centre; i.e. 2-amido-indoline 21 and indandione 22,
were inactive (Fig. 4b). We thus propose that of all the apparently
active indandione derivatives fragment to give ninhydrin 23, which
is the active species.
p300 inhibitor [34]. The core, electrophilic, ETP functionality has
been shown to be sufficient for activity, with a number of analogues
showing similar activity to the natural products [35e37]. Subse-
quent work determined that chetomin and other ETPs work, at
least in part, by Zn(II) ejection from the CH1 domain of p300, thus
disrupting its structure, ablating the interaction with HIF-1a [38].
Modes of action involving cysteine modification and/or zinc ejec-
tion are likely inherently unselective, with the ETPs, such as
chaetocin, showing inhibition against thioredoxin reductase and a
number of histone methyl transferases [39e41].
In a search for inhibitors for the HIF-1a/p300 interaction we
conducted an HTS of 10,000 natural product-based structures using
a similar ELISA competition assay (Fig. 2). The results led to the
identification of electrophilic inhibitors of the HIF-1a/p300 PPI.
2. Results and discussion
To further investigate the mode of action of these electrophilic
compounds we tested whether they caused Zn(II) ejection from
jumonji domain 2A histone demethylase (KDM4A), for which
treatment with other Zn(II) ejectors has been shown to be inhibi-
tory [50e52]. In the catalytic domain of KDM4A a Zn(II) ion is
bound to three cysteines and one histidine in an analogous fashion
to the coordination observed in the CH1 domain of p300 (Fig. 6b
and c). Ebselen, a known zinc-ejector for KDM4A was used as a
positive control, with the dye FluoZin-3™ (FZ-3) providing a
measure of the unbound zinc concentration [50]. Compounds 8, 10,
16 and ninhydrin 23, which were active in the competition binding
assay also caused loss of Zn(II) from KDM4A in a dose and time
dependent manner (Fig. 7b). Despite being less effective than
ninhydrin 23 in the competition-binding assay, quinone 8 and
reduced quinone 10 showed comparable KDM4A activity to 23.
Analogous studies on p300 yielded similar results, although the
high basal levels of Zn(II), added to p300 such that it folds correctly,
results in poorer resolution (Fig. 7a, Supp. Info e Fig. S2). The lack of
selectivity observed by the quinones and indandiones identified in
our initial screen suggest that they are likely not selective for
different zinc binding sites [53,54].
The output of the screen led to the identification of two distinct
compound classes that showed promising activity: benzoquinones
a)
b)
When 8 and 23 were tested in a HeLa cell viability assay, sig-
nificant dose-dependent cytotoxicity was observed after 48 h
(Supp. Info e Fig. S3). The inactive compound 21 was not cytotoxic
Fig. 2. Inhibitors of the HIF-1
compound screen. IC50 values
indandiones.
a
/p300 interaction identified in a natural product-like
M) are in parentheses; (a) benzoquinones; (b)
(m
Please cite this article in press as: M.K.P. Jayatunga, et al., Inhibition of the HIF1
a-p300 interaction by quinone- and indandione-mediated
