A fluorescent histone deacetylase (HDAC) inhibitor for cellular imaging

Article abstract of DOI:10.1039/c5cc02059j

Fluorescence microscopy studies using 4-morpholinoscriptaid (4MS) demonstrated rapid cellular uptake of this scriptaid analogue into the cytoplasm but no nuclear penetration. As 4MS and scriptaid have the same in vitro activity against HDACs and KASUMI-1 cells; 4MS exemplifies a rational approach to subtly modify 'profluorogenic' substrates for intracellular studies.

Full text of DOI:10.1039/c5cc02059j

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A fluorescent histone deacetylase (HDAC) inhibitor for
cellular imaging
Cite this: DOI: 10.1039/x0xx00000x
Cassandra L. Fleming,a Trent D. Ashton,a Cameron Nowell,b Mark Devlin,c Anthony
Natoli,c Jeannette Schreudersc and Frederick M. Pfeffera
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
Fluorescence microscopy studies using 4-morpholinoscriptaid
(4MS) demonstrated rapid cellular uptake of this scriptaid
analogue into the cytoplasm but no nuclear penetration. As
4MS and Scriptaid have the same in vitro activity against
HDACs and KASUMI-1 cells; 4MS exemplifies a rational
approach to subtly modify ‘profluorogenic’ substrates for
intracellular studies.
Fluorescent tagging of bioactives for cell based microscopy studies
is conventionally achieved by conjugating, by means of a spacer, a
bulky fluorophore to the drug of interest.1 While fluorescent, the
extraneous conjugation to a fluorophore invariably leads to an
increase in molecular weight and has an impact on cLogP— Figure 1. Structure of Scriptaid (
1
) and the fluorescent 4MS. Regions of the HDAC
pharmacophore are also indicated.
parameters that medicinal chemists spend considerable effort
optimising. Indeed, the behaviour of the ‘tagged’ compound in cells
might not accurately reflect that of the parent.
While SAR studies of Scriptaid do not extend to modifications
of the ‘capping’ group, we reasoned that, given the non-critical role
of the capping group with respect to HDAC inhibition, introduction
of a small amino moiety would not have deleterious effects on
HDAC activity. Indeed, calculated properties (cLogP and PSA) of
Scriptaid (1) and 4MS showed only subtle differences (see ESI for
full details) and thus it was anticipated that other than fluorescence,
4MS would behave in a near identical fashion to the parent.
Ideally, only subtle structural modifications would be performed
on a bioactive to render it fluorescent. Despite the seemingly
obvious benefits of such an approach, no examples could be found.‡
While this strategy is limited somewhat in that only aromatic
compounds can be considered, the dominance of highly aromatic
scaffolds in drug discovery makes it complementary to the
conventional ‘tagging’ approach. A bioactive that is identified as
being structurally related to an existing fluorophore might be termed
‘profluorogenic’; for example naphthalene is a common component
of biologically active compounds2 and is the core of several highly
fluorescent entities including the 4-aminonaphthalimides.3
Scriptaid (1, Figure 1) is a well-studied histone deacetylase
(HDAC) inhibitor with established anticancer activity.4–5 More
recently, Scriptaid (1) has been shown to have beneficial effects in
the treatment of HIV and neurodegenerative disorders.6 Scriptaid (1)
possesses the typical pharmacophore of HDAC inhibitors which
includes (i) zinc binding group (chelates the Zn2+ ion of the active
site, see Figure 1) (ii) linker (occupies a hydrophobic tunnel) which
connects to the (iii) capping group (solvent exposed).7
Recognising the structural similarities of the 4-amino-1,8-
naphthalimide fluorophore and the known HDAC inhibitor Scriptaid
(1), the fluorescent Scriptaid analogue 4MS was developed (Figure
1); the key feature being the introduction of the relatively small 4-
morpholino substituent (MW = 87).
Scheme 1. Synthesis of 4MS
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Table 1. Inhibition of individual HDAC isoforms, IC50 (µM)
HDAC Isoform (SF), SF = Selectivity Factor vs HDAC6
Compound
KASUMI-1
1
3
6
8
11
0.36 ± 0.03
(30)
0.29 ± 0.02
(24)
1.74 ± 0.06a
(145)
0.37 ± 0.06
(31)
1.52 ± 0.01
(127)
Scriptaid (1)
0.012 ± 0.0026
0.49b
0.29
1.43 ± 0.13
(119)
0.32 ± 0.04
(27)
1.81 ± 0.17
(151)
4MS
0.012 ± 0.0019
a Values represent the average of two measurements (n = 2). b Error < 1% (n = 3).
To construct 4MS, 4-bromonaphthalimide 2 (see ESI for
synthesis) was converted to the corresponding 4-morpholino
derivative
3 using a palladium mediated Buchwald–Hartwig
approach (Scheme 1).8 Treatment of 4-bromonaphthalimide 3 with
morpholine (3 equiv), Pd2(dba)3·CHCl3 (4 mol%) and the
commercially available ligand, Xantphos (4 mol%) in the presence
of Cs2CO3 (3 equiv) at 40 °C for 24 h gave the desired aryl amine 3
in excellent yield (90%). Carboxylic acid 4 was obtained in high
yield (91%) using excess LiOH·H2O. The free acid was converted to
the mixed anhydride (using ClO2Et and Et3N) then in situ treated
with hydroxylamine (in methanol) to give the desired hydroxamic
acid 4MS in good yield (67%, 50% over 4 steps).
A
B
C
D
E
F
Compound 4MS was then assessed for inhibitory activity against
HDAC isoforms 1, 3 and 8 (class I), 6 (class IIb) and HDAC 11
(class IV). The introduction of the morpholine group was not
deleterious towards HDAC inhibition. Indeed, when compared to
Figure 3. Confocal microscopy images of MDA-MB-231 cells showing the rapid
cellular uptake of 4MS at 1.0 µM. Images are of the following time points: (A) 0 s
(B) 10 s (C) 20 s (D) 30 s (E) 40 s (F) 50 s.
Scriptaid (1) 4MS had similar activity against both HDAC6 (IC50
12 nM, Table 1) and HDAC3 (4MS, IC50 = 0.32 µM; cf 1, IC50
0.37 µM) and was slightly more efficacious against HDAC1 (IC50
=
=
=
suited for fluorescence microscopy. In DMSO, the absorption
maximum (Figure 2) was 399 nm with an emission maximum at 534
nm (Stokes shift = 135 nm). This compares favourably with
Scriptaid (1), which presents no visible fluorescence.
1.43 µM) than Scriptaid (1, IC50 = 1.74 µM). Consequentially, 4MS
still displayed similar selectivity as Scriptaid (1) for HDAC6 over
HDAC1 and HDAC3. For the other class I isoform investigated
(HDAC8) 4MS inhibited with an IC50 of 1.81 µM compared with
1.52 µM for 1 which corresponds to improved selectivity (4MS, SF
= 151; cf 1, SF = 127). A moderate improvement in activity was also
obtained at HDAC11 (4MS, IC50 = 0.29 µM; cf 1, IC50 = 0.36 µM).
Both Scriptaid (1) and 4MS exhibit modest selectivity towards
cytosolic HDAC6, which is known to modulate a number of
processes that can lead to cell stress and apoptosis including α-
tubulin deacetylation and binding to the stress granule protein,
G3PB1.9 Indeed, HDAC6 inhibitors have been proposed as therapies
for acute myeloid leukemia,10 a hematological cancer represented by
KASUMI-1 cells. As such the ability of 4MS to decrease cell
growth was evaluated in KASUMI-1 cell lines (Table 1) and 4MS
inhibited the growth of the leukemia cell line with an IC50 of 0.29
µM, which is comparable with Scriptaid (1, IC50 = 0.49 µM).
To demonstrate the potential of 4MS as a tool to provide
pharmacokinetic information, confocal microscopy was used to
visualise the cellular penetration and subcellular location of 4MS in
the human breast cancer MDA-MB-231 cell line. The cells were
treated in situ with a 1.0 μM solution of 4MS and images were taken
at 10 second time points (Figure 3A–F). At each time point, a
significant increase in emission intensity from the treated cells was
evident, clearly indicating rapid cellular uptake. Cellular uptake of
biologically active molecules is currently generally monitored by
radio-labelling or fluorescent tagging.12 While radio-labelling can be
achieved through isotopic replacement it requires specialised
equipment and precautions. As mentioned, fluorescent tagging often
leads to a significant increase in the molecular weight of the known
biologically active compound. Large increases in molecular weights
may result in poor absorption and cell permeation of the fluorescent
therapeutic.13 By utilising the inherent fluorescent properties of
4MS, rapid cellular uptake was visualised in less than 50 seconds.
Additional time course studies were conducted at lower
concentrations (0.025–0.30 µM), in which 4MS was incubated in
MDA-MB-231 cells for 48 h and co-stained with propidium iodide
to monitor cell death (Figure 4). No fluorescence was observed in
the nucleus, suggesting that 4MS did not penetrate the nuclear
envelope. It is possible that the lack of observed fluorescence in the
nucleus was due to DNA mediated fluorescence quenching rather
than lack of penetration therefore titrations of 4MS with calf thymus
(ct)-DNA were conducted. While a slight decrease in the emission
intensity of 4MS was observed upon the addition of ct-DNA in
phosphate buffer (10 mM at pH 7.4), the emission band of 4MS was
still clearly visible after a large excess of ct-DNA had been added
(see ESI). These results indicate that 4MS was not entering the
nucleus and that the lack of fluorescence emission from the nucleus
was not a result of DNA binding/quenching. The observed
cytoplasmic accumulation is interesting given the concentration of
The photophysical properties of 4MS were typical of 4-
dialkylamino-1,8-naphthalimide derivatives11 and proved to be well
Figure 2. Normalised excitation and emission spectra of 4MS in DMSO.
2 | J. Name., 2012, 00, 1-3
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c
Incubation Time (h)
24
Peter MacCallum, Cancer Centre, St Andrews Place, East Melbourne,
Victoria, 3002, Australia.
0
48
†
Electronic Supplementary Information (ESI) available: [Detailed
1
experimental procedures, H NMR and 13C NMR spectra of compounds,
as
DOI: 10.1039/c000000x/
comprehensive literature search involving combinations of
well
as
photophysical
results
are
provided.].
See
A
‡
A
keywords including “theranostics”, “fluorescent drugs”, tagging” and
“bioactive” gave no relevant results.
B
1
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Figure 4. Confocal microscopy images of 4MS within MDA-MB-231 cells at (A)
0.025 µM (B) 0.1 µM (C) 0.3 µM. Cells were costained with propidium iodide to
monitor cell death and fluorescent images were taken at the following time
points: 0, 24 and 48 h. Green = 4MS. Red = propidium iodide.
3
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HDACs in the nucleus and may ultimately prove advantageous as
the class II HDAC isoforms, such as HDAC6, are known to shuttle
between the nucleus and the cytoplasm.14 Based on our results, it
might be possible to design selective inhibitors for class II HDAC by
refining the localisation profile of the therapeutic rather than
focussing solely on enhancing its specific enzyme binding.
Upon incubation of MDA-MB-231 cells with the fluorescent
4MS at 0.025 and 0.1 µM no significant changes were observed
(Figure 4A and B, respectively). However, the treatment of MDA-
MB-231 cells with 0.3 µM 4MS, cell death was evident within 48 h
(Figure 4C).
In summary, the rational design and synthesis of 4MS has been
performed. Structural changes from Scriptaid (1) were minimal and
were not detrimental to isoform selectivity or anticancer activity
(KASUMI-1). The strategic location of the morpholino substituent at
the 4-position of the naphthalimide imparted photophysical
properties and enabled the study of these compounds in cells using
fluorescence microscopy; demonstrated with the visualisation of
rapid cellular uptake and subsequent distribution in MDA-MB-231
cells. This fluorescent analogue has very quickly contributed to our
understanding of how the well-studied Scriptaid (1) acts at a cellular
level and is likely to be a value to the many researchers currently
investigating Scriptaid (1) as a potential neuroprotectant and
anticancer agent.
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CLF thanks the Research Centre for Chemistry and
Biotechnology for a top-up scholarship. The authors would like to
acknowledge the Australian Research Council for funding Deakin
University’s Magnetic Resonance Facility through LIEF grant
LE110100141, as well as for additional equipment support related to
this project (LE120100213).
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Environmental Sciences, Deakin University, Waurn Ponds, Victoria,
3216, Australia. Email: fred.pfeffer@deakin.edu.au
b
Monash Institute of Pharmaceutical Science, Royal Parade, Parkville,
Victoria, 3052, Australia.
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