1. Synthesis and Optimization of Dual kinase/bromodomain Inhibitors
Shiva Dastjerdi, Dennis L. Buckley, Jun Qi, Justin M. Roberts, Anthony Varca, James E. Bradner*
Dana-Farber/Harvard Cancer Center, Department of Medical Oncology, Harvard Medical School, Boston, MA
Bromodomains (BRDs) are a class of epigenetic reader proteins that
recognize acetyl-lysine residues on chromatin. The deregulation of
bromodomains and extra terminal (BET) family (which consists of BRD2,
BRD3, BRD4 and BRDT) is observed in various cancers, including NUT
midline carcinoma, neuroblastoma, leukemia, etc. Bromodomain testis-
specific (BRDT) is a potential contraceptive target essential for
chromatin remodeling during spermatogenesis. The first published
inhibitor of BET bromodomains, JQ1, was found at our lab at Dana-
Farber Cancer Institute and showed potent inhibition of BRD4 and BRDT
and has lead to interest in the development of novel BET bromodomain
inhibitors. An unexpected source of bromodomain inhibitors has been
compounds with previously described kinase inhibitory activity.
Recently, over 14 previously described kinase inhibitors have been
shown to also inhibit BRD4 . This research aims to generate small
molecule inhibitors with increased potency for bromodomains and
kinases. For this purpose, we sought to synthesis a small targeted library
of derivatives of known dual kinase/bromodomain inhibitors which
also have activity against the non-BET bromodomains P300, CREBBP,
TAF1 and TAF1L. We succeeded in synthesizing a library of 90
bromodomain inhibitors that shared the same dihydropteridinone
scaffold using our lab’s Cap-Scanning technology that was previously
used to develop selective HDAC inhibitors. We then tested these
inhibitors for activity against BRD4 and BRDT in an ALPHA assay and
were able to find a number of derivatives with improved activity over
the parent compound. ALPHA Screen
(Amplified Luminescent Proximity Homogeneous Assay Screen) is a
bead-based proximity assay. Singlet oxygen molecules, generated by
high energy irradiation of Donor beads, travel over a constrained
distance to Acceptor beads. This results in excitation of a cascading
series of chemical reactions, causing generation of a chemiluminescent
signal. Since the lifetime of the singlet oxygen reactive species in
aqueous solutions is very short, donor and acceptor beads need to be
bound to one another in order to generate a signal.
We plan to synthesize a larger library using slightly different scaffolds,
test our library for activity against non-BET bromodomains, and
continue to optimize the hits we have found already.
Introduction
The goal is to make a library of 90 bromodomain inhibitors using the
cap-scan technology and to test their activity against BRD4 and BRDT using
ALPHA screening.
Purpose
Result and discussionSynthesis of Bromodomain Inhibitors
Conclusion and Future work
Reference
Acknowledgements
Figure 1: Synthesis of Dihydropteridinone and Pyrimidobenzdiazepine scaffolds using
Buchwald-Hartwig amination and using cap-scanning technology for each scaffold.
Figure 2: Synthesizing pyrimidodiazepine scaffold followed by the addition of aminobenzoic acid.
After the iron reduction reaction , we could not go any further with the product because of
the low percent yield due to the presence of the excess iron, therefore we used
dihydropteridinone scaffold to synthesize and apply the cap-scan technology and
ALPHAScreening on. Our future plan would be to improve the synthesis of pyrimidodiazepine
and to do the cap-scanning and ALPHA screening on it.
Figure 3: Synthesizing of dihydropteridinone scaffold followed by addition of aminobenzoic acid
Figure 4: JQ1 which appeared first in the graph, is the most potent inhibitor for BRD4 and BRDT. The blue bar shows the potency of the dihydropteridinone scaffold with no
aldehyde attached to it.
Results of the cap-scanning and ALPHAScreening of the 96-well plate show how each well containing different aldehydes show potency against
BRD4 and BRDT comparing to the JQ1 inhibitor (the red bar) that has the maximum potency against them.
As figure 4 shows the luminescence released by the acceptor bead for 96 different compounds. There an increase of luminescence from left
to right which shows decreasing of the inhibitions of the compounds. JQ1 which is shown as a red bar is appeared as the first inhibitor with
the lowest luminescence. BRD4 cap-scan plate on the left shows 8 potential inhibitors (black bars) between JQ1 and the dihydropteridinone
scaffold. Those inhibitors were selected for being spot-checked by LC-MS to confirm their molecular weight accuracy based on their
structures and were selected for further synthesis in the future. Figure 5 shows the 8 selected inhibitors after the ALPHAScreening.
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•Dr. James E. Bradner’s lab members for their support.
•The National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM076321
•NIH-funded Initiative for Maximizing Student Development (IMSD)