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Inhibition of Monocytic Leukaemia Zinc Finger (MOZ) Acetyltransferase to Target Epigenetically Driven Leukaemias

Hendrik Falk1,2,3, Theresa Connor1, Hong Yang1,3, Karen J Loft1,3, Joanne L. Alcindor1,2,3, George Nikolakopoulos1,2,3, Regina N Surjadi4, John D Bentley4, Meghan K Hattarki4, Olan Dolezal4, James M Murphy1,2, Brendon J Monahan4, Thomas S Peat4, Tim Thomas1,2, Jonathan B Baell1,2,3, John P Parisot1,2,3, Ian P Street1,2,3
1 The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia
2 Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia
3 Cancer Therapeutics CRC P/L, High Throughput Screening Group, 4 Research Avenue, Bundoora, Victoria, 3083, Australia
4 Commonwealth Scientific and Industrial Research Organisation (CSIRO), Materials Science and Engineering Division, 343 Royal Parade, Parkville VIC 3052, Australia


Advances in understanding the formation and persistence of cancers have led to increased treatment options using inhibitors of G-protein-coupled receptors (GPCR) and kinases. Nevertheless, many types of cancer have remained refractory to drug treatment and new classes of druggable targets need to be found. Increasingly, epigenetic aberrations are regarded as key factors in cancer progression. Nucleosome modifications provide additional levels of regulation of cellular functions.
Monocytic leukaemia zinc finger protein (MOZ) is member of the MYST family of histone acetyl transferases (HAT). Its members play a key role in regulating patterns of gene expression in proliferation, differentiation, and apoptosis. MOZ in particular has been implicated in the formation of haematopoietic stem cells during foetal development and in the self-renewal of adult haematopoietic stem cells. Deregulation of these processes is known to give rise to the formation of leukemic stem cells, rendering MOZ an excellent candidate to target for treating myeloid leukaemia. Currently no small molecule MOZ-HAT inhibitors are available.
We have implemented a hit discovery campaign to identify small molecule inhibitors of MOZ-HAT activity. We developed a homogenous assay for detecting the MOZ-catalysed acetylation of synthetic substrate peptides based on histone N-terminal sequences. Antibodies specifically recognising the acetylated lysine product were used for detection and this signal was amplified with AlphascreenTM technology. This method delivered a high signal-to-background ratio and could be miniaturised easily. Importantly, the assay was able to measure activities not only of MOZ but also of related MYST family HAT proteins.
The assay produced reliable results in a primary screening campaign testing 243,000 small molecule lead-like compounds. We identified inhibitory compounds from several chemical classes. Several types of secondary assays were used to eliminate assay-interfering compounds and prioritise the hits. In addition, Surface plasmon resonance (SPR) demonstrated the reversibility of binding and competition with the substrate acetyl coenzyme A. A radiometric assay using purified histone proteins confirmed the inhibitory activity in the low micromolar range. In summary, this study establishes a new high-throughput assay for HAT activity and provides the foundations for the development of a new class of drugs for the treatment of leukaemias.

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