Investigating Ash1L Histone Methyltransferase using Chemical Biology Approaches

Ash1L (absent, small, or homeotic-like 1) is a histone lysine methyltransferase that catalyzes mono- and dimethylation of histone H3 lysine 36. Previous studies showed that Ash1L is implicated in a variety of cancers. Remarkably Ash1L is involved in the pathogenesis of acute leukemias with chromosomal translocations of the Mixed Lineage Leukemia (MLL) gene, which present in 5-10% of adult acute leukemias and up to 70% of infant leukemias with poor prognosis. Hence Ash1L is a validated target for therapeutic intervention, and the availability of potent small molecule inhibitors would be invaluable to investigate the roles of Ash1L in leukemias and other diseases. We previously employed fragment-based drug discovery and identified compounds that bind to Ash1L. Through optimization of the fragment hit, we developed inhibitors that exhibit sub-millimolar activity against Ash1L. In this work, we aimed to further improve the inhibitory activity of compounds and present the development of potent Ash1L inhibitors. We conducted extensive medicinal chemistry optimization and structure-based design. These efforts yielded a series of potent compounds that selectively inhibit Ash1L in biochemical assays and exhibit on-target activity in human leukemia cells. Moreover, we obtained multiple high resolution crystal structures of Ash1L in complex with inhibitors, and these structures reveal that compounds bind to a pocket in the vicinity of the autoinhibitory loop in the SET domain. To our knowledge, compounds we developed represent first-in-class inhibitory chemical probes targeting Ash1L. Based on the design of Ash1L inhibitors we made a fluorescent tool compound and developed a fluorescence polarization assay. Employing this assay we performed high-throughput screening (HTS) of 23,500 compounds and identified a hit compound that binds to Ash1L. The discovery of this compound from HTS provides a promising direction for developing a new class of Ash1L inhibitors. Lastly, we used Ash1L inhibitors to probe the conformational dynamics of Ash1L SET domain and studied the protein-protein interaction between Ash1L and Mrg15. We discovered that binding of inhibitor partially stabilizes the conformation of the SET domain but the autoinhibitory loop region experiences enhanced dynamics in solution. We also validated that Mrg15 binds to Ash1L and activates its enzymatic activity. However, we found that the activities of Ash1L inhibitors are not affected by the presence of Mrg15. In conclusion, our studies describe multiple approaches of using well-characterized chemical probes to investigate inhibition and functions of Ash1L histone methyltransferase.

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