Fragments vs ASH1L: a chemical probe

It has been a while since Practical Fragments last covered epigenetic targets that bind to or remodel chromatin. The protein ASH1L (absent, small, or homeotic-like 1) is a molecular leviathan containing some 3000 amino acids and multiple domains, including three “readers” such as bromodomains that bind to acetylated lysine residues. ASH1L also contains a SET “writer” domain that transfers a methyl group to specific histone lysine residues. This domain is the subject of a new Nat. Commun. paper by Tomasz Cierpicki, Jolanta Grembecka, and a large group of collaborators at University of Michigan Ann Arbor and elsewhere.

 

Previous work implicated ASH1L in a particularly lethal type of leukemia with chromosomal translocations in a protein called MLL1. The researchers found that the ASH1L SET domain was essential for leukemogenesis, suggesting that a small molecule inhibitor could be an effective anticancer strategy.

 

A two-dimensional ¹H-¹⁵N TROSY-HSQC NMR screen of 1600 fragments in pools of 20, each at 250 µM, was conducted against the ASH1L SET domain. Compound 1 binds weakly, but chemical shift perturbations mapped the binding site near an autoinhibitory loop, suggesting the molecule could lock the protein in an inactive conformation. Fragment growing led to compound 2.

 

Further fragment growing led to AS-5, with low micromolar affinity as assessed by isothermal titration calorimetry (ITC) as well as functional activity in an enzymatic assay. At this point the researchers obtained a crystal structure, which confirmed binding of AS-5 near the autoinhibitory loop. The thioamide forms a chalcogen bond to a backbone carbonyl, a relatively uncommon interaction. Further structure-based design ultimately led to AS-99, with high nanomolar activity as assessed both biochemically and by ITC. The molecule is selective against a panel of 20 histone methyltransferases and 30 kinases.

 

Despite having relatively modest affinity, AS-99 inhibited growth in MLL leukemia cells and caused them to differentiate. Further mechanistic studies confirmed that this was due to blocking enzymatic activity of ASH1L and downregulation of target genes. Importantly, a negative control molecule in which the sulfur of the thioamide was replaced with an oxygen showed dramatically lower activity both biochemically and in cells. AS-99 has sufficiently favorable pharmacokinetic parameters for i.p. dosing, and a mouse xenograft model showed reduced tumor growth.

 

This paper is a nice example of academic lead discovery, and AS-99 looks to be a useful chemical probe. There is still much to do to optimize the molecule; the thioamide in particular will raise eyebrows among medicinal chemists. Nonetheless, as the researchers point out, AS-99 is a first-in-class molecule that should facilitate further pharmacological understanding of the role of ASH1L in leukemia.