Fig. 2: Development and characterization of ASH1L inhibitors.

a Structure of the fragment hit, compound 1, and its binding to ASH1L SET domain. Superposition of the ¹H-¹⁵N TROSY-HSQC spectra of 100 µM ASH1L with 5% DMSO (black) with 500 µM (orange) or 1 mM (blue) of compound 1. b Chemical shift perturbations upon binding of 1 to ASH1L mapped on the crystal structure of ASH1L SET domain (PDB code 4YNM). Residues experiencing chemical shift perturbations calculated as \({\triangle }_{{\rm{HN}}}=\sqrt{({{\rm{\delta }}}_{{\rm{HN}}}^{2}+{{\rm{\delta }}}_{{\rm{N}}}^{2})}\) larger than 15 Hz are colored and encircled in violet. Residues unobserved or unassigned are colored and encircled in orange. c Chemical structures and activities for selected ASH1L inhibitors. IC₅₀ values represent mean ± s.d. from two independent experiments. d Superposition of the ¹H-¹⁵N TROSY-HSQC spectra of 100 µM ASH1L with 5% DMSO (black) and with 500 µM (orange) or 100 µM (blue) of AS-5. e Titration curves from the HMT assay for ASH1L with compounds presented in panel (c) mean ± s.d. Representative curves are shown from two independent experiments, each performed in duplicates. f Binding isotherm from the ITC experiment performed for the binding of AS-5 to ASH1L. Data are mean ± s.d. from two independent experiments. A representative binding isotherm is shown. N represents stoichiometry of binding.