Compound (SMILES): Nc1ncnc2c1ncn2[C@@H]1O[C@H](CSCC[C@H](N)OC=O)[C@@H](O)[C@H]1O
InChIKey: HIGCALLWUWOHCV-BAYCTPFLSA-N
Target: Histone-lysine N-methyltransferase, H3 lysine-79 specific (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TEK3
Assay: Assays of Enzymatic Activity: Unless otherwise indicated, assays of DOT1L enzymatic activity were performed under balanced conditions (all substrates present at concentrations equal to their respective KM values) using a radiometric assay of S-[methyl-3H] adenosyl-L-methionine transfer from SAM to chicken erythrocyte nucleosomes. Reactions were initiated by addition of S-[methyl-3H] adenosyl-L-methionine and allowed to run at room temperature for 120 minutes before being quenched by the addition of 800 μM cold SAM.Compound IC50 values were determined from assays of enzymatic activity in which compound was titrated into reaction mixtures by 3-fold serial dilution from DMSO stocks. For each titration, 10 concentrations of inhibitor were used along with 100% inhibition (2.5 μM SAH) and 0% inhibition (1 μL of neat DMSO per well) controls. Plots of residual enzyme velocity as a function of inhibitor concentration were fit to a standard Langmuir isotherm equation (12) to derive estimates of the IC50 value of the compound. As described herein, the inhibition modality of key compounds within the aminonucleoside series were tested and always found to be competitive with SAM and noncompetitive with respect to nucleosome substrate. For most compounds, the Ki value was calculated from the IC50 value using the appropriate equation for competitive inhibition with respect to SAM.For selected compounds, the inhibition modality with respect to the two substrates (SAM and nucleosomes) were determined by dual titration of compound and varied substrate concentration while holding the other substrate fixed at its KM value. Plots of velocity as a function of varied substrate at multiple inhibitor concentrations were globally fit to a general equation for enzyme inhibition using Graphpad Prism. Selection of the modality for each data set was done by evaluating the value of a, a term related to the degree of cooperative or anticooperative interaction between substrate and inhibitor binding, as previously described. A value of α≥10 was taken as consistent with competitive inhibition, while a value of α≤0.1 was taken as consistent with uncompetitive inhibition. Values of α between 10 and 0.1 were considered to be consistent with noncompetitive inhibition.Compounds that displayed an IC50 value within 50-fold of the enzyme concentration used in initial assays ([E]=0.25 nM) were treated as tight binding inhibitors. In this case, the Ki value was determined by measuring the IC50 value of the compound (vide supra) at varying concentrations of enzyme from 5 nM to 0.25 nM. A plot of IC50 as a function of enzyme concentration was fit to a linear equation and the y-intercept value was equivalent to Ki(1+[S]/KM) where [S] and KM refer to SAM, the substrate with which these inhibitors compete. Knowing the values of [S] and KM used in the assay, the Ki value was then calculated from the y-intercept value.Determination of Ligand Association and Dissociation Rate ConstantsLigand association and dissociation rate constants were determined by surface plasmon resonance (SPR). DOT1L was stored in 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.8 and immobilized by direct amine coupling, diluting enzyme into coupling buffer containing 10 mM Hepes pH 7.4, 1 mM TCEP. Immobilization run buffer contained 10 mM Hepes pH 7.4, 150 mM NaCl, 500 μM TCEP, and approximately 10,000 RUs (response units) of DOT1L was captured. A reference channel of a surface that was activated in parallel and blocked was created in a second flow cell was also created. Data was captured on either a Biacore 4000 (chip CM5) or a Biorad ProteOn (chip GLM).Kd determinations were determined using run buffer containing 20 mM Tris pH 8.0, 10 mM NaCl, 100 mM KCl, 0.002% Tween-20, 500 μM TCEP, 2% DMSO, with the following injection parameters: 30 μl/min flow rate, with a 30 second association phase followed by monitoring dissociation for 30 seconds. Exp
Measurement: Ki = 320 nM (pChEMBL 6.5)
Descriptors: MW 384.42, ALogP -1.39, HBA 12, HBD 4, PSA 171.63, QED 0.24