Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non‐Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B‐cell lymphoma cells harboring gain‐of‐function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL‐AF9,MLL‐AF4, and AML1‐ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.

A wide variety of compound libraries are currently available to obtain active compounds for drug target proteins, but the affinities of initially screened compounds are usually too low and have to be improved by chemical modification of the compounds. In such cases, pharmacophore information of the compounds plays a key role for the next modification step. Structure determination of the protein-compound complex is too time-consuming to be applied for such situations. Therefore, many chemists would highly appreciate simple and accurate experimental procedures to obtain pharmacophore information.From this viewpoint, various ligand-observed NMR spectroscopy experiments have been proposed to characterize protein-ligand interactions.[1] Among them, experiments exploiting nuclear Overhauser effects (NOEs; transferred NOE, saturation transfer difference (STD), pumped NOE, water-ligand observed by gradient spectroscopy (water-LOGSY), etc.) are widely used and are also utilized as a ligand pharmacophore (or epitope) mapping technique. [2] However, it has recently been revealed that the difference of the longitudinal relaxation of each ligand proton severely interferes with the derived pharmacophore mapping result, [3] and it is crucial to evaluate intermolecular cross-relaxation terms for accurate pharmacophore mapping. With this in mind, pharmacophore mapping by diffusion NMR spectroscopy, [4] adiabatic fast passage NOESY (AFP-NOESY), [5] and group epitope mapping considering relaxation of the ligand (GEM-CRL) [6] quantitatively exploit the obtained intermolecular cross-relaxation effect.Herein, we propose a simple and rapid approach for pharmacophore mapping experiments, which utilizes the difference between the longitudinal relaxation rates of ligand protons with and without irradiation of the protons of the target protein.The longitudinal relaxation of ligand proton I is represented by the modified Bloch equation [Eq. (1)]where DI z = I z ÀI z 0 (I z represents the instantaneous longitudinal magnetization, I z 0 represents the thermal equilibrium values of I z ); DS z and DX z for protons S and X are defined correspondingly. R I represents the auto-relaxation rate constant of ligand proton I, s IS represents the intramolecular cross-relaxation rate constant between ligand proton I and another ligand proton S, and s IX represents the intermolecular cross-relaxation rate constant between ligand proton I and proton X of the target protein. In the case that longitudinal relaxation rates are measured by using an inversion-recovery method, and the initial 1808 inversion pulse is replaced with two consecutive 908 pulses with an appropriate phase cycling, [7] the thermal equilibrium term can be omitted from Equation (1) to give Equation (2).

Cyclorasins 9A5 and 9A54 are 11‐mer cyclic peptides that inhibit the Ras‐Raf protein interaction. The peptides share a cell‐penetrating peptide (CPP)‐like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell‐based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three‐dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino‐acid sidechains, Tle‐2 and dVal‐3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.

Cryptic ligand binding sites, which are not evident in the unligated structures, are beneficial in tackling with difficult but attractive drug targets, such as protein-protein interactions (PPIs). However, cryptic sites have thus far not been rationally pursued in the early stages of drug development. Here, we demonstrated by nuclear magnetic resonance that the cryptic site in Bcl-xL exists in a conformational equilibrium between the open and closed conformations under the unligated condition. While the fraction of the open conformation in the unligated wild-type Bcl-xL is estimated to be low, F143W mutation that is distal from the ligand binding site can substantially elevate the population. The F143W mutant showed a higher hit rate in a phage-display peptide screening, and the hit peptide bound to the cryptic site of the wild-type Bcl-xL. Therefore, by controlling the conformational equilibrium in the cryptic site, the opportunity to identify a PPI inhibitor could be improved.

The thermodynamic properties of a ligand in the bound state affect its binding specificity. Strict binding specificity can be achieved by introducing multiple spatially defined interactions, such as hydrogen bonds and van der Waals interactions, into the ligand-receptor interface. These introduced interactions are characterized by restricted local dynamics and improved surface complementarity in the bound state. In this study, we experimentally evaluated the local dynamics and the surface complementarity of weak-affinity ligands in the receptor-bound state by forbidden coherence transfer analysis in free-bound exchange systems (Ex-FCT), using the interaction between a ligand, a myocyte-enhancer factor 2A (MEF2A) docking peptide, and a receptor, p38α, as a model system. The Ex-FCT analyses successfully provided information for the rational design of a ligand with higher affinity and preferable thermodynamic properties for p38α.