Mechanisms of action and resistance in histone methylation-targeted therapy.

Epigenomes enable the rectification of disordered cancer gene expression, thereby providing new targets for pharmacological interventions. The clinical utility of targeting histone H3 lysine trimethylation (H3K27me3) as an epigenetic hallmark has been demonstrated1-7. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumour cells remain unclear. Here we show the potency and mechanisms of action and resistance of the EZH1-EZH2 dual inhibitor valemetostat in clinical trials of patients with adult T cell leukaemia/lymphoma. Administration of valemetostat reduced tumour size and demonstrated durable clinical response in aggressive lymphomas with multiple genetic mutations. Integrative single-cell analyses showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, including tumour suppressor genes. Nevertheless, subsequent long-term treatment encounters the emergence of resistant clones with reconstructed aggregate chromatin that closely resemble the pre-dose state. Acquired mutations at the PRC2-compound interface result in the propagation of clones with increased H3K27me3 expression. In patients free of PRC2 mutations, TET2 mutation or elevated DNMT3A expression causes similar chromatin recondensation through de novo DNA methylation in the H3K27me3-associated regions. We identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Targeting epigenetic drivers and chromatin homeostasis may provide opportunities for further sustained epigenetic cancer therapies.

Synthesis, activity, and their relationships of 2,4-diaminonicotinamide derivatives as EGFR inhibitors targeting C797S mutation.

The C797S mutation is one of the major factors behind resistance to the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. Herein, we describe the discovery of the 2,4-diaminonicotinamide derivative 5j, which shows potent inhibitory activity against EGFR del19/T790M/C797S and L858R/T790M/C797S. We also report the structure-activity relationship of the 2,4-diaminonicotinamide derivatives and the co-crystal structure of 5j and EGFR del19/T790M/C797S.

Flexibility and Cell Permeability of Cyclic Ras-Inhibitor Peptides Revealed by the Coupled Nosé-Hoover Equation.

Quantifying the cell permeability of cyclic peptides is crucial for their rational drug design. However, the reasons remain unclear why a minor chemical modification, such as the difference between Ras inhibitors cyclorasin 9A5 and 9A54, can substantially change a peptide's permeability. To address this question, we performed enhanced sampling simulations of these two 11-mer peptides using the coupled Nosé-Hoover equation (cNH) we recently developed. The present cNH simulations realized temperature fluctuations over a wide range (240-600 K) in a dynamic manner, allowing structural samplings that were well validated by nuclear Overhauser effect measurements. The derived structural ensembles were comprehensively analyzed by all-atom structural clustering, mapping the derived clusters onto principal components (PCs) that characterize the cyclic structure, and calculating cluster-dependent geometric and chemical properties. The planar-open conformation was dominant in aqueous solvent, owing to inclusion of the Trp side chain in the main-chain ring, while the compact-closed conformation, which favors cell permeation due to its compactness and high polarity, was also accessible. Conformation-dependent cell permeability was observed in one of the derived PCs, demonstrating that decreased cell permeability in 9A54 is due to the high free energy barrier separating the two conformations. The origin of the change in free energy surface was determined to be loss of flexibility in the modified residues 2-3, resulting from the increased bulkiness of their side chains. The derived molecular mechanism of cell permeability highlights the significance of complete structural dynamics surveys for accelerating drug development with cyclic peptides.

Discovery of DS79932728: A Potent, Orally Available G9a/GLP Inhibitor for Treating ß-Thalassemia and Sickle Cell Disease.

Therapeutic reactivation of the γ-globin genes for fetal hemoglobin (HbF) production is an attractive strategy for treating ß-thalassemia and sickle cell disease. It was reported that genetic knockdown of the histone lysine methyltransferase EHMT2/1 (G9a/GLP) is sufficient to induce HbF production. The aim of the present work was to acquire a G9a/GLP inhibitor that induces HbF production sufficiently. It was revealed that tetrahydroazepine has versatility as a side chain in various skeletons. We ultimately obtained a promising aminoindole derivative (DS79932728), a potent and orally bioavailable G9a/GLP inhibitor that was found to induce γ-globin production in a phlebotomized cynomolgus monkey model. This work could facilitate the development of effective new approaches for treating ß-thalassemia and sickle cell disease.

Discovery of novel histone lysine methyltransferase G9a/GLP (EHMT2/1) inhibitors: Design, synthesis, and structure-activity relationships of 2,4-diamino-6-methylpyrimidines.

The discovery and optimization of a novel series of G9a/GLP (EHMT2/1) inhibitors are described. Starting from known G9a/GLP inhibitor 5, efforts to explore the structure-activity relationship and optimize drug properties led to a novel compound 13, the side chain of which was converted to tetrahydroazepine. Compound 13 showed increased G9a/GLP inhibitory activity compared with compound 5. In addition, compound 13 exhibited improved human ether-a-go-go related gene (hERG) inhibitory activity over compound 5 and also improved pharmacokinetic profile in mice (oral bioavailability: 17 to 40%). Finally, the co-crystal structure of G9a in complex with compound 13 provides the basis for the further development of tetrahydroazepine-based G9a/GLP inhibitors.

Discovery of Novel Pyrido-pyridazinone Derivatives as FER Tyrosine Kinase Inhibitors with Antitumor Activity.

To obtain a new anticancer drug, we focused on FER tyrosine kinase. Starting with high-throughput screening with our in-house chemical library, compound 1, which has a pyridine moiety, was found. Referring to their X-ray crystal structure with FES proto-oncogene tyrosine kinase, as a surrogate of FER followed by chemical modification including scaffold hopping of the pyridine template, we discovered pyrido-pyridazinone derivatives with potent FER kinase inhibitory activity. Here, we disclose the structure-activity relationship on the scaffold and representative compound 21 (DS21360717), which showed in vivo antitumor efficacy in a subcutaneous tumor model.

A novel inhibitor stabilizes the inactive conformation of MAPK-interacting kinase 1.

Mitogen-activated protein kinase (MAPK)-interacting kinases 1 (Mnk1) and 2 (Mnk2) modulate translation initiation through the phosphorylation of eukaryotic translation initiation factor 4E, which promotes tumorigenesis. However, Mnk1 and Mnk2 are dispensable in normal cells, suggesting that the inhibition of Mnk1 and Mnk2 could be effective in cancer therapy. To provide a structural basis for Mnk1 inhibition, a novel Mnk1 inhibitor was discovered and the crystal structure of Mnk1 in complex with this inhibitor was determined. The crystal structure revealed that the inhibitor binds to the autoinhibited state of Mnk1, stabilizing the Mnk-specific DFD motif in the DFD-out conformation, thus preventing Mnk1 from switching to the active conformation and thereby inhibiting the kinase activity. These results provide a valuable platform for the structure-guided design of Mnk1 inhibitors.

Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor.

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.

Discovery and structure-guided fragment-linking of 4-(2,3-dichlorobenzoyl)-1-methyl-pyrrole-2-carboxamide as a pyruvate kinase M2 activator.

Tumor cells switch glucose metabolism to aerobic glycolysis by expressing the pyruvate kinase M2 isoform (PKM2) in a low active form, providing glycolytic intermediates as building blocks for biosynthetic processes, and thereby supporting cell proliferation. Activation of PKM2 should invert aerobic glycolysis to an oxidative metabolism and prevent cancer growth. Thus, PKM2 has gained attention as a promising cancer therapy target. To obtain novel PKM2 activators, we conducted a high-throughput screening (HTS). Among several hit compounds, a fragment-like hit compound with low potency but high ligand efficiency was identified. Two molecules of the hit compound bound at one activator binding site, and the molecules were linked based on the crystal structure. Since this linkage succeeded in maintaining the original position of the hit compound, the obtained compound exhibited highly improved potency in an in vitro assay. The linked compound also showed PKM2 activating activity in a cell based assay, and cellular growth inhibition of the A549 cancer cell line. Discovery of this novel scaffold and binding mode of the linked compound provides a valuable platform for the structure-guided design of PKM2 activators.

Quantitative Structure-activity Relationship (QSAR) Models for Docking Score Correction.

In order to improve docking score correction, we developed several structure-based quantitative structure activity relationship (QSAR) models by protein-drug docking simulations and applied these models to public affinity data. The prediction models used descriptor-based regression, and the compound descriptor was a set of docking scores against multiple (∼600) proteins including nontargets. The binding free energy that corresponded to the docking score was approximated by a weighted average of docking scores for multiple proteins, and we tried linear, weighted linear and polynomial regression models considering the compound similarities. In addition, we tried a combination of these regression models for individual data sets such as IC50 , Ki , and %inhibition values. The cross-validation results showed that the weighted linear model was more accurate than the simple linear regression model. Thus, the QSAR approaches based on the affinity data of public databases should improve docking scores.

Antitumor effects of novel highly hydrophilic and non-ATP-competitive MEK1/2 inhibitor, SMK-17.

The mitogen-activated protein kinase (MAPK) signal pathway plays a central role in regulating tumor cell proliferation, survival, and differentiation. The components of this pathway, Ras/Raf/MEK/ERK, are frequently activated in human cancers. Targeting this pathway is considered to be a promising anticancer strategy. In particular, MEK is an attractive drug target because of its high selectivity to ERK. We can expect potent growth inhibitory and proapoptotic effects by inhibiting MEK. Here, we report derivatives of N-[2-(2-chloro-4-iodo-phenylamino)-3,4-difluorophenyl]-methanesulfonamide as novel MEK1/2 inhibitors. Among these compounds, we found SMK-17 to be a potent MEK1/2 inhibitor with high aqueous solubility. The in-silico docking study suggested that SMK-17 is bound to an allosteric pocket of MEK1. The kinetic study and the kinase profiler analysis confirmed the allosteric nature of SMK-17. SMK-17 inhibited MEK1 kinase activity in a non-ATP-competitive manner and it was highly selective to MEK1 and 2. SMK-17 inhibited the growth of tumor cell lines in vitro. Especially, it seemed that cell lines harboring highly phosphorylated MEK1/2 and ERK1/2 were highly sensitive to SMK-17. Moreover, unlike previously reported MEK inhibitors, PD184352 or U0126, SMK-17 did not inhibit the phosphorylation of ERK5. In vivo, SMK-17 exhibited potent antitumor activity in animal models on oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways, which resulted in significant antitumor efficacy without notable side effects. These findings suggest that SMK-17, an exquisitely selective, orally available MEK1/2 inhibitor, is a useful chemical biology tool for characterizing the function of MEK/MAPK signaling both in vitro and in vivo.

Discovery of imidazo[1,2-b]pyridazines as IKKß inhibitors. Part 2: improvement of potency in vitro and in vivo.

We have increased the potency of imidazo[1,2-b]pyridazine derivatives as IKKß inhibitors with two strategies. One is to enhance the activity in cell-based assay by adjusting the polarity of molecules to improve permeability. Another is to increase the affinity for IKKß by the introduction of additional substituents based on the hypothesis derived from an interaction model study. These improved compounds showed inhibitory activity of TNFα production in mice.

Discovery of imidazo[1,2-b]pyridazine derivatives as IKKbeta inhibitors. Part 1: Hit-to-lead study and structure-activity relationship.

Imidazo[1,2-b]pyridazine derivatives from high-throughput screening were developed as IKKbeta inhibitors. By the optimization of the 3- and 6-position of imidazo[1,2-b]pyridazine scaffold, cell-free IKKbeta inhibitory activity and TNFalpha inhibitory activity in THP-1 cell increased. Also, these compounds showed high kinase selectivity. The structure-activity relationship was revealed and the interaction model of imidazo[1,2-b]pyridazine compounds with IKKbeta was constructed.