Dissecting the Kinetic Mechanism of Human Lysine Methyltransferase 2D and Its Interactions with the WRAD2 Complex.

Human lysine methyltransferase 2D (hKMT2D) is an epigenetic writer catalyzing the methylation of histone 3 lysine 4. hKMT2D by itself has little catalytic activity and reaches full activation as part of the WRAD2 complex, additionally comprising binding partners WDR5, RbBP5, Ash2L, and DPY30. Here, a detailed mechanistic study of the hKMT2D SET domain and its WRAD2 interactions is described. We characterized the WRAD2 subcomplexes containing full-length components and the hKMT2D SET domain. By performing steady-state analysis as a function of WRAD2 concentration, we identified the inner stoichiometry and determined the binding affinities for complex formation. Ash2L and RbBP5 were identified as the binding partners critical for the full catalytic activity of the SET domain. Contrary to a previous report, product and dead-end inhibitor studies identified hKMT2D as a rapid equilibrium random Bi-Bi mechanism with EAP and EBQ dead-end complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) analysis showed that hKMT2D uses a distributive mechanism and gives further insights into how the WRAD2 components affect mono-, di-, and trimethylation. We also conclude that the Win motif of hKMT2D is not essential in complex formation, unlike other hKMT2 proteins.

Novel potent and selective pyrazolylpyrimidine-based SYK inhibitors.

Hybridisation of amino-pyrimidine based SYK inhibitors (e.g. 1a) with previously reported diamine-based SYK inhibitors (e.g. TAK-659) led to the identification and optimisation of a novel pyrimidine-based series of potent and selective SYK inhibitors, where the original aminomethylene group was replaced by a 3,4-diaminotetrahydropyran group. The initial compound 5 achieved excellent SYK potency. However, it suffered from poor permeability and modest kinase selectivity. Further modifications of the 3,4-diaminotetrahydropyran group were identified and the interactions of those groups with Asp512 were characterised by protein X-ray crystallography. Further optimisation of this series saw mixed results where permeability and kinase selectivity were increased and oral bioavailability was achieved in the series, but at the expense of potent hERG inhibition.

Identification of a novel series of azabenzimidazole-derived inhibitors of spleen tyrosine kinase.

Spleen Tyrosine Kinase (SYK) is a well-studied enzyme with therapeutic applications in oncology and autoimmune diseases. We identified an azabenzimidazole (ABI) series of SYK inhibitors by mining activity data of 86,000 compounds from legacy biochemical assays with SYK and other homologous kinases as target enzymes. A structure-based design and hybridization approach was then used to improve the potency and kinase selectivity of the hits. Lead compound 23 from this novel ABI series has a SYK IC50 = 0.21 nM in a biochemical assay and inhibits growth of SUDHL-4 cells at a GI50 = 210 nM.

Correlative cryo-soft X-ray tomography and cryo-structured illumination microscopy reveal changes to lysosomes in amyloid-ß-treated neurons.

Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer's disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology.

Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases.

Novel substituted benzylidene-1,3-thiazolidine-2,4-diones (TZDs) have been identified as potent and highly selective inhibitors of the PIM kinases. The synthesis and SAR of these compounds are described, along with X-ray crystallographic, anti-proliferative, and selectivity data.

Sources of 2,5-diaminoimidazolone lesions in DNA damage initiated by hydroxyl radical attack.

The present study reports radiation-chemical yields of 2.5-diaminoimidazolone (Iz) derivatives in X-irradiated phosphate-buffered solutions of guanosine and double-stranded DNA. Various gassing conditions (air, N20/O2 (4:1), N2O, vacuum) were employed to elucidate the contribution of several alternative pathways leading to Iz in reactions initiated by hydroxyl radical attack on guanine. In all systems, Iz was identified as the second by abundance guanine degradation product after 8-oxoguanine, formed in 1:5 (guanosine) and 1:3.3 (DNA) ratio to the latter in air-saturated solutions. Experimental data strongly suggest that the addition of molecular oxygen to the neutral guanine radical G(-H)• plays a major in Iz production in oxygenated solutions of double-stranded DNA while in other systems it may compete with recombination of G(-H)• with superoxide and/or alkyl peroxyl radicals. The production of Iz through hydroxyl radical attack on 8-oxoguanine was also shown to take place although the chemical yield of Iz (ca 6%) in this process is too low to compete with the other pathways. The linearity of Iz accumulation with dose also indicates a negligible contribution of this channel to its yield in all systems.

Structural insights into the critical DNA damage sensors DNA-PKcs, ATM and ATR.

ATM, ATR and DNA-PKCs are key effectors of DNA Damage response and have been extensively linked to tumourigenesis and survival of cancer cells after radio/chemotherapy. Despite numerous efforts, the structures of these proteins remained elusive until very recently. The resolution revolution in Cryo-EM allowed for molecular details of these proteins to be seen for the first time. Here we provide a comprehensive review of the structures of ATM, ATR and DNA-PKcs and their complexes and expand with observations springing from our own cryo-EM studies. These observations include a novel conformation of ATR and novel dimeric arrangements of DNA-PKcs.

Structures of closed and open conformations of dimeric human ATM.

ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase-related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, insulin signaling, and neurogenesis. Our electron cryomicroscopy (cryo-EM) suggests that human ATM is in a dynamic equilibrium between closed and open dimers. In the closed state, the PIKK regulatory domain blocks the peptide substrate-binding site, suggesting that this conformation may represent an inactive or basally active enzyme. The active site is held in this closed conformation by interaction with a long helical hairpin in the TRD3 (tetratricopeptide repeats domain 3) domain of the symmetry-related molecule. The open dimer has two protomers with only a limited contact interface, and it lacks the intermolecular interactions that block the peptide-binding site in the closed dimer. This suggests that the open conformation may be more active. The ATM structure shows the detailed topology of the regulator-interacting N-terminal helical solenoid. The ATM conformational dynamics shown by the structures represent an important step in understanding the enzyme regulation.

Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.

Potent and Selective Inhibitors of CK2 Kinase Identified through Structure-Guided Hybridization.

In this paper we describe a series of 3-cyano-5-aryl-7-aminopyrazolo[1,5-a]pyrimidine hits identified by kinase-focused subset screening as starting points for the structure-based design of conformationally constrained 6-acetamido-indole inhibitors of CK2. The synthesis, SAR, and effects of this novel series on Akt signaling and cell proliferation in vitro are described.

Structural basis of substrate methylation and inhibition of SMYD2.

Protein lysine methyltransferases are important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine to specific acceptor lysines on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate nonhistone protein substrates, revealing an additional mechanism to regulate cellular physiology. The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target. Here we report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. We also present the crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505. This substrate competitive inhibitor is bound in the peptide binding groove of SMYD2. These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class.

Discovery of 5-chloro-N2-[(1S)-1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (AZD1480) as a novel inhibitor of the Jak/Stat pathway.

The myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis are a heterogeneous but related group of hematological malignancies characterized by clonal expansion of one or more myeloid lineages. The discovery of the Jak2 V617F gain of function mutation highlighted Jak2 as a potential therapeutic target in the MPNs. Herein, we disclose the discovery of a series of pyrazol-3-yl pyrimidin-4-amines and the identification of 9e (AZD1480) as a potent Jak2 inhibitor. 9e inhibits signaling and proliferation of Jak2 V617F cell lines in vitro, demonstrates in vivo efficacy in a TEL-Jak2 model, has excellent physical properties and preclinical pharmacokinetics, and is currently being evaluated in Phase I clinical trials.