Effect of bet missense mutations on bromodomain function, inhibitor binding and stability

Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of attention in the oncology field resulting in the rapid translation of early BET bromodomain inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism or found in cancer on BET bromodomain function and stability and the influence of these mutants on inhibitor binding. We found that most BET missense mutations localize to peripheral residues in the two terminal helices. Crystal structures showed that the three dimensional structure is not compromised by these mutations but mutations located in close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding. Most mutations affect significantly protein stability and tertiary structure in solution, suggesting new interactions and an alternative network of protein-protein interconnection as a consequence of single amino acid substitution. To our knowledge this is the first report studying the effect of mutations on bromodomain function and inhibitor binding

Direct Observation of Reductive Coupling Mechanism between Oxygen and Iron/Nickel in Cobalt-Free Li-Rich Cathode Material: An in Operando X-Ray Absorption Spectroscopy Study

Li-rich cathodes possess high capacity and are promising candidates in next-generation high-energy density Li-ion batteries. This high capacity is partly attributed to its poorly understood oxygen-redox activity. The present Li-rich cathodes contain expensive and environmentally-incompatible cobalt as a main transition metal. In this work, cobalt-free, iron-containing Li-rich cathode material (nominal composition Li$_{1.2}$Mn$_{0.56}$Ni$_{0.16}$Fe$_{0.08}$O$_{2}$) is synthesized, which exhibits excellent discharge capacity (≈250 mAh g$^{-1}$ and cycling stability. In operando, X-ray absorption spectroscopy at Mn, Fe, and Ni K edges reveals its electrochemical mechanism. X-ray absorption near edge structure (XANES) features of Fe and Ni K edges show unusual behavior: when an electrode is charged to 4.5 V, Fe and Ni K edges’ XANES features shift to higher energies, evidence for Fe$^{3+}$→Fe$^{4+}$ and Ni$^{2+}$→Ni$^{4+}$ oxidation. However, when charged above 4.5 V, XANES features of Fe and Ni K edges shift back to lower energies, indicating Fe$^{4+}$→Fe$^{3+}$ and Ni$^{4+}$→Ni$^{3+}$ reduction. This behavior can be linked to a reductive coupling mechanism between oxygen and Fe/Ni. Though this mechanism is observed in Fe-containing Li-rich materials, the only electrochemically active metal in such cases is Fe. Li$_{1.2}$Mn$_{0.56}$Ni$_{0.16}$Fe$_{0.08}$O$_{2}$ has multiple electrochemically active metal ions; Fe and Ni, which are investigated simultaneously and the obtained results will assist tailoring of cost-effective Li-rich materials

A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B

Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions

Diverse Levels of Sequence Selectivity and Catalytic Efficiency of Protein-Tyrosine Phosphatases

The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences, but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >105-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >105-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3–18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A co-crystal structure of PTP1B bound with a nephrin pY1193 peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed

Pharmacoproteomic characterisation of human colon and rectal cancer

Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials. © 2017 The Authors. Published under the terms of the CC BY 4.0 licens

Bromodomains as therapeutic targets

Acetylation of lysine residues is a post-translational modification with broad relevance to cellular signalling and disease biology. Enzymes that ‘write’ (histone acetyltransferases, HATs) and ‘erase’ (histone deacetylases, HDACs) acetylation sites are an area of extensive research in current drug development, but very few potent inhibitors that modulate the ‘reading process’ mediated by acetyl lysines have been described. The principal readers of ɛ-N-acetyl lysine (Kac) marks are bromodomains (BRDs), which are a diverse family of evolutionary conserved protein-interaction modules. The conserved BRD fold contains a deep, largely hydrophobic acetyl lysine binding site, which represents an attractive pocket for the development of small, pharmaceutically active molecules. Proteins that contain BRDs have been implicated in the development of a large variety of diseases. Recently, two highly potent and selective inhibitors that target BRDs of the BET (bromodomains and extra-terminal) family provided compelling data supporting targeting of these BRDs in inflammation and in an aggressive type of squamous cell carcinoma. It is likely that BRDs will emerge alongside HATs and HDACs as interesting targets for drug development for the large number of diseases that are caused by aberrant acetylation of lysine residues

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Structural and biophysical studies reveal how CaMKII kinases, which are important for cellular learning and memory, are switched on by binding of Ca2+/calmodulin