Chemical Biology Publications 2010
Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.
IMPORTANCE OF THE FIELD: Inflammatory diseases are one of the major health issues and have become a major focus in the pharmaceutical and biotech industries. To date, drugs prescribed for treatment of these diseases target enzymes that are not specific to the immune system resulting in adverse effects. The main challenge of this research field is, therefore, identifying targets that act specifically on the diseased tissue. AREAS COVERED IN THIS REVIEW: This review summarizes drug discovery efforts on kinases that have been identified as key players mediating inflammation and autoimmune disorders. In particular, we discuss recent developments on well-established targets such as mammalian target of rapamycin, JAK3, spleen tyrosine kinase, p38α and lymphocyte specific kinase but provide also a perspective on emerging targets. WHAT THE READER WILL GAIN: The reader will obtain an overview of drug discovery efforts on kinases in inflammation, recent clinical and preclinical data and developed inhibitor scaffolds. In addition, the reader will be updated on issues in target validation of current drug targets and the potential of selected novel kinase targets in this important disease area. TAKE HOME MESSAGE: Cellular signaling networks that regulate inflammatory response are still poorly understood making rational selection of targets challenging. Recent data suggest that kinase targets that are specific to the immune system and mediate signals immediately downstream of surface receptors are most efficacious in the clinic.
SUMMARY: The structural genomics of histone tail recognition web server is an open access resource that presents within mini articles all publicly available experimental structures of histone tails in complex with human proteins. Each article is composed of interactive 3D slides that dissect the structural mechanism underlying the recognition of specific sequences and histone marks. A concise text html-linked to interactive graphics guides the reader through the main features of the interaction. This resource can be used to analyze and compare binding modes across multiple histone recognition modules, to evaluate the chemical tractability of binding sites involved in epigenetic signaling and design small molecule inhibitors. AVAILABILITY: http://www.thesgc.org/resources/histone_tails/ CONTACT: firstname.lastname@example.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 2402010. Small-molecule bromodomain inhibitors for cancer therapy
Understanding of the molecular basis of long-term fear memory (fear LTM) formation provides targets in the treatment of emotional disorders. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is one of the key synaptic molecules involved in fear LTM formation. There are two endogenous inhibitor proteins of CaMKII, CaMKII N alpha and N beta, which can regulate CaMKII activity in vitro. However, the physiological role of these endogenous inhibitors is not known. Here, we have investigated whether CaMKII N beta protein expression is regulated after contextual fear conditioning or exposure to a novel context. Using a novel CaMKII N beta-specific antibody, CaMKII N beta expression was analysed in the naïve mouse brain as well as in the amygdala and hippocampus after conditioning and context exposure. We show that in naïve mouse forebrain CaMKII N beta protein is expressed at its highest levels in olfactory bulb, prefrontal and piriform cortices, amygdala and thalamus. The protein is expressed both in dendrites and cell bodies. CaMKII N beta expression is rapidly and transiently up-regulated in the hippocampus after context exposure. In the amygdala, its expression is regulated only by contextual fear conditioning and not by exposure to a novel context. In conclusion, we show that CaMKII N beta expression is differentially regulated by novelty and contextual fear conditioning, providing further insight into molecular basis of fear LTM.
The identification as cooperating targets of Proviral Integrations of Moloney virus in murine lymphomas suggested early on that PIM serine/threonine kinases play an important role in cancer biology. Whereas elevated levels of PIM1 and PIM2 were mostly found in hematologic malignancies and prostate cancer, increased PIM3 expression was observed in different solid tumors. PIM kinases are constitutively active and their activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates including several cell cycle regulators and apoptosis mediators. PIM1 but not PIM2 seems also to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression. Knockdown experiments by RNA interference or dominant-negative acting mutants suggested that PIM kinases are important for maintenance of a transformed phenotype and therefore potential therapeutic targets. Determination of the protein structure facilitated identification of an increasing number of potent small molecule PIM kinase inhibitors with in vitro and in vivo anticancer activity. Ongoing efforts aim to identify isoform-specific PIM inhibitors that would not only help to dissect the kinase function but hopefully also provide targeted therapeutics. Here, we summarize the current knowledge about the role of PIM serine/threonine kinases for the pathogenesis and therapy of hematologic malignancies and solid cancers, and we highlight structural principles and recent progress on small molecule PIM kinase inhibitors that are on their way into first clinical trials.
Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.
Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase with evidence of implication in growth dysregulation and apoptosis resistance, making it a relevant target for cancer therapy. Several CK2 inhibitors have been developed showing variable efficiency, emphasizing the need to expand the chemical diversity of those inhibitors. We report the identification and characterization of 2,8-difurandicarboxylic acid derivatives as a new class of nanomolar ATP-competitive inhibitors. Selectivity profiling pointed out proviral insertion Moloney virus kinases (Pim kinases) as the only other kinases that are significantly inhibited. By combining structure-activity relationship analysis with structural determination, we were able to determine the binding mode of these inhibitors for both kinases and to explain their strong inhibitory potency. Essential chemical features necessary for activity on both kinases were then identified. The described compounds are not cell permeable: however, they could provide a lead for developing novel inhibitors usable also in vivo. Given the similar but not redundant pathophysiological functions of CK2 and Pim family members, such inhibitors would provide new attractive leads for targeted cancer therapy. This work highlights that 2 functionally related kinases from different kinome branches display exquisite sensitivity to a common inhibitor.
Nat Chem Biol, 6 (3), pp. 166-169. | Citations: 167 (Scopus) | Read more2010. The (un)targeted cancer kinome.
UNLABELLED: Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.
BACKGROUND: The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains. PRINCIPAL FINDINGS: We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family. SIGNIFICANCE: The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
Protein tyrosine phosphatases (PTPs) are essential regulators of signal transduction pathways, and control, together with protein tyrosine kinases (PTKs), the reversible phosphorylation of tyrosine residues. PTPs are, from a structural biology viewpoint, one of the most comprehensively covered protein families. This chapter focuses on the structural comparison of catalytic domains of the major PTP families, and summarizes implications of the available structural data for the understanding of PTP regulation and for the design of specific PTP inhibitors. Modular domains outside the catalytic domain also significantly regulate PTP function. Inactivation of PTPs by receptor dimerization is discussed as a key regulatory mechanism of PTP activity. This control mechanism was first suggested based on structural studies of the membrane proximal D1 domain of RPTP α. More structural data regarding phosphatases in complex with their substrates and regulators will be available in the near future, which will allow a more detailed insight into the regulation and substrate recognition of this diverse class of enzymes. © 2010 Elsevier Inc. All rights reserved.
Protein structure determination of soluble globular protein domains has developed into an efficient routine technology which can now be applied to generate and analyze structures of entire human protein families. In the kinase area, several kinase families still lack comprehensive structural analysis. Nevertheless, Structural Genomics (SG) efforts contributed more than 40 kinase catalytic domain structures during the past 4 years providing a rich resource of information for large scale comparisons of kinase active sites. Moreover, many of the released structures are inhibitor complexes that offer chemical starting points for development of selective and potent inhibitors. Here we discuss the currently available structural data and strategies that can be utilized for the development of highly selective inhibitors.
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