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Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer. While long drug-target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors have been reported. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM., The arginine methyltransferase PRMT5 is over-expressed in cancer and has a role in the maintenance of stem cells. Here, the authors show that PRMT5 inhibitors can block the growth of patient derived glioblastoma stem cell cultures in vitro and in vivo, suggesting that PRMT5 inhibition may be a useful therapeutic strategy

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif (UHM), a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3’ splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology (GO) analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome, but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 200 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on gene expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Finally, the functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration of the cells. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.

Graphical abstract, Kinases are signalling proteins which have proven to be successful targets for the treatment of a variety of diseases, predominantly in cancers. However, only a small proportion of kinases (

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint securing proper chromosome segregation. It is being evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer with several reversible inhibitors currently undergoing clinical trials. While long drug–target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors are known. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor RMS-07 targeting a cysteine (Cys604) in the kinase's hinge region present only in few other protein kinases. The compound showed excellent MPS1 inhibitory potency and high selectivity against all protein kinases harboring an equivalent cysteine as well as in a larger differential scanning fluorimetry-based screening panel. Covalent binding was confirmed by mass spectrometry and X-ray crystal structure. We expect this tool compound to open new avenues for the design of MPS1-specific covalent chemical probes or drugs.

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections., Author summary Target-based drug discovery strategies hold the promise to discover safer and more effective treatments for Neglected Tropical Diseases (NTDs). Genetic manipulation techniques have been used to successfully identify essential genes in eukaryotic parasites. Unfortunately, the fact that a gene is essential under controlled laboratory conditions does not automatically make the corresponding gene-product vulnerable to pharmacological intervention in a clinical setting within the human host. To allow the discovery and development of small molecule tool compounds that can be used to validate pharmacologically vulnerable targets, one must first establish compound screening assays and obtain structural information for the candidate target. Eukaryotic cells lacking deoxyhypusine synthase (DHS) function are not viable. DHS catalyzes the first step in a post-translational modification that is critical for the function of eIF5A. Presence of mature eIF5A is also essential for eukaryotic cell viability. Here we reported compound screening assays (yeast-based for Brugia malayi and Leishmania major; in vitro for B. malayi only) and provided further regulatory and structural insights we hope will aid in the identification and development of inhibitors for the DHS enzymes from two NTD-causing organisms—B. malayi, the causative agent of lymphatic filariasis and L. major, the causative agent of cutaneous leishmaniasis.

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.

The control of mRNA translation has key roles in the regulation of gene expression and biological processes such as mammalian cellular differentiation and identity. Methodological advances in the last decade have resulted in considerable progress towards understanding how translational control contributes to the regulation of diverse biological phenomena. In this review, we discuss recent findings in the involvement of translational control in the mammalian neocortex development and neuronal biology. We focus on regulatory mechanisms that modulate translational efficiency during neural stem cells self-renewal and differentiation, as well as in neuronal-related processes such as synapse, plasticity, and memory.

Alterations in microRNA (miRNA) processing have been previously linked to aging. Here we used the small molecule enoxacin to pharmacologically interfere with miRNA biogenesis and study how it affects aging in C. elegans. Enoxacin extended worm lifespan and promoted survival under normal and oxidative stress conditions. Enoxacin-induced longevity required the transcription factor SKN-1/Nrf2 and was blunted by the antioxidant N-acetyl-cysteine, suggesting a prooxidant-mediated mitohormetic response. The longevity effects of enoxacin were also dependent on the miRNA pathway, consistent with changes in miRNA expression elicited by the drug. Among these differentially expressed miRNAs, the widely conserved miR-34-5p was found to play an important role in enoxacin-mediated longevity. Enoxacin treatment down-regulated miR-34-5p and did not further extend lifespan of long-lived mir-34 mutants. Moreover, N-acetyl-cysteine abrogated mir-34(gk437)-induced longevity. Evidence also points to double-stranded RNA-specific adenosine deaminases (ADARs) as new targets of enoxacin since ADAR loss-of-function abrogates enoxacin-induced lifespan extension. Thus, enoxacin increases lifespan by reducing miR-34-5p levels, interfering with the redox balance and promoting healthspan., Highlights • Enoxacin extends lifespan and promotes healthspan in C. elegans. • Enoxacin downregulates miR-34-5p to prolong lifespan. • Longevity induced by enoxacin and mir-34 loss of function involves a mitohormetic response. • Double-stranded RNA-specific adenosine deaminases (ADARs) are potential targets of enoxacin.

Recent studies have suggested abnormal brain network organization in subjects with Autism Spectrum Disorders (ASD). Here we applied spectral clustering algorithm, diverse centrality measures (betweenness (BC), clustering (CC), eigenvector (EC), and degree (DC)), and also the network entropy (NE) to identify brain sub-systems associated with ASD. We have found that BC increases in the following ASD clusters: in the somatomotor, default-mode, cerebellar, and fronto-parietal. On the other hand, CC, EC, and DC decrease in the somatomotor, default-mode, and cerebellar clusters. Additionally, NE decreases in ASD in the cerebellar cluster. These findings reinforce the hypothesis of under-connectivity in ASD and suggest that the difference in the network organization is more prominent in the cerebellar system. The cerebellar cluster presents reduced NE in ASD, which relates to a more regular organization of the networks. These results might be important to improve current understanding about the etiological processes and the development of potential tools supporting diagnosis and therapeutic interventions.

The Pre-mRNA processing factor 4B Kinase is a serine/threonine kinase responsible for the phosphorylation of essentials proteins of the tri-snRNP complex in the B Complex of the spliceosome in mammals, being that the overexpression of PRP4K has being associated with certain ovary cancer forms, Paclitaxel and Carboplatin resistance³. The understanding of the kinase domain is crucial for the ligand-based rational drug design, and our research group have being working with the obtainment of high resolution crystallographic structures of this protein domain. In this work we present some advances in the protein PRP4K kinase domain production and purification, and also the attempts of protein overexpression in mammals cells.

Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their therapeutic potential, there are no inhibitors available for VRK1 or VRK2. We report here the discovery and elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compounds displayed KD values of 190 nM and 401 nM for VRK1 and VRK2, respectively. Differences in compound binding mode and substituent preferences between the two VRKs were identified by the series structure-activity relationship combined with the crystallographic analysis of key compounds. We expect that our results will serve as a starting point for the design of specific and potent inhibitors against each of the two VRKs based on a pyridine scaffold.

Background Plants reprogram metabolism and development to rapidly adapt to biotic and abiotic stress. Protein kinases play a significant role in this process by phosphorylating protein substrates that activate or inactivate signaling cascades that regulate cellular and metabolic adaptations. Despite their importance in plant biology, a notably small fraction of the plant kinomes has been studied to date. Results In this report, we describe ZmDRIK1, a stress-responsive receptor-like pseudokinase whose expression is downregulated under water restriction. We show the structural features and molecular basis of the absence of ATP binding exhibited by ZmDRIK1. The ZmDRIK1 kinase domain lacks conserved amino acids that are essential for phosphorylation activity. The crystal structure of the ZmDRIK1 kinase domain revealed the presence of a spine formed by the side chain of the triad Leu240, Tyr363, and Leu375 that occludes the ATP binding pocket. Although ZmDRIK1 is unable to bind nucleotides, it does bind the small molecule ENMD-2076 which, in a cocrystal structure, revealed the potential to serve as a ZmDRIK1 inhibitor. Conclusion ZmDRIK1 is a novel receptor-like pseudokinase responsive to biotic and abiotic stress. The absence of ATP binding and consequently, the absence of phosphorylation activity, was proven by the crystal structure of the apo form of the protein kinase domain. The expression profiling of the gene encoding ZmDRIK1 suggests this kinase may play a role in downregulating the expression of stress responsive genes that are not necessary under normal conditions. Under biotic and abiotic stress, ZmDRIK1 is down-regulated to release the expression of these stress-responsive genes.

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s - known as SAPKs (Stress/ABA-activated Protein Kinases). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2-box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2-box and ABA-box. Surprisingly, mutations in the SnRK2-box did not drastically affect sugarcane SnRK2 activity, in contrast to previous observations for the homologous proteins in Arabidopsis. Also, we found that the ABA-box might have a role in SnRK2 activation in the absence of PP2C phosphatase. Taken together, our results demonstrate that both C-terminal domains of sugarcane SnRK2 proteins play a fundamental role in protein activation and activity.

RNA interference (RNAi) is a valuable technique to determine gene function. In Caenorhabditis elegans, RNAi can be achieved by feeding worms bacteria carrying a plasmid expressing double-stranded RNA (dsRNA) targeting a gene of interest. The most commonly used plasmid vector for this purpose is L4440. However, it has been noticed that sequences within L4440 may elicit unspecific effects. Here, we provide a comprehensive characterization of these effects and their mechanisms and describe new unexpected phenotypes uncovered by the administration of unspecific exogenous dsRNA. An example involves dsRNA produced by the multiple cloning site (MCS) of L4440, which shares complementary sequences with some widely used reporter vectors and induces partial transgene silencing via the canonical and antiviral RNAi pathway. Going beyond transgene silencing, we found that the reduced embryonic viability of mir-35-41(gk262) mutants is partially reversed by exogenous dsRNA via a mechanism that involves canonical RNAi. These results indicate cross-regulation between different small RNA pathways in C. elegans to regulate embryonic viability. Recognition of the possible unspecific effects elicited by RNAi vectors is important for rigorous interpretation of results from RNAi-based experiments.

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no specific small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homologue, CAMKK1, are thought to have overlapping biological roles. Here we present six novel co-structures of CAMKK2 bound to potent ligands identified from a library of ATP-competitive kinase inhibitors. Isothermal titration calorimetry (ITC) revealed that binding to some of these molecules is enthalpy driven. We expect our results to further advance current efforts to discover small molecule kinase inhibitors specific to each human CAMKK.

A regulação de splicing por proteínas kinases é uma área emergente de pesquisa devido ao seu potencial como alvo de drogas, especialmente em casos de câncer e doenças neurológicas. Uma das kinases associadas à regulação de proteínas de splicing, a Pre-mRNA processing factor 4B kinase (PRPF4), foi também relacionada à progressão do câncer de mama. Tal proteína tem função e mecanismo ainda desconhecidos. Esse projeto visa a contribuir para o entendimento dos níveis endógenos da proteína PRPF4 em células de câncer de mama que foram relacionadas com o marcador tumoral de mama HER2 (receptor-2 do fator de crescimento epidérmico humano). Além disso, existem poucas informações estruturais sobre a proteína PRPF4 e um ponto de partida é o entendimento da estrutura do domínio de kinase por cristalografia. Por isso, propusemos expressar e purificar o domínio de kinase da proteína PRPF4 utilizando sistema de células de inseto. Assim, podemos contribuir para o próximo passo de desenvolvimento de moléculas inibidoras dessa kinase.

Made available in DSpace on 2019-10-04T12:42:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-15 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundacao Arthur Bernardes (FUNARBE - Program FUNARBE/FUNARPEX) The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (-50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated Delta N Delta S-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain. (C) 2019 Published by Elsevier B.V. Univ Fed Vicosa, Dept Bioquim & Biol Mol, Vicosa, MG, Brazil Univ Sao Paulo, Inst Quim Sao Carlos, Sao Carlos, SP, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil Univ Estadual Campinas, Inst Biol, Dept Genet Evolucao Microbiol & Imunol, Campinas, SP, Brazil Univ Fed Vicosa, Dept Vet, Vicosa, MG, Brazil Univ Estadual Campinas, Inst Biol, Dept Bioquim & Biol Tecidual, Campinas, SP, Brazil Univ Estadual Campinas, Fac Ciencias Farmaceut, Campinas, SP, Brazil Univ Estadual Campinas, Struct Genom Consortium, Av Dr Andre Tosello 550, Campinas, SP, Brazil Univ Oxford, Struct Genom Consortium, Old Rd Campus,Res Bldg,Roosevelt Dr, Oxford OX3 7DQ, England Univ Estadual Campinas, CBMEG, Ctr Mol Biol & Genet Engn, Campinas, SP, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil CNPq: 485011/2012-3 CNPq: 420648/2016-0 CNPq: 471415/2013-8 CNPq: 303129/2015-8 FAPEMIG: CBB-APQ-01637-13 FAPEMIG: CBB-APQ-02556-15 FAPEMIG: RED-00140-16 FAPESP: 2011/23110-0 FAPESP: 2012/50161-8 FAPESP: 2014/07206-6 FAPESP: 2017/07335-9 FAPESP: 2012/00195-3 FAPESP: 13/50724-5 FAPESP: 2017/03489-1 CAPES: 001

The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities.

The PR-1 proteins (pathogenesis-related protein 1) are involved in plant defense mechanisms against various pathogens. The genome of cacao (Theobroma cacao) encodes 14 PR-1 proteins, named TcPR-1a to TcPR-1n. Two of them, TcPR-1f and TcPR-1g, have a C-terminal expansion with high similarity to protein kinase domains, suggesting a receptor-like kinase (RLK) protein architecture. Moreover, TcPR-1g is highly expressed during cacao response to Witches' Broom Disease, caused by the fungus Moniliopthora perniciosa. Here we describe a structural genomics approach to clone, express and purify the kinase domains of TcPR-1f and TcPR-1g. Escherichia coli BL21(DE3)-R3 cells were used for protein expression and co-expression of Lambda Protein Phosphatase was critical for successfully obtaining soluble recombinant protein. We expect that the ability to express and purify the kinase domains of TcPR-1f and TcPR-1g will further our understanding of the role these proteins play during cacao defense response.

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

RNA-binding proteins affect cellular metabolic programs through development and in response to cellular stimuli. Though much work has been done to elucidate the roles of a handful of RNA-binding proteins and their effect on RNA metabolism, the progress of studies to understand the effects of post-translational modifications of this class of proteins is far from complete. This chapter summarizes the work that has been done to identify the consequence of post-translational modifications to some RNA-binding proteins. The effects of these modifications have been shown to increase the panoply of functions that a given RNA-binding protein can assume. We will survey the experimental methods that are used to identify the presence of several protein modifications and methods that attempt to discern the consequence of these modifications.

RNA-binding proteins affect cellular metabolic programs through development and in response to cellular stimuli. Though much work has been done to elucidate the roles of a handful of RNA-binding proteins and their effect on RNA metabolism, the progress of studies to understand the effects of post-translational modifications of this class of proteins is far from complete. This chapter summarizes the work that has been done to identify the consequence of post-translational modifications to some RNA-binding proteins. The effects of these modifications have been shown to increase the panoply of functions that a given RNA-binding protein can assume. We will survey the experimental methods that are used to identify the presence of several protein modifications and methods that attempt to discern the consequence of these modifications.

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

The C. elegans microRNA let-7 regulates developmental progression, and human let-7 has been implicated in disease. Examination of endogenous let-7 expression in C. elegans now reveals a complex regulation process whereby pri-let-7 is regulated transcriptionally with oscillations during each larval stage, and co-transcriptionally by LIN-28 which prevents mature microRNA accumulation during early larval stages.

The adult mammalian brain contains self-renewable, multipotent neural stem cells (NSCs) that are responsible for neurogenesis and plasticity in specific regions of the adult brain. Extracellular matrix, vasculature, glial cells, and other neurons are components of the niche where NSCs are located. This surrounding environment is the source of extrinsic signals that instruct NSCs to either self-renew or differentiate. Additionally, factors such as the intracellular epigenetics state and retrotransposition events can influence the decision of NSC's fate into neurons or glia. Extrinsic and intrinsic factors form an intricate signaling network, which is not completely understood. These factors altogether reflect a few of the key players characterized so far in the new field of NSC research and are covered in this review.

MicroRNAs (miRNAs) constitute an abundant family of 22-nucleotide RNAs that base-pair to target mRNAs and typically inhibit their expression. To assess the global impact of animal miRNAs on gene regulation, the expression of predicted targets and their cognate miRNAs was extensively analyzed in mammals and Drosophila.1,2 In general, targets are co-expressed at relatively low or undetectable levels in the same tissues as the miRNAs predicted to regulate them. Additionally, genes that are highly co-expressed with miRNAs usually lack target sites. The authors conclude that many animal genes are under evolutionary pressure to maintain or avoid complementary sites to miRNAs.1,2 Thus, the miRNA pathway broadly contributes to the complex gene regulatory networks that shape animal tissue development and identity. BioEssays 28: 449–452, 2006. © 2006 Wiley Periodicals, Inc.

Summary MicroRNAs (miRNAs) are ∼22 nucleotide RNAs that negatively regulate the expression of protein-coding genes. In a present model of miRNA function in animals, miRNAs that form imperfect duplexes with their targets inhibit protein expression without affecting mRNA levels. Here, we report that in C. elegans , regulation by the let-7 miRNA results in degradation of its lin-41 target mRNA, despite the fact that its 3′UTR regulatory sequences can only partially base-pair with the miRNA. Furthermore, lin-14 and lin-28 are targets of the lin-4 miRNA, and we show that the mRNA levels for these protein-coding genes significantly decrease in response to lin-4 expression. This study reveals that mRNAs containing partial miRNA complementary sites can be targeted for degradation in vivo, raising the possibility that regulation at the level of mRNA stability may be more common than previously appreciated for the miRNA pathway.

The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.

RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo–RNAi) or natural endogenous (endo–RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo–RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo–siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs., Author Summary RNA interference (RNAi) has become a widely used approach for silencing genes of interest. This tool is possible because endogenous RNA silencing pathways exist broadly across organisms, including humans, worms, and plants. The general RNAi pathway utilizes small ∼21-nucleotide RNAs to target specific protein-coding genes through base-pairing interactions. Since RNAs from exogenous sources require some of the same factors as endogenous small RNAs to silence gene expression, there can be competition between the pathways. Thus, perturbations in the endogenous RNAi pathway can result in enhanced silencing efficiency by exogenous small RNAs. MicroRNAs (miRNAs) comprise another endogenous small RNA pathway, but their biogenesis and mechanism of gene silencing are distinct in many ways from RNAi pathways. Here we show that a family of miRNAs regulates the effectiveness of RNAi in Caenorhabditis elegans. Loss of mir-35-41 results in enhanced RNAi by exogenous RNAs and reduced silencing of endogenous RNAi targets. The embryonic miR-35-41 miRNAs regulate the sensitivity to RNAi through lin-35/Rb, a homolog of the human Retinoblastoma tumor suppressor gene previously shown to regulate RNAi effectiveness in C. elegans. Additionally, we show that this sensitivity can be passed on to the next generation of worms, demonstrating a far-reaching effect of the miR-35-41 miRNAs on gene regulation by other small RNA pathways.

Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha 2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/beta-actin-mRNA ratio (mean +/- SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 +/- 0.15; N = 5; P0.05) compared to non-responders (0.35 +/- 0.17; N = 12) and controls (0.30 +/- 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

A recent study by Massirer et al. in the nematode C. elegans has shown that a family of microRNAs (miRNAs), miR-35-41, regulates the efficiency of RNA interference (RNAi), revealing a new connection between these small RNA pathways. In this commentary, we discuss the potential mechanisms for cross regulation in the miRNA and RNAi pathways and the implications for gene expression. While miRNAs are genetically encoded, the small interfering RNAs (siRNAs) that function in RNAi can originate from processing of exogenous dsRNA (exo-RNAi) or from the production of siRNAs from endogenous transcripts (endo-RNAi). These small RNA pathways involve Dicer and Argonaute proteins and typically use antisense base pairing to target mRNAs for downregulated expression. The discovery that loss of miR-35–41 results in enhanced exo-RNAi sensitivity and reduced endo-RNAi effectiveness suggests that these miRNAs normally help balance the RNAi pathways. The effect of mir-35–41 on RNAi is largely through lin-35, the C. elegans homolog of the tumor suppressor Retinoblastoma (Rb) gene. lin-35/Rb previously has been shown to regulate RNAi sensitivity through unclear mechanisms and the new finding that accumulation of LIN-35/Rb protein is dependent on miR-35–41 adds another layer of complexity to this process. The utilization of miRNAs to control the responsiveness of RNAi exemplifies the cross-regulation embedded in small RNA-directed pathways.

The application of graph analysis methods to the topological organization of brain connectivity has been a useful tool in the characterization of brain related disorders. However, the availability of tools, which enable researchers to investigate functional brain networks, is still a major challenge. Most of the studies evaluating brain images are based on centrality and segregation measurements of complex networks. In this study, we applied the concept of graph spectral entropy (GSE) to quantify the complexity in the organization of brain networks. In addition, to enhance interpretability, we also combined graph spectral clustering to investigate the topological organization of sub-network's modules. We illustrate the usefulness of the proposed approach by comparing brain networks between attention deficit hyperactivity disorder (ADHD) patients and the brain networks of typical developing (TD) controls. The main findings highlighted that GSE involving sub-networks comprising the areas mostly bilateral pre and post central cortex, superior temporal gyrus, and inferior frontal gyri were statistically different (p-value=0.002) between ADHD patients and TD controls. In the same conditions, the other conventional graph descriptors (betweenness centrality, clustering coefficient, and shortest path length) commonly used to identify connectivity abnormalities did not show statistical significant difference. We conclude that analysis of topological organization of brain sub-networks based on GSE can identify networks between brain regions previously unobserved to be in association with ADHD.

Large-scale genome projects have generated a rapidly increasing number of DNA sequences. Therefore, development of computational methods to rapidly analyze these sequences is essential for progress in genomic research. Here we present an automatic annotation system for preliminary analysis of DNA sequences. The gene annotation tool (GATO) is a Bioinformatics pipeline designed to facilitate routine functional annotation and easy access to annotated genes. It was designed in view of the frequent need of genomic researchers to access data pertaining to a common set of genes. In the GATO system, annotation is generated by querying some of the Web-accessible resources and the information is stored in a local database, which keeps a record of all previous annotation results. GATO may be accessed from everywhere through the internet or may be run locally if a large number of sequences are going to be annotated. It is implemented in PHP and Perl and may be run on any suitable Web server. Usually, installation and application of annotation systems require experience and are time consuming, but GATO is simple and practical, allowing anyone with basic skills in informatics to access it without any special training. GATO can be downloaded at [http://mariwork.iq.usp.br/gato/]. Minimum computer free space required is 2 MB.