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The recognition of target DNA sequences during the interference phase of prokaryotic CRISPR-Cas immunity relies on Protospacer-Adjacent Motif (PAM) sequences, specific for each Cas effector. PAM identification is a laborious and time consuming process that requires multiple stages including in vitro and in vivo cleavage assays followed by Next Generation Sequencing of targets that withstood cleavage. Determining PAM is an essential step of characterisation of any novel Cas9 ortholog and determines the likelihood of its potential use. This study investigates the potential of machine learning to predict PAM sequences for a given Cas9 ortholog based on the results of cleavage experiments and employing an Active Learning process akin to Reinforcement Learning with Human Feedback. Machine learning-facilitated PAM identification would streamline and accelerate existing pipelines for describing novel Cas proteins. We demonstrate that simple models with a small amount of data are sufficient for confident PAM predictions when training is effectively orchestrated.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

The Tunka Radio Extension (Tunka-Rex) is a digital antenna array (63 antennas distributed over 1km^2) co-located with the TAIGA observatory in Eastern Siberia. Tunka-Rex measures radio emission of air-showers induced by ultra-high energy cosmic rays in the frequency band of 30-80 MHz. Air-shower signal is a short (tens of nanoseconds) broadband pulse. Using time positions and amplitudes of these pulses, we reconstruct parameters of air showers and primary cosmic rays. The amplitudes of low-energy event (E<10^17 eV) cannot be used for successful reconstruction due to the domination of background. To lower the energy threshold of the detection and increase the efficiency, we use autoencoder neural network which removes noise from the measured data. This work describes our approach to denoising raw data and further reconstruction of air-shower parameters. We also present results of the low-energy events reconstruction with autoencoder.

Статья ориентирована на освещение проблем развития информационно-коммуникационной культуры учащихся и определение путей их решения. Выделены факторы, влияющие на организацию развития данного качества у учащихся. В качестве значимого фактора выделено взаимодействие субъектов развития учащегося, в частности, в интересах определения содержания программ обучения. Предложены подходы к организации формирования специальных умений в рамках нормативных ограничений. Исследовательская работа в школе определена как значимое направление деятельности педагогов, в том числе во взаимодействии с представителями высших учебных заведений: профессорско-преподавательским составом, аспирантами, студентами. Выработан комплекс предложений к организации работы по формированию культуры реализации цифровых технологий.

Статья ориентирована на освещение проблем, обусловленных информатизацией и технологизацией процессов жизнедеятельности человека. Выделены проблемы, характерные для развития детей в цифровом мире. Выводы основаны на анализе результатов собеседования с детьми дошкольного возраста и учащимися школы и их родителями. Предложены подходы к гармоничному развитию детей в современных условиях развития общества. Проанализированы основные факторы, связанные с развитием личности в современном обществе, общественными тенденциями. Выработаны направления деятельности учителя при использовании информационно-коммуникационных средств в деятельности ребенка.

В статье обращено внимание на необходимость формирования у личности качеств, необходимых для жизнедеятельности в современном обществе. При этом акцент сделан на развитии у детей информационно-коммуникационной компетентности, для решения этой задачи важно обеспечить усвоение ими фундаментальных знаний, формирование у них навыков саморазвития. При этом школа выделена в качестве основного звена в цепи непрерывного обучения и воспитания личности, отвечающих требованиям современного общества. Выработаны подходы к решению задач развития личности, требования к субъектам развития личности, в том числе к организации их взаимодействия в интересах формирования содержания её развития. Определены факторы, опираясь на которые, следует определять компоненты развития личности.

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, the conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggest that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.

A data life cycle (DLC) is a high-level data processing pipeline that involves data acquisition, event reconstruction, data analysis, publication, archiving, and sharing. For astroparticle physics a DLC is particularly important due to the geographical and content diversity of the research field. A dedicated and experiment spanning analysis and data centre would ensure that multi-messenger analyses can be carried out using state-of-the-art methods. The German-Russian Astroparticle Data Life Cycle Initiative (GRADLCI) is a joint project of the KASCADE-Grande and TAIGA collaborations, aimed at developing a concept and creating a DLC prototype that takes into account the data processing features specific for the research field. An open science system based on the KASCADE Cosmic Ray Data Centre (KCDC), which is a web-based platform to provide the astroparticle physics data for the general public, must also include effective methods for distributed data storage algorithms and techniques to allow the community to perform simulations and analyses with sophisticated machine learning methods. The aim is to achieve more efficient analyses of the data collected in different, globally dispersed observatories, as well as a modern education to Big Data Scientist in the synergy between basic research and the information society. The contribution covers the status and future plans of the initiative., Comment: presented at the 36th ICRC (Madison, WI; 2019)

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.

By screening an epigenetic compound library, we identified that UNC0638, a highly potent inhibitor of the histone methyltransferases G9a and GLP, was a weak inhibitor of SPIN1 (spindlin 1), a methyllysine reader protein. Our optimization of this weak hit resulted in the discovery of a potent, selective, and cell-active SPIN1 inhibitor, compound 3 (MS31). Compound 3 potently inhibited binding of trimethyllysine-containing peptides to SPIN1, displayed high binding affinity, was highly selective for SPIN1 over other epigenetic readers and writers, directly engaged SPIN1 in cells, and was not toxic to nontumorigenic cells. The crystal structure of the SPIN1–compound 3 complex indicated that it selectively binds tudor domain II of SPIN1. We also designed a structurally similar but inactive compound 4 (MS31N) as a negative control. Our results have demonstrated for the first time that potent, selective, and cell-active fragment-like inhibitors can be generated by targeting a single tudor domain.

By screening an epigenetic compound library, we identified that UNC0638, a highly potent inhibitor of the histone methyltransferases G9a and GLP, was a weak inhibitor of SPIN1 (spindlin 1), a methyllysine reader protein. Our optimization of this weak hit resulted in the discovery of a potent, selective, and cell-active SPIN1 inhibitor, compound 3 (MS31). Compound 3 potently inhibited binding of trimethyllysine-containing peptides to SPIN1, displayed high binding affinity, was highly selective for SPIN1 over other epigenetic readers and writers, directly engaged SPIN1 in cells, and was not toxic to nontumorigenic cells. The crystal structure of the SPIN1–compound 3 complex indicated that it selectively binds tudor domain II of SPIN1. We also designed a structurally similar but inactive compound 4 (MS31N) as a negative control. Our results have demonstrated for the first time that potent, selective, and cell-active fragment-like inhibitors can be generated by targeting a single tudor domain.

YEATS-domain-containing MLLT1 is an acetyl/acyl-lysine reader domain, which is structurally distinct from well-studied bromodomains and has been strongly associated in development of cancer. Here, we characterized piperazine-urea derivatives as an acetyl/acyl-lysine mimetic moiety for MLLT1. Crystal structures revealed distinct interaction mechanisms of this chemotype compared to the recently described benzimidazole-amide based inhibitors, exploiting different binding pockets within the protein. Thus, the piperazine-urea scaffold offers an alternative strategy for targeting the YEATS domain family.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.

A data life cycle (DLC) is a high-level data processing pipeline that involves data acquisition, event reconstruction, data analysis, publication, archiving, and sharing. For astroparticle physics a DLC is particularly important due to the geographical and content diversity of the research field. A dedicated and experiment spanning analysis and data centre would ensure that multi-messenger analyses can be carried out using state-of-the-art methods. The German-Russian Astroparticle Data Life Cycle Initiative (GRADLCI) is a joint project of the KASCADE-Grande and TAIGA collaborations, aimed at developing a concept and creating a DLC prototype that takes into account the data processing features specific for the research field. An open science system based on the KASCADE Cosmic Ray Data Centre (KCDC), which is a web-based platform to provide the astroparticle physics data for the general public, must also include effective methods for distributed data storage algorithms and techniques to allow the community to perform simulations and analyses with sophisticated machine learning methods. The aim is to achieve more efficient analyses of the data collected in different, globally dispersed observatories, as well as a modern education to Big Data Scientist in the synergy between basic research and the information society. The contribution covers the status and future plans of the initiative., Comment: presented at the 36th ICRC (Madison, WI; 2019)

Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.

SGC Oxford has expressed, purified and crystallized the second bromodomain of PHIP as part of the probe programme. Fragment screening and X-ray crystallography identified binders, some of which optimised to uM affinity. However, molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.

In the frame of the Karlsruhe-Russian Astroparticle Data Life Cycle Initiative it was proposed to deploy an educational resource astroparticle.online for the training of students in the field of astroparticle physics. This resource is based on HUBzero, which is an open-source software platform for building powerful websites, which supports scientific discovery, learning, and collaboration. HUBzero has been deployed on the servers of Matrosov Institute for System Dynamics and Control Theory. The educational resource astroparticle.online is being filled with the information covering cosmic messengers, astroparticle physics experiments and educational courses and schools on astroparticle physics. Furthermore, the educational resource astroparticle.online can be used for online collaboration. We present the current status of this project and our first experience of application of this service as a collaboration framework., Comment: 5 pages, 1 figure

A distributed data warehouse system is one of the actual issues in the field of astroparticle physics. Famous experiments, such as TAIGA, KASCADE-Grande, produce tens of terabytes of data measured by their instruments. It is critical to have a smart data warehouse system on-site to store the collected data for further distribution effectively. It is also vital to provide scientists with a handy and user-friendly interface to access the collected data with proper permissions not only on-site but also online. The latter case is handy when scientists need to combine data from different experiments for analysis. In this work, we describe an approach to implementing a distributed data warehouse system that allows scientists to acquire just the necessary data from different experiments via the Internet on demand. The implementation is based on CernVM-FS with additional components developed by us to search through the whole available data sets and deliver their subsets to users' computers., Comment: 5 pages, 3 figures, The 8th International Conference "Distributed Computing and Grid-technologies in Science and Education" (GRID 2018)

Modern large-scale astroparticle setups measure high-energy particles, gamma rays, neutrinos, radio waves, and the recently discovered gravitational waves. Ongoing and future experiments are located worldwide. The data acquired have different formats, storage concepts, and publication policies. Such differences are a crucial point in the era of Big Data and of multi-messenger analysis in astroparticle physics. We propose an open science web platform called ASTROPARTICLE.ONLINE which enables us to publish, store, search, select, and analyze astroparticle data. In the first stage of the project, the following components of a full data life cycle concept are under development: describing, storing, and reusing astroparticle data; software to perform multi-messenger analysis using deep learning; and outreach for students, post-graduate students, and others who are interested in astroparticle physics. Here we describe the concepts of the web platform and the first obtained results, including the meta data structure for astroparticle data, data analysis by using convolution neural networks, description of the binary data, and the outreach platform for those interested in astroparticle physics. The KASCADE-Grande and TAIGA cosmic-ray experiments were chosen as pilot examples., Comment: Published in Data Journal

In the frame of the Karlsruhe-Russian Astroparticle Data Life Cycle Initiative it was proposed to deploy an educational resource astroparticle.online for the training of students in the field of astroparticle physics. This resource is based on HUBzero, which is an open-source software platform for building powerful websites, which supports scientific discovery, learning, and collaboration. HUBzero has been deployed on the servers of Matrosov Institute for System Dynamics and Control Theory. The educational resource astroparticle.online is being filled with the information covering cosmic messengers, astroparticle physics experiments and educational courses and schools on astroparticle physics. Furthermore, the educational resource astroparticle.online can be used for online collaboration. We present the current status of this project and our first experience of application of this service as a collaboration framework., Comment: 5 pages, 1 figure

A distributed data warehouse system is one of the actual issues in the field of astroparticle physics. Famous experiments, such as TAIGA, KASCADE-Grande, produce tens of terabytes of data measured by their instruments. It is critical to have a smart data warehouse system on-site to store the collected data for further distribution effectively. It is also vital to provide scientists with a handy and user-friendly interface to access the collected data with proper permissions not only on-site but also online. The latter case is handy when scientists need to combine data from different experiments for analysis. In this work, we describe an approach to implementing a distributed data warehouse system that allows scientists to acquire just the necessary data from different experiments via the Internet on demand. The implementation is based on CernVM-FS with additional components developed by us to search through the whole available data sets and deliver their subsets to users' computers., Comment: 5 pages, 3 figures, The 8th International Conference "Distributed Computing and Grid-technologies in Science and Education" (GRID 2018)

Modern large-scale astroparticle setups measure high-energy particles, gamma rays, neutrinos, radio waves, and the recently discovered gravitational waves. Ongoing and future experiments are located worldwide. The data acquired have different formats, storage concepts, and publication policies. Such differences are a crucial point in the era of Big Data and of multi-messenger analysis in astroparticle physics. We propose an open science web platform called ASTROPARTICLE.ONLINE which enables us to publish, store, search, select, and analyze astroparticle data. In the first stage of the project, the following components of a full data life cycle concept are under development: describing, storing, and reusing astroparticle data; software to perform multi-messenger analysis using deep learning; and outreach for students, post-graduate students, and others who are interested in astroparticle physics. Here we describe the concepts of the web platform and the first obtained results, including the meta data structure for astroparticle data, data analysis by using convolution neural networks, description of the binary data, and the outreach platform for those interested in astroparticle physics. The KASCADE-Grande and TAIGA cosmic-ray experiments were chosen as pilot examples., Comment: Published in Data Journal

Background: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. Results: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. Conclusions: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wi

The p300/CBP‐associated factor (PCAF) and related GCN5 bromodomain‐containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine‐based L‐45 (dubbed L‐Moses) as the first potent, selective, and cell‐active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)‐(−)‐norephedrine furnished L‐45 in enantiopure form. L‐45 was shown to disrupt PCAF‐Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co‐crystal structure of L‐45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L‐45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell‐permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Gastric cancer is one of the most common malignancies and a leading cause of cancer death worldwide. The prognosis of stomach cancer is generally poor as this cancer is not very sensitive to commonly used chemotherapies. Epigenetic modifications play a key role in gastric cancer and contribute to the development and progression of this malignancy. In order to explore new treatment options in this target area we have screened a library of epigenetic inhibitors against gastric cancer cell lines and identified inhibitors for the BET family of bromodomains as potent inhibitors of gastric cancer cell proliferations. Here we show that both the pan-BET inhibitor (+)-JQ1 as well as a newly developed specific isoxazole inhibitor, PNZ5, showed potent inhibition of gastric cancer cell growth. Intriguingly, we found differences in the antiproliferative response between gastric cancer cells tested derived from Brazilian patients as compared to those from Asian patients, the latter being largely resistant to BET inhibition. As BET inhibitors are entering clinical trials these findings provide the first starting point for future therapies targeting gastric cancer.

The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.

A combination of conventional organic synthesis, remotely monitored flow synthesis and bioassay platforms, were used for the evaluation of novel inhibitors targeting bromodomains outside the well-studied bromodomain and extra terminal (BET) family, here exemplified by activity measurements on the bromodomain of BRD9 protein, a component of some tissue-specific SWi/SNF chromatin remodelling complexes. The Frontal Affinity Chromatography combined with Mass Spectrometry (FAC-MS) method proved to be reliable and results correlated well with an independent thermal shift assay.

Background Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase. Results The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC50 values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation. Conclusions Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and

Background Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase. Results The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC50 values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation. Conclusions Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and

There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).

There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).

There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).