Populus spp. clonal response of growth in Argiudoll and Hapludoll soils in Buenos Aires Province, Argentina

The aim of this study was to assess the growth of clones of Populus spp. in the soil types Hapludolls and Argiudolls, of the mainland in the Buenos Aires province. The study took place in Alberti (34°50’ S, 60°30’ W, 35 m.a.s.l.), Buenos Aires, Argentina. Five clones of Populus spp. were studied: ‘Stoneville 66’ (‘Delta Gold’); ‘Conti 12’, ‘568-1’, ‘610-31’ and ‘564-17’. The experimental design consisted of randomized complete blocks with sub-samples. There were three blocks on three types of soil: a Typic Hapludoll, Entic Hapludoll, and a Typic Argiudoll. Physical and chemical characterizations of each soil type were performed. The average values of total basal area and height were recorded. The results were subjected to analysis of variance, and means were separated according to Tukey’s test. An analysis of genotype-soil interaction was performed. No significant interaction between clone factor and soil factor was obtained. Clones ‘Stoneville 66’, and ‘568-1’ did not differ significantly among themselves and displayed better growth. Therefore, no specific associations between these clones and the soils existed. These results should be valid and applicable for a large amount of land because of the wide distribution of Argiudoll and Hapludoll soils.El objetivo del presente trabajo fue evaluar el crecimiento de clones de Populus spp. en suelos de tipo Hapludoles y Argiudoles de la zona continental de la Provincia de Buenos Aires. El estudio se localizó en Alberti (34°50’ S, 60°30’ O, 35 m.s.n.m.), Buenos Aires, Argentina y se utilizó Populus deltoides (‘Stoneville 66’ (‘Delta Gold’), ‘A-610-31’ y ‘A-564-17’) y P. x canadensis (‘I-Conti 12’ y ‘A-568-1’). El diseño experimental fue de bloques completos al azar con submuestreo. Se instalaron tres bloques, sobre tres tipos de suelo: Hapludol típico, Hapludol éntico y Argiudol típico. Se realizó una caracterización físico-química de cada suelo, se registraron los valores medios de área basal y altura total de los árboles. Los resultados se sometieron a análisis de la varianza y los promedios se separaron según Tukey (p ≤ 0,05). Se realizó un análisis de interacción genotipo-suelo que no arrojó interacciones significativas. Los clones ‘Stoneville 66’ y ‘568-1’ tuvieron el mejor crecimiento, sin diferencias significativas entre sí, por lo que no existirían asociaciones específicas entre esos clones y los suelos considerados. Los resultados obtenidos en este estudio serían válidos para una importante superficie de tierras debido a la amplia distribución areal de los suelos Argiudoles y Hapludoles dentro de la unidad climática considerada.Facultad de Ciencias Agrarias y Forestale

An Allosteric Inhibitor of Protein Arginine Methyltransferase 3

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets

RPRD1A and RPRD1B Are Human RNA Polymerase II C-Terminal Domain Scaffolds for Ser5 Dephosphorylation

The RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) heptapeptide repeats (Y1-S2-P3-T4-S5-P6-S7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD interaction domains (CIDs) with CTD repeats phosphorylated at S2 and S7. Our high resolution crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an interesting example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2

Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite

Small-molecule ligands of methyl-lysine binding proteins: Optimization of selectivity for L3MBTL3

Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design

Discovery of a 2,4-Diamino-7-aminoalkoxyquinazoline as a Potent and Selective Inhibitor of Histone Lysine Methyltransferase G9a

SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template led to the discovery of 8 (UNC0224) as a potent and selective G9a inhibitor. A high resolution X-ray crystal structure of the G9a-8 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. The co-crystal structure validated our binding hypothesis and will enable structure-based design of novel inhibitors. 8 is a useful tool for investigating the biology of G9a and its roles in chromatin remodeling

Protein Lysine Methyltransferase G9a Inhibitors: Design, Synthesis, and Structure Activity Relationships of 2,4-Diamino-7-aminoalkoxy-quinazolines.

Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is over expressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth and the di-methylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. Based on the structural insights revealed by this co-crystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison Ki = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date

Exploring the Trypanosoma brucei Hsp83 Potential as a Target for Structure Guided Drug Design

Human African trypanosomiasis is a neglected parasitic disease that is fatal if untreated. The current drugs available to eliminate the causative agent Trypanosoma brucei have multiple liabilities, including toxicity, increasing problems due to treatment failure and limited efficacy. There are two approaches to discover novel antimicrobial drugs--whole-cell screening and target-based discovery. In the latter case, there is a need to identify and validate novel drug targets in Trypanosoma parasites. The heat shock proteins (Hsp), while best known as cancer targets with a number of drug candidates in clinical development, are a family of emerging targets for infectious diseases. In this paper, we report the exploration of T. brucei Hsp83--a homolog of human Hsp90--as a drug target using multiple biophysical and biochemical techniques. Our approach included the characterization of the chemical sensitivity of the parasitic chaperone against a library of known Hsp90 inhibitors by means of differential scanning fluorimetry (DSF). Several compounds identified by this screening procedure were further studied using isothermal titration calorimetry (ITC) and X-ray crystallography, as well as tested in parasite growth inhibitions assays. These experiments led us to the identification of a benzamide derivative compound capable of interacting with TbHsp83 more strongly than with its human homologs and structural rationalization of this selectivity. The results highlight the opportunities created by subtle structural differences to develop new series of compounds to selectively target the Trypanosoma brucei chaperone and effectively kill the sleeping sickness parasite

The Cryptosporidium parvum Kinome

<p>Abstract</p> <p>Background</p> <p>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 <it>Cryptosporidium parvum </it>kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.</p> <p>Results</p> <p>The <it>C</it>. <it>parvum </it>kinome comprises over 70 members, some of which may be promising drug targets. These <it>C. parvum </it>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 <it>Cryptosporidium spp</it>. Comparison of specific kinases with their <it>Plasmodium falciparum </it>and <it>Toxoplasma gondii </it>orthologues revealed some distinct characteristics within the <it>C. parvum </it>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 <it>Cp</it>CDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of <it>Cp</it>CDPK1. In addition, structural analysis of <it>Cp</it>CDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.</p> <p>Conclusions</p> <p>Identification and comparison of the <it>C. parvum </it>protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.</p

Populus spp., respuestas clonales de crecimiento en suelos Argiudoles y Hapludoles, de la Provincia de Buenos Aires, Argentina

The aim of this study was to assess the growth of clones of Populus spp. in the soil types Hapludolls and Argiudolls, of the mainland in the Buenos Aires province. The study took place inAlberti (34°50' S, 60°30'W, 35m.a.s.l.), Buenos Aires , Argentina. Five clones oí Populus spp. were studied: ' Stoneville 66' ('Delta Gold'); 'Conti 12', '568-1', '610-31' and '564-17'. The experimental design consisted of randomized complete blocks with sub-samples. There were three blocks on three types of soil: a Typic Hapludoll, Entic Hapludoll, and a Typic Argiudoll. Physical and chemical characterizations of each soil type were performed. The average valúes of total basal área and height were recorded. The results were subjected to analysis of variance, and means were separated according to Tukey's test. An analysis of genotype-soil interaction was performed. No significant interaction between clone factor and soil factor was obtained. Clones 'Stoneville 66', and '568-1' did not differ significantly among themselves and displayed better growth. Therefore, no specific associations between these clones and the soils existed. These results should be valid and applicable for a large amount of land because of the wide distribution of Argiudoll and Hapludoll soils.El objetivo del presente trabajo fue evaluar el crecimiento de clones de Populus spp. en suelos de tipo Hapludoles y Argiudoles de la zona continental de la Provincia de Buenos Aires. El estudio se localizó en Alberti (34°50' S, 60°30' O, 35 m.s.n.m.), Buenos Aires, Argentina y se utilizó Populus deltoides ('Stoneville 66' ('Delta Gold'), 'A-610-31' y 'A-564-17') y P. x canadensis ('I-Conti 12' y 'A-568-1'). El diseño experimental fue de bloques completos al azar con submuestreo. Se instalaron tres bloques, sobre tres tipos de suelo: Hapludol típico, Hapludol éntico y Argiudol típico. Se realizó una caracterización físico-química de cada suelo, se registraron los valores medios de área basal y altura total de los árboles. Los resultados se sometieron a análisis de la varianza y los promedios se separaron según Tukey (p ≤ 0,05). Se realizó un análisis de interacción genotipo-suelo que no arrojó interacciones significativas. Los clones 'Stoneville 66' y '568-1' tuvieron el mejor crecimiento, sin diferencias significativas entre sí, por lo que no existirían asociaciones específicas entre esos clones y los suelos considerados. Los resultados obtenidos en este estudio serían válidos para una importante superficie de tierras debido a la amplia distribución areal de los suelos Argiudoles y Hapludoles dentro de la unidad climática considerada