scholarly journals Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

2021 ◽  
Author(s):  
David Dilworth ◽  
Ronan P. Hanley ◽  
Renato Ferreira de Freitas ◽  
Abdellah Allali-Hassani ◽  
Mengqi Zhou ◽  
...  
Keyword(s):  
Cooperative Control ◽  
Binding Pocket ◽  
Negative Control ◽  
Protein Isoforms ◽  
Nucleolar Localization ◽  
Chemical Probe ◽  
Structural Determinants ◽  
Pwwp Domain ◽  
Exact Function ◽  
In Cells

AbstractNSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 ± 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.

A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization

2021 ◽  
Author(s):  
David Dilworth ◽  
Ronan P. Hanley ◽  
Renato Ferreira de Freitas ◽  
Abdellah Allali-Hassani ◽  
Mengqi Zhou ◽  
...  
Keyword(s):  
Nucleolar Localization ◽  
Chemical Probe ◽  
Pwwp Domain

scholarly journals Cytoplasmic Dynein Nucleates Microtubules to Organize Them into Radial Arrays In Vivo

2004 ◽  
Vol 15 (6) ◽  
pp. 2742-2749 ◽  
Author(s):  
Viacheslav Malikov ◽  
Anna Kashina ◽  
Vladimir Rodionov
Keyword(s):  
Cytoplasmic Dynein ◽  
Exact Function ◽  
Necessary And Sufficient ◽  
Stimulation Of ◽  
In Cells

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.

scholarly journals Synthesis of adenosine analogues with indole moiety as human adenosine A 3 receptor ligands

2018 ◽  
Vol 5 (2) ◽  
pp. 171596 ◽  
Author(s):  
Yan Xia ◽  
Xiliang Zheng ◽  
Erkang Wang ◽  
Dongfeng Li ◽  
Ruibin Hou ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Binding Mode ◽  
Receptor Interaction ◽  
Receptor Ligands ◽  
Structural Determinants ◽  
Endogenous Modulator ◽  
Anti Cancer ◽  
Adenosine Derivatives ◽  
G Protein Coupled ◽  
Modelling Approach

Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A 1 , A 2A , A 2B and A 3 , which belong to the G-protein-coupled receptor superfamily. The human A 3 AR (hA 3 AR) subtype is implicated in several cytoprotective functions. Therefore, hA 3 AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anti-cancer and cardioprotective agents. Here, we prepared novel adenosine derivatives with indole moiety as hA 3 AR ligands. According to the biological assay, we found that 2-substituents 11 were critical structural determinants for A 3 AR ligands ( K i  = 111 nM). The observed structure–affinity relationships of this class of ligands were also exhaustively rationalized using the molecular modelling approach. This allows the investigation on the binding mode of the potential compound in the ligand-binding pocket of the human A 3 receptor. The results demonstrated that 11 can interact with the ASN250, GLN167, PHE168 and VAL178 through hydrogen bonding, which are shown to be important for ligand–receptor interaction.

scholarly journals The X-Ray Structure of the Haloalcohol Dehalogenase HheA from Arthrobacter sp. Strain AD2: Insight into Enantioselectivity and Halide Binding in the Haloalcohol Dehalogenase Family

2006 ◽  
Vol 188 (11) ◽  
pp. 4051-4056 ◽  
Author(s):  
René M. de Jong ◽  
Kor H. Kalk ◽  
Lixia Tang ◽  
Dick B. Janssen ◽  
Bauke W. Dijkstra
Keyword(s):  
Tertiary Structure ◽  
Halogen Bond ◽  
Environmental Pollutants ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Side Chain ◽  
Structural Determinants ◽  
Substrate Binding Pocket ◽  
X Ray ◽  
Insight Into

ABSTRACT Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheAAD2 from Arthrobacter sp. strain AD2 at 2.0-Å resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 shows that HheAAD2 has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheAAD2 can bind both enantiomers of the haloalcohol substrate 1-p-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheAAD2. Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheAAD2, in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.

scholarly journals Golgi Recruitment Assay for Visualizing Small-Molecule Ligand–target Engagement in Cells

2020 ◽  
Author(s):  
Sachio Suzuki ◽  
Masahiro Ikuta ◽  
Tatsuyuki Yoshii ◽  
Akinobu Nakamura ◽  
Keiko Kuwata ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Ligand Binding ◽  
Drug Target ◽  
Chemical Biology ◽  
Protein Isoforms ◽  
Target Engagement ◽  
Ligand Affinity ◽  
Important Challenge ◽  
Small Molecule Ligand ◽  
In Cells

<b>The development of methods that allow detection of ligand–target engagement in cells is an important challenge in chemical biology and drug discovery. Here, we present a Golgi recruitment (G-REC) assay in which the ligand binding to the target protein can be visualized as Golgi-localized fluorescence signals. We show that the G-REC assay is applicable to the detection of various ligand–target interactions, ligand affinity comparison among distinct protein isoforms, and the monitoring of unmodified drug–target engagement in cells.</b>

scholarly journals The Metalloid Reductase of Pseudomonas Moravenis Stanleyae Conveys Nanoparticle Mediated Metalloid Tolerance

2018 ◽  
Author(s):  
Richard Nemeth ◽  
Mackenzie Neubert ◽  
Thomas Ni ◽  
Christopher J. Ackerson
Keyword(s):  
Structural Analysis ◽  
Substrate Specificity ◽  
Glutathione Reductase ◽  
Cell Lines ◽  
Bacterial Cell ◽  
Binding Pocket ◽  
Oxidized Glutathione ◽  
Se Nanoparticles ◽  
In Cells

In the present work we have identified a glutathione reductase like metalloid reductase (GRLMR) responsible for mediating selenite tolerance in <i>Pseudomonas moravenis</i> stanleyae through the enzymatic generation of Se(0) nanoparticles. This enzyme has an unprecedented substrate specificity for selenodiglutathione (K<sub>m</sub>= 336 μM) over oxidized glutathione (K<sub>m</sub>=8.22 mM). This enzyme was able to induce selenite tolerance in foreign bacterial cell lines by increasing the IC<sub>90</sub> for selenite from 1.9 mM in cell lacking the GRLMR gene to 21.3 mM for cells containing the GRLMR gene. It was later confirmed by STEM and EDS that Se nanoparticles were absent in control cells and present in cells expressing GRLMR. Structural analysis suggests the lack of a sulfur residue in the substrate/product binding pocket may be responsible for this unique substrate specificity.

scholarly journals A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5

2020 ◽  
Author(s):  
Thomas Hanke ◽  
Jong Fu Wong ◽  
Benedict-Tilmann Berger ◽  
Ismahan Abdi ◽  
Lena Marie Berger ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Heart Diseases ◽  
Negative Control ◽  
Cellular Systems ◽  
Receptor Protein ◽  
Chemical Probe ◽  
Activin Receptor ◽  
Control Compound

AbstractThe transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homolog 2). Thus, this chemical probe offers an excellent tool for mechanistic studies on the ALK4/5 signaling pathway and the contribution of these targets to disease.

scholarly journals In silico analysis of alternative splicing on drug-target gene interactions

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yanrong Ji ◽  
Rama K. Mishra ◽  
Ramana V. Davuluri
Keyword(s):  
Alternative Splicing ◽  
Drug Discovery ◽  
Drug Target ◽  
In Silico Analysis ◽  
Binding Pocket ◽  
Protein Isoforms ◽  
Cancer Drug ◽  
Discovery Research ◽  
Structure Level ◽  
Drug Discovery Research

AbstractIdentifying and evaluating the right target are the most important factors in early drug discovery phase. Most studies focus on one protein ignoring the multiple splice-variant or protein-isoforms, which might contribute to unexpected therapeutic activity or adverse side effects. Here, we present computational analysis of cancer drug-target interactions affected by alternative splicing. By integrating information from publicly available databases, we curated 883 FDA approved or investigational stage small molecule cancer drugs that target 1,434 different genes, with an average of 5.22 protein isoforms per gene. Of these, 618 genes have ≥5 annotated protein-isoforms. By analyzing the interactions with binding pocket information, we found that 76% of drugs either miss a potential target isoform or target other isoforms with varied expression in multiple normal tissues. We present sequence and structure level alignments at isoform-level and make this information publicly available for all the curated drugs. Structure-level analysis showed ligand binding pocket architectures differences in size, shape and electrostatic parameters between isoforms. Our results emphasize how potentially important isoform-level interactions could be missed by solely focusing on the canonical isoform, and suggest that on- and off-target effects at isoform-level should be investigated to enhance the productivity of drug-discovery research.

scholarly journals Specificity Controls for Immunocytochemical Methods

2000 ◽  
Vol 48 (2) ◽  
pp. 163-165 ◽  
Author(s):  
Richard W. Burry
Keyword(s):  
Good Control ◽  
Positive Control ◽  
Negative Control ◽  
Primary Antibody ◽  
Antibody Specificity ◽  
Antibody Localization ◽  
In Cells

Immunocytochemistry is used for antibody localization of proteins in cells and tissues. The specificity of the results depends on two independent criteria: the specificity of the antibody and of the method used. The antibody specificity is best determined by immunoblot and or immunoprecipitation. Absorption of the antibody with a protein does not determine that the antibody would have bound to the same protein in the tissue, and therefore is not a good control for antibody specificity. The specificity of the method is best determined by both a negative control, replacing the primary antibody with serum, and a positive control, using the antibody with cells known to contain the protein. With the increasing use of immunocytochemistry, it is important to be aware of the appropriate controls needed to show specificity of the labeling.

scholarly journals Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding

2013 ◽  
Vol 452 (3) ◽  
pp. 423-432 ◽  
Author(s):  
Radika Soysa ◽  
Hanka Venselaar ◽  
Jacqueline Poston ◽  
Buddy Ullman ◽  
Marie-Pierre Hasne
Keyword(s):  
Trypanosoma Cruzi ◽  
Structural Model ◽  
Three Dimensional ◽  
Salt Bridge ◽  
Homology Model ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Helical Structures ◽  
Structural Determinants ◽  
Key Residues

The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247–Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.

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