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1.
Cell ; 162(3): 478-87, 2015 Jul 30.
Article in English | MEDLINE | ID: mdl-26232220

ABSTRACT

Solute carrier (SLC) membrane transport proteins control essential physiological functions, including nutrient uptake, ion transport, and waste removal. SLCs interact with several important drugs, and a quarter of the more than 400 SLC genes are associated with human diseases. Yet, compared to other gene families of similar stature, SLCs are relatively understudied. The time is right for a systematic attack on SLC structure, specificity, and function, taking into account kinship and expression, as well as the dependencies that arise from the common metabolic space.


Subject(s)
Membrane Transport Proteins/metabolism , Animals , Biomedical Research , Drug Discovery , Gene Expression , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics
2.
Nucleic Acids Res ; 51(D1): D1492-D1502, 2023 01 06.
Article in English | MEDLINE | ID: mdl-36268860

ABSTRACT

We describe the Chemical Probes Portal (https://www.chemicalprobes.org/), an expert review-based public resource to empower chemical probe assessment, selection and use. Chemical probes are high-quality small-molecule reagents, often inhibitors, that are important for exploring protein function and biological mechanisms, and for validating targets for drug discovery. The publication, dissemination and use of chemical probes provide an important means to accelerate the functional annotation of proteins, the study of proteins in cell biology, physiology, and disease pathology, and to inform and enable subsequent pioneering drug discovery and development efforts. However, the widespread use of small-molecule compounds that are claimed as chemical probes but are lacking sufficient quality, especially being inadequately selective for the desired target or even broadly promiscuous in behaviour, has resulted in many erroneous conclusions in the biomedical literature. The Chemical Probes Portal was established as a public resource to aid the selection and best-practice use of chemical probes in basic and translational biomedical research. We describe the background, principles and content of the Portal and its technical development, as well as examples of its applications and use. The Chemical Probes Portal is a community resource and we therefore describe how researchers can be involved in its content and development.


Subject(s)
Molecular Probes , Proteins , Drug Discovery , Proteins/chemistry , Proteins/metabolism , Databases, Chemical
3.
Annu Rev Biochem ; 78: 541-68, 2009.
Article in English | MEDLINE | ID: mdl-19489729

ABSTRACT

The large-scale structural biology projects that target human proteins focus predominantly on the catalytic domains of potential therapeutic targets and the domains of human proteins that mediate protein-protein and protein-small-molecule interactions. Their main scientific objective is to elucidate the molecular basis for specificity and selectivity of function within large protein families of therapeutic interest, such as kinases, phosphatases, and proteins involved in epigenetic regulation. Half of the unique human protein structures determined in the past three years derive from these initiatives.


Subject(s)
Proteins/chemistry , Proteome/chemistry , Databases, Protein , Human Genome Project , Humans , Proteins/genetics , Proteins/metabolism
4.
J Chem Inf Model ; 60(12): 5727-5729, 2020 12 28.
Article in English | MEDLINE | ID: mdl-32914973

ABSTRACT

Massive drug repurposing (or repositioning) campaigns are trying to find potential antiviral treatments for COVID-19. Many involve experimental or virtual screening of libraries of compounds previously proven safe in humans-"old drugs". In 20 years of these efforts in many other diseases, never has a new therapeutic hypothesis derived from screening of old drugs in a lab led to the drug being approved for the new indication.


Subject(s)
Antiviral Agents/chemistry , COVID-19 Drug Treatment , Drug Repositioning/methods , SARS-CoV-2/drug effects , Antiviral Agents/pharmacology , Drug Design , Humans , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology
5.
Nat Methods ; 12(8): 725-31, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26121405

ABSTRACT

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibody Specificity , Chromatin/chemistry , Immunoprecipitation/methods , Proteomics/methods , Cloning, Molecular , Computational Biology/methods , Escherichia coli/metabolism , HEK293 Cells , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin G/chemistry , Mass Spectrometry/methods , Peptide Library , Proteins/chemistry , Proteome , Reproducibility of Results
6.
Bioorg Med Chem ; 25(5): 1672-1680, 2017 03 01.
Article in English | MEDLINE | ID: mdl-28162900

ABSTRACT

FIKKs are parasite-specific protein kinases with distinctive sequence motifs and their biological roles have not been completely elucidated. Here, we report the first potent Cryptosporidium FIKK (CpFIKK) inhibitor. We identified 4b as a potent (IC50=0.2nM) inhibitor of CpFIKK catalytic activity. In addition, we identified both CpCDPK1 selective as well as dually acting CpFIKK-CDPK1 inhibitors from the same structural class of compounds. We evaluated these CpFIKK inhibitors for inhibition of parasite growth in vitro. The observed effects on parasite growth did not correlate with CpFIKK inhibition, suggesting that CpFIKK may not be involved in parasite growth.


Subject(s)
Cryptosporidium/enzymology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Amino Acid Sequence , Cryptosporidium/growth & development , Drug Discovery , Humans , Sequence Homology, Amino Acid , Spectrum Analysis/methods , Structure-Activity Relationship
9.
Genes Dev ; 23(23): 2711-6, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19952106

ABSTRACT

Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. Here we show that the Drosophila DHR96 nuclear receptor binds cholesterol and is required for the coordinate transcriptional response of genes that are regulated by cholesterol and involved in cholesterol uptake, trafficking, and storage. DHR96 mutants die when grown on low levels of cholesterol and accumulate excess cholesterol when maintained on a high-cholesterol diet. The cholesterol accumulation phenotype can be attributed to misregulation of npc1b, an ortholog of the mammalian Niemann-Pick C1-like 1 gene NPC1L1, which is essential for dietary cholesterol uptake. These studies define DHR96 as a central regulator of cholesterol homeostasis.


Subject(s)
Cholesterol/metabolism , Drosophila melanogaster/metabolism , Gene Expression Regulation , Homeostasis/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Cholesterol, Dietary/metabolism , Diet , Dietary Fats/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Homeostasis/genetics , Models, Animal , Mutation/genetics , Mutation/immunology , Receptors, Cytoplasmic and Nuclear/genetics , Survival Analysis
10.
J Biol Chem ; 290(30): 18678-98, 2015 Jul 24.
Article in English | MEDLINE | ID: mdl-26071590

ABSTRACT

The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members.


Subject(s)
Evolution, Molecular , Hydrolases/chemistry , Hydrolases/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence/genetics , Binding Sites , Catalysis , Catalytic Domain/genetics , Crystallography, X-Ray , Genome, Fungal , Hydrolases/genetics , Kinetics , Phylogeny , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity
11.
Proc Natl Acad Sci U S A ; 110(24): 9710-5, 2013 Jun 11.
Article in English | MEDLINE | ID: mdl-23716676

ABSTRACT

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Molecular Conformation , Nucleotides/chemistry , Protein Structure, Tertiary , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Crystallography, X-Ray , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Mutation , Nucleotides/metabolism , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Protein Binding , Sequence Homology, Amino Acid , Sf9 Cells
12.
J Struct Funct Genomics ; 15(4): 215-22, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25306867

ABSTRACT

This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga maritima MSB8 determined at a resolution of 2.3 Å. In comparative sequence-clustering analysis, TM0543 displays similarity to NatAB-like proteins, which are components of the ABC-type Na(+) efflux pump permease. However, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of ABC-type efflux transporters solved to date. The structure of the putative TM0543 domain also exhibits different dimer architecture and topology of its presumed ATP binding pocket, which may indicate that it does not bind nucleotide at all. Structural analysis of calcium ion binding sites found at the interface between TM0543 dimer subunits suggests that protein may be involved in ion-transporting activity. A detailed analysis of the protein sequence and structure is presented and discussed.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Bacterial Proteins/chemistry , Thermotoga maritima/chemistry , Binding Sites , Crystallography, X-Ray , Protein Structure, Quaternary , Protein Structure, Tertiary , Structure-Activity Relationship
13.
Proteomics ; 14(9): 975-88, 2014 May.
Article in English | MEDLINE | ID: mdl-24596128

ABSTRACT

At the 12th Annual HUPO World Congress of Proteomics in Japan, the Human Proteome Project (HPP) presented 16 scientific workshop sessions. Here we summarize highlights of ten workshops from the Biology and Disease-driven HPP (B/D-HPP) teams and three from the HPP Resource Pillars. Highlights of the three Chromosome-centric HPP sessions appeared in the many articles of the 2014 C-HPP special issue of the Journal of Proteome Research .


Subject(s)
Databases, Protein , Proteomics , Computational Biology , Humans , Japan
15.
Nat Commun ; 15(1): 5640, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38965235

ABSTRACT

The Structural Genomics Consortium is an international open science research organization with a focus on accelerating early-stage drug discovery, namely hit discovery and optimization. We, as many others, believe that artificial intelligence (AI) is poised to be a main accelerator in the field. The question is then how to best benefit from recent advances in AI and how to generate, format and disseminate data to enable future breakthroughs in AI-guided drug discovery. We present here the recommendations of a working group composed of experts from both the public and private sectors. Robust data management requires precise ontologies and standardized vocabulary while a centralized database architecture across laboratories facilitates data integration into high-value datasets. Lab automation and opening electronic lab notebooks to data mining push the boundaries of data sharing and data modeling. Important considerations for building robust machine-learning models include transparent and reproducible data processing, choosing the most relevant data representation, defining the right training and test sets, and estimating prediction uncertainty. Beyond data-sharing, cloud-based computing can be harnessed to build and disseminate machine-learning models. Important vectors of acceleration for hit and chemical probe discovery will be (1) the real-time integration of experimental data generation and modeling workflows within design-make-test-analyze (DMTA) cycles openly, and at scale and (2) the adoption of a mindset where data scientists and experimentalists work as a unified team, and where data science is incorporated into the experimental design.


Subject(s)
Data Science , Drug Discovery , Machine Learning , Drug Discovery/methods , Data Science/methods , Humans , Artificial Intelligence , Information Dissemination/methods , Data Mining/methods , Cloud Computing , Databases, Factual
16.
J Bacteriol ; 195(24): 5461-8, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24097944

ABSTRACT

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the "minimal" myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis.


Subject(s)
Bacteriophage P2/chemistry , Bacteriophage P2/physiology , Escherichia coli/virology , Viral Tail Proteins/chemistry , Viral Tail Proteins/metabolism , Bacteriophage P2/ultrastructure , Glycoproteins/chemistry , Glycoproteins/metabolism , Magnetic Resonance Spectroscopy , Microscopy, Electron , Protein Conformation , Virion/chemistry , Virion/ultrastructure
17.
J Proteome Res ; 12(1): 23-7, 2013 Jan 04.
Article in English | MEDLINE | ID: mdl-23259511

ABSTRACT

The biology and disease oriented branch of the Human Proteome Project (B/D-HPP) was established by the Human Proteome Organization (HUPO) with the main goal of supporting the broad application of state-of the-art measurements of proteins and proteomes by life scientists studying the molecular mechanisms of biological processes and human disease. This will be accomplished through the generation of research and informational resources that will support the routine and definitive measurement of the process or disease relevant proteins. The B/D-HPP is highly complementary to the C-HPP and will provide datasets and biological characterization useful to the C-HPP teams. In this manuscript we describe the goals, the plans, and the current status of the of the B/D-HPP.


Subject(s)
Disease/genetics , Proteome , Biological Science Disciplines , Disease/classification , Gene Expression , Genome, Human , Human Genome Project , Humans , Mass Spectrometry , Proteome/genetics , Proteome/metabolism
18.
J Biol Chem ; 287(38): 32085-95, 2012 Sep 14.
Article in English | MEDLINE | ID: mdl-22801427

ABSTRACT

One of the final steps in the morphogenetic pathway of phage λ is the packaging of a single genome into a preformed empty head structure. In addition to the terminase enzyme, the packaging chaperone, FI protein (gpFI), is required for efficient DNA packaging. In this study, we demonstrate an interaction between gpFI and the major head protein, gpE. Amino acid substitutions in gpFI that reduced the strength of this interaction also decreased the biological activity of gpFI, implying that this head binding activity is essential for the function of gpFI. We also show that gpFI is a two-domain protein, and the C-terminal domain is responsible for the head binding activity. Using nuclear magnetic resonance spectroscopy, we determined the three-dimensional structure of the C-terminal domain and characterized the helical nature of the N-terminal domain. Through structural comparisons, we were able to identify two previously unannotated prophage-encoded proteins with tertiary structures similar to gpFI, although they lack significant pairwise sequence identity. Sequence analysis of these diverse homologues led us to identify related proteins in a variety of myo- and siphophages, revealing that gpFI function has a more highly conserved role in phage morphogenesis than was previously appreciated. Finally, we present a novel model for the mechanism of gpFI chaperone activity in the DNA packaging reaction of phage λ.


Subject(s)
Bacteriophage lambda/chemistry , Bacteriophage lambda/enzymology , Endodeoxyribonucleases/chemistry , Amino Acid Sequence , Catalysis , Chromatography, Gel , DNA Packaging , DNA, Viral/genetics , Genome, Viral , Magnetic Resonance Spectroscopy/methods , Molecular Sequence Data , Protein Binding/genetics , Protein Conformation , Protein Interaction Mapping , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid
19.
Nat Chem Biol ; 7(8): 566-74, 2011 Jul 10.
Article in English | MEDLINE | ID: mdl-21743462

ABSTRACT

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.


Subject(s)
Enzyme Inhibitors/pharmacology , Histone-Lysine N-Methyltransferase/metabolism , Quinazolines/pharmacology , Animals , Cell Line , Gene Silencing , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/genetics , Humans , Mice , Molecular Structure
20.
Alzheimers Dement (N Y) ; 9(2): e12394, 2023.
Article in English | MEDLINE | ID: mdl-37215505

ABSTRACT

Alzheimer's disease (AD) drug discovery has focused on a set of highly studied therapeutic hypotheses, with limited success. The heterogeneous nature of AD processes suggests that a more diverse, systems-integrated strategy may identify new therapeutic hypotheses. Although many target hypotheses have arisen from systems-level modeling of human disease, in practice and for many reasons, it has proven challenging to translate them into drug discovery pipelines. First, many hypotheses implicate protein targets and/or biological mechanisms that are under-studied, meaning there is a paucity of evidence to inform experimental strategies as well as high-quality reagents to perform them. Second, systems-level targets are predicted to act in concert, requiring adaptations in how we characterize new drug targets. Here we posit that the development and open distribution of high-quality experimental reagents and informatic outputs-termed target enabling packages (TEPs)-will catalyze rapid evaluation of emerging systems-integrated targets in AD by enabling parallel, independent, and unencumbered research.

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