[Show abstract][Hide abstract] ABSTRACT: The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-N-(1-(pyridin-2-yl)ethyl)propanamide, 1, has previously been identified as a potent but unselective inhibitor of diphtheria toxin-like ADP-ribosyltransferase 3 (ARTD3). Herein we describe synthesis and evaluation of 55 compounds in this class. It was found that the stereochemistry is of great importance for both selectivity and potency, and that substituents on the phenyl resulted in poor solubility. Certain variations at the meso position were tolerated and caused a large shift in the binding pose. Changes to the ethylene linker that connects the quinazolinone to the amide were also investigated but proved detrimental to the binding. By combining synthetic organic chemistry and structure-based design two selective inhibitors of ARTD3 were discovered.
No preview · Article · Nov 2013 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at sub-micromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.
No preview · Article · Jun 2013 · ACS Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: ADP-ribosyltransferases (ARTs) catalyze the transfer of ADP-ribose from NAD onto substrates. Some ARTs generate in an iterative process ADP-ribose polymers that serve as adaptors for distinct protein domains. Other ARTs, exemplified by ARTD10, function as mono-ADP-ribosyltransferases, but it has been unclear whether this modification occurs in cells and how it is read. We observed that ARTD10 colocalized with ARTD8 and defined its macrodomains 2 and 3 as readers of mono-ADP-ribosylation both in vitro and in cells. The crystal structures of these two ARTD8 macrodomains and isothermal titration calorimetry confirmed their interaction with ADP-ribose. These macrodomains recognized mono-ADP-ribosylated ARTD10, but not poly-ADP-ribosylated ARTD1. This distinguished them from the macrodomain of macroH2A1.1, which interacted with poly- but not mono-ADP-ribosylated substrates. Moreover, Ran, an ARTD10 substrate, was also read by ARTD8 macrodomains. This identifies readers of mono-ADP-ribosylated proteins, defines their structures, and demonstrates the presence of this modification in cells.
[Show abstract][Hide abstract] ABSTRACT: The diphtheria toxin-like ADP-ribosyltransferases (ARTDs) are an enzyme family that catalyzes the transfer of ADP-ribose units onto substrate proteins by using nicotinamide adenine dinucleotide (NAD(+)) as a cosubstrate. They have a documented role in chromatin remodelling and DNA repair, and inhibitors of ARTD1 and 2 (PARP1 and 2) are currently in clinical trials for the treatment of cancer. The detailed function of most other ARTDs is still unknown. By using virtual screening, we identified small ligands of ARTD7 (PARP15/BAL3) and ARTD8 (PARP14/BAL2). Thermal-shift assays confirmed that 16 compounds, belonging to eight structural classes, bound to ARTD7/ARTD8. Affinity measurements with isothermal titration calorimetry for two isomers of the most promising hit compound confirmed binding in the low micromolar range to ARTD8. Crystal structures showed anchoring of the hits in the nicotinamide pocket. These results form a starting point in the development of chemical tools for the study of the role and function of ARTD7 and ARTD8.
No preview · Article · Jul 2012 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: ADP-ribosylation is involved in the regulation of DNA repair, transcription, and other processes. The 18 human ADP-ribose
transferases with diphtheria toxin homology include ARTD1/PARP1, a cancer drug target. Knowledge of other family members may
guide therapeutics development and help evaluate potential drug side effects. Here, we present the crystal structure of human
ARTD15/PARP16, a previously uncharacterized enzyme. ARTD15 features an α-helical domain that packs against its transferase
domain without making direct contact with the NAD+-binding crevice or the donor loop. Thus, this novel domain does not resemble the regulatory domain of ARTD1. ARTD15 displays
auto-mono(ADP-ribosylation) activity and is affected by canonical poly(ADP-ribose) polymerase inhibitors. These results add
to a framework that will facilitate research on a medically important family of enzymes.
Preview · Article · Jun 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Inhibitors of poly-ADP-ribose polymerase (PARP) family proteins are currently in clinical trials as cancer therapeutics, yet the specificity of many of these compounds is unknown. Here we evaluated a series of 185 small-molecule inhibitors, including research reagents and compounds being tested clinically, for the ability to bind to the catalytic domains of 13 of the 17 human PARP family members including the tankyrases, TNKS1 and TNKS2. Many of the best-known inhibitors, including TIQ-A, 6(5H)-phenanthridinone, olaparib, ABT-888 and rucaparib, bound to several PARP family members, suggesting that these molecules lack specificity and have promiscuous inhibitory activity. We also determined X-ray crystal structures for five TNKS2 ligand complexes and four PARP14 ligand complexes. In addition to showing that the majority of PARP inhibitors bind multiple targets, these results provide insight into the design of new inhibitors.
Full-text · Article · Feb 2012 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.
We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.
Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.
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[Show abstract][Hide abstract] ABSTRACT: Perturbed cell adhesion mechanisms are crucial for tumor invasion and metastasis. A cell adhesion protein, TSLC1 (tumor suppressor in lung cancer 1), is inactivated in a majority of metastatic cancers. DAL-1 (differentially expressed in adenocarcinoma of the lung protein), another tumor suppressor, binds through its FERM domain to the TSLC1 C-terminal, 4.1 glycophorin C-like, cytoplasmic domain. However, the molecular basis for this interaction is unknown. Here, we describe the crystal structure of a complex between the DAL-1 FERM domain and a portion of the TSLC1 cytoplasmic domain. DAL-1 binds to TSLC1 through conserved residues in a well defined hydrophobic pocket in the structural C-lobe of the DAL-1 FERM domain. From the crystal structure, it is apparent that Tyr(406) and Thr(408) in the TSLC1 cytoplasmic domain form the most important interactions with DAL-1, and this was also confirmed by surface plasmon resonance studies. Our results refute earlier exon deletion experiments that indicated that glycophorin C interacts with the α-lobe of 4.1 FERM domains.
Preview · Article · Feb 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.
[Show abstract][Hide abstract] ABSTRACT: Altered inositol metabolism is implicated in a number of diabetic complications. The first committed step in mammalian inositol catabolism is performed by myo-inositol oxygenase (MIOX), which catalyzes a unique four-electron dioxygen-dependent ring cleavage of myo-inositol to D-glucuronate. Here, we present the crystal structure of human MIOX in complex with myo-inosose-1 bound in a terminal mode to the MIOX diiron cluster site. Furthermore, from biochemical and biophysical results from N-terminal deletion mutagenesis we show that the N terminus is important, through coordination of a set of loops covering the active site, in shielding the active site during catalysis. EPR spectroscopy of the unliganded enzyme displays a two-component spectrum that we can relate to an open and a closed active site conformation. Furthermore, based on site-directed mutagenesis in combination with biochemical and biophysical data, we propose a novel role for Lys(127) in governing access to the diiron cluster.
Preview · Article · Jun 2008 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Phosphatases are a diverse group of enzymes that regulate numerous cellular processes. Much of what is known relates to the
tyrosine, threonine, and serine phosphatases, whereas the histidine phosphatases have not been studied as much. The structure
of phosphohistidine phosphatase (PHPT1), the first identified eukaryotic-protein histidine phosphatase, has been determined
to a resolution of 1.9Å using multiple-wavelength anomalous dispersion methods. This enzyme can dephosphorylate a variety
of proteins (e.g. ATP-citrate lyase and the β-subunit of G proteins). A putative active site has been identified by its electrostatic character,
ion binding, and conserved protein residues. Histidine 53 is proposed to play a major role in histidine dephosphorylation
based on these observations and previous mutational studies. Models of peptide binding are discussed to suggest possible mechanisms
for substrate recognition.
No preview · Article · Dec 2006 · Journal of Biological Chemistry