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1.
J Med Chem ; 58(16): 6574-88, 2015 Aug 27.
Article in English | MEDLINE | ID: mdl-26218264

ABSTRACT

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein-ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.


Subject(s)
Antineoplastic Agents/pharmacology , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Inhibitor of Apoptosis Proteins/drug effects , Peptide Fragments/pharmacology , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Cell Proliferation/drug effects , Computational Biology , Drug Design , Drug Discovery , High-Throughput Screening Assays , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacokinetics , Piperazines/chemical synthesis , Piperazines/pharmacology , Xenograft Model Antitumor Assays
2.
MAbs ; 6(2): 533-46, 2014.
Article in English | MEDLINE | ID: mdl-24492308

ABSTRACT

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.


Subject(s)
Antibodies, Monoclonal/metabolism , Interleukins/immunology , Macrophage Colony-Stimulating Factor/metabolism , Receptor, Macrophage Colony-Stimulating Factor/immunology , Recombinant Fusion Proteins/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Computational Biology , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Humans , Immunoglobulin Variable Region/chemistry , Macrophage Colony-Stimulating Factor/immunology , Mice , Models, Chemical , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Sequence Alignment
3.
FASEB J ; 24(11): 4523-34, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20634350

ABSTRACT

The transcriptional activity of nuclear retinoic acid receptors (RARs) relies on the association/dissociation of coregulators at the ligand-binding domain. However, we determined that the N-terminal domain (NTD) also plays a role through its phosphorylation, and we isolated vinexinß, a cytoskeleton protein with three SH3 domains, as a new partner of the RARγ NTD. Here we deciphered the mechanism of the interaction and its role in RARγ-mediated transcription. By combining molecular and biophysical (surface plasmon resonance, NMR, and fluorescence resonance energy transfer) approaches, we demonstrated that the third SH3 domain of vinexinß interacts with a proline-rich domain (PRD) located in RARγ NTD and that phosphorylation at a serine located in the PRD abrogates the interaction. The affinity of the interaction was also evaluated. In vivo, vinexinß represses RARγ-mediated transcription and we dissected the underlying mechanism in chromatin immunoprecipitation experiments performed with F9 cells expressing RARγ wild type or mutated at the phosphorylation site. In the absence of retinoic acid (RA), vinexinß does not occupy RARγ target gene promoters and sequesters nonphosphorylated RARγ out of promoters. In response to RA, RARγ becomes phosphorylated and dissociates from vinexinß. This separation allows RARγ to occupy promoters. This is the first report of an RAR corepressor association/dissociation out of promoters and regulated by phosphorylation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Receptors, Retinoic Acid/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Mice , Molecular Sequence Data , Mutation/genetics , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Receptors, Retinoic Acid/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Retinoic Acid Receptor gamma
4.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 7): 745-55, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20606254

ABSTRACT

Tfb5 interacts with the Tfb2 subunit of the general transcription factor TFIIH to ensure efficient nucleotide-excision repair in eukaryotes. The crystal structure of the complex between Tfb5 and the C-terminal region of Tfb2 (Tfb2C) from Saccharomyces cerevisiae has recently been reported. Here, the structure-determination process is described as a case study. Although crystals were obtained readily, it was not possible to determine experimental phases from a first crystal form (Tfb2(412-513)-Tfb5(2-72)) that diffracted to 2.6 A resolution. Shortening of the Tfb2C from its N-terminus was decisive and modified the crystal packing, leading to a second crystal form (Tfb2(435-513)-Tfb5(2-72)). These crystals diffracted to 1.7 A resolution with excellent mosaicity and allowed structure determination by conventional approaches using heavy atoms. The refined structure from the second crystal form was used to solve the structure of the first crystal form by molecular replacement. Comparison of the two structures revealed that the N-terminal region of Tfb2C and (to a lesser extent) the C-terminal region of Tfb5 contributed to the crystal packing. A detailed analysis illustrates how variation in domain boundaries influences crystal packing and quality.


Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Transcription Factor TFIIH/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Transcription Factor TFIIH/metabolism
5.
Biochimie ; 91(8): 1003-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19467292

ABSTRACT

Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.


Subject(s)
RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Selenoproteins/biosynthesis , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Protein Denaturation , Rats , Sequence Analysis, DNA
6.
Nat Struct Mol Biol ; 15(9): 980-4, 2008 Sep.
Article in English | MEDLINE | ID: mdl-19172752

ABSTRACT

Patients with the rare neurodevelopmental repair syndrome known as group A trichothiodystrophy (TTD-A) carry mutations in the gene encoding the p8 subunit of the transcription and DNA repair factor TFIIH. Here we describe the crystal structure of a minimal complex between Tfb5, the yeast ortholog of p8, and the C-terminal domain of Tfb2, the yeast p52 subunit of TFIIH. The structure revealed that these two polypeptides adopt the same fold, forming a compact pseudosymmetric heterodimer via a beta-strand addition and coiled coils interactions between terminal alpha-helices. Furthermore, Tfb5 protects a hydrophobic surface in Tfb2 from solvent, providing a rationale for the influence of p8 in the stabilization of p52 and explaining why mutations that weaken p8-p52 interactions lead to a reduced intracellular TFIIH concentration and a defect in nucleotide-excision repair, a common feature of TTD cells.


Subject(s)
Trichothiodystrophy Syndromes/metabolism , Crystallography, X-Ray , DNA Repair , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Mutation , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factor TFIIH/chemistry , Transcription Factor TFIIH/genetics , Transcription Factor TFIIH/metabolism , Transcription, Genetic , Trichothiodystrophy Syndromes/classification , Trichothiodystrophy Syndromes/genetics
7.
J Mol Biol ; 368(2): 473-80, 2007 Apr 27.
Article in English | MEDLINE | ID: mdl-17350038

ABSTRACT

Trichothiodystrophy (TTD) is a rare hereditary multi-system disorder associated with defects in nucleotide excision repair (NER) and transcription as consequences of mutations in XPB, XPD and p8/TTD-A subunits of transcription factor IIH (TFIIH). Here, we report the solution structure of the p8/TTD-A protein, a small alpha/beta protein built around an antiparallel beta-sheet that forms a homodimer with an extended interface. In order to characterize the dimer interface, we have introduced a mutation at position 44, which destabilizes the dimeric form of the protein. We have shown that this mutation has no effect on the intrinsic ability of p8/TTD-A to stimulate NER in vitro, but affects the capacity of p8/TTD-A to restore TFIIH concentration in TTD-A fibroblasts. Point mutations found in TTD-A patients are discussed on the basis of the present structure.


Subject(s)
Abnormalities, Multiple/metabolism , Protein Subunits/chemistry , Transcription Factor TFIIH/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Chromatography, Gel , DNA Repair , Dimerization , Humans , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Solutions , Structure-Activity Relationship
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