Publications by authors named "Sandrine Thieffine"

6 Publications

  • Page 1 of 1

Insights into PARP Inhibitors' Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays.

J Biomol Screen 2014 Sep 10;19(8):1212-9. Epub 2014 Jun 10.

Nerviano Medical Sciences S.r.l., Nerviano, Italy

PARP inhibitors are an exciting new class of antineoplastic drugs that have been proven to be efficacious as single agents in cancer settings with inherent DNA repair defects, as well as in combination with DNA-damaging chemotherapeutics. Currently, they are designed to target the catalytic domain of PARP-1, the most studied member of the family, with a key role in the DNA-damage repair process. Because PARP inhibitors are substrate (NAD(+)) competitors, there is a need for a deeper understanding of their cross-reactivity. This is particularly relevant for PARP-2, the PARP-1 closest homologue, for which an embryonic lethal phenotype has been observed in double knockout mice. In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD(+)binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicable to other members of the PARP family.
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http://dx.doi.org/10.1177/1087057114538319DOI Listing
September 2014

Development of biochemical assays for the identification of eIF4E-specific inhibitors.

J Biomol Screen 2012 Jun 5;17(5):581-92. Epub 2012 Mar 5.

Biotechnology Department, Nerviano Medical Sciences, Nerviano (MI), Italy.

Control of mRNA translation plays a critical role in cell growth, proliferation, and differentiation and is tightly regulated by AKT and RAS oncogenic pathways. A key player in the regulation of this process is the mRNA 5' cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E). eIF4E contributes to malignancy by selectively enabling the translation of a limited pool of mRNAs that generally encode key proteins involved in cell cycle progression, angiogenesis, and metastasis. Several data indicate that the inhibition of eIF4E in tumor cell lines and xenograft models impairs tumor growth and induces apoptosis; eIF4E, therefore, can be considered a valuable target for cancer therapy. Targeting the cap-binding pocket of eIF4E should represent a way to inhibit all the eIF4E cellular functions. We present here the development and validation of different biochemical assays based on fluorescence polarization and surface plasmon resonance techniques. These assays could support high-throughput screening, further refinement, and characterization of eIF4E inhibitors, as well as selectivity assessment against CBP80/CBP20, the other major cap-binding complex of eukaryotic cells, overall providing a robust roadmap for development of eIF4E-specific inhibitors.
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http://dx.doi.org/10.1177/1087057112438554DOI Listing
June 2012

Identification of candidate substrates for poly(ADP-ribose) polymerase-2 (PARP2) in the absence of DNA damage using high-density protein microarrays.

FEBS J 2011 Oct 6;278(19):3676-87. Epub 2011 Sep 6.

Department of Biotechnology, BU Oncology, Nerviano Medical Sciences Srl, Nerviano (MI), Italy.

Poly(ADP-ribose) polymerase-2 (PARP2) belongs to the ADP-ribosyltransferase family of enzymes that catalyze the addition of ADP-ribose units to acceptor proteins, thus affecting many diverse cellular processes. In particular, PARP2 shares with PARP1 and, as recently highlighted, PARP3 the sole property of being catalytically activated by DNA-strand breaks, implying key downstream functions in the cellular response to DNA damage for both enzymes. However, evidence from several studies suggests unique functions for PARP2 in additional processes, possibly mediated through its basal, DNA-damage unstimulated ADP-ribosylating activity. Here, we describe the development and application of a protein microarray-based approach tailored to identify proteins that are ADP-ribosylated by PARP2 in the absence of DNA damage mimetics and might thus represent useful entry points to the exploration of novel PARP2 functions. Several candidate substrates for PARP2 were identified and global hit enrichment analysis showed a clear enrichment in translation initiation and RNA helicase molecular functions. In addition, the top scoring candidates FK506-binding protein 3 and SH3 and cysteine-rich domain-containing protein 1 were selected and confirmed in a complementary assay format as substrates for unstimulated PARP2.
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http://dx.doi.org/10.1111/j.1742-4658.2011.08286.xDOI Listing
October 2011

Structural basis for CARM1 inhibition by indole and pyrazole inhibitors.

Biochem J 2011 Jun;436(2):331-9

Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, NJ 08543-4000, USA.

CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.
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http://dx.doi.org/10.1042/BJ20102161DOI Listing
June 2011

Cell division cycle 7 kinase inhibitors: 1H-pyrrolo[2,3-b]pyridines, synthesis and structure-activity relationships.

J Med Chem 2009 Jul;52(14):4380-90

Nerviano Medical Sciences, 20014 Nerviano, Milano, Italy.

Cdc7 kinase has recently emerged as an attractive target for cancer therapy and low-molecular-weight inhibitors of Cdc7 kinase have been found to be effective in the inhibition of tumor growth in animal models. In this paper, we describe synthesis and structure-activity relationships of new 1H-pyrrolo[2,3-b]pyridine derivatives identified as inhibitors of Cdc7 kinase. Progress from (Z)-2-phenyl-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethylene)-3,5-dihydro-4H-imidazol-4-one (1) to [(Z)-2-(benzylamino)-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethylene)-1,3-thiazol-4(5H)-one] (42), a potent ATP mimetic inhibitor of Cdc7 kinase with IC(50) value of 7 nM, is also reported.
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http://dx.doi.org/10.1021/jm900248gDOI Listing
July 2009

Optimization of pyrazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1).

Bioorg Med Chem Lett 2009 Jun 22;19(11):2924-7. Epub 2009 Apr 22.

Bristol-Myers Squibb Pharmaceutical Research and Development, Princeton, NJ 08543, United States.

Design, synthesis, and SAR development led to the identification of the potent, novel, and selective pyrazole based inhibitor (7f) of Coactivator Associated Arginine Methyltransferase (CARM1).
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http://dx.doi.org/10.1016/j.bmcl.2009.04.075DOI Listing
June 2009