Emmanuelle Braud

Development of Novel Thiazolopyrimidines as CDC25B Phosphatase Inhibitors

CDC25 inhibition by thiazolopyrimidines: CDC25 is an attractive target for cancer therapy, as it is overexpressed in numerous cancers and is often associated with tumor aggressiveness and poor prognosis. Based on an in silico/in vitro screen, we developed a series of thiazolopyrimidines to further improve inhibitory activity and cytotoxic properties.

Rapid Discovery of Triazolobenzylidene-Thiazolopyrimidines (TBTP) as CDC25 Phosphatase Inhibitors by Parallel Click Chemistry and in Situ Screening

UniVersite Paul Sabatier Toulouse III, Faculte des Sciences Pharmaceutiques, Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox, UMR152 IRD-UT3, 31062 Toulouse cedex 09, France, UniVersite Paris Descartes, UFR Biomedicale, Laboratoire de Pharmacochimie Moleculaire et Cellulaire, 45 rue des Saints-Peres, 75270 Paris, cedex 06, France, and INSERM U648, 45 rue des Saints-Peres, 75270 Paris, cedex 06, France

Novel naphthoquinone and quinolinedione inhibitors of CDC25 phosphatase activity with antiproliferative properties.

CDC25 phosphatases are considered as attractive targets for anti-cancer therapy. To date, quinone derivatives are among the most potent inhibitors of CDC25 phosphatase activity. We present in this paper the synthesis and the biological evaluation of new quinolinedione and naphthoquinone derivatives, containing carboxylic or malonic acids groups introduced to mimic the role of the phosphate moieties of Cyclin-Dependent Kinase complexes. The most efficient compounds show inhibitory activity against CDC25B with IC(50) values in the 10 microM range, and are cytotoxic against HeLa cells.

Design and synthesis of novel bis-thiazolone derivatives as micromolar CDC25 phosphatase inhibitors: effect of dimerisation on phosphatase inhibition.

CDC25 phosphatases are involved in deregulated cell cycle progression and tumor development with poor prognosis. Among the most potent CDC25 inhibitors, quinonoid-based derivatives have been extensively studied. Dimerisation of heterocyclic quinones has led to IRC-083864, a bis-quinone compound with increased CDC25B inhibitory activity. Thirty-one bis-thiazolone derivatives were synthesized and assayed for CDC25 inhibitory activity. Most of the dimers displayed enhanced inhibitory activities with micromolar IC(50) values lower than that observed for each thiazolone scaffold separately. Moreover, most of these compounds were selective CDC25 inhibitors. Dimer 40 showed an IC(50) value of 2.9 μM and could inhibit CDC25 activity without generating reactive oxygen species which is likely to occur with quinone-based inhibitors. Molecular docking studies suggested that the dimers could bind simultaneously to the active site and the inhibitor binding pocket.

Study of the docking-dependent PLK1 phosphorylation of the CDC25B phosphatase.

CDC25 (A, B and C) phosphatases control cell cycle progression through the timely dephosphorylation and activation of cyclin-dependent kinases (CDK). At mitosis the CDC25B phosphatase activity is dependent on its phosphorylation by multiple kinases impinging on its localisation, stability and catalytic activity. Here we report that prior phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro, and we also show that phosphorylated S50 serves as a docking site for PLK1. Using a sophisticated strategy based on the sequential phosphorylation of CDC25B with (16)O and (18)O ATP prior to nanoLC-MS/MS analysis we identified 13 sites phosphorylated by PLK1. This study illustrates the complexity of the phosphorylation pattern and of the subsequent regulation of CDC25B activity.

5-Substituted [1]pyrindine derivatives with antiproliferative activity

We report herein the synthesis of 5-substituted [1]pyrindine derivatives and the evaluation of their antiproliferative properties on HeLa cells, a cervical carcinoma tumor cell line, and on the melanoma A2058 cell line. The most efficient compounds display cytotoxicity against tumor cells in the micromolar range but have interestingly no effect against the normal human fibroblasts CRL-2796. Generally, these pyrindines are active on both tumor cell lines. Compounds bearing large substituents with structural rigidity at position 5 such as phenyl-furyl show no inhibition of cell growth.

Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D-Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by β-Lactamase BlaC.

Combinations of β-lactams of the carbapenem class, such as meropenem, with clavulanate, a β-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual β-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum β-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the β-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide-alkyne Huisgen cycloaddition reaction catalyzed by copper(I) is fully compatible with the highly unstable β-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the L,D-transpeptidase LdtMt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of LdtMt1 and reduced hydrolysis by BlaC.

Synthesis of 3′-Fluoro-tRNA Analogues for Exploring Non-ribosomal Peptide Synthesis in Bacteria

Aminoacyl‐tRNAs (aa‐tRNAs) participate in a vast repertoire of metabolic pathways, including the synthesis of the peptidoglycan network in the cell walls of bacterial pathogens. Synthesis of aminoacyl‐tRNA analogues is critical for further understanding the mechanisms of these reactions. Here we report the semi‐synthesis of 3′‐fluoro analogues of Ala‐tRNAAla. The presence of fluorine in the 3′‐position blocks Ala at the 2′‐position by preventing spontaneous migration of the residue between positions 2′ and 3′. NMR analyses showed that substitution of the 3′‐hydroxy group by fluorine in the ribo configuration favours the S‐type conformation of the furanose ring of terminal adenosine A76. In contrast, the N‐type conformation is favoured by the presence of fluorine in the xylo configuration. Thus, introduction of fluorine in the ribo and xylo configurations affects the conformation of the furanose ring in reciprocal ways. These compounds should provide insight into substrate recognition by Fem transferases and the Ala‐tRNA synthetases.

Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d-Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis

The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by l,d-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the l,d-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essential role of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.

Insights into the interaction of high potency inhibitor IRC-083864 with phosphatase CDC25

CDC25 phosphatases play a crucial role in cell cycle regulation. They have been found to be over‐expressed in various human tumours and to be valuable targets for cancer treatment. Here, we report the first model of binding of the most potent CDC25 inhibitor to date, the bis‐quinone IRC‐083864, into CDC25B obtained by combining molecular modeling and NMR studies. Our study provides new insights into key interactions of the catalytic site inhibitor and CDC25B in the absence of any available experimental structure of CDC25 with a bound catalytic site inhibitor. The docking model reveals that IRC‐083864 occupies both the active site and the inhibitor binding pocket of the CDC25B catalytic domain. NMR saturation transfer difference and WaterLOGSY data indicate the binding zones of the inhibitor and support the docking model. Probing interactions of analogues of the two quinone units of IRC‐083864 with CDC25B demonstrate that IRC‐083864 competes with each monomer. Proteins 2017; 85:593–601.