Catherine Guillou

Catalytic enantioselective vinylogous Mukaiyama-Michael addition of 2-silyloxyfurans to cyclic unsaturated oxo esters.

The copper-catalyzed asymmetric addition of 2-silyloxyfurans to cyclic unsaturated oxo esters is reported. The reaction proceeds with excellent diastereocontrol (usually dr 99:1) and modest to high enantioselectivity, depending on the nature of the ester group and the substitution of the cyclic oxo ester. We have shown that these substrates can be transformed into a variety of building blocks bearing a γ-butenolide or γ-lactone connected to a cycloalkane or cycoalkene moiety.

Relocation of Aurora B and Survivin from Centromeres to the Central Spindle Impaired by a Kinesin‐Specific MKLP‐2 Inhibitor

Mitotic kinesin-like protein 2 (MKLP-2), a member of the kinesin-6 family, is essential for cytokinesis. We screened 8900 small molecules for inhibition of the ATPase activity of MKLP-2 and identified the first inhibitor, (Z)-2-(1H-indol-3yl)-3-(pyridin-3-yl)acrylonitrile, which we named paprotrain (PAssenger PROteins TRAnsport INhibitor; Figure 1b). Paprotrain is a reversible inhibitor uncompetitive with ATP (inhibitor constant Ki = (3.4 0.1) mm) and noncompetitive with microtubules (MTs; Ki = (1.6 0.1) mm). It is specific for MKLP-2 as it does not inhibit other members of the kinesin superfamily. Paprotrain is cell permeable and incubation with 10 to 50 mm inhibitor results in binucleated cells, a characteristic phenotype similar to that observed following RNA interference (RNAi)-mediated depletion of MKLP-2. Additional mitotic spindle defects were also observed. Notably, paprotrain perturbs MKLP-2-mediated relocation of the chromosome passenger proteins Aurora B and survivin from the centromeres to the central spindle. Lack of relocation of passenger proteins to the central spindle is associated with failure of cytokinesis and generation of binucleated cells. In contrast, paprotrain does not impair kinesin family member 4 (Kif4)-mediated translocation of protein regulating cytokinesis 1 (PRC1), which emphasizes its specificity for MKLP-2. We conclude that paprotrain is a new lead compound targeting MKLP-2 by a novel mechanism of action. The motor domain of human MKLP-2 (residues 1–519) was cloned, expressed, and purified (Figure 1a). Gel filtration and analytical ultracentrifugation showed that the protein is monomeric (data not shown) with an estimated molecular mass of (77 5) kDa (see Table S1 in the Supporting Information). The basal ATPase activity was (0.034 0.012) s , with a KM,ATP of 59.4 mm. The activity decreased with increasing NaCl concentration and inhibitor screening was therefore performed in the absence of salt. The MT-stimulated ATPase activity was 1.8 s , which corresponded to an estimated 53fold stimulation compared to the basal activity. The K0.5,MT was 1.6 mm (see Figure S1 and Table S2 in the Supporting Information). We measured the inhibition of the basal and MTstimulated ATPase activity in the presence of paprotrain (Figure 2). Calculated IC50 values were (1.35 0.2) mm in basal (Figure 2a) and (0.83 0.1) mm in MT-stimulated ATPase assays (Figure 2b). The measured rates of ATP hydrolysis in the presence of varying concentrations of paprotrain and MTs demonstrate that the inhibitor acts through a mixed noncompetitive mechanism with respect to MTs with an inhibitor constant Ki = (1.6 0.07) mm (Figure 2c). We also analyzed the ATP hydrolysis rate at different ATP and inhibitor concentrations. Paprotrain is an ATP uncompetitive inhibitor with Ki = (3.36 0.09) mm (Figure 2d). The ATPase activity of a panel of 12 other kinesins 3] was not inhibited by paprotrain, thus demonstrating high specificity within the kinesin superfamily (see Figure S2 a in the Supporting Information). Notably, paprotrain does not inhibit Figure 1. a) Bar diagram of human MKLP-2 and the protein construct used for inhibitor screening. MKLP-2 has an N-terminal motor domain (amino acids (aa) 56 to 505), an a-helical region (aa 519 to 821), and a C-terminal tail domain (aa 821 to 890). MKLP-2 contains an approximately 100-residue insertion (aa 189 to 288) in the motor domain that is unique for members of the kinesin-6 family. b) Chemical formula of paprotrain. Trx = thioredoxin.

A Phenotypic Assay to Identify Chikungunya Virus Inhibitors Targeting the Nonstructural Protein nsP2

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen responsible for an acute infection of abrupt onset, characterized by high fever, polyarthralgia, myalgia, headaches, chills, and rash. In 2006, CHIKV was responsible for an epidemic outbreak of unprecedented magnitude in the Indian Ocean, stressing the need for therapeutic approaches. Since then, we have acquired a better understanding of CHIKV biology, but we are still missing active molecules against this reemerging pathogen. We recently reported that the nonstructural nsP2 protein of CHIKV induces a transcriptional shutoff that allows the virus to block cellular antiviral response. This was demonstrated using various luciferase-based reporter gene assays, including a trans-reporter system where Gal4 DNA binding domain is fused to Fos transcription factor. Here, we turned this assay into a high-throughput screening system to identify small molecules targeting nsP2-mediated shutoff. Among 3040 molecules tested, we identified one natural compound that partially blocks nsP2 activity and inhibits CHIKV replication in vitro. This proof of concept suggests that similar functional assays could be developed to target other viral proteins mediating a cellular shutoff and identify innovative therapeutic molecules.

Iminosugars as a new class of cholinesterase inhibitors

To further extend the scope of iminosugar biological activity, a systematic structure-activity relationship investigation has been performed by synthesizing and evaluating as cholinesterase inhibitors a library of twenty-three iminoalditols with different substitutions and stereochemistry patterns. These compounds have been evaluated in vitro for the inhibition of cholinesterases (different sources of acetylcholinesterase and butyrylcholinesterase). Some compounds have IC50 values in the micromolar range and display significant inhibition selectivity for butyrylcholinesterase over acetylcholinesterase. These are the first examples of iminosugar-based inhibitors of cholinesterases.

At a supra-physiological concentration, human sexual hormones act as quorum-sensing inhibitors.

N-Acylhomoserine lactone (AHL)-mediated quorum-sensing (QS) regulates virulence functions in plant and animal pathogens such as Agrobacterium tumefaciens and Pseudomonas aeruginosa. A chemolibrary of more than 3500 compounds was screened using two bacterial AHL-biosensors to identify QS-inhibitors (QSIs). The purity and structure of 15 QSIs selected through this screening were verified using HPLC MS/MS tools and their activity tested on the A. tumefaciens and P. aeruginosa bacterial models. The IC50 value of the identified QSIs ranged from 2.5 to 90 µg/ml, values that are in the same range as those reported for the previously identified QSI 4-nitropyridine-N-oxide (IC50 24 µg/ml). Under the tested culture conditions, most of the identified QSIs did not exhibit bacteriostatic or bactericidal activities. One third of the tested QSIs, including the plant compound hordenine and the human sexual hormone estrone, decreased the frequency of the QS-regulated horizontal transfer of the tumor-inducing (Ti) plasmid in A. tumefaciens. Hordenine, estrone as well as its structural relatives estriol and estradiol, also decreased AHL accumulation and the expression of six QS-regulated genes (lasI, lasR, lasB, rhlI, rhlR, and rhlA) in cultures of the opportunist pathogen P. aeruginosa. Moreover, the ectopic expression of the AHL-receptors RhlR and LasR of P. aeruginosa in E. coli showed that their gene-regulatory activity was affected by the QSIs. Finally, modeling of the structural interactions between the human hormones and AHL-receptors LasR of P. aeruginosa and TraR of A. tumefaciens confirmed the competitive binding capability of the human sexual hormones. This work indicates potential interferences between bacterial and eukaryotic hormonal communications.

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6–Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.Actin and microtubules play important roles in Golgi structure and function but how they are connected is poorly understood. Here the authors show that KIF20A is involved in the fission process and, in association with Myosin II, serves to anchor RAB6 on Golgi/TGN membranes near microtubules nucleating sites.

Abstract 4140: Identification of a selective MKLP2/KIF20A inhibitor with high in-vivo antitumor activity

Mitotic kinesins are essential regulators of cancer cell replication and migration. The mitotic kinesin MKLP2/KIF20A, a member of the kinesin-6 family, plays an essential role during cytokinesis and was identified as a potential new target for cancer chemotherapy1. We have previously identified Paprotrain, a new synthetic compound, as the first selective MKLP2 inhibitor2. Recently, we obtained Paprotrain analogues with higher potency on MKLP23. Herein we describe the identification and characterization of BKS0349, a new potent analogue of Paprotrain. BKS0349 compound is 10 times more potent than paprotain on MKLP2 inhibition and has shown an even more restricted specificity profile when tested on a large set of kinases. In-vitro this compound is highly cytotoxic on a wide panel of human cancer cell lines (IC50 ranged 10-70 nM, which corresponds to 1000 fold improvement of paprotrain potency) while no toxicity is observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50µM). BKS0349 compound is well tolerated in mice using repeated intravenous administrations (200 mg/kg, twice a week for 4 weeks). In xenografted nude mice, in-vivo treatment with BKS0349 compound demonstrates a high antitumor activity against various human cancer cell models, either sensitive or resistant to some standard-of-care treatments. In addition, human cancer cells treated in-vitro as well as in-vivo with BSK0349 compound display Golgi scattering and a mitotic arrest leading to cell death, as hallmarks of BKS0349’ mode of action. These findings show that MKLP2 is a potential new target for cancer chemotherapy and BKS0349 a good candidate to be developed for cancer treatment. 1Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance. P. Khongkow et al. Oncogene, 2015, 1-13. 2Relocation of Aurora B and Survivin from centromeres to the central spindle impaired by a kinesin-specific MKLP-2 inhibitor. S. Tcherniuk et al. Angew. Chem. Int. Ed., 2010, 49:8228-8231. 3New MKLP-2 inhibitors in the paprotrain series: design, synthesis and biological evaluations. C. Labriere et al. Bioorg and Med Chem, 2016, 24:731-734. Note: This abstract was not presented at the meeting. Citation Format: Yves Collette, Stephanie Miserey-Lenkei, Catherine Guillou, Denis Carniato, Bernard Pau, Bruno Goud, Norbert Vey, Cecile E. Bougeret. Identification of a selective MKLP2/KIF20A inhibitor with high in-vivo antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4140. doi:10.1158/1538-7445.AM2017-4140