Jérôme Courcambeck

Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations

HIV-1 drug resistance mutations are often inversely correlated with viral fitness, which remains poorly described at the molecular level. Some resistance mutations can also suppress resistance caused by other resistance mutations. We report the molecular mechanisms by which a virus resistant to lamivudine with the M184V reverse transcriptase mutation shows increased susceptibility to tenofovir and can suppress the effects of the tenofovir resistance mutation K65R. Additionally, we report how the decreased viral replication capacity of resistant viruses is directly linked to their decreased ability to use natural nucleotide substrates and that combination of the K65R and M184V resistance mutations leads to greater decreases in viral replication capacity. All together, these results define at the molecular level how nucleoside-resistant viruses can be driven to reduced viral fitness.

Mutation rate in hepatitis C virus NS3 protease is not influenced by HIV-1 protease inhibitor therapy

HIV-1 Tat is known to influence several intracellular functions including its ability to activate long terminal repeat (LTR) promoter-mediated transactivation and cause apoptosis [1–3]. Although a number of studies have been performed with subtype B gene products, relatively little information is available for subtype C, which is responsible for causing more than 50% infections worldwide, including India where it is the major subtype. In the present study, we constructed several recombinant clones of Tat-B (derived from pNL4-3) [4] and Tat-C (derived from an Indian isolate which is 95% similar to the consensus subtype C based on its amino acid sequence [5] (clone 93IN905, GenBank Accession no. AF067158) [6] with respect to the two exons for fine functional domain analysis. It is worth noting that most subtype C isolates possess QGD compared with RGD in the second exon of Tat gene in the same position (78–80 amino acids) (a known cell adhesion motif), which is associated with integrin-mediated signaling and cell adhesion, etc., besides other changes throughout the gene. We reasoned that these changes might modulate its ability to transactivate LTR promoters and apoptosis. Therefore, we made constructs of subtypes B and C Tat that consisted of either the RGD or QGD motif and also swapped the first and second exons of the Tat gene. Precise gene fusion technology was used to generate such chimeric constructs as described by one of us earlier [7] and confirmed by sequencing. The various constructs made are indicated at the bottom of Fig. 1a with Tat-B and Tat-C with RGD/QGD domains. An internal reporter gene control (pSV-b-gal; Promega, Madison, Wisconsin, USA) was always included to ensure uniform transfection efficiency.

Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors

Herein, we report the synthesis and structure-activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c (EC50=0.8 μM).

Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model

AIM To evaluate the antiviral potency of a new anti-hepatitis C virus (HCV) antiviral agent targeting the cellular autophagy machinery. METHODS Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices (2.7 × 10(6) cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant (multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription - polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. RESULTS The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dose-dependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect (compared to hydroxychloroquine EC50 = 1.17 μmol/L). CONCLUSION Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dose-dependent manner without cytotoxic effect.

Abstract 1914: GNS561 is a new quinoline derivative with high efficacy on cancer stem cells from colorectal liver metastasis and hepatocellular carcinoma

Background: In spite of wide application of sorafenib for advanced hepatocellular carcinoma (HCC) treatment, and systemic chemotherapy cocktails (5-fluorouracil, irinotecan, and oxaliplatin) for metastatic colorectal cancer, the prognostic for both cancers remains poor. In recent years, highly tumorigenic sub-populations of cancer cells named Cancer Stem Cells (CSCs) have been claimed as responsible of some tumor recurrences. Indeed, CSCs are resistant to chemotherapy, and they have the ability to regenerate all the tumor bulk with its heterogeneous cell type populations. For this reason, new drugs with original mechanism of action which target CSC properties would likely improve cancer treatment Material and methods: Antitumor activity of GNS561 was tested on a panel of cancer cell lines and primary tumors. GNS561 impact on CSCs subpopulation in patient derived cells from colorectal hepatic metastatic tumors was assessed by flow cytometry (ALDH activity). In HCC, the effect of this drug was evaluated by sphere formation assay as readout to estimate CSC survival. Tolerance and plasma and liver pharmacokinetic were evaluated after single and repeated dosing in mice and rats. In vivo GNS561 activity was tested in orthotopic mouse model Results: GNS561 demonstrated multiple cellular effects such as inhibition of autophagy, induction of apoptosis and cell cycle modulation. It showed antitumor activity against several human cancer cell lines. Furthermore, GNS561 was effective against a panel of HCC tumors even from patients harboring sorafenib resistance. GNS561 showed nonetheless an original dose-response cytotoxic activity against the whole tumor populations but also against a subpopulation displaying high ALDH activity in three CRC patient-derived cell lines established from fresh liver metastasis biopsies. Consequently, on the same model this compound induced a striking decrease of sphere formation. In HCC cell lines GNS561 was active on both whole populations (mean EC50 2µM) and subpopulations displaying CSC features (Epcam high). In addition, in the opposite of sorafenib, GNS561 decreases the HCC cell capacity to form spheroids. In mouse, GNS561 was found well tolerated and highly selectively trapped in the liver (exposure ratio liver/plasma about 170 animals). In HCC PDX mouse model, tumor growth was significantly reduced by GNS561 with a dose-response manner, this tumor regression was associated with AFP level decreases by 72% with GNS561 (p=0.002) and 54% with sorafenib (p=0.046) compared to control Conclusions: GNS561 is a liver selective drug which offers great promise for HCC and liver metastatic tumors treatment. By simultaneously targeting the cancer stem cell subpopulation and tumor bulk, both cell heterogeneity, plasticity and recurrences could be overcome at least in colorectal cancer and HCC. GNS 561 is now aimed to further reach clinical development in patients in 2017 Note: This abstract was not presented at the meeting. Citation Format: Firas Bassissi, Elena Patricia Gifu, Sonia Brun, Jerome Courcambeck, Antoine Beret, Jean Marc Pascussi, Julie Pannequin, Eric Raymond, Philippe Halfon, Philippe Merle, Claude Caron de Fromentel. GNS561 is a new quinoline derivative with high efficacy on cancer stem cells from colorectal liver metastasis and hepatocellular carcinoma [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 1914. doi:10.1158/1538-7445.AM2017-1914

Abstract 5124: GNS561 a new quinoline derivative inhibits the growth of hepatocellular carcinoma in a cirrhotic rat and human PDX orthotopic mouse models

Background: Hepatocellular carcinoma (HCC) remains a major health problem, often diagnosed at late stages with limited number of therapeutic options. New drugs with original mechanisms of action are urgently aimed to improve current armamentarium in HCC patients. Quinoline derivatives are novel class of oral small molecules inducing multiple cellular effects such as inhibition of autophagy, induction of apoptosis, and cell cycle modulation. The aim of these studies was to assess tolerance and efficacy of a new quinolone derivative GNS561. Material and methods: In vitro experiments were realized with viability, apoptosis and migration in tumor cells in HCC cell lines and primary tumor. Drug tolerance and plasma and liver pharmacokinetic were evaluated after single and repeated dosing in mice and rat. GNS561 and sorafenib efficacy in vivo were evaluated in a PDX orthotopic BALB/c-nu mouse model and in a diethylnitrosamine (DEN)-induced HCC cirrhotic rat model. AFP, cell proliferation and tumor weight and size were assessed in mice. In rat tumor progression was followed by MRI, pathological analysis (tumor size and number), immunohistochemistry and PCR analysis after 6weeks of treatment. Results: GNS 561 shows potent anti-proliferative activity when assayed against a panel of human tumor cell lines and notably against a panel of HCC patient primry tumors even in those with sorafenib resistance (Mean EC50 3µM vs 11µM for sorafenib). GNS561 is highly selectively trapped in the liver. Plasma and liver PK in mice and rats after single and repeated doses confirm this selectivity with good tolerance and oral bioavailability. In PDX mouse model, tumor growth was significantly reduced by GNS561 with a dose-response manner, this tumor regression was associated with AFP level decreases by 72% with GNS561 (p=0.002) and 54% with sorafenib (p=0.046) compared to control. In rat model, mean number of tumors was significantly lower in GNS561 at 15mg/kg group (n=50.6), in sorafanib at 10mg/kg (n=65.1) and in combination group (n=40.6), when compared to control (n=100.4; p=0.0024, p= 0.029 and p=0.0002). Tumor decrease measured by MRI was associated with a significantly reduced proliferation of tumor cells particularly in GNS561 group (70%) and combination (84%) compared to control, whereas the effect of sorafenib alone on proliferation was modest (30%). Conclusions: GNS561 is a liver selective drug with good tolerance and promising efficacy in different HCC animal models. GNS 561 was more efficient than sorafenib to control tumor growth in preclinical models. Based on its safe toxicity profile and potent activity in rodent models, GNS 561 is now aimed to further reach clinical development in patients with HCC in 2017. Citation Format: Firas BASSISSI, Zuzana Macek Jilkova, Sonia Brun, Jerome Courcambeck, Jennifer Tracz, Keerthi Kurma, Gael S. Roth, Cindy Khaldi, Corinne Chaimbault, Benoit Quentin, Emilie Asseraf, Antoine Beret, Eric Raymond, Philippe Halfon, Thomas Decaens. GNS561 a new quinoline derivative inhibits the growth of hepatocellular carcinoma in a cirrhotic rat and human PDX orthotopic mouse models [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 5124. doi:10.1158/1538-7445.AM2017-5124

Résistance aux analogues de nucléosides dans le traitement de l’hépatite chronique B

Resume Propos. – Le traitement classique des hepatites virales chroniques B (VHB) repose sur le traitement par interferon alpha. Toutefois une reponse prolongee et durable apres l’arret du traitement par interferon alpha n’est observee que chez 30 % des patients. Actualites et points forts. – Le developpement de nouvelles molecules, analogues de nucleosides, a ete favorise par des modeles d’etudes de la replication virale B qui permettent d’evaluer l’efficacite de nouveaux agents antiviraux tels la lamivudine, le famciclovir, l’adefovir, l’entecavir ou la clevudine. Cependant, a l’instar du VIH, des souches VHB resistantes au traitement sont apparues au decours des traitements par la plupart de ces analogues nucleosidiques. Cette mise au point a pour but d’aborder les mecanismes de resistance et de caracterisation des mutants du VHB afin d’optimiser les choix therapeutiques. Perspectives et projets. – La determination des resistances aux differentes molecules actives contre le VHB apparait comme preponderante pour guider les choix therapeutiques et maintenir une replication virale B indetectable. Le traitement de l’hepatite B, comme celui de la plupart des maladies virales chroniques, reposera a terme sur des associations therapeutiques dont l’efficacite sera controlee par la surveillance de l’emergence de souches resistantes.