GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.

GNS561, a New Autophagy Inhibitor Active against Cancer Stem Cells in Hepatocellular Carcinoma and Hepatic Metastasis from Colorectal Cancer.

Patients with advanced hepatocellular carcinoma (HCC) or metastatic colorectal cancer (mCRC) have a very poor prognosis due to the lack of efficient treatments. As observed in several other tumors, the effectiveness of treatments is mainly hampered by the presence of a highly tumorigenic sub-population of cancer cells called cancer stem cells (CSCs). Indeed, CSCs are resistant to chemotherapy and radiotherapy and can regenerate the tumor bulk. Hence, innovative drugs that are efficient against both bulk tumor cells and CSCs would likely improve cancer treatment. In this study, we demonstrated that GNS561, a new autophagy inhibitor that induces lysosomal cell death, showed significant activity against not only the whole tumor population but also a sub-population displaying CSC features (high ALDH activity and tumorsphere formation ability) in HCC and in liver mCRC cell lines. These results were confirmed in vivo in HCC from a DEN-induced cirrhotic rat model in which GNS561 decreased tumor growth and reduced the frequency of CSCs (CD90+CD45-). Thus, GNS561 offers great promise for cancer therapy by exterminating both the tumor bulk and the CSC sub-population. Accordingly, a global phase 1b clinical trial in liver cancers was recently completed.

GNS561 acts as a potent anti-fibrotic and pro-fibrolytic agent in liver fibrosis through TGF-ß1 inhibition.

BACKGROUND: Hepatic fibrosis is the result of chronic liver injury that can progress to cirrhosis and lead to liver failure. Nevertheless, there are no anti-fibrotic drugs licensed for human use. Here, we investigated the anti-fibrotic activity of GNS561, a new lysosomotropic molecule with high liver tropism. METHODS: The anti-fibrotic effect of GNS561 was determined in vitro using LX-2 hepatic stellate cells (HSCs) and primary human HSCs by studying cell viability, activity of caspases 3/7, autophagic flux, cathepsin maturation and activity, HSC activation and transforming growth factor-ß1 (TGF-ß1) maturation and signaling. The contribution of GNS561 lysosomotropism to its anti-fibrotic activity was assessed by increasing lysosomal pH. The potency of GNS561 on fibrosis was evaluated in vivo in a rat model of diethylnitrosamine-induced liver fibrosis. RESULTS: GNS561 significantly decreased cell viability and promoted apoptosis. Disrupting the lysosomal pH gradient impaired its pharmacological effects, suggesting that GNS561 lysosomotropism mediated cell death. GNS561 impaired cathepsin activity, leading to defective TGF-ß1 maturation and autophagic processes. Moreover, GNS561 decreased HSC activation and extracellular matrix deposition by downregulating TGF-ß1/Smad and mitogen-activated proteine kinase signaling and inducing fibrolysis. Finally, oral administration of GNS561 (15 mg/kg per day) was well tolerated and attenuated diethylnitrosamine-induced liver fibrosis in this rat model (decrease of collagen deposition and of pro-fibrotic markers and increase of fibrolysis). CONCLUSION: GNS561 is a new potent lysosomotropic compound that could represent a valid medicinal option for hepatic fibrosis treatment through both its anti-fibrotic and its pro-fibrolytic effects. In addition, this study provides a rationale for targeting lysosomes as a promising therapeutic strategy in liver fibrosis.

GNS561, a new lysosomotropic small molecule, for the treatment of intrahepatic cholangiocarcinoma.

Among the acquired modifications in cancer cells, changes in lysosomal phenotype and functions are well described, making lysosomes a potential target for novel therapies. Some weak base lipophilic drugs have a particular affinity towards lysosomes, taking benefits from lysosomal trapping to exert anticancer activity. Here, we have developed a new lysosomotropic small molecule, GNS561, and assessed its activity in multiple in vitro intrahepatic cholangiocarcinoma models (HuCCT1 and RBE cell lines and patient-derived cells) and in a chicken chorioallantoic membrane xenograft model. GNS561 significantly reduced cell viability in two intrahepatic cholangiocarcinoma cell lines (IC50 of 1.5 ± 0.2 µM in HuCCT1 and IC50 of 1.7 ± 0.1 µM in RBE cells) and induced apoptosis as measured by caspases activation. We confirmed that GNS561-mediated cell death was related to its lysosomotropic properties. GNS561 induced lysosomal dysregulation as proven by inhibition of late-stage autophagy and induction of a dose-dependent build-up of enlarged lysosomes. In patient-derived cells, GNS561 was more potent than cisplatin and gemcitabine in 2/5 and 1/5 of the patient-derived cells models, respectively. Moreover, in these models, GNS561 was potent in models with low sensitivity to gemcitabine. GNS561 was also efficient in vivo against a human intrahepatic cholangiocarcinoma cell line in a chicken chorioallantoic membrane xenograft model, with a good tolerance at doses high enough to induce an antitumor effect in this model. In summary, GNS561 is a new lysosomotropic agent, with an anticancer activity against intrahepatic cholangiocarcinoma. Further investigations are currently ongoing to fully elucidate its mechanism of action.

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.

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).

Resistance of hepatitis C virus to NS3-4A protease inhibitors: mechanisms of drug resistance induced by R155Q, A156T, D168A and D168V mutations.

BACKGROUND/AIMS: One of the main issues in the development of antiviral therapy is the emergence of drug-resistant viruses. In the case of hepatitis C virus (HCV), selection of drug-resistant mutants was evidenced by in vitro studies on protease inhibitors (PIs); for example, BILN-2061, VX-950 and SCH-6. Four mutations in the HCV protease (R155Q, A156T, D168A and D168V) have been identified in vitro in the HCV replicon system that confer resistance to BILN-2061 (a reference inhibitor). However, the molecular mechanism of drug resistance is still unknown. The aim of this study is to unravel, using an molecular modelling strategy, the structural basis of such molecular mechanism of HCV resistance to PIs. We focused on protease mutations conferring HCV resistance to BILN-2061 and described for the first time such mechanism at a molecular level. METHODS: The structures of drug-resistant NS3 proteases were obtained by mutation of selected residues (R155Q, A156T, D168A and D168V) and the ternary complexes formed between NS3-4A and BILN-2061 were optimized using GenMol software (www.3dgenoscience.com; Genoscience, Marseille, France). RESULTS: Two mechanisms were evidenced for viral resistance to BILN-2061. A 'direct' resistance mechanism is based on contacts between the mutated R155Q and A156T protease residues and its inhibitor. In the 'indirect' resistance mechanism, the mutated D168A/V residue is not in close contact with the drug itself but interacts with other residues connected to the drug. CONCLUSIONS: These data provide new insights in the understanding of the mechanisms of HCV drug escape, and may allow predicting potential cross-resistance phenomenon with other PIs. This approach can be used as a basis for future rational PI drug design candidates.

Synthesis and antiviral evaluation of cis-substituted cyclohexenyl and cyclohexanyl nucleosides.

Starting from commercially available (rac)-3-cyclohexene-1-carboxylic acid, a series of purine and pyrimidine cis-substituted cyclohexenyl and cyclohexanyl nucleosides were synthesized through a key Mitsunobu reaction. Antiviral evaluations were performed on HIV, coxsackie B3, and herpes viruses (HSV-1, HSV-2, VZV, HCMV). Three compounds showed moderate activity against HSV-1 and coxsackie viruses. Specific computer modeling studies were performed on HSV-1 thymidine kinase in order to understand the enzyme activation of an analogue showing moderate antiviral activity.

A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions.

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k(pol): 22-fold (from 7.2 to 0.33 s(-1)) for K65R RT and 84-fold (from 7.2 to 0.086 s(-1)) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.

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.

Hepatitis C virus RNA load in relapsed patients: week two of treatment is the best time to predict the complete response.

OBJECTIVE: An early virological response has been shown to be predictive of a sustained virological response to antiviral treatment in chronic hepatitis C infection. The aim of the study was to analyse viral kinetics during the first 6 weeks of treatment (interferon plus ribavirin) in 18 relapsed hepatitis C patients after a first course of interferon monotherapy. METHODS: We studied 18 relapsed patients treated with interferon and ribavirin. A sustained virological response (negative HCV RNA measured by polymerase chain reaction 6 months after the end of therapy) was obtained in 12 patients. Samples were obtained before therapy and each week for 6 weeks during therapy; HCV RNA levels were determined using quantitative bDNA. RESULTS: At the end of week two, a viral-load drop of more than 2.20 log was observed in all the 12 patients with a sustained virological response and in none of the six other patients. When we considered the median of the viral load reduction from baseline for each week of treatment, week two appeared to be the time point most predictive of a sustained viral response (positive predictive value 83%; negative predictive value 92%). CONCLUSION: During treatment with interferon plus ribavirin in relapsed hepatitis C patients, viral kinetics showed that the second week of treatment appeared to be the time point most predictive of a sustained viral response.

Synthesis and antiviral activity of new anti-HIV amprenavir bioisosteres.

Starting from the chemical structure of the recent FDA-approved anti-HIV drug Amprenavir (Agenerase), a potent HIV-protease inhibitor, we have designed new series of Amprenavir bioisoteres in which the methylene group of the benzyl group was replaced by a sulfur atom. This structural modification has required an original multistep synthesis. Unfortunately, introduction of the sulfur atom abolished or drastically decreased both inhibitory activity on recombinant HIV protease and HIV infection protection on MT4 cell cultures.