A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome.

Broad-spectrum antibiotic therapy decimates the gut microbiome, resulting in a variety of negative health consequences. Debio 1452 is a staphylococcus-selective enoyl-acyl carrier protein reductase (FabI) inhibitor under clinical development and was used to determine whether treatment with pathogen-selective antibiotics would minimize disturbance to the microbiome. The effect of oral Debio 1452 on the microbiota of mice was compared to the effects of four commonly used broad-spectrum oral antibiotics. During the 10 days of oral Debio 1452 treatment, there was minimal disturbance to the gut bacterial abundance and composition, with only the unclassified S24-7 taxon reduced at days 6 and 10. In comparison, broad-spectrum oral antibiotics caused ∼100- to 4,000-fold decreases in gut bacterial abundance and severely altered the microbial composition. The gut bacterial abundance and composition of Debio 1452-treated mice were indistinguishable from those of untreated mice 2 days after the antibiotic treatment was stopped. In contrast, the bacterial abundance in broad-spectrum-antibiotic-treated mice took up to 7 days to recover, and the gut composition of the broad-spectrum-antibiotic-treated mice remained different from that of the control group 20 days after the cessation of antibiotic treatment. These results illustrate that a pathogen-selective approach to antibiotic development will minimize disturbance to the gut microbiome.

Cyclophilin inhibitors block arterivirus replication by interfering with viral RNA synthesis.

Virus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC(50)) of 5.2 µM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC(50) of 0.95 µM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC(50) that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC(50) similar to that of CsA. The addition of 4 µM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in an in vitro assay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.

Debio 0932, a new oral Hsp90 inhibitor, alleviates psoriasis in a xenograft transplantation model.

Debio 0932 is a novel oral heat shock protein 90 (Hsp90) inhibitor developed for anti-cancer therapy. Surprising-ly, during the first clinical trial, one psoriasis patient experienced complete remission of his skin manifestation. However, a possible therapeutic utility of Hsp90 in psoriasis has not previously been reported. The objective of the present study was to explore the ability of Debio 0932 to alleviate psoriasis in a preclinical model. A psoriasis xenograft transplantation model was employed where skin from 5 psoriasis patients was transplanted onto immunodeficient mice (8 xenografts per donor). Debio 0932 was administered perorally daily for 3 weeks and resulted in significant clinical alleviation of psoriasis by day 11 and reduced epidermal thickness evaluated post-treatment. Alleviation of psoriasis in the psoriasis xenograft transplantation model, which may be due to Hsp90's involvement in signalling pathways that are up-regulated in psoriasis, substantiates a potential role of Debio 0932 in psoriasis treatment.

The cyclophilin inhibitor alisporivir prevents hepatitis C virus-mediated mitochondrial dysfunction.

UNLABELLED: Alisporivir (Debio-025) is an analogue of cyclosporine A and represents the prototype of a new class of non-immunosuppressive cyclophilin inhibitors. In vitro and in vivo studies have shown that alisporivir inhibits hepatitis C virus (HCV) replication, and ongoing clinical trials are exploring its therapeutic potential in patients with chronic hepatitis C. Recent data suggest that the antiviral effect is mediated by inhibition of cyclophilin A, which is an essential host factor in the HCV life cycle. However, alisporivir also inhibits mitochondrial permeability transition by binding to cyclophilin D. Because HCV is known to affect mitochondrial function, we explored the effect of alisporivir on HCV protein-mediated mitochondrial dysfunction. Through the use of inducible cell lines, which allow to investigate the effects of HCV polyprotein expression independent from viral RNA replication and which recapitulate the major alterations of mitochondrial bioenergetics observed in infectious cell systems, we show that alisporivir prevents HCV protein-mediated decrease of cell respiration, collapse of mitochondrial membrane potential, overproduction of reactive oxygen species and mitochondrial calcium overload. Strikingly, some of the HCV-mediated mitochondrial dysfunctions could even be rescued by alisporivir. CONCLUSION: These observations provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C.

Exploratory window-of-opportunity trial to investigate the tumor pharmacokinetics/pharmacodynamics of the IAP antagonist Debio 1143 in patients with head and neck cancer.

Inhibitor of apoptosis proteins (IAPs) regulate apoptosis and modulate NF-κB signaling thereby driving expression of genes involved in immune/inflammatory responses. The orally available IAP antagonist Debio 1143 has potential to enhance tumor response to chemoradiotherapy and/or immunotherapy. Patients with pre-operative squamous cell carcinomas of the head and neck (SCCHN) received: Debio 1143 monotherapy (200 mg/day [D]1-15 +/- 2); Debio 1143 (200 mg/day D1-15 +/- 2) plus cisplatin (40 mg/m2 D 1 and 8); cisplatin alone (40 mg/m2 D 1 and 8; EudraCT: 2014-004655-31). Pharmacokinetic/pharmacodynamic effects were assessed in plasma and resected tumors. Primary end point; effect of Debio 1143 on cellular IAP-1 (cIAP-1). Levels of cIAP-1/-2, X-linked inhibitor of apoptosis protein (XIAP), tumor infiltrating lymphocytes (TILs), including CD8+ T cells, programmed cell death protein 1 (PD-1), PD-ligand 1 (PD-L1), and gene expression were also analyzed. Twenty-three of 26 patients completed treatment. In the Debio 1143 monotherapy cohort (n = 13), mean tumor concentrations of Debio 1143 were 18-fold (maximum 55.2-fold) greater than in plasma, exceeding the half-maximal inhibitory concentration for cIAPs and XIAP by 100 to 1000-fold, with significant engagement/degradation of cIAP-1 (p < 0.05). Overall, levels of CD8+ TILs, PD-1, and PD-L1 positive immune cells increased significantly (p < 0.05) following Debio 1143 treatment. Changes were observed in the expression of genes related to NF-κB signaling. Treatments were well-tolerated. Debio 1143 penetrated SCCHN tumors, engaged cIAP-1, and induced immune inflammatory changes in the tumor microenvironment. Based on the mode of action demonstrated here and in previous studies, these data support future combinations of Debio 1143 with immune-checkpoint agents.

Structural basis for the non-immunosuppressive character of the cyclosporin A analogue Debio 025.

Debio 025 is a cyclosporin A (CsA) analogue that interferes strongly with the hepatitis C viral life cycle. Compared to CsA, Debio 025 has an additional methyl group at position 3 of the cyclic undecapeptide and an N-ethylvaline instead of an N-methylleucine at position 4. Unlike CsA, Debio 025 lacks immunosuppressive activity in vitro and in vivo. We show here that, in vitro, the cyclophilin A (CypA)-Debio 025 complex cannot interact any longer with calcineurin (CaN), a determinant for the immunosuppressive activity of CsA. We further use NMR spectroscopy to investigate at the molecular level the interaction of Debio 025 with CypA and thereby understand the basis for this loss of CaN interaction. NMR data and molecular modeling indicate that Debio 025 optimally interacts with CypA, which underlies the anti-HCV properties of Debio 025. However, the interaction between CaN and the CypA-Debio 025 complex is impeded by sterical hindrance of the CaN with the side chain of its Val4 residue. This is in sharp contrast with the case for the CypA-CsA-CaN ternary complex, where the Leu4 side chain can enter a hydrophobic cavity at the CaN interface. The structure of the CypA-Debio 025 complex thus provides a rational explanation for the non-immunosuppressive character of Debio 025.

The isomerase active site of cyclophilin A is critical for hepatitis C virus replication.

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors.

BACKGROUND & AIMS: The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A. METHODS: We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays. RESULTS: We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A. CONCLUSIONS: Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells.

BACKGROUND: Hyperactivity of the epithelial sodium (Na+) channel (ENaC) and increased Na+ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na+ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown. METHODS: We evaluated by short-circuit current (Isc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients. RESULTS: Neither hNE nor EPI-hNE4 treatments did modify Isc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased Isc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate Isc, an effect which was blocked by EPI-hNE4. CONCLUSIONS: These results indicate that hNE does activate ENaC and transepithelial Na+ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.

The inhibitor of apoptosis proteins antagonist Debio 1143 promotes the PD-1 blockade-mediated HIV load reduction in blood and tissues of humanized mice.

The immune checkpoint programmed cell death protein 1 (PD-1) plays a major role in T cell exhaustion in cancer and chronic HIV infection. The inhibitor of apoptosis protein antagonist Debio 1143 (D1143) enhances tumor cell death and synergizes with anti-PD-1 agents to promote tumor immunity and displayed HIV latency reversal activity in vitro. We asked in this study whether D1143 would stimulate the potency of an anti-human PD-1 monoclonal antibody (mAb) to reduce HIV loads in humanized mice. Anti-PD-1 mAb treatment decreased PD-1+ CD8+ cell population by 32.3% after interruption of four weeks treatment, and D1143 co-treatment further reduced it from 32.3 to 73%. Anti-PD-1 mAb administration reduced HIV load in blood by 94%, and addition of D1143 further enhanced this reduction from 94 to 97%. D1143 also more profoundly promoted with the anti-PD-1-mediated reduction of HIV loads in all tissues analyzed including spleen (71 to 96.4%), lymph nodes (64.3 to 80%), liver (64.2 to 94.4), lung (64.3 to 80.1%) and thymic organoid (78.2 to 98.2%), achieving a >5 log reduction of HIV loads in CD4+ cells isolated from tissues 2 weeks after drug treatment interruption. Ex vivo anti-CD3/CD28 stimulation increased the ability to activate exhausted CD8+ T cells in infected mice having received in vivo anti-PD-1 treatment by 7.9-fold (5 to 39.6%), and an additional increase by 1.7-fold upon D1143 co-treatment (39.6 to 67.3%). These findings demonstrate for the first time that an inhibitor of apoptosis protein antagonist enhances in a statistically manner the effects of an immune check point inhibitor on antiviral immunity and on HIV load reduction in tissues of humanized mice, suggesting that the combination of two distinct classes of immunomodulatory agents constitutes a promising anti-HIV immunotherapeutic approach.

Phase I Trial of Debio 1143, an Antagonist of Inhibitor of Apoptosis Proteins, Combined with Cisplatin Chemoradiotherapy in Patients with Locally Advanced Squamous Cell Carcinoma of the Head and Neck.

PURPOSE: Debio 1143 is an oral antagonist of inhibitor of apoptosis proteins, which enhances tumor response with concomitant chemoradiotherapy. Addition of Debio 1143 to cisplatin-based chemoradiotherapy in locally advanced squamous cell carcinomas of the head and neck (LA-SCCHN) was evaluated in a phase I/II study to determine the MTD and recommended phase II dose (RP2D). Here, phase I results are reported. PATIENTS AND METHODS: Treatment-naïve patients with LA-SCCHN (stages III/IVA/IVB) received Debio 1143 (100, 200, 300 mg/day), for 14 days every 3 weeks, with cisplatin (100 mg/m², every 3 weeks), for three cycles, and concomitant conventional fractionation radiotherapy (70 Gy/7 weeks). Dose-limiting toxicity (DLT) was evaluated over 9 weeks using continual reassessment. RESULTS: Fourteen patients were treated/evaluable for DLT. Median age was 64.5 years, and all patients were current/former smokers. Primary tumors were hypopharynx, oropharynx (all human papillomavirus/p16 negative), larynx, and oral cavity. Two of six patients at 200 mg/day had DLT (grade 3 tubular necrosis, grade 3 aspartate aminotransferase/alanine aminotransferase increase, grade 4 febrile neutropenia, and grade 3 lipase increase), which was considered the MTD and RP2D. Common grade 3-4 adverse events were dysphagia (36%) and mucositis (29%). Laboratory abnormalities were frequent and generally mild, including anemia, white blood cell decrease, and increased creatinine. Addition of Debio 1143 did not compromise chemotherapy administration. Overall locoregional control rate at 18 months was 85%. Overall response rate was 85%, including 69% complete responses. Progression-free survival rate at 24 months was 74%. CONCLUSIONS: The RP2D of Debio 1143 is 200 mg/day for 14 days, every 3 weeks, when combined with concomitant high-dose cisplatin chemoradiotherapy in LA-SCCHN. Debio 1143 addition to chemoradiotherapy was safe and manageable. Preliminary efficacy is encouraging and supports further development.

Debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis C virus (HCV) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for HCV (STAT-C) inhibitors.

Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J. Paeshuyse et al., Hepatology 43:761-770, 2006). In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 3.6 log(10) units (R. Flisiak et al., Hepatology 47:817-826, 2008), whereas a reduction of 4.6 log(10) units was obtained in phase II studies when Debio 025 was combined with interferon (R. Flisiak et al., J. Hepatol., 48:S62, 2008). We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025. The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays. Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors. Debio 025, when it was used at concentrations that have been observed in human plasma (0.1 or 0.5 muM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors. Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.

SMAC Mimetic Debio 1143 and Ablative Radiation Therapy Synergize to Enhance Antitumor Immunity against Lung Cancer.

PURPOSE: Adaptive antitumor immunity following ablative radiotherapy (ART) is attenuated by host myeloid-derived suppressor cell (MDSC), tumor-associated macrophage (TAM), and regulatory T-cell (Treg) infiltrates. We hypothesized treatment with ART and a secondary mitochondrial-derived activators of caspase (SMAC) mimetic could reverse the immunosuppressive lung cancer microenvironment to favor adaptive immunity. EXPERIMENTAL DESIGN: To evaluate for synergy between ART and the SMAC mimetic Debio 1143 and the dependence upon CD8+ T cells and TNFα, we used LLC-OVA syngeneic mouse model of lung cancer and treated them with Debio 1143 and/or ART (30 Gy) with or without anti-CD8, anti-TNFα, or anti-IFNγ antibodies. Tumor-infiltrating OVA-specific CD8+ T cells, Tc1 effector cells, MDSCs, TAMs, and Tregs, were quantified by flow cytometry. Tc1-promoting cytokines TNFα, IFNγ, and IL1ß and the immunosuppressive IL10 and Arg-1 within LLC-OVA tumor tissue or mouse serum were measured by RT-PCR and ELISA. RESULTS: ART delayed tumor growth, and the addition of Debio 1143 greatly enhanced its efficacy, which included several complete responses. These complete responders rejected an LLC-OVA tumor rechallenge. ART and Debio 1143 synergistically induced a tumor-specific, Tc1 cellular and cytokine response while eliminating immunosuppressive cells and cytokines from the tumor microenvironment. Depletion of CD8+ cells, TNFα, and IFNγ with blocking antibody abrogated synergy between ART and Debio 1143 and partially restored tumor-infiltrating MDSCs. CONCLUSIONS: Debio 1143 augments the tumor-specific adaptive immunity induced by ART, while reversing host immunosuppressive cell infiltrates in the tumor microenvironment in a TNFα, IFNγ, and CD8+ T-cell-dependent manner. This provides a novel strategy to enhance the immunogenicity of ART.

The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate.

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).

Nuclear Imaging Study of the Pharmacodynamic Effects of Debio 1143, an Antagonist of Multiple Inhibitor of Apoptosis Proteins (IAPs), in a Triple-Negative Breast Cancer Model.

Background: Debio 1143, a potent orally available SMAC mimetic, targets inhibitors of apoptosis proteins (IAPs) members and is currently in clinical trials. In this study, nuclear imaging evaluated the effects of Debio 1143 on tumor cell death and metabolism in a triple-negative breast cancer (TNBC) cell line (MDA-MB-231)-based animal model. Methods: Apoptosis induced by Debio 1143 was assessed by FACS (caspase-3, annexin 5 (A5)), binding of 99mTc-HYNIC-Annexin V, and a cell proliferation assay. 99mTc-HYNIC-Annexin V SPECT and [18F]-FDG PET were also performed in mice xenografted with MDA-MB-231 cells. Results: Debio 1143 induced early apoptosis both in vitro and in vivo 6 h after treatment. Debio 1143 inhibited tumor growth, which was associated with a decreased tumor [18F]-FDG uptake when measured during treatment. Conclusions: This imaging study combining SPECT and PET showed the early proapoptotic effects of Debio 1143 resulting in a robust antitumor activity in a preclinical TNBC model. These imaging biomarkers represent valuable noninvasive tools for translational and clinical research in TNBC.

DEBIO 1143, an IAP inhibitor, reverses carboplatin resistance in ovarian cancer cells and triggers apoptotic or necroptotic cell death.

The poor prognosis of ovarian cancer (it is the leading cause of death from gynecological cancers) is mainly due to the acquisition of resistance to carboplatin. Among the possible resistance pathways, resistance to apoptosis and especially the overexpression of inhibitor of apoptosis proteins (IAP) cIAP1 and X-linked IAP (XIAP), have been implicated. DEBIO 1143, a SMAC (second mitochondria-derived activator of caspase) mimetic, belongs to a new class of targeted agents currently being evaluated in clinical trials, which activate apoptotic cell death and block pro-survival signaling in cancer cells. Here, we demonstrate that DEBIO 1143 in vitro inhibits the cell viability of two carboplatin-sensitive cell lines (IGROV-1 and A2780S) as well as three carboplatin-resistant cell lines (A2780R, SKOV-3 and EFO-21). Of note, DEBIO 1143 is able to reverse resistance to carboplatin by inducing cell death either by apoptosis or necroptosis depending on the cell lines. To identify a biomarker able to predict the sensitivity of the cell lines to DEBIO 1143 treatment we analyzed the expression of the DEBIO 1143 targets cIAP1 and XIAP, and one of their downstream targets, caspase 9. These proteins did not constitute a marker of DEBIO 1143 sensitivity/resistance. Importantly, we confirmed these findings in vivo in SKOV-3 xenograft models where DEBIO 1143 highly potentiated carboplatin treatment.

HCV1b genome evolution under selective pressure of the cyclophilin inhibitor alisporivir during the DEB-025-HCV-203 phase II clinical trial.

Major advances have revolutionized the HCV antiviral treatment field, with interferon-free combinations of direct-acting antivirals (DAAs) resulting into success rates of >90% for all HCV genotypes. Nevertheless, viral eradication at a global level stills remains challenging, stimulating the continued search for new affordable pan-genotypic drugs. To overcome selection of drug resistant variants, targeting host proteins can be an attractive mechanism of action. Alisporivir (Debio 025) is a potent pan-genotypic host-targeting antiviral agent, acting on cyclophilin A, which is necessary for HCV replication. The efficacy and safety of three different oral doses of alisporivir in combination with pegylated interferon-α2a given over a period of four weeks, was investigated in a randomized, double-blind and placebo-controlled phase IIa clinical trial, in 90 treatment-naïve subjects infected with chronic hepatitis C, wherefrom 58 HCV1b samples were selected for genetic sequencing purposes. Sequencing results were used to study the HCV genome for amino acid changes potentially related with selective pressure and resistance to alisporivir. By comparing baseline and on-treatment sequences, a large variation in proportion of amino acid changes was detected in all treatment arms. The NS5A variant D320E, which was previously identified during in vitro resistance selection and resulted in 3.6-fold reduced alisporivir susceptibility, emerged in two subjects in the alisporivir monotherapy arm. However, emergence of D320E appeared to be associated only with concurrent viral load rebound in one subject with 0.8log10IU/ml increase in HCV RNA. In general, for all datasets, low numbers of positions under positive selective pressure were observed, with no significant differences between naïve and treated sequences. Additionally, incomplete sequence information for some of the 22 patients and the low number of individuals per treatment arm, is limiting the power to assess the association of alisporivir or interferon treatment with the observed amino acid changes.

Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes.

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I(Na)) by a GPI-anchored serine protease (mouse channel-activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I(Na) 6-10-fold in the Xenopus oocyte expression system. I(Na) and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I(125)-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I(Na) 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP(o)). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I(Na) by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP(o). Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I(Na) (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I(Na) was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.

The radiosensitizing activity of the SMAC-mimetic, Debio 1143, is TNFα-mediated in head and neck squamous cell carcinoma.

BACKGROUND AND PURPOSE: Second mitochondria-derived activator of caspase (SMAC)-mimetics are a new class of targeted drugs that specifically induce apoptotic cancer cell death and block pro-survival signaling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. MATERIAL AND METHODS: The present study was designed to investigate the radiosensitizing effect and optimal sequence of administration of the novel SMAC-mimetic Debio 1143 in vitro and in vivo. Apoptosis, alteration of DNA damage repair (DDR), and tumor necrosis factor-alpha (TNF-α) signaling were examined. RESULTS: In vitro, Debio 1143 displayed anti-proliferative activity and enhanced intrinsic radiation sensitivity in 5/6 head and neck squamous cell carcinoma (HNSCC) cell lines in a synergistic manner. In vivo, Debio 1143 dose-dependently radio-sensitized FaDu and SQ20B xenografts, resulting in complete tumor regression in 8/10 FaDu-xenografted mice at the high dose level. At the molecular level, Debio 1143 combined with radiotherapy (RT) induced enhancement of caspase-3 activity, increase in Annexin V-positive cells and karyopyknosis, and increase in TNF-α mRNA levels. Finally, in a neutralization experiment using a TNF-α-blocking antibody and a caspase inhibitor, it was shown that the radiosensitizing effect of Debio 1143 is mediated by caspases and TNF-α. CONCLUSIONS: These results demonstrate that the novel SMAC-mimetic Debio 1143 is a radiosensitizing agent that is worthy of further investigation in clinical trials in combination with radiotherapy.

SMAC mimetic Debio 1143 synergizes with taxanes, topoisomerase inhibitors and bromodomain inhibitors to impede growth of lung adenocarcinoma cells.

Targeting anti-apoptotic proteins can sensitize tumor cells to conventional chemotherapies or other targeted agents. Antagonizing the Inhibitor of Apoptosis Proteins (IAPs) with mimetics of the pro-apoptotic protein SMAC is one such approach. We used sensitization compound screening to uncover possible agents with the potential to further sensitize lung adenocarcinoma cells to the SMAC mimetic Debio 1143. Several compounds in combination with Debio 1143, including taxanes, topoisomerase inhibitors, and bromodomain inhibitors, super-additively inhibited growth and clonogenicity of lung adenocarcinoma cells. Co-treatment with Debio 1143 and the bromodomain inhibitor JQ1 suppresses the expression of c-IAP1, c-IAP2, and XIAP. Non-canonical NF-κB signaling is also activated following Debio 1143 treatment, and Debio 1143 induces the formation of the ripoptosome in Debio 1143-sensitive cell lines. Sensitivity to Debio 1143 and JQ1 co-treatment was associated with baseline caspase-8 expression. In vivo treatment of lung adenocarcinoma xenografts with Debio 1143 in combination with JQ1 or docetaxel reduced tumor volume more than either single agent alone. As Debio 1143-containing combinations effectively inhibited both in vitro and in vivo growth of lung adenocarcinoma cells, these data provide a rationale for Debio 1143 combinations currently being evaluated in ongoing clinical trials and suggest potential utility of other combinations identified here.