Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide1, 2. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis3, 4, 5, several of which are currently in clinical trials6, 7, 8. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis

Etude multimodale des interactions entre le virus de l’hépatite B et la cyclic AMP-GMP synthase, cGAS

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this PhD work, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an HBV infection-susceptible cell culture model. Collectively, our data show that (1) the cGAS-STING pathway exhibits robust antiviral activity against HBV infection including reduction of viral cccDNA levels; (2) naked HBV genomic rcDNA is sensed in a cGAS-dependent manner whereas packaging of the viral genome during infection abolishes host cell recognition of viral nucleic acids; (3) HBV infection down-regulates the cGAS/STING pathway actors as well as innate immune effector gene expression in vitro and vivo. Overall, this work led to describing new aspects of the complex interaction between HBV and the DNA sensor cGAS in hepatocytes.Le virus de l’hépatite B (HBV) est l’agent étiologique de l’hépatite B. Ce virus est responsable d’hépatite chronique B, de cirrhose et de cancer du foie au niveau mondial. L’absence d’activation de la voie Interféron (IFN) suite à l’infection par HBV est encore mal comprise. Récemment, le senseur cellulaire cytosolic GMP-AMP synthase (cGAS) a été décrit comme un senseur efficace de DNA double brin possédant également une activité antivirale envers des virus à ADN et à ARN. Le but de mes travaux de thèse a été de contribuer à la compréhension des relations existants entre le HBV et cGAS, à des stades précoces et tardifs de l’infection HBV en utilisant des expériences de perte- et gain- de function ainsi que du profilage génomique des génes apparentés à cGAS dans un modéle cellulaire permissif au HBV. Mes travaux ont démontré (1) que cGAS exerce une forte activité antivirale envers le HBV incluant une réduction de la forme nucléaire du génome, le cccDNA; (2) alors que le rcDNA génomique nu est reconnu par la voie cGAS/STING et induit une réponse IFN efficace, la nucléocapside virale protège le DNA génomique viral et l’empêche d’être détecté par la réponse immunitaire innée; et (3) que l’infection par HBV diminue l’expression des acteurs de la voie cGAS-STING et des gènes impliqués dans la réponse immunitaire innée in vitro et in vivo. Ce dernier point met en lumière le rôle de cGAS dans un nouveau mécanisme d’échappement du HBV au système immunitaire inné dans les cellules hépatocytaires et dans ce mécanisme

Nonstructural 5A Protein of Hepatitis C Virus Interacts with Pyruvate Carboxylase and Modulates Viral Propagation

<div><p>Hepatitis C virus (HCV) is highly dependent on cellular factors for its own propagation. By employing tandem affinity purification method, we identified pyruvate carboxylase (PC) as a cellular partner for NS5A protein. NS5A interacted with PC through the N-terminal region of NS5A and the biotin carboxylase domain of PC. PC expression was decreased in cells expressing NS5A and HCV-infected cells. Promoter activity of PC was also decreased by NS5A protein. However, FAS expression was increased in cells expressing NS5A and cell culture grown HCV (HCVcc)-infected cells. Silencing of PC promoted fatty acid synthase (FAS) expression level. These data suggest HCV may modulate PC via NS5A protein for its own propagation.</p></div

PC interacts and colocalizes with the HCV NS5A protein.

<p>PC interacts with NS5A derived from both genotype 1b and 2a. Either Huh7.5 cells (A) or HEK293T cells (B) were cotransfected with Myc-tagged NS5A of genotype 1b or 2a in the absence or presence of Flag-tagged PC. Cell lysates harvested at 24 h after transfection were immunoprecipitated with anti-Myc antibody and then bound proteins were immunoblotted with anti-Flag antibody. Protein expressions of NS5A and PC were verified using the same cell lysates by immunoblotting with anti-Myc antibody and anti-Flag antibody, respectively. (C) The biotin carboxylase domain of PC is the binding site for NS5A. Schematic diagram of both wild type and mutants of PC (upper panel). HEK293T cells were cotransfected with Myc-tagged NS5A and Flag-tagged mutant constructs of PC. Cells were harvested at 24 h after transfection and were immunoprecipitated with anti-Myc antibody. The coprecipitated proteins were immunoblotted with anti-Flag antibody (lower panel). BC, biotin carboxylase; CT, carboxyltransferase; PT, PC tetramerization; BCCP, biotin-carboxyl carrier protein. (D) PC interacts with the domain I of NS5A. Schematic diagram shows both wild type and mutants of NS5A (upper panel). HEK293T cells were cotransfected with Flag-tagged PC and Myc-tagged constructs of NS5A. Cell lysates harvested at 24 h after transfection were immunoprecipitated with anti-Flag antibody and then bound proteins were immunoblotted with anti-Myc antibody. Protein expressions of both NS5A and PC were confirmed using the same lysates by immunoblotting with anti-Myc and anti-Flag antibodies, respectively. (E) PC colocalizes with NS5A. Huh7.5 cells were infected with HCV Jc1 for 4 h. At two days postinfection, cells were fixed and then incubated with anti-NS5A, anti-PC antibody, and anti-VDAC antibody, respectively. Cells were further incubated with the appropriated secondary antibodies and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. (F) NS5A localizes to mitochondria. Huh7.5 cells were infected with Jc1 for 4 h and were cultured for 2 days. The cells were fractionated into cytosol, mitochondria, nuclei & cell debris, and microsomal fraction. The purities of the each subcellular fraction were confirmed by using GAPDH, VDAC, and Lamin A antibody, respectively.</p

HCV proteins modulate transcriptional activity of PC.

<p>(A) Schematic diagram of two PC promoters, P1 and P2, used in this study. (B) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with the indicated HCV protein expression plasmid. At 24 h after transfection, cells were harvested and luciferase activities were determined (top panels). Protein expressions were determined by anti-Myc antibody as shown in arrows (bottom panels). Vector indicates pGL3 empty vector. (-) denotes pGL3-PC in the absence of HCV protein (C) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with increasing amounts of NS5A expression plasmid (genotype 1b). Cells were harvested at 24 h after transfection and then luciferase activities were determined (top panels). NS5A expressions were determined by anti-NS5A antibody (bottom panels). (D) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with Myc-tagged mutant constructs of NS5A. At 24 h after transfection, cells were harvested and luciferase activities were determined. (E) HEK293T cells were transfected with either P1-Luc (left panel) or P2-Luc (right panel) reporter plasmid together with NS5A plasmid derived from either genotype 1b or 2a. At 24 h after transfection, cells were harvested and luciferase activities were determined. (F) Huh7.5 cells were either mock infected or infected with HCV Jc1 for 4 h. At 5 days postinfection, cells were transfected with either vector or P1-Luc (left panel), or vector or P2-Luc (right panel) reporter plasmid. Cells were further cultured for 2 days and then luciferase activities were determined. Protein expressions were verified by immunoblot analysis using anti-NS3 and anti-actin antibody, respectively. Luciferase activities were normalized based on β-galactosidase activities. Asterisks indicate significant differences (*, <i>P</i><0.05, **, <i>P</i><0.01) from the activity for the control.</p

NS5A regulates PC expression level.

<p>(A–C) PC expression level is decreased, whereas FAS expression level is increased in cells expressing NS5A protein. (A) Total cell lysates harvested from either IFN-cured cells or replicon cells were immunoblotted with the indicated antibodies (top panel). Total RNAs isolated from either IFN-cured cells or replicon cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel) by qRT-PCR. (B) Huh7.5 cells were either mock infected or infected with Jc1. Total cell lysates harvested at 5 days postinfection were immunoblotted with the indicated antibodies (top panel). Total RNAs isolated from either mock infected or Jc1 infected cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel) by qRT-PCR using data from three independent experiments. Asterisks indicate significant differences (*, <i>P</i><0.05) from the value for the control. Error bars indicate standard deviations. (C) Equal amounts of cell lysates harvested from either vector stable or NS5A stable cells derived from genotype 1b or 2a were immunoblotted with the indicated antibodies (top panel). Total RNAs isolated from the indicated stable cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel). Asterisks indicate significant differences (*, <i>P</i><0.05) from the value for the control. (D) Huh7.5 cells were transfected with siRNAs of either negative or PC. At 3 days after transfection, cells were harvested and analyzed by immunoblotting with the indicated antibodies (top panel). Actin was used as a loading control. Total RNAs isolated from the indicated cells were quantitated for PC mRNA levels (middle panel) and FAS mRNA levels (bottom panel). Asterisks indicate significant differences (*, <i>P</i><0.05) from the value for the control. (E) Huh7.5 cells were transiently transfected with either pEF6 empty vector or pEF6-NS5A-Myc expression plasmid. Cell lysates harvested at 48 h or 72 h after transfection were immunoblotted with the indicated antibodies.</p

Silencing of PC impairs production of infectious HCV.

<p>(A) Knockdown of PC had no effect on viral protein levels in HCV replicon cells. Huh7 cells harboring HCV replicon were transfected with 10 nM of negative (Neg), positive (Pos), or the indicated siRNA duplexes for 3 days. Total RNAs were extracted and intracellular HCV RNA was analyzed by qRT-PCR. Negative, irrelevant siRNA pool; positive, HCV-specific siRNAs. (B) Total replicon cell lysates harvested at 3 days after siRNA transfection were immunoblotted with the indicated antibodies. (C) Huh7.5 cells were transfected with 10 nM of the indicated siRNA. At 2 days after siRNA transfection, cells were infected with Jc1 for 4 h. At 48 h postinfection, cell proliferation was assessed by the MTT assay. Huh7.5 cells were treated as described in (C). At 48 h postinfection, both intracellular HCV RNA (D) and extracellular HCV RNA isolated from culture supernatants (F) were analyzed by qRT-PCR. (E) Total cell lysates harvested at 48 h after Jc1 infection were immunoblotted with the indicated antibodies. (G) Intracellular infectious HCV particles were prepared by 4 rounds of freeze and thaw treatments from cells treated as in figure legend to C. Neg indicates cells infected with intracellular HCV isolated from the Negative siRNA-transfected cells. PC denotes cells infected with intracellular HCV isolated from the PC siRNA-transfected cells. Pos denotes cells infected with intracellular HCV isolated from the Positive siRNA-transfected cells. (H) Extracellular infectious HCV particles were prepared from the culture media in cells treated as in figure legend to C. Neg indicates cells infected with extracellular HCV isolated from the Negative siRNA-transfected cells. PC denotes cells infected with extracellular HCV isolated from the PC siRNA-transfected cells. Pos denotes cells infected with extracellular HCV isolated from the Positive siRNA-transfected cells. (G, H) Naïve Huh7.5 cells were then infected with intracellular infectious HCV (G) and extracellular infectious HCV (H). Total cell lysates harvested at 2 days postinfection were immunoblotted to determine the indicated protein levels, and HCV RNA levels were analyzed by qRT-PCR (top panels). Naïve Huh7.5 cells treated as described above were analyzed for immunofluorescence using anti-NS5A antibody (bottom panels). Cells were counterstained with DAPI to label nuclei. Samples were analyzed for immunofluorescence staining using a Zeiss LSM 700 laser confocal microscopy system. Asterisks indicate significant differences (*, <i>P</i><0.05, **, <i>P</i><0.01) from the value for the negative control. Error bars indicate standard deviations.</p

Usefulness of whole-blood interferon-gamma assay and interferon-gamma enzyme-linked immunospot assay in the diagnosis of active pulmonary tuberculosis

PURPOSES: The aim of this study was to evaluate the usefulness of the whole-blood interferon-gamma assay (enzyme-linked immunosorbent assay [ELISA]) and interferon-gamma enzyme-linked immunospot assay (ELISPOT) based on early secretory antigenic target 6 and culture filtrate protein 10 in the diagnosis of active pulmonary tuberculosis (TB) in routine clinical practice. METHOD: We conducted a prospective study enrolling 144 participants with suspected pulmonary TB in a tertiary referral hospital in Seoul, South Korea, to investigate the diagnostic sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of these tests. Clinical assessment, tuberculin skin test (TST), whole-blood interferon-gamma ELISA (QuantiFERON-TB Gold [QFT-G]; Cellestis Ltd; Victoria, Australia), and an ELISPOT assay (T SPOT.TB; Oxford Immunotec; Oxford, UK) were performed. Test results were compared with the final confirmed diagnoses. RESULTS: Active pulmonary TB was diagnosed in 67 of 144 participants (47%). Sensitivities of QFT-G and T SPOT.TB for active pulmonary TB were 89% (95% confidence interval [CI], 79 to 96%) and 92% (95% CI, 83 to 97%), respectively; and specificities were 49% (95% CI, 37 to 61%) and 47% (95% CI, 36 to 59%). NPVs of QFT-G (84%; 95% CI, 69 to 93%) and T SPOT.TB (87%; 95% CI, 73 to 96%) were higher than that of TST (64%; 95% CI, 51 to 76%) [p = 0.001 and p < 0.001, respectively]. CONCLUSION: High NPVs of QFT-G and T SPOT.TB for the diagnosis of active TB suggest the supplementary role of these tests for the diagnostic exclusion of active TB, although the low PPV limits their usefulness in routine clinical practice in South Korea, where the prevalence of latent TB infection is considerable

Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin-p65 interaction

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics. Thus, there is a significant demand for new chemical entities (NCEs) targeting neuroinflammation. Herein, we rediscovered benzopyran-embedded tubulin inhibitor 1 as an anti-neuroinflammatory agent via phenotype-based screening. A competitive photoaffinity labeling study revealed that compound 1 binds to tubulin at the colchicine-binding site. Structure-activity relationship analysis of 1&apos;s analogs identified SB26019 as a lead compound with enhanced anti-neuroinflammatory efficacy. Mechanistic studies revealed that upregulation of the tubulin monomer was critical for the anti-neuroinflammatory activity of SB26019. We serendipitously found that the tubulin monomer recruits p65, inhibiting its translocation from the cytosol to the nucleus and blocking NF-kappa B-mediated inflammatory pathways. Further in vivo validation using a neuroinflammation mouse model demonstrated that SB26019 suppressed microglial activation by downregulating lba-1 and proinflammatory cytokines. Intraperitoneal administration of SB26019 showed its therapeutic potential as an NCE for successful anti-neuroinflammatory regulation. Along with the recent growing demands on tubulin modulators for treating various inflammatory diseases, our results suggest that colchicine-binding site-specific modulation of tubulins can be a potential strategy for preventing neuroinflammation and treating CNS diseases.Y

Hepatitis B Virus Evasion From Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase Sensing in Human Hepatocytes

International audienceChronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) was identified as a DNA sensor. In this study, we investigated the functional role of cGAS in sensing HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss-of-function and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes, and HBV-infected human liver chimeric mice. Here, we show that cGAS is expressed in the human liver, primary human hepatocytes, and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral covalently closed circular DNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. Conclusion: HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways