Combinatorial Anti-arenaviral Therapy with the Small Molecule SKI-1/S1P Inhibitor PF-429242 and Ribavirin

Arenaviruses are enveloped negative strand viruses that cause acute and chronic infections. Several Arenaviruses can cause severe hemorrhagic fever in humans. In West Africa Lassa virus causes several hundred thousand infections per year, while Junin, Machupo, Guanarito, and Sabia virus have emerged in South America. So far, only one drug is licensed against arenaviruses, the nucleoside analogue Ribavirin (Rib), which is effective when given early in disease, but shows only minor therapeutic effects in late stages of the infection. Previous works demonstrated that processing of the arenavirus glycoprotein precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexinisozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infectionand production of infectious virus. Recently, the SKI-1/S1P inhibitor PF-429242wasshownto inhibit Old World arenavirusGPCprocessing, cell-to-cell propagation, and infectious virus production. In the present study, we assessed the activity of PF-429242 against processing of the GPCs of the genetically and structurally more distant New World arenaviruses and found potent inhibition of processing of the GPCs of Junin, Machupo, and Guanarito virus. Using the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we studied the potency of PF-429242 in the context of acute and chronic infection. In line with published data, PF-429242 potently inhibited acute LCMV infection. PF-429242 was also highly active against chronic infection and drug treatment resulted in rapid extinction of the virus without emergence of drug-resistant variants. In a combinatorial drug approach, we found that PF-429242 potentiated the anti-viral effect of Rib in treatment of acute andchronic infection. Taken together, we showed that the SKI-1/S1P inhibitor PF-429242 is broadly active against GPC processing of all major human pathogenic arenaviruses. Apart from being potent in acute infection, the drug is remarkably active in clearing chronic infection and potentiated the anti-arenaviral activity of Rib.

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

Gender difference in the response to an angiotensin-converting enzyme inhibitor and a diurectic in hypertensive patients of African descent

Résumé de la thèse en français Titre : Différence entre hommes et femmes dans la réponse à un inhibiteur de l'enzyme de conversion de l'angiotensine et un diurétique chez des patients hypertendus d'origine Africaine. Introduction: L'efficacité des inhibiteurs de l'enzyme de conversion de l'angiotensine (ACEI) dans le traitement de l'hypertension artérielle chez les patients africains est controversée. Objectif: Nous avons examiné la baisse de la tension artérielle ambulatoire (ABP) en réponse à un diurétique et un ACEI chez des patients hypertendus d'origine africaine et nous avons évalué les différentes caractéristiques déterminant l'efficacité du traitement. Méthodes: Etude en simple-aveugle randomisée, en crossover AB/BA. Arrangement Familles hypertendues d'origine africaine de la population générale des Seychelles. Participants : 52 patients (29 hommes et 23 femmes) sur 62 patients hypertendus éligibles ont été inclus. Le principal résultat était la mesure de la réponse de l'ABP à 20 mg de lisinopril (LIS) ou 25 mg d'hydrochlorothiazide (HCT) quotidiennement pendant quatre semaines. Résultats: Le jour, la réponse systolique/diastolique de l'ABP sous HCT était de 4.9 (95% intervalle de confiance (IC) 1.2-8.6)/3.6 (1.0-6.2) mm Hg pour les hommes et 12.9 (9.216.6)/6.3 (3.7-8.8) mm Hg pour les femmes. Sous LIS, la réponse était de 18.8 (15.022.5)/14.6 (12.0-17.1) mm Hg pour les hommes et de 12.4 (8.7-16.2)/7.7 (5.1-10.2) mm Hg pour les femmes. La nuit, la réponse systolique/diastolique sous HCT était de 5.0 (0.6-9.4)/2.7 ((-0.4)-5.7) mm Hg pour les hommes et de 11.5 (7.1-16.0)/5.7 (2.6-8.8) mm Hg pour les femmes, et sous LIS était de 18.7 (14.2-22.1)/15.4 (12.4-18.5) mm Hg pour les hommes et de 3.5 ((-1.0)-7.9)/2.3 ((-0.8)-5.4) mm Hg pour les femmes. L'analyse de régression linéaire multiple a montré que le sexe est un prédicteur indépendant de la réponse tensionnelle à l'HCT et au LIS. Conclusions : Les patients hypertendus d'origine africaine ont présenté une baisse tensionnelle plus grande en réponse au LIS qu'à l'HCT. Les hommes ont mieux répondu au LIS qu'à l'HCT alors que les femmes ont répondu de manière similaire aux deux traitements.

A proline-rich TGF-beta-responsive transcriptional activator interacts with histone H3.

The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-beta (TGF-beta) have been analyzed. We show that TGF-beta specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors. A TGF-beta-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-1 was shown to mediate TGF-beta induction in NIH-3T3 cells. Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-1-interacting protein. A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-1-interacting protein in yeast. Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells. This interaction requires the TRD, and it appears to be upregulated by TGF-beta in vivo. Moreover, point mutations in the TRD that inhibit TGF-beta induction also reduce interaction with histone H3. In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA. Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3. These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.

Sustained 24-hour blockade of the renin-angiotensin system: a high dose of a long-acting blocker is as effective as a lower dose combined with an angiotensin-converting enzyme inhibitor.

Whether a higher dose of a long-acting angiotensin II receptor blocker (ARB) can provide as much blockade of the renin-angiotensin system over a 24-hour period as the combination of an angiotensin-converting enzyme inhibitor and a lower dose of ARB has not been formally demonstrated so far. In this randomized double-blind study we investigated renin-angiotensin system blockade obtained with 3 doses of olmesartan medoxomil (20, 40, and 80 mg every day) in 30 normal subjects and compared it with that obtained with lisinopril alone (20 mg every day) or combined with olmesartan medoxomil (20 or 40 mg). Each subject received 2 dose regimens for 1 week according to a crossover design with a 1-week washout period between doses. The primary endpoint was the degree of blockade of the systolic blood pressure response to angiotensin I 24 hours after the last dose after 1 week of administration. At trough, the systolic blood pressure response to exogenous angiotensin I was 58% +/- 19% with 20 mg lisinopril (mean +/- SD), 58% +/- 11% with 20 mg olmesartan medoxomil, 62% +/- 16% with 40 mg olmesartan medoxomil, and 76% +/- 12% with the highest dose of olmesartan medoxomil (80 mg) (P = .016 versus 20 mg lisinopril and P = .0015 versus 20 mg olmesartan medoxomil). With the combinations, blockade was 80% +/- 22% with 20 mg lisinopril plus 20 mg olmesartan medoxomil and 83% +/- 9% with 20 mg lisinopril plus 40 mg olmesartan medoxomil (P = .3 versus 80 mg olmesartan medoxomil alone). These data demonstrate that a higher dose of the long-acting ARB olmesartan medoxomil can produce an almost complete 24-hour blockade of the blood pressure response to exogenous angiotensin in normal subjects. Hence, a higher dose of a long-acting ARB is as effective as a lower dose of the same compound combined with an angiotensin-converting enzyme inhibitor in terms of blockade of the vascular effects of angiotensin.

Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

The protective capacities of histone H1 against experimental murine cutaneous leishmaniasis.

In a murine model of experimental cutaneous leishmaniasis, we investigated the protection elicited by injection of histone H1 isolated from parasites by perchloric extraction, of a H1 recombinant protein produced in E. coli, and of H1 long and short synthetic peptides, against infection by L. major. Partial protection was achieved in most of the animals as shown by reduction in lesion size, upon immunization with histone H1 or its peptides, provided that the region 1-60 was present in the molecule. These observations argue in favor of a thorough examination of the possibility of including histone H1 described here in a cocktail vaccine against human leishmaniasis.

Linking epigenetics to lipid metabolism: focus on histone deacetylases.

A number of recent studies revealed that epigenetic modifications play a central role in the regulation of lipid and of other metabolic pathways such as cholesterol homeostasis, bile acid synthesis, glucose and energy metabolism. Epigenetics refers to aspects of genome functions regulated in a DNA sequence-independent fashion. Chromatin structure is controlled by epigenetic mechanisms through DNA methylation and histone modifications. The main modifications are histone acetylation and deacetylation on specific lysine residues operated by two different classes of enzymes: Histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The interaction between these enzymes and histones can activate or repress gene transcription: Histone acetylation opens and activates chromatin, while deacetylation of histones and DNA methylation compact chromatin making it transcriptionally silent. The new evidences on the importance of HDACs in the regulation of lipid and other metabolic pathways will open new perspectives in the comprehension of the pathophysiology of metabolic disorders.

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Pathway analysis of glioblastoma tissue after preoperative treatment with the EGFR tyrosine kinase inhibitor gefitinib--a phase II trial.

Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 μg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation.

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Expression pattern of sirtuins in innate immune cells and influence of Sirtuin 1/2 inhibition on innate immune responses to Toll-like receptor ligands and to infection

Purpose/Objective: The family of histone deacetylases comprises 18 members in mammals, among which seven sirtuins (SIRT1-7). Sirtuins are NADP-dependent enzymes that have been involved in the control of cell metabolism, proliferation and survival. The expression pattern of sirtuins and their influence on host response to microbial infection remain largely unknown. The aim of the study was to analyze the expression of SIRT1-7 and to address the effects of SIRT1/2 inhibition on innate immune responses in vitro and in vivo.. Materials and methods: in vitro: Bone marrow (BM), BM-derived macrophages (BMDMs) and dendritic cells (BMDCs) and RAW 264.7 and J774.1 macrophage cell lines were stimulated for 0, 2, 6 and 18 h with LPS, Pam3CSK4 and CpG ODN. SIRT1-7 mRNA was quantified by real time-PCR. TNF was measured by ELISA. In vivo: BALB/c mice were challenged with LPS (350 lg i.p.) with or without a SIRT1/2 inhibitor. Blood and organs were collected after 0, 1, 4, 8 and 24 h to quantify SIRT1-7 and TNF. Mortality was assessed daily. Results: Bone marrow, macrophages and DCs express, in order of abundance, SIRT2 > > SIRT1, SIRT3 and SIRT6 > SIRT4, SIRT5 and SIRT7. Microbial products decrease the expression of all sirtuins except SIRT6 in a time dependent manner in BMDMs (0_24 h). SIRT2 is the most expressed sirtuin also in the liver, kidney (together with SIRT3) and spleen. Upon LPS challenge, SIRT1, SIRT3, SIRT4 and SIRT7 mRNA levels decrease in the liver (from 4 h to 24 h), whereas SIRT1-7 mRNA levels decrease within 1 h in both kidney and spleen. Pharmacological inhibition of SIRT1/2 decreases TNF production by macrophages stimulated with LPS, Pam3CSK4 and CpG ODN (n = 6; P < 0.001). In agreement, prophylactic treatment with a SIRT1/2 inhibitor decreases TNF production (n = 8; P = 0.04) and increases survival (n = 13, P = 0.03) of mice challenged with LPS. Conclusions: Sirtuins are expressed in innate immune cells. Inhibition of SIRT1/2 activity decreases cytokine production by macrophages and protects from endotoxemia, suggesting that sirtuin inhibitors may represent novel adjunctive therapy for treating inflammatory disorders such as sepsis.

Absence of mTOR Inhibitor Effect on Hepatic Cyst Growth: A Case Report of a Kidney Transplant Recipient with Autosomal Dominant Polycystic Kidney Disease.

Some experimental studies have suggested a beneficial effect of the mammalian target of rapamycin (mTOR) inhibitor use on hepatic and renal cyst growth in patients with autosomal dominant polycystic kidney disease (ADPKD). However, the results of clinical studies are conflicting and the role of mTOR inhibitors is still uncertain. We report the case of a patient with ADPKD who underwent deceased kidney transplantation because of an end-stage renal disease. The evolution was uneventful with an excellent graft function under cyclosporine (CsA) monotherapy. Some years later, the patient developed a symptomatic hepatomegaly due to growth of cysts. CsA was replaced by sirolimus, an mTOR inhibitor, in order to reduce or control the increase in the cyst and liver volume. Despite the switch, the hepatic volume increased by 25% in two years. Finally sirolimus was stopped because of the lack of effect on hepatic cyst growth and the presence of sirolimus side effects. The interest of our case resides in the followup by MRI imaging during the mTOR inhibitor treatment and 15 months after the restart of the initial immunosuppressive therapy. This observation indicates that mTOR inhibitors did not have significant effect on cyst-associated hepatic growth in our patient, which is consistent with some results of recent large clinical studies.