Convenient synthesis of C75, an inhibitor of FAS and CPT1

C75 is a synthetic racemic α-methylene-γ-butyrolactone exhibiting anti-tumoral properties in vitro and in vivo as well as inducing hypophagia and weight loss in rodents. These interesting properties are thought to be a consequence of the inhibition of the key enzymes FAS and CPT1 involved in lipid metabolism. The need for larger amounts of this compound for biological evaluation prompted us to develop a convenient and reliable route to multigram quantities of C75 from easily available ethyl penta-3,4-dienoate 6. A recently described protocol for the addition of 6 to a mixture of dicyclohexylborane and nonanal followed by acidic treatment of the crude afforded lactone 8, as a mixture of cis and trans isomers, in good yield. The DBU-catalyzed isomerization of the methyl esters 9 arising from 8 gave a 10:1 trans/cis mixture from which the trans isomer was isolated and easily transformed into C75. The temporary transformation of C75 into a phenylseleno ether derivative makes its purification, manipulation and storage easier.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model.

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation.

Interleukin-1β (IL-1β) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1β activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1β that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1β by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1β and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.

Convenient synthesis of C75, an inhibitor of FAS and CPT1

C75 is a synthetic racemic α-methylene-γ-butyrolactone exhibiting anti-tumoral properties in vitro and in vivo as well as inducing hypophagia and weight loss in rodents. These interesting properties are thought to be a consequence of the inhibition of the key enzymes FAS and CPT1 involved in lipid metabolism. The need for larger amounts of this compound for biological evaluation prompted us to develop a convenient and reliable route to multigram quantities of C75 from easily available ethyl penta-3,4-dienoate 6. A recently described protocol for the addition of 6 to a mixture of dicyclohexylborane and nonanal followed by acidic treatment of the crude afforded lactone 8, as a mixture of cis and trans isomers, in good yield. The DBU-catalyzed isomerization of the methyl esters 9 arising from 8 gave a 10:1 trans/cis mixture from which the trans isomer was isolated and easily transformed into C75. The temporary transformation of C75 into a phenylseleno ether derivative makes its purification, manipulation and storage easier.

The JNK inhibitor XG-102 protects from ischemic damage with delayed intravenous administration also in the presence of recombinant tissue plasminogen activator.

BACKGROUND: XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide which selectively inhibits the c-Jun N-terminal kinase, is a powerful neuroprotectant in mouse models of middle cerebral artery occlusion (MCAo) with delayed intracerebroventricular injection. We aimed to determine whether this neuroprotection could also be achieved by intravenous injection of XG-102, which is a more feasible approach for future use in stroke patients. We also tested the compatibility of the compound with recombinant tissue plasminogen activator (rtPA), commonly used for intravenous thrombolysis and known to enhance excitotoxicity. METHODS: Male ICR-CD1 mice were subjected to a 30-min-suture MCAo. XG-102 was injected intravenously in a single dose, 6 h after ischemia. Hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mM) deprivation for 30 min. rtPA was added after ischemia and before XG-102 administration, both in vitro and in vivo. RESULTS: The lowest intravenous dose achieving neuroprotection was 0.0003 mg/kg, which reduced the infarct volume after 48 h from 62 +/- 19 mm(3) (n = 18) for the vehicle-treated group to 18 +/- 9 mm(3) (n = 5, p &lt; 0.01). The behavioral outcome was also significantly improved at two doses. Addition of rtPA after ischemia enhanced the ischemic damage both in vitro and in vivo, but XG-102 was still able to induce a significant neuroprotection. CONCLUSIONS: A single intravenous administration of XG-102 several hours after ischemia induces a powerful neuroprotection. XG-102 protects from ischemic damage in the presence of rtPA. The feasibility of systemic administration of this promising compound and its compatibility with rtPA are important steps for its development as a drug candidate in ischemic stroke.

Minor protease inhibitor mutations at baseline do not increase the risk for a virological failure in HIV-1 subtype B infected patients.

BACKGROUND: Minor protease inhibitor (PI) mutations often exist as polymorphisms in HIV-1 sequences from treatment-naïve patients. Previous studies showed that their presence impairs the antiretroviral treatment (ART) response. Evaluating these findings in a larger cohort is essential. METHODS: To study the impact of minor PI mutations on time to viral suppression and time to virological failure, we included patients from the Swiss HIV Cohort Study infected with HIV-1 subtype B who started first-line ART with a PI and two nucleoside reverse transcriptase inhibitors. Cox regression models were performed to compare the outcomes among patients with 0 and ≥ 1 minor PI mutation. Models were adjusted for baseline HIV-1 RNA, CD4 cell count, sex, transmission category, age, ethnicity, year of ART start, the presence of nucleoside reverse transcriptase inhibitor mutations, and stratified for the administered PIs. RESULTS: We included 1199 patients of whom 944 (78.7%) received a boosted PI. Minor PI mutations associated with the administered PI were common: 41.7%, 16.1%, 4.7% and 1.9% had 1, 2, 3 or ≥ 4 mutations, respectively. The time to viral suppression was similar between patients with 0 (reference) and ≥ 1 minor PI mutation (multivariable hazard ratio (HR): 1.1 [95% confidence interval (CI): 1.0-1.3], P = .196). The time to virological failure was also similar (multivariable HR:.9 [95% CI:.5-1.6], P = .765). In addition, the impact of each single minor PI mutation was analyzed separately: none was significantly associated with the treatment outcome. CONCLUSIONS: The presence of minor PI mutations at baseline has no effect on the therapy outcome in HIV infected individuals.

The Selective Phosphodiesterase 4 Inhibitor Roflumilast and Phosphodiesterase 3/4 Inhibitor Pumafentrine Reduce Clinical Score and TNF Expression in Experimental Colitis in Mice.

OBJECTIVE: The specific inhibition of phosphodiesterase (PDE)4 and dual inhibition of PDE3 and PDE4 has been shown to decrease inflammation by suppression of pro-inflammatory cytokine synthesis. We examined the effect of roflumilast, a selective PDE4 inhibitor marketed for severe COPD, and the investigational compound pumafentrine, a dual PDE3/PDE4 inhibitor, in the preventive dextran sodium sulfate (DSS)-induced colitis model. METHODS: The clinical score, colon length, histologic score and colon cytokine production from mice with DSS-induced colitis (3.5% DSS in drinking water for 11 days) receiving either roflumilast (1 or 5 mg/kg body weight/d p.o.) or pumafentrine (1.5 or 5 mg/kg/d p.o.) were determined and compared to vehicle treated control mice. In the pumafentrine-treated animals, splenocytes were analyzed for interferon-γ (IFNγ) production and CD69 expression. RESULTS: Roflumilast treatment resulted in dose-dependent improvements of clinical score (weight loss, stool consistency and bleeding), colon length, and local tumor necrosis factor-α (TNFα) production in the colonic tissue. These findings, however, were not associated with an improvement of the histologic score. Administration of pumafentrine at 5 mg/kg/d alleviated the clinical score, the colon length shortening, and local TNFα production. In vitro stimulated splenocytes after in vivo treatment with pumafentrine showed a significantly lower state of activation and production of IFNγ compared to no treatment in vivo. CONCLUSIONS: These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice.

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Eosinophilic oesophagitis and proton pump inhibitor-responsive oesophageal eosinophilia have similar clinical, endoscopic and histological findings.

BACKGROUND: Some patients with a phenotypic appearance of eosinophilic oesophagitis (EoE) respond histologically to PPI, and are described as having PPI-responsive oesophageal eosinophilia (PPI-REE). It is unclear if PPI-REE is a GERD-related phenomenon, a subtype of EoE, or a completely unique entity. AIM: To compare demographic, clinical and histological features of EoE and PPI-REE. METHODS: Two databases were reviewed from the Walter Reed and Swiss EoE databases. Patients were stratified into two groups, EoE and PPI-REE, based on recent EoE consensus guidelines. Response to PPI was defined as achieving less than 15 eos/hpf and a 50% decrease from baseline following at least a 6-week course of treatment. RESULTS: One hundred and three patients were identified (63 EoE and 40 PPI-REE; mean age 40.2 years, 75% male and 89% Caucasian). The two cohorts had similar dysphagia (97% vs. 100%, P = 0.520), food impaction (43% vs. 35%, P = 0.536), and heartburn (33% vs. 32%, P = 1.000) and a similar duration of symptoms (6.0 years vs. 5.8 years, P = 0.850). Endoscopic features were also similar between EoE and PPI-REE; rings (68% vs. 68%, P = 1.000), furrows (70% vs. 70%, P = 1.000), plaques (19% vs. 10%, P = 0.272), strictures (49% vs. 30%, P = 0.066). EoE and PPI-REE were similar in the number of proximal (39 eos/hpf vs. 38 eos/hpf, P = 0.919) and distal eosinophils (50 vs. 43 eos/hpf, P = 0.285). CONCLUSIONS: EoE and PPI-responsive oesophageal eosinophilia are similar in clinical, histological and endoscopic features and therefore are indistinguishable without a PPI trial. Further studies are needed to determine why a subset of patients with oesophageal eosinophilia respond to PPI.

The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways.

BACKGROUND: Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response. RESULTS: Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2. CONCLUSIONS: We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.

Plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2 in various disease states.

The association of increased PA-inhibitor (PAI) activity and of PAI-1 and PAI-2 antigen levels with different pathological conditions was studied in a collective of over 300 patients. PAI-1 and PAI-2 levels were measured by specific radioimmunoassays. A good correlation was observed of PAI activity with PAI-1 antigen (r = 0.718; p less than 0.0001) but not with PAI-2 (r = 0.070; n.s.). Both in the controls and in the patients, PAI activity and PAI-1 antigen showed an extremely large range of values. PAI activity ranged from 0.5 to 68 U/ml and PAI-1 antigen from 6 to 600 ng/ml. Increased PAI activity and PAI-1 antigen was observed in patients with malignant tumors, cardiovascular or thromboembolic disease, in the postoperative phase, with hepatic insufficiency, after trauma and after extracorporeal circulation. The large spectrum of disease states with increased PAI activity and PAI-1 antigen reinforces previous suggestions that PAI-1 is an acute phase reactant. After extracorporeal circulation, PAI activity and PAI-1 concentrations strongly increased within one hour, remained elevated for at least one week and returned to preoperation values within 7 days. PAI-2 values ranged from below detection limit (15 ng/ml), observed in half of the plasmas, to 485 ng/ml in a pregnant woman. High values of PAI-2 were only observed in pregnancy.

Hemodynamic and humoral effects of the new renin inhibitor enalkiren in normal humans.

The effect of the renin inhibitor enalkiren (Abbott-64662) was evaluated in eight normal volunteer subjects on a standardized sodium diet (100 mmol/day) by measurement of various components of the renin-angiotensin system and drug levels in plasma. On day 1, vehicle and doses of 0.001, 0.003, and 0.01 mg/kg i.v. were administered within 2 minutes at 90-minute intervals. On day 2, vehicle and doses of 0.01, 0.03, and 0.1 mg/kg i.v. were given. With the higher doses, blood pressure tended to decrease slightly with no change in heart rate. Plasma renin activity and plasma angiotensin-(1-8)octapeptide (angiotensin II) fell markedly in a dose-dependent manner. Inhibition of plasma renin activity was maximal 5 minutes after administration of the drug and persisted 90 minutes after the doses of 0.03 and 0.1 mg/kg. Not surprisingly, there was a close correlation between plasma renin activity and plasma angiotensin II levels (r = 0.81, n = 28, p less than 0.001). In contrast, active and total renin measured directly by monoclonal antibodies rose in dose-related fashion in response to renin inhibition. Pharmacokinetic parameters were calculated using the plasma drug concentrations obtained up to 6 hours after the 0.1 mg/kg dose. By means of a two-compartment model, plasma mean half-life of the drug was estimated at 1.60 +/- 0.43 hours.

Fructose Rich Diet-Induced High Plasminogen Activator Inhibitor-1 (PAI-1) Production in the Adult Female Rat: Protective Effect of Progesterone.

The effect of progesterone (P4) on fructose rich diet (FRD) intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT) dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT) or containing P4 (12 mg/kg). Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND) or containing fructose (10% w/v; CT-FRD and P4-FRD). At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND) rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND) plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1). Lipidemia and adiponectinemia were high (vs. P4-ND) in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production.