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Chagas disease is a chronic infection caused by Trypanosoma cruzi. The first-line drugs approved for treatment have several limitations and are associated with toxicity. The recombinant TSA-1-C4 and Tc24-C4 proteins have been profiled as promising candidates for the formulation of therapeutic vaccines, leading us to propose them in combination as a bivalent recombinant protein strategy. In this study, we evaluated the immunomodulatory effect of TSA-1-C4 plus Tc24-C4 recombinant protein combination by in-vitro assays. Macrophages from naïve BALB/c mice were isolated and stimulated with TSA-1-C4+Tc24-C4 proteins, supernatants were recovered to measure NO, H O , and, TNF-α, IL-1β, IL-6 and IL-10 cytokines. Stimulated macrophages were co-cultured with CD8 T cells from naïve mice, and inflammatory cytokine-profile was measured from supernatants. Additionally, by experimental T. cruzi acute infection model, cytotoxicity was evaluated in mice after the treatment with TSA-1-C4+Tc24-C4 proteins in presence of a TLR-4 agonist adjuvant, E6020-SE. We observed that the bivalent recombinant protein strategy activates macrophages by NO and H O production, also induced a significant Th1 immune-response compared to either TSA-1-C4 or Tc24-C4 stimulated macrophages. Moreover, naïve CD8 T cells in presence of TSA-1-C4+Tc24-C4 stimulated-macrophages similarly boosted Th1 profile by significant production of IFN-γ and TNF-α cytokines. These results support the synergistic effect of the bivalent recombinant protein strategy, which leads activation of peritoneal macrophages and CD8 T cells eliciting the Th1 immune response. Although the administration of TSA-1-C4+Tc24-C4+E6020-SE showed cytotoxic activity in T. cruzi-infected mice, there was not a benefit compared to stimulate with TSA-1-C4 or Tc24-C4 antigens formulated with E6020-SE adjuvant.

Post-infectious conditions, where clinical symptoms fail to resolve even after pathogen clearance, present major health burdens. However, the mechanisms involved remain poorly understood. In Chagas disease (CD), caused by the parasite Trypanosoma cruzi, antiparasitic agents can clear T. cruzi but late-stage treatment does not improve clinical cardiac outcomes. In this study, we revealed differential metabolic trajectories of cardiac regions during T. cruzi infection, matching sites of clinical symptoms. Incomplete, region-specific, cardiac metabolic restoration was observed in animals treated with the antiparasitic benznidazole, even though parasites were successfully cleared. In contrast, superior metabolic restoration was observed for a combination treatment of reduced-dose benznidazole plus an immunotherapy (Tc24-C4 T. cruzi flagellar protein and TLR4 agonist adjuvant), even though parasite burden reduction was lower. Overall, these results provide a mechanism to explain prior clinical treatment failures in CD and to test novel candidate treatment regimens. More broadly, our results demonstrate a link between persistent metabolic perturbation and post-infectious conditions, with broad implications for our understanding of post-infectious disease sequelae.

World Health Assembly Resolution 50.29, adopted in 1997, committed the World Health Organization (WHO) and its member states to eliminate lymphatic filariasis (LF) as a public health problem. In 2000, to support this ambitious goal and the health ministries in the >70 LF-endemic countries, the Global Programme to Eliminate Lymphatic Filariasis (GPELF) was created. The resulting WHO elimination strategy consists of two main components: to stop the spread of infection by interrupting transmission and to alleviate the suffering of affected populations (by controlling morbidity). The GPELF has brought together a broad global partnership of public and private actors, including three pharmaceutical companies with headquarters in three different continents. The medicine donations programmes from GlaxoSmithKline, MSD (trade name of Merck & Co. Inc., Kenilworth, NJ, USA) and Eisai have enabled significant achievements during the first 20 y of the GPELF and are positioned to provide essential contributions to the GPELF's goals for the next decade. As we celebrate the progress towards LF elimination during the GPELF's first 20 y, this article reflects on the factors that led to the creation of the three donation programmes, the contributions these programmes have made and some lessons learned along the way. We close by emphasizing our continued commitments to LF elimination and perspectives on the next decade.

BackgroundChagas disease (CD) is caused by Trypanosoma cruzi and affects 6-7 million people worldwide. Approximately 30% of chronic patients develop chronic chagasic cardiomyopathy (CCC) after decades. Benznidazole (BNZ), one of the first-line chemotherapy approved for CD, induces toxicity and fails to halt the progression of CCC in chronic patients. The recombinant parasite-derived antigens, including Tc24, Tc24-C4, TSA-1, and TSA-1-C4 with Toll-like receptor 4 (TLR-4) agonist-adjuvants reduce cardiac parasite burdens, heart inflammation, and fibrosis, leading us to envision their use as immunotherapy together with BNZ. Given genetic immunization (DNA vaccines) encoding Tc24 and TSA-1 induce protective immunity in mice and dogs, we propose that immunization with the corresponding recombinant proteins offers an alternative and feasible strategy to develop these antigens as a bivalent human vaccine. We hypothesized that a low dose of BNZ in combination with a therapeutic vaccine (TSA-1-C4 and Tc24-C4 antigens formulated with a synthetic TLR-4 agonist-adjuvant, E6020-SE) could provide antigen-specific T cell immunity and prevent cardiac fibrosis progression.Methodology/ Principal findingsWe evaluated the therapeutic vaccine candidate plus BNZ (25 mg/kg/day/7 days) given at days 72 and 79 post-infection (p.i) (early chronic phase). Fibrosis, inflammation, and parasite burden were quantified in heart tissue at day 200 p.i. (late chronic phase). Further, spleen cells were collected to evaluate antigen-specific CD4+ and CD8+ T cell immune responses, using flow cytometry. We found that vaccine-linked BNZ treated mice had lower cardiac fibrosis compared to the infected untreated control group. Moreover, cells from mice that received the immunotherapy had higher stimulation index of antigen-specific CD8+Perforin+ T cells as well as antigen-specific central memory T cells compared to infected untreated control.ConclusionsOur results suggest that the bivalent immunotherapy together with BNZ treatment protects mice against cardiac fibrosis and activates strong CD8+ T cell responses by in vitro restimulation, evidencing the induction of a long-lasting T. cruzi-immunity.Author summaryChagas disease (CD) is a neglected tropical disease caused by the parasite Trypanosoma cruzi, transmitted through contact with infected feces of vectors bugs. CD can induce cardiac abnormalities including the development of fibrosis and eventually death. Benznidazole (BNZ) is the first-line drug approved against CD, however, its toxicity and lack of efficacy in the chronic phase have limited its use. Previous studies have demonstrated the feasibility of reducing doses of BNZ given in combination with therapeutic vaccines during the acute phase of CD, which increases its tolerability and reduces adverse side effects. Considering that patients are often diagnosed until prolonged stages of the disease, its necessary to evaluate therapies given in the chronic phase of CD. In this study, we evaluated a vaccine formulated with the recombinant T. cruzi-antigens TSA-1-C4 and Tc24-C4 and the adjuvant E6020-SE in combination with a low dose of BNZ given during the chronic phase of T. cruzi-infection using a murine model. The authors found that the combination therapy protects mice against cardiac fibrosis, allow the activation of a CD8+ T cell response and induce a prolonged memory response against T. cruzi. This study supports the development of the vaccine-linked chemotherapy approach in order to prevent T. cruzi chronic infection.

Global dispersion of multidrug resistant bacteria is very common and evolution of antibiotic‐resistance is occurring at an alarming rate, presenting a formidable challenge for humanity. The development of new therapeuthics with novel molecular targets is urgently needed. Current drugs primarily affect protein, nucleic acid, and cell wall synthesis. Metabolic pathways, including those involved in amino acid biosynthesis, have recently sparked interest in the drug discovery community as potential reservoirs of such novel targets. Tryptophan biosynthesis, utilized by bacteria but absent in humans, represents one of the currently studied processes with a therapeutic focus. It has been shown that tryptophan synthase (TrpAB) is required for survival of Mycobacterium tuberculosis in macrophages and for evading host defense, and therefore is a promising drug target. Here we present crystal structures of TrpAB with two allosteric inhibitors of M. tuberculosis tryptophan synthase that belong to sulfolane and indole‐5‐sulfonamide chemical scaffolds. We compare our results with previously reported structural and biochemical studies of another, azetidine‐containing M. tuberculosis tryptophan synthase inhibitor. This work shows how structurally distinct ligands can occupy the same allosteric site and make specific interactions. It also highlights the potential benefit of targeting more variable allosteric sites of important metabolic enzymes.

The Tuberculosis Drug Accelerator, an experiment designed to facilitate collaboration in tuberculosis drug discovery by breaking down barriers among competing labs and institutions, has reached a 10-year landmark. We review the consortium’s achievements, advantages and limitations and advocate for the application of similar models to other diseases.

E6020 is a synthetic agonist of Toll-like receptor-4 (TLR4). The purpose of this study was to evaluate the effect of different doses of E6020-SE on Trypanosoma cruzi-specific immune responses and its ability to confer protection against acute lethal infection in mice. Forty female BALB/c were infected with 500 trypomastigotes of T cruzi H1 strain, divided into four groups (n = 10) and treated at 7- and 14-day post-infection (dpi) with different doses of E6020-SE or PBS (control). Survival was followed for 51 days, mice were euthanized and hearts were collected to evaluate parasite burden, inflammation and fibrosis. We found significantly higher survival and lower parasite burdens in mice injected with E6020-SE at all doses compared to the control group. However, E6020-SE treatment did not significantly reduce cardiac inflammation or fibrosis. On the other hand, E6020-SE modulated Th1 and Th2 cytokines, decreasing IFN-γ and IL-4 in a dose-dependent manner after stimulation with parasite antigens. We conclude that E6020-SE alone increased survival by decreasing cardiac parasite burdens in BALB/c mice acutely infected with T cruzi but failed to prevent cardiac damage. Our results suggest that for optimal protection, a vaccine antigen is necessary to balance and orient a protective immune response.

Summary Background (+)-SJ000557733 (SJ733) is a novel, orally bioavailable inhibitor of Plasmodium falciparum ATP4. In this first-in-human and induced blood-stage malaria phase 1a/b trial, we investigated the safety, tolerability, pharmacokinetics, and antimalarial activity of SJ733 in humans. Methods The phase 1a was a single-centre, dose-escalation, first-in-human study of SJ733 allowing modifications to dose increments and dose-cohort size on the basis of safety and pharmacokinetic results. The phase 1a took place at St Jude Children's Research Hospital and at the University of Tennessee Clinical Research Center (Memphis, TN, USA). Enrolment in more than one non-consecutive dose cohort was allowed with at least 14 days required between doses. Participants were fasted in seven dose cohorts and fed in one 600 mg dose cohort. Single ascending doses of SJ733 (75, 150, 300, 600, 900, or 1200 mg) were administered to participants, who were followed up for 14 days after SJ733 dosing. Phase 1a primary endpoints were safety, tolerability, and pharmacokinetics of SJ733, and identification of an SJ733 dose to test in the induced blood-stage malaria model. The phase 1b was a single-centre, open-label, volunteer infection study using the induced blood-stage malaria model in which fasted participants were intravenously infected with blood-stage P falciparum and subsequently treated with a single dose of SJ733. Phase 1b took place at Q-Pharm (Herston, QLD, Australia) and was initiated only after phase 1a showed that exposure exceeding the threshold minimum exposure could be safely achieved in humans. Participants were inoculated on day 0 with P falciparum-infected human erythrocytes (around 2800 parasites in the 150 mg dose cohort and around 2300 parasites in the 600 mg dose cohort), and parasitaemia was monitored before malaria inoculation, after inoculation, immediately before SJ733 dosing, and then post-dose. Participants were treated with SJ733 within 24 h of reaching 5000 parasites per mL or at a clinical score higher than 6. Phase 1b primary endpoints were calculation of a parasite reduction ratio (PRR48) and parasite clearance half-life, and safety and tolerability of SJ733 (incidence, severity, and drug-relatedness of adverse events). In both phases of the trial, SJ733 hydrochloride salt was formulated as a powder blend in capsules containing 75 mg or 300 mg for oral administration. Healthy men and women (of non-childbearing potential) aged 18–55 years were eligible for both studies. Both studies are registered with ClinicalTrials.gov ( NCT02661373 for the phase 1a and NCT02867059 for the phase 1b). Findings In the phase 1a, 23 healthy participants were enrolled and received one to three non-consecutive doses of SJ733 between March 14 and Dec 7, 2016. SJ733 was safe and well tolerated at all doses and in fasted and fed conditions. 119 adverse events were recorded: 54 (45%) were unrelated, 63 (53%) unlikely to be related, and two (2%) possibly related to SJ733. In the phase 1b, 17 malaria-naive, healthy participants were enrolled. Seven participants in the 150 mg dose cohort were inoculated and dosed with SJ733. Eight participants in the 600 mg dose cohort were inoculated, but two participants could not be dosed with SJ733. Two additional participants were subsequently inoculated and dosed with SJ733. SJ733 exposure increased proportional to the dose through to the 600 mg dose, then was saturable at higher doses. Fasted participants receiving 600 mg exceeded the target area under the concentration curve extrapolated to infinity (AUC0–∞) of 13 000 μg × h/L (median AUC0–∞ 24 283 [IQR 16 135–31 311] μg × h/L, median terminal half-life 17·4 h [IQR 16·1–24·0], and median timepoint at which peak plasma concentration is reached 1·0 h [0·6–1·3]), and this dose was tested in the phase 1b. All 15 participants dosed with SJ733 had at least one adverse event. Of the 172 adverse events recorded, 128 (74%) were mild. The only adverse event attributed to SJ733 was mild bilateral foot paraesthesia that lasted 3·75 h and resolved spontaneously. The most common adverse events were related to malaria. Based on parasite clearance half-life, the derived log10 PRR48 and corresponding parasite clearance half-lives were 2·2 (95% CI 2·0–2·5) and 6·47 h (95% CI 5·88–7·18) for 150 mg, and 4·1 (3·7–4·4) and 3·56 h (3·29–3·88) for 600 mg. Interpretation The favourable pharmacokinetic, tolerability, and safety profile of SJ733, and rapid antiparasitic effect support its development as a fast-acting component of combination antimalarial therapy. Funding Global Health Innovative Technology Fund, Medicines for Malaria Venture, and the American Lebanese Syrian Associated Charities.

Chagas disease, caused by the parasite Trypanosoma cruzi, develops into chronic Chagas’ cardiomyopathy in ~30% of infected individuals, characterized by conduction disorders, arrhythmias, heart failure, and even sudden cardiac death. Current anti-parasitic treatments are plagued by significant side effects and poor efficacy in the chronic phase of disease; thus, there is a pressing need for new treatment options. A therapeutic vaccine could bolster the protective TH1-mediated immune response, thereby slowing or halting the progression of chronic Chagas’ cardiomyopathy. Prior work in mice has demonstrated therapeutic efficacy of a Tc24 recombinant protein vaccine in the acute phase of Chagas disease. However, it is anticipated that humans will be vaccinated therapeutically when in the chronic phase of disease. This study investigates the therapeutic efficacy of a vaccine prototype containing recombinant protein Tc24, formulated with an emulsion containing the Toll-like receptor 4 agonist E6020 as an immunomodulatory adjuvant in a mouse model of chronic T. cruzi infection. Among outbred ICR mice vaccinated during chronic T. cruzi infection, there is a significant increase in the number of animals with undetectable systemic parasitemia (60% of vaccinated mice compared to 0% in the sham vaccine control group), and a two-fold reduction in cardiac fibrosis over the control group. The vaccinated mice produce a robust protective TH1-biased immune response to the vaccine, as demonstrated by a significant increase in antigen-specific IFNγ-production, the number of antigen-specific IFNγ-producing cells, and IgG2a antibody titers. Importantly, therapeutic vaccination significantly reduced cardiac fibrosis in chronically infected mice. This is a first study demonstrating therapeutic efficacy of the prototype Tc24 recombinant protein and E6020 stable emulsion vaccine against cardiac fibrosis in a mouse model of chronic T. cruzi infection., Author summary Chagas disease is a parasitic infection that can cause severe heart disease. Current treatments do not work well and have significant side effects. Because of this, the authors created a new vaccine prototype with the goal that it could be given to infected people to prevent Chagas-associated heart disease. The vaccine contains a manufactured protein identical to a protein in the parasite (called Tc24) as well as a component to help the body produce a protective immune response (a vaccine adjuvant called E6020). The vaccine would boost the body’s natural immune response to the parasite infection, reducing the number of parasites in the body, and protecting the heart. Frequently, people are not diagnosed until later in the infection, because the early (or acute) stage of disease can be mistaken for a common cold. Because of this, it is important to test the vaccine when given in the later (or chronic) stage of infection. The authors tested the vaccine in a mouse model of chronic T. cruzi infection and found that the vaccinated mice had lower levels of parasites in their body and less damage to their hearts. This research shows promising value of a therapeutic vaccine to prevent Chagas-associated heart disease in a mouse model, with the hope that the same effect could be found in humans one day.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Significance Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are neglected tropical diseases that cause severe disability and affect more than 157 million people globally. Current control efforts are hindered by the lack of a safe macrofilaricidal drug that can eliminate the parasitic adult nematodes safely. A clinically validated approach for delivering macrofilaricidal activity is to target the Wolbachia bacterial endosymbiont of the causative nematodes. This first-in-class and highly potent and specific anti-Wolbachia preclinical candidate molecule, AWZ1066S, has the potential to significantly impact current global onchocerciasis and lymphatic filariasis elimination programs and reduce elimination time frames from decades to years., Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Chagas disease affects 6 to 7 million people worldwide, resulting in significant disease burdens and health care costs in countries of endemicity. Chemotherapeutic treatment is restricted to two parasiticidal drugs, benznidazole and nifurtimox. Both drugs are highly effective during acute disease but are only minimally effective during chronic disease and fraught with significant adverse clinical effects. In experimental models, vaccines can be used to induce parasite-specific balanced T H 1/T H 2 immune responses that effectively reduce parasite burdens and associated inflammation while minimizing adverse effects. The objective of this study was to determine the feasibility of vaccine-linked chemotherapy for reducing the amount of benznidazole required to significantly reduce blood and tissue parasite burdens. In this study, we were able to achieve a 4-fold reduction in the amount of benznidazole required to significantly reduce blood and tissue parasite burdens by combining the low-dose benznidazole with a recombinant vaccine candidate, Tc24 C4, formulated with a synthetic Toll-like 4 receptor agonist, E6020, in a squalene oil-in-water emulsion. Additionally, vaccination induced a robust parasite-specific balanced T H 1/T H 2 immune response. We concluded that vaccine-linked chemotherapy is a feasible option for advancement to clinical use for improving the tolerability and efficacy of benznidazole.

Despite the generally accepted immunostimulatory effect of Toll-Like Receptor 4 (TLR4) agonists and their value as vaccine adjuvants, there remains a demand for fast and easy quantification assays for these TLR4 agonists in order to accelerate and improve vaccine formulation studies. A new medium-throughput method was developed for the quantification of the TLR4 agonist, E6020, independent of the formulation composition. The assay uses a fluorescent hydrazide (DCCH) to label the synthetic lipopolysaccharide (LPS) analog E6020 through its diketone groups. This novel, low-cost, and fluorescence based assay may obviate the need for traditional approaches that primarily rely on Fourier transform infrared spectroscopy (FTIR) or mass spectrometry. The experiments were performed in a wide diversity of vaccine formulations containing E6020 to assess method robustness and accuracy. The assay was also expanded to evaluate the loading efficiency of E6020 in poly(lactic-co-glycolic acid) (PLGA) micro-particles.

Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.

E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione)] is a novel anti-inflammatory agent that has potent inhibitory effects on the production of proinflammatory cytokines from leukocytes and antiproliferative activity on keratinocytes. To characterize the in vivo pharmacological activity of E6201, topically administered E6201 was evaluated in several different animal models of dermatitis. E6201 formulated as an ointment or cream showed dose-dependent inhibition of croton oil-induced acute edema formation and neutrophil infiltration into mouse skin. In addition, E6201 cream inhibited the 1-fluoro-2,4-dinitrobenzene-induced contact hypersensitivity reaction mediated by T cells in mice. In this model, E6201 cream also suppressed the migration of neutrophils and lymphocytes into the inflammatory site. Pretreatment with E6201 cream attenuated phorbol-12 myristate 13-acetate-induced ornithine decarboxylase activity, a marker of proliferation in epidermis. Furthermore, E6201 ointment showed inhibitory effects on both mezerein-induced and interleukin (IL)-23-induced epidermal hyperplasia. E6201 also suppressed T cell receptor-stimulated IL-17 production from human T cells. These results indicate that topically administered E6201 may be a useful agent for the prevention and treatment of cutaneous inflammatory and hyperproliferative diseases such as psoriasis.

Background and purpose: Rheumatoid arthritis (RA) is an autoimmune disorder involving subsets of activated T cells, in particular T helper (Th) 1 and Th17 cells, which infiltrate and damage tissues and induce inflammation. Prostaglandin E2 (PGE2) enhances the Th17 response, exacerbates collagen-induced arthritis (CIA) and promotes inflammatory pain. The current study investigated whether selective antagonism of the PGE2 EP4 receptor would suppress Th1/Th17 cell development and inflammatory arthritis in animal models of RA. Experimental approach: Effects of PGE2 and a novel EP4 receptor antagonist ER-819762 on Th1 differentiation, interleukin-23 (IL-23) production by dendritic cells (DCs), and Th17 development were assessed in vitro. The effect of ER-819762 was evaluated in CIA and glucose-6-phosphate isomerase (GPI)-induced arthritis models. In addition, the effects of ER-819762 on pain were evaluated in a model of chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in the rat. Key results: Stimulation of the EP4 receptor enhanced Th1 differentiation via phosphatidylinositol 3 kinase signalling, selectively promoted Th17 cell expansion, and induced IL-23 secretion by activated DCs, effects suppressed by ER-819762 or anti-PGE2 antibody. Oral administration of ER-19762 suppressed Th1 and Th17 cytokine production, suppressed disease in collagen- and GPI-induced arthritis in mice, and suppressed CFA-induced inflammatory pain in rats. Conclusion and implications: PGE2 stimulates EP4 receptors to promote Th1 differentiation and Th17 expansion and is critically involved in development of arthritis in two animal models. Selective suppression of EP4 receptor signalling may have therapeutic value in RA both by modifying inflammatory arthritis and by relieving pain.

The goal of this study is to identify a novel inhibitor with anti-inflammatory and antiproliferative properties for the treatment of psoriasis. Compound f152A1 \[(3 S ,5 Z ,8 S ,11 E )-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,9,10-tetrahydro-1 H -benzo[c\]\[1\]oxacyclotetradecine1,7(8 H )-dione] was identified as the main active metabolite with strong inhibitory activity against tumor necrosis factor-α (TNFα) transcription in a fraction originated from the fermentation broth of the fungus Curvularia verruculosa. Although active in cell-based assays, f152A1 was unstable in plasma and liver microsome preparations, thus limiting its pharmaceutical utilization. To improve the metabolic properties of f152A1, a medicinal chemistry program was undertaken, resulting in the generation of over 400 analogs of f152A1. Eventually, E6201 [(3 S ,4 R ,5 Z ,8 S ,9 S ,11 E )-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1 H -2-benzoxacyclotetradecine-1,7(8 H )-dione] was identified as a promising analog in this series. In the present study, we characterized the in vitro activities of E6201 and discovered that the compound inhibits lipopolysaccharide-activated TNFα reporter activity in THP-1-33 cells with an IC50 value of 50 nM and selectively inhibits mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1 and MEK kinase-1 in cell-free biochemical assays. In addition, E6201 showed inhibitory activity in several other cell-based systems: 1) phosphorylation of c-jun N-terminal kinase and p38 MAPKs; 2) nuclear factor-κB and activated protein-1 activation in various cell types; 3) interleukin (IL)-2 production from human lymphocytes; 4) hyperproliferation of human keratinocytes; 5) IL-8 production from human keratinocytes; and 6) proinflammatory cytokine production from human peripheral blood mononuclear cells. Based on the data presented here, E6201 may be beneficial for treatment of inflammatory and hyperproliferative diseases such as psoriasis through its anti-inflammatory activities on immune cells and antihyperproliferative activities on keratinocytes.

Alpha-D-glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-alpha and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 microM. E5564 blocked LPS-mediated activation of nuclear factor-kappaB in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to (6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-beta-O-phosphono-alpha-D-glucopyranose tetrasodium salt (E5531), another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose- and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.

A series of novel, synthetic compounds containing lipids linked to a phosphate-containing acyclic backbone are shown to have similar biological properties to lipopolysaccharide (LPS). These compounds showed intrinsic agonistic properties when tested for their ability to stimulate tumor necrosis factor-alpha in human whole blood and interleukin-6 in U373 human glioblastoma cells without added LPS coreceptor CD14. The presence of the LPS antagonist E5564 completely blocked responses, suggesting that the novel compounds and LPS share a common mechanism of cell activation. Stereoselectivity of the molecules was observed in vitro; compounds with an R,R,R,R-configuration were strongly agonistic, whereas compounds with an R,S,S,R-configuration were much weaker in their activity on human whole blood and U373 cells. We also tested the effect of the compounds in cells transfected with the LPS receptor Toll-like receptor 4 (TLR4), with similar results, further supporting a shared mechanism with LPS. This was confirmed in vivo where the agonists failed to elicit cytokine responses in C3H/HeJ mice lacking TLR4 signaling. Because LPS-like molecules enhance immune responses, the compounds were mixed with tetanus toxoid and administered to mice in an immunization protocol to test for adjuvant activity. They enhanced the generation of specific antibodies against tetanus toxoid. Our results indicate that these unique compounds behave as agonists of TLR4, resulting in responses similar to those elicited by LPS. They display adjuvant activity in vivo and may be useful for the development of vaccine therapies.

In her News Feature “Out of bounds” (27 January p. [339][1]), A. McCook strove to present an objective narrative of the situation in which a student at Harvard University was forced to undergo a psychiatric evaluation and then filed a court order against scientist Dr. Lee Rubin. However, McCook failed to take into consideration Rubin's longstanding reputation as a scientist and a mentor. We are scientists who have worked with, been mentored by, or collaborated with Rubin. Some are current members of his laboratory, some have known him for many years, and a few have known him for more than 30 years. We could not let this story stand without adding our perspective. Rubin's scientific reputation can be readily appreciated simply by skimming through the list of his stellar publications. However, a dry bibliography fails to illuminate the quality of the scientist behind the science. Rubin has always upheld the highest standards of scientific and personal integrity. His kindness and concern for the personal well-being and professional development of his graduate students, and indeed everyone in his laboratory, has always been clearly evident. Rubin has always had our backs, whether as a mentor, a colleague, a boss, or a friend. He upholds the highest standards of scientific rigor while providing compassionate mentorship. His commitment to leading by example means we place the highest value on our friendship and connections with him. We have sought out his scientific counsel and, in several cases, engaged in collaborations that span many years. We feel fortunate to have had the opportunity to know and to work with him. It is our collective experience that besides being an outstanding scientist, he is a warm and caring scientific mentor who does not deserve to have the reputation he has built over 40 years or more besmirched. [1]: /lookup/doi/10.1126/science.355.6323.339

Inspired by natural product, LL-Z1640-2, clinical candidate, E6201 (22) was discovered in a medicinal chemistry effort through total synthesis. The modification on C14-position to N-alkyl substitution showed to be potent in vitro and orally active in vivo in anti-inflammatory assays.

The potent in vitro lead compound, ER-803064 (2), a MEK1 and MEKK1 inhibitor inspired from natural product LL-Z1640-2 (f152A1), was further optimized to improve in vitro and in vivo potency. The modifications on C14 position led to discovery of the lead compounds 28 and 29, which regained full in vitro potency of f152A1 and showed higher in vivo potency by iv administration.

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)-containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti-double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.

The synthetic antagonists of lipopolysaccharide (LPS), E5531 and E5564, are analogs of the lipid A portion of LPS that not only lack agonistic activity but also inhibit the biological effects of LPS both in vitro and in vivo. The effects of LPS and these synthetic antagonists have been localized to the recently described Toll-like receptor 4 (TLR4). A recent report indicated that the naturally occurring LPS antagonist Rhodobacter sphaeroides LPS loses its antagonist properties and gains pro-inflammatory qualities in the presence of chlorpromazine and other amphipathic drugs. To determine whether these reported actions occur with our chemically defined LPS antagonists, we examined the effects of chlorpromazine, fluphenazine, trifluoperazine, and lidocaine on the antagonism elicited by RsLPS and E5531 in U373 cells, which produce IL-6 in response to LPS. We also tested the effects of these amphipathic molecules on the LPS-neutralizing activity of RsLPS and E5564 on LPS-induced TNF-α release in human whole blood. The results indicate that neither chlorpromazine, fluphenazine, trifluoperazine nor lidocaine alter the activity of E5531 or E5564 in an in vitro cell system or human whole blood. Furthermore, chlorpromazine did not affect the antagonistic activity of RsLPS or E5564 on IL-6 generation by peripheral blood mononuclear cells. Thus, based on these data, our purified synthetic LPS-antagonists do not appear to lose their antagonistic properties and/or become agonists in the presence of amphipathic agents or drugs.

Lipopolysaccharide (LPS) stimulates multiple signaling events, including nuclear factor-kappaB (NF-kappaB) activity and the mitogen-activated protein (MAP) kinases, ERK, JNK, and p38 in LPS-responsive cells, resulting in transcriptional activation and cytokine generation. LPS-induced signaling via toll-like receptor 4 (TLR4) results in the activation of the transcription factor NF-kappaB. Since LPS activates other signaling cascades in responsive cells, the objective of this study was to determine whether such events are mediated by TLR4 in response to LPS. We generated human embryonic kidney cells (HEK293) that stably express TLR4 (HEK-TLR4) and examined their responsiveness to LPS by measuring NF-kappaB activity and production of interleukin-8 (IL-8). A trans-reporting system was used to measure the activity of Elk-1, an ETS-domain transcription factor targeted by MAP kinase pathways. LPS stimulated NF-kappaB reporter activity and IL-8 production but not Elk-1 activity in HEK-TLR4 cells. When MD-2, a protein associated with the extracellular domain of TLR4, was expressed in these cells, there was a marked increase in Elk-1 activity as well as ERK, JNK, and p38 MAP kinase phosphorylation in response to LPS. TLR4-mediated NF-kappaB reporter activity and IL-8 production was enhanced by the expression of MD-2. This study demonstrates that expression of both TLR4 and MD-2 is required for LPS to activate or augment the MAP kinase pathways, Elk-1 stimulation, and IL-8 generation.

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4(-/-) mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)-a potent, well-tolerated, synthetic TLR4 antagonist-blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

Maitotoxin elicited a marked influx of 45Ca2+ into NIH 3T3 fibroblast cells. The influx was blocked by imidazoles (econazole, miconazole, SKF 96365, clotrimazole, calmidazolium) with ic 50 values from 0.56 to 3 μM. Phenylalkylamines (verapamil, methoxyverapamil) and nitrendipine were less potent, and diltiazem was very weak. Among other calcium blockers, the diphenylbutylpiperidines fluspirilene and penfluridol, the diphenylpropylpiperidine loperamide, and the local anesthetic proadifen were quite active with ic 50 values of 2–4 μM. The pattern of inhibition of maitotoxin-elicited calcium influx did not correspond to the ability of the agents to block elevation of calcium that ensues through calcium-release activated calcium (CRAC) channels after activation of phosphoinositide breakdown by ATP in HL-60 cells. The imidazoles did block CRAC channels, but fluspirilene, penfluridol, loperamide and proadifen were ineffective. Loperamide actually appeared to enhance influx of calcium via the activated CRAC channels. The imidazoles, in particular calmidazolium, caused an apparent influx of calcium and caused a stimulation of phosphoinositide breakdown in HL-60 cells.

Ca 2+ influx triggered by antigen binding to T cell receptors (TCR) is an early event in T cell activation. An additional (Ca 2+ influx induced by depletion of intracellular Ca 2+ (CDCI) has been characterized in human Jurkat T cells that is both temporally and mechanistically distinct from TCR-mediated Ca 2+ influx (TCRCI). Both TCRCI and CDCI were insensitive to voltage-gated Ca 2+ channel antagonists (e.g., nifedipine, verapamil, and ω-conotoxin G) and pertussis toxin, yet were voltage-sensitive and inhibited by SKF 96365 (a receptorgated Ca 2+ channel blocker) and cholera toxin. However, TCRCI but not CDCI was associated with a significant increase in inositol phosphate (IP x ) levels and inhibited by phorbol ester, while CDCI but not TCRCI was inhibited by Sr 2+ , forskolin (FSK), and 1,9-dideoxy FSK in a cAMP-independent fashion. Moreover, TCR stimulation did not deplete thapsigargin-sensitive Ca 2+ stores, suggesting that TCRCI is not merely a consequence of Ca 2+ depletion. These results indicate that Ca 2+ entry following the depletion of intracellular Ca 2+ stores or TCR stimulation occur through distinct cellular mechanisms coexisting in Jurkat T cells.

We previously reported that TLR4(-/-) mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist Eritoran blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post infection. Herein we extend these findings: anti-TLR4- or -TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h before PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4(-/-) mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks high-mobility group B1 (HMGB1)-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI; (iii) interleukin (IL)-1β contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the antiviral drug oseltamivir were administered starting 4 days post infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.

Synthetic routes to difluorinated analogs of the adrenergic agonists, norepinephrine (NE), epinephrine (E), and phenylephrine (PE) have been developed. The syntheses were based on elaboration of the ethanolamine side chains from the appropriately polyfunctionalized benzaldehydes. The benzaldehydes were prepared from precursor difluorinated benzenes by sequential regioselective lithiations and reaction with electrophiles to introduce hydroxyl and carboxaldehyde functionalities. Binding and functional assay data demonstrate that the 2,6-difluorinated analogs are relatively inactive at both alpha- and beta-adrenergic receptors. These results are consistent with earlier observations that 2-fluoro substitution of adrenergic agonists decreases alpha-adrenergic activity whereas 6-fluoro substitution decreases beta-adrenergic activity.

In Chinese hamster ovary cells transfected with m5 muscarinic receptors, carbachol stimulates both calcium influx and calcium release from intracellular stores. The marine toxin maitotoxin (MTX) elicits a similar response on calcium influx. Carbachol- and MTX-induced calcium influx can be inhibited by the proposed blockers of receptor-operated calcium channels (ROCC), CAI and SK&F 96365. Both carbachol and MTX induce a significant increase in total protein tyrosine phosphorylation, which is dependent on extracellular calcium and can be inhibited by CAI and SK&F 96365. Phospholipase C-gamma was identified as one of the substrates subject to calcium-dependent tyrosine phosphorylation following carbachol or MTX stimulation. Carbachol-induced [3H]inositol trisphosphate formation was partially inhibited by an inhibitor of tyrosine kinases, by removal of extracellular calcium, and by the inhibitor of receptor-operated calcium channels CAI suggesting that phosphorylation of phospholipase C-gamma plays a role in the muscarinic activation of phosphoinositide breakdown. Such an effect of carbachol is reminiscent of effects observed with peptide growth factors and represents a novel alternative signaling pathway for a muscarinic G protein-coupled receptor.

A frog used for "hunting magic" by several groups of Panoan-speaking Indians in the borderline between Brazil and Peru is identified as Phyllomedusa bicolor. This frog's skin secretion, which the Indians introduce into the body through fresh burns, is rich in peptides. These include vasoactive peptides, opioid peptides, and a peptide that we have named adenoregulin, with the sequence GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAV as determined from mass spectrometry and Edman degradation. The natural peptide may contain a D amino acid residue, since it is not identical in chromatographic properties to the synthetic peptide. Adenoregulin enhances binding of agonists to A1 adenosine receptors; it is accompanied in the skin secretion by peptides that inhibit binding. The vasoactive peptide sauvagine, the opioid peptides, and adenoregulin and related peptides affect behavior in mice and presumably contribute to the behavioral sequelae observed in humans.

Incubation of the C6 cells with 10 microM idazoxan (an alpha 2-adrenoceptor antagonist and putative antidepressant) for 5 days in vitro resulted in a 23% reduction of beta-adrenoceptor number and a 37% decrease in isoproterenol-induced cyclic AMP accumulation. In contrast, post-receptor stimulated cyclic AMP accumulation (by the use of forskolin or cholera toxin) was unaffected. The desensitization of the beta-adrenoceptor was accompanied by an increase in the KL/KH ratio for this receptor. Chronic in vitro treatment of C6 glioma cells with idazoxan did not significantly affect cholera or pertussis toxin catalyzed ribosylation of Gs and Gi/Go in these cells. Similarly, idazoxan did not alter either the basal levels of protein kinase C (PKC) alpha, or its cytoplasm to membrane translocation. These results suggest that idazoxan may have direct postsynaptic effects, the site of which may be at the level of receptor/G protein interaction.

The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.

The goal of this study is to identify a novel inhibitor with anti-inflammatory and antiproliferative properties for the treatment of psoriasis. Compound f152A1 [(3S,5Z,8S,11E)-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,9,10-tetrahydro-1H-benzo[c][1]oxacyclotetradecine1,7(8H)-dione] was identified as the main active metabolite with strong inhibitory activity against tumor necrosis factor-alpha (TNFalpha) transcription in a fraction originated from the fermentation broth of the fungus Curvularia verruculosa. Although active in cell-based assays, f152A1 was unstable in plasma and liver microsome preparations, thus limiting its pharmaceutical utilization. To improve the metabolic properties of f152A1, a medicinal chemistry program was undertaken, resulting in the generation of over 400 analogs of f152A1. Eventually, E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] was identified as a promising analog in this series. In the present study, we characterized the in vitro activities of E6201 and discovered that the compound inhibits lipopolysaccharide-activated TNFalpha reporter activity in THP-1-33 cells with an IC(50) value of 50 nM and selectively inhibits mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1 and MEK kinase-1 in cell-free biochemical assays. In addition, E6201 showed inhibitory activity in several other cell-based systems: 1) phosphorylation of c-jun N-terminal kinase and p38 MAPKs; 2) nuclear factor-kappaB and activated protein-1 activation in various cell types; 3) interleukin (IL)-2 production from human lymphocytes; 4) hyperproliferation of human keratinocytes; 5) IL-8 production from human keratinocytes; and 6) proinflammatory cytokine production from human peripheral blood mononuclear cells. Based on the data presented here, E6201 may be beneficial for treatment of inflammatory and hyperproliferative diseases such as psoriasis through its anti-inflammatory activities on immune cells and antihyperproliferative activities on keratinocytes.

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

Incubation of rat glioma C6 cells with 10 microM desipramine for five days in vitro resulted in a 31% reduction of beta-adrenoceptors and a 38% reduction in isoproterenol-stimulated cyclic AMP accumulation. In contrast, forskolin or cholera toxin-stimulated cyclic AMP was unaffected by desipramine. Surprisingly, the beta-adrenoceptor desensitization was accompanied by an increase in the ratio of dissociation constants (KL/KH) for the low and high affinity states of the beta-adrenoceptor respectively and supports the concept of a complex interaction between the receptor and Gs protein.