An inhibitor of RORγ for chronic pulmonary obstructive disease treatment.

The role of RORγ as a transcription factor for Th17 cell differentiation and thereby regulation of IL-17 levels is well known. Increased RORγ expression along with IL-17A levels was observed in animal models, immune cells and BAL fluid of COPD patients. Increased IL-17A levels in severe COPD patients are positively correlated with decreased lung functions and increased severity symptoms and emphysema, supporting an urgency to develop novel therapies modulating IL-17 or RORγ for COPD treatment. We identified a potent RORγ inhibitor, PCCR-1 using hit to lead identification followed by extensive lead optimization by structure-activity relationship. PCCR-1 resulted in RORγ inhibition with a high degree of specificity in a biochemical assay, with > 300-fold selectivity over other isoforms of ROR. Our data suggest promising potency for IL-17A inhibition in human and canine PBMCs and mouse splenocytes with no significant impact on Th1 and Th2 cytokines. In vivo, PCCR-1 exhibited significant efficacy in the acute CS model with dose-dependent inhibition of the PD biomarkers that correlated well with the drug concentration in lung and BAL fluid, demonstrating an acceptable safety profile. This inhibitor effectively inhibited IL-17A release in whole blood and BALf samples from COPD patients. Overall, we identified a selective inhibitor of RORγ to pursue further development of novel scaffolds for COPD treatment.

Design, Synthesis, and Pharmacological Evaluation of 5,6-Disubstituted Pyridin-2(1H)-one Derivatives as Phosphodiesterase 10A (PDE10A) Antagonists.

We report the design and synthesis of novel 5,6-diarylated pyridin-2(1H)-one derivatives as pharmacophoric PDE10A inhibitors. This highly potent molecular scaffold was developed from an inactive diarylpyridine-2-amine derivative 3b by extensive and systematic analogue synthesis and SAR analysis. Further optimization of the scaffold resulted in identification of pyridin-2(1H)-one 18b as a lead compound with good potency (IC50 = 1.6 nM) and selectivity (>6000-fold) over other related PDEs but with a poor pharmacokinetic profile. Careful metabolite profiling of 18b revealed that poor systemic exposure in rats (Cmax = 44 ng/mL; AUC0-t = 359 ng · h/mL) at 10 mg/kg was due to the formation of O-glucuronide conjugate by phase 2 metabolism. The structure of the glucuronide metabolite was confirmed by retention time and LC-MS/MS fragmentation matching with the synthetic glucuronide 26. The problem of low exposure of 18b was effectively addressed by its conversion to an acetate prodrug 25b, which upon oral dosing resulted in an improved pharmacokinetic profile (Cmax = 359 ng.h/mL; AUC0-t = 2436 ng.h/mL) and a desirable brain to plasma ratio of 1.2. The prodrug 25b showed good efficacy in selected rodent models of psychosis.

Design, synthesis and pharmacological evaluation of novel polycyclic heteroarene ethers as PDE10A inhibitors: part II.

We report the design and synthesis of novel pyrrolo[3,2-b]quinoline containing heteroarene ethers as PDE10A inhibitors with good to excellent potency, selectivity and metabolic stability. Further optimization of this primary series resulted in the identification of 1-methyl-3-(4-{[3-(pyridine-4-yl)pyrazin-2-yl]oxy}phenyl)-1H-pyrrolo[3,2-b]pyridine 13a with good hPDE10A potency (IC50: 6.3 nM), excellent selectivity over other related PDEs and desirable physicochemical properties. The compound exhibited high peripheral and adequate brain levels upon oral dosing in rodents. The compound also showed excellent efficacy in multiple preclinical animal models related to psychiatric disorders, particularly schizophrenia.

Design, synthesis and pharmacological evaluation of novel polycyclic heteroarene ethers as PDE10A inhibitors: Part I.

We report analogue-based rational design and synthesis of two novel series of polycyclic heteroarenes, pyrrolo[3,2-b]quinolines and pyrido[2,3-b]indoles, tethered to a biaryl system by a methyl-, ethyl- or propyl ether as PDE10A inhibitors. A number of analogues were prepared with variable chain length and evaluated for their ability to block PDE10A enzyme using a radiometric assay. Detailed SAR analyses revealed that compounds with an ethyl ether linker are superior in potency compared to compounds with methyl or propyl ether linkers. These compounds, in general, showed poor metabolic stability in rat and human liver microsomes. The metabolic profile of one of the potent compounds was studied in detail to identify metabolic liabilities of these compounds. Structural modifications were carried out that resulted in improved metabolic stability without significant loss of potency.

Discovery of benzo[d]imidazo[5,1-b]thiazole as a new class of phosphodiesterase 10A inhibitors.

The design, synthesis and structure activity relationship studies of a series of compounds from benzo[d]imidazo[5,1-b]thiazole scaffold as phosphodiesterase 10A (PDE10A) inhibitors are discussed. Several potent analogs with heteroaromatic substitutions (9a-d) were identified. The anticipated binding mode of these analogs was confirmed by performing the in silico docking experiments. Later, the heteroaromatics were substituted with saturated heteroalkyl groups which provided a tool compound 9e with excellent PDE10A activity, PDE selectivity, CNS penetrability and with favorable pharmacokinetic profile in rats. Furthermore, the compound 9e was shown to be efficacious in the MK-801 induced psychosis model and in the CAR model of psychosis.

Imidazopyridazinones as novel PDE7 inhibitors: SAR and in vivo studies in Parkinson's disease model.

The synthesis and structure-activity relationship studies of a series of compounds from imidazopyridazinone scaffold as PDE7 inhibitors are disclosed. Potent analogs such as compounds 7 (31nM), 8 (27nM), and 9 (12nM) were identified. The PDE selectivity and pharmacokinetic profile of compounds 7, 8 and 9 are also disclosed. The adequate CNS penetration of compound 7 in mice allowed it to be tested in the MPTP induced PD model and haloperidol induced catalepsy model to probe the differential pharmacology of PDE7 in the striatal pathway.

Isothiazole and isoxazole fused pyrimidones as PDE7 inhibitors: SAR and pharmacokinetic evaluation.

The synthesis and structure-activity relationship studies of isothiazole and isoxazole fused pyrimidones as PDE7 inhibitors are discussed. The pharmacokinetic profile of 10 and 21 with adequate CNS penetration, required for in vivo Parkinson's disease models, are disclosed.

CH-1504, a metabolically inert antifolate for the potential treatment of rheumatoid arthritis.

CH-1504, being developed by Chelsea Therapeutics Inc under license from the University of South Alabama, is an orally available, metabolically inert antifolate, for the potential treatment of rheumatoid arthritis (RA). CH-1504 is an analog of methotrexate (MTX) but differs from the classical antifolates because of an improved safety and tolerability profile. A significant proportion of the toxicity profile of MTX can be attributed to its polyglutamylated and hydroxylated metabolites; therefore, metabolism-blocked antifolates, such as CH-1504, have been designed to prevent the accumulation of toxic metabolites. Preclinical studies and phase II clinical trials indicated that CH-1504 and MTX inhibit dihydrofolate reductase activity with equal potency. In a phase II, proof-of-concept trial in patients with RA, CH-1504 was associated with improved tolerability and reduced hepatotoxicity as compared with MTX; in addition, improvements in the American College of Rheumatology response rates were similar following treatment with either CH-1504 or MTX. Furthermore, Chelsea Therapeutics are developing the L-isomer of CH-1504, CH-4051, which displays improved in vitro potency over with racemate and appears to be Chelsea Therapeutics' preferred candidate for future development. Inert antifolates appear to be a promising drug class for the treatment of RA because the disease-modifying properties of MTX are retained, but the therapeutic window of the inert antifolates is improved. However, further trials are required to establish the efficacy and long-term safety in a wider population of patients with RA.

Pharmacological profile of AW-814141, a novel, potent, selective and orally active inhibitor of p38 MAP kinase.

The p38 mitogen activated protein kinase (MAPK) is a key signaling molecule that plays a crucial role in the progression of various inflammatory diseases such as rheumatoid arthritis (RA), asthma and chronic obstructive pulmonary disease. The objective of the present study was to evaluate the anti-inflammatory activity of a p38 MAPK inhibitor, AW-814141. AW-814141 inhibited enzymatic activity of recombinant p38-alpha and beta isoforms with IC(50) value of 100nM and 158nM, respectively. AW-814141 also inhibited the release of tumor necrosis factor (TNF)-alpha by lipopolysaccharide (LPS) treated human peripheral blood mononuclear cells with an IC(50) value of 212nM and demonstrated selectivity against a panel of few kinases. Oral administration of AW-814141 (10mpk) in LPS-injected mice resulted in a significant reduction in TNF-alpha production in the circulation. In a carrageenan-induced rat paw edema model and collagen-induced arthritis model (CIA), AW-814141 dose dependently inhibited paw swelling. In different in vivo efficacy models, efficacy of AW-814141 was found to be better as compared to the reference compounds (Vx-745 and BIRB-796). This study demonstrated that AW-814141 is a novel p38 MAPK inhibitor and it displays promising in vitro and in vivo anti-inflammatory activities and can be used for the treatment of rheumatoid arthritis.

Fostamatinib, a Syk inhibitor prodrug for the treatment of inflammatory diseases.

Rigel Pharmaceuticals Inc is developing fostamatinib, a prodrug of the spleen tyrosine kinase (Syk) inhibitor R-406, for the potential treatment of autoimmune diseases such as rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP) and B-cell lymphomas. Syk is a key mediator of Fc and B-cell receptor signaling in inflammatory cells, such as B-cells, mast cells, macrophages and neutrophils. Preclinical studies of R-406 or fostamatinib demonstrated a significant reduction in major inflammatory mediators such as TNFalpha, IL-1, IL-6 and IL-18, leading to reduced inflammation and bone degradation in models of RA. In a phase II clinical trial, fostamatinib treatment effectively improved American College of Rheumatology response rates in patients with RA. Preclinical studies and phase II trials also suggested the potential of using fostamatinib for the treatment of ITP and B-cell lymphomas, by increasing platelet counts and inducing response rates, respectively. Fostamatinib is orally bioavailable and was well tolerated in phase I and II trials, with the most common side effect being gastrointestinal symptoms. At the time of publication, phase II trials for fostamatinib were ongoing in patients with RA, ITP and B-cell lymphomas. The Syk inhibitor appears to be a promising therapeutic for immunological diseases, but further data are required to establish the efficacy and long-term safety of the drug in humans.

MK2: a novel molecular target for anti-inflammatory therapy.

BACKGROUND: Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK. MK2 is stimulated in a wide range of inflammatory conditions and its catalytic activity is required for cytokine production, cell migration and is a potential drug target for inflammatory diseases. Disruption of MK2 leads to a reduction in TNF-alpha production. MK2-mediated pro-inflammatory cytokine production has been demonstrated in several inflammatory conditions where TNF-alpha plays a role. OBJECTIVE/METHODS: We discuss the development of specific MK2 inhibitors for the treatment of inflammatory diseases. RESULTS/CONCLUSION: Inhibition of the p38 MAPK pathway may have therapeutic uses for inflammatory diseases. However, blocking p38 MAPK activation in vivo is not advisable due to toxicity, significant off-target effects, and lack of oral bioavailability. This concern may be countered by the use of MK2 inhibitors that can dissect the pathways downstream of p38 without affecting additional cellular functions.

An improved zinc cocktail-mediated fluorescence polarization-based kinase assay for high-throughput screening of kinase inhibitors.

During the past few years, high-throughput screening (HTS) has provided a useful resource to researchers involved in the development of kinase inhibitors as a novel therapeutic modality. However, with all the choices among kinase assays, there is not yet a one-size-fits-all assay. Therefore, selection of a specific kinase assay is a daunting task. HTS assays should be homogeneous, cost effective, use nonradioactive reagents, generic and not time consuming. Here, we report an improved method of assaying protein kinase activity using a zinc cocktail in a fluorescence polarization-(FP) based format. Assay conditions were standardized manually and validated in a HTS format using a liquid handler. We validated this assay for both serine/threonine and tyrosine (receptor/nonreceptor) kinases. The results obtained in the HTS assay system were comparable to the commercially available fluorescence-based assay. We suggest that the reported assay is a cost-effective alternative to the IMAP-based generic kinase assay.

Spleen tyrosine kinase: a novel target for therapeutic intervention of rheumatoid arthritis.

BACKGROUND: In the last few years, significant progress has been made in understanding the pathogenic mechanisms and in defining the role of relevant cells and molecules in the pathophysiology of rheumatoid arthritis (RA). Various therapies, both biological (anti-TNF, anti-interleukins [e.g., IL-1]) and small molecule inhibitors have been explored for the treatment of RA. OBJECTIVE: To date, no single signaling pathway inhibitor as wide acting as the corticosteroids, is known. However, treatment with corticosteroids is also associated with allied side effects. Despite a lot of efforts in the category of small molecule inhibitors, no inhibitor is available to deal with RA at both fronts (inflammation and tissue damage), without causing immense side effects. METHOD: This present review explores the role of spleen tyrosine kinase (Syk) in the pathogenesis of RA and also discusses how it may meet the present day therapeutic requirements for the treatment of RA. This review gives an in-depth discussion on the role of Syk signaling in RA, the possibilities of using Syk as a target and also discusses the possible side effects that could be associated with its inhibition. CONCLUSION: We propose Syk inhibition as a potential therapeutic approach for the treatment of RA.

Development of a cell death-based method for the screening of nuclear factor-kappaB inhibitors.

Nuclear factor kappa B (NF-kappaB) plays a significant role in immunity and inflammation and represents a first choice as pharmacological target for anti-inflammatory therapy. However, research in this field has been hampered by the fact that no convenient assay suitable for large-scale screening procedures is available. The present study provides a cell death-based assay method for screening of nuclear factor-kappaB inhibitors. In this study, we observed that four distinct pharmacologic inhibitors of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), N-tosyl-L-lysyl chloromethyl ketone (TPCK), genistein and BAY11-7082, resulted in the cell death of murine macrophages, J774A.1. DNA-binding experiments showed that lethal doses were consistent with those required for NF-kappaB inhibition. DNA fragmentation analysis showed that cell death is apoptotic in nature. Further studies suggested that NF-kappaB inhibitors induced apoptosis is independent of the involvement of other markers of cell death such as caspases and p38 MAP (Mitogen activated protein) kinase. From this study, we conclude that NF-kappaB activation may represent an important survival mechanism in macrophages. This study also provides a new cell-based screening method, as any compound that will inhibit NF-kappaB activity will result in the death of macrophages.

Identification and characterization of RHOA-interacting proteins in bovine spermatozoa.

In somatic cells, RHOA mediates actin dynamics through a GNA13-mediated signaling cascade involving RHO kinase (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be negatively regulated by protein kinase A (PRKA), and it interacts with members of the A-kinase anchoring (AKAP) family via intermediary proteins. In spermatozoa, actin polymerization precedes the acrosome reaction, which is necessary for normal fertility. The present study was undertaken to determine whether the GNA13-mediated RHOA signaling pathway may be involved in acrosome reaction in bovine caudal sperm, and whether AKAPs may be involved in its targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and cofilin were all detected by Western blot in bovine caudal sperm. Overlay, immunoprecipitation, and subsequent mass spectrometry analysis identified several RHOA-interacting proteins, including proacrosin, angiotensin-converting enzyme, tubulin, aldolase C, and AKAP4. Using overlay and pulldown techniques, we demonstrate that phosphorylation of AKAP3 increases its interaction with the RHOA-interacting proteins PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). Varying calcium concentrations in pulldown assays did not significantly alter binding to R2D2 proteins. These data suggest that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK-cofilin pathway is present in bovine spermatozoa, that RHOA interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and ROPN1 is regulated by phosphorylation in vitro.

Enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) in Escherichia coli.

The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme.

Biomarkers of leukocyte traffic and activation in the vaginal mucosa.

Development of novel vaginal spermicides and anti-human immunodeficiency virus (HIV) microbicides requires careful assessment of their potential to recruit and activate CD4+ HIV-1 host cells in the female genital tract mucosa, two events that facilitate HIV-1 infection. Leukocyte traffic and activation are mediated by proinflammatory cytokines and chemokines, e.g. interleukin (IL)-1, IL-6 and IL-8, which have been detected in vaginal secretions in association with epithelial damage and infections. These proinflammatory mediators, however, have bidirectional, destructive as well as beneficial, effects on the mucosal barrier, and may be counterbalanced by endogenous inhibitors. Here we propose additional biomarkers for the evaluation of compound-induced cervicovaginal mucosal inflammation. Displaying different temporal patterns of detection, the levels of soluble E-selectin, vascular adhesion molecule-1, CD14 and myeloperoxidase in vaginal secretions reflected the mucosal leukocyte reaction to proinflammatory compounds being evaluated for safety in an improved rabbit vaginal irritation model. These biomarkers, which were also detected in human vaginal secretions, may be used to enhance the characterization of mucosal safety of vaginally applied compounds, both in animal as well as clinical studies.

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa.

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.

Role of energy metabolism in the pregnancy interceptive action of Ferula assafoetida and Melia azedarach extracts in rat.

Ethanolic extract of Ferula assafoetida and chloroform fraction of Melia azedarach, both devoid of estrogenic activity, were examined for their pregnancy interceptive property. Treatment of rats from days 1 to 7 of pregnancy with either of the plant extracts resulted in pregnancy failure in about 65-85% of the animals. The possible role of energy metabolism in the antifertility action was investigated by measuring changes in activities of the key enzymes of carbohydrate metabolism in uterus on day 7 of pregnancy. It was observed that on the day 7 of pregnancy, one key enzyme of glycolytic pathway (phosphofructokinase) was significantly reduced in the uteri of treated rats as compared to controls. Hexosemonophosphate pathway also appeared to be sensitive to treatment with the plant extracts and showed an inhibitory effect on the enzyme activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Oxidative energy metabolism through tricarboxylic acid cycle, which is considered to be the main source of energy to the uterus at this stage, was maximally affected by the treatment with several enzymes showing significant inhibition. The two plant materials appeared to interrupt the latter metabolic pathway more significantly. It is thus concluded that plants lacking phytoestrogens may intercept pregnancy by their ability to disrupt energy metabolism in rat uterus during implantation, especially the oxidative pathway.