Identification and synthesis of novel inhibitors of mycobacterium ATP synthase.

A non-diaryl quinoline scaffold 6,7-dihydropyrazolo[1,5-a]pyrazin-4-one was identified by screening of diverse set of compounds against M. smegmatis ATP synthase. Herein, we disclose our efforts to develop the structure activity relationship against Mycobacterium tuberculosis (Mtb.H37Rv strain) around the identified hit 1. A scaffold hopping approach was used to identify compounds 14a, 14b and 24a with improved activity against MTb.H37Rv.

Pharmacology of a novel, orally active PDE4 inhibitor.

BACKGROUND: Intracellular cyclic adenosine monophosphate (cAMP) in inflammatory cells and airway smooth muscle is critical to the modulation of inflammatory response generation. Phosphodiesterase 4 (PDE4), an enzyme that catalyzes cAMP degradation, is therefore being actively explored as a molecular target for the treatment of airway inflammation, particularly asthma and chronic obstructive pulmonary disease. The field has undergone major advances in optimizing generation of compounds with a safe therapeutic margin; however, most PDE4 inhibitors tested so far have unacceptable side effects, particularly nausea and vomiting. METHODS: We evaluated NIS-62949 in a wide range of in vitro and ex vivo cell-based assays to ascertain its anti-inflammatory potential. The compound was evaluated in murine models of lipopolysaccharide-induced endotoxemia and pulmonary neutrophilia. Parameters of airway inflammation, airway hyperreactivity and bronchoconstriction were evaluated in a guinea pig model of antigen-induced allergy. In order to assess the emetic potential, the compound was evaluated biochemically for binding to high-affinity rolipram-binding site. Subsequently, the compound was tested in a surrogate model for emesis, and the results obtained were correlated directly to tests conducted in a Beagle dog model. RESULTS: NIS-62949 is a potent, highly selective PDE4 inhibitor. The compound demonstrated potent ability to inhibit tumor necrosis factor-alpha release from human peripheral blood mononuclear cells, lymphocyte proliferation and cytokine production. The in vitro profile of NIS-62949 prompted further evaluation of the compound in vivo and the compound was found to be comparable to roflumilast in several experimental models of pulmonary inflammation. Importantly, NIS-62949 displayed a safer profile compared to roflumilast. CONCLUSIONS: Our results report the development of a promising, novel PDE4 inhibitor, NIS-62949, with a wider therapeutic window as compared to second-generation PDE4 inhibitors such as roflumilast.

Functional screening of adrenergic receptors by measuring intracellular calcium using the FlexStation scanning fluorimeter.

In this study we test whether functional screening of compounds to adrenergic G protein-coupled receptors (GPCRs) would provide data that correlated significantly with radiolabeled binding data, thereby permitting researchers to replace expensive radioligand-binding experiments with non-radioactive screening. An increase in intracellular calcium levels represents an important second messenger signal for several recombinant GPCRs. In this study, we describe the affinities of three alpha adrenoceptor antagonists (terazosin, tamsulosin and alfuzosin), determined by monitoring the changes in intracellular calcium levels and comparing them with their radioligand-binding affinities. In addition to determining the functional affinities of the three alpha adrenoceptor antagonists, we evaluate their binding at two alpha adrenoceptor subtypes and optimized the assay for high-throughput screening.

Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer.

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are "gold standard" because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the "mix & measure" assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca+2 measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compared the IC50 values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca+2 mobilization assays can be an alternate to radioligand binding assays.

RBx 6198: a novel alpha1-adrenoceptor antagonist for the treatment of benign prostatic hyperplasia.

The present study, investigates the effect of RBx 6198, 2-{3-[4-(2-Isopropoxy-phenyl)-piperazin-1-yl]-propyl}-3a, 4, 7, 7a-tetrahydro-isoindole-1, 3,-dione, a novel alpha(1)-adrenoceptor antagonist, in both in vitro and in vivo test systems. RBx 6198 is a potent (nanomolar affinity) alpha(1A)-adrenoceptor antagonist with demonstrable uroselectivity in anaesthesized dog model. In radioligand binding studies using human recombinant receptors, RBx 6198 exhibited high selectivity (approximately 50 fold) for the alpha(1A)-adrenoceptor subtype as compared to alpha(1B)-adrenoceptor subtype. In order to assess tissue selectivity, the antagonistic effect of RBx 6198 on the phenylephrine induced contractile response of isolated rat prostate, spleen and aorta was characterized. RBx 6198 was 8 fold more potent in inhibiting phenylephrine-evoked contractions of isolated tissues compared to tamsulosin. However, the compound was non-selective for alpha(1A) vs. alpha(1D)-adrenoceptor like tamsulosin. In anaesthetized beagle dogs RBx 6198 suppressed the intraurethral pressure response to phenylephrine to a greater extent than the mean arterial pressure response thereby demonstrating uroselectivity consistent with in vitro binding and functional data. RBx 6198 was 6.4 fold more uroselective as compared to tamsulosin after i.v. route dose administration. Taken together all results from preclinical studies, it is suggested that RBx 6198 is a novel alpha(1)-adrenoceptor antagonist that exhibited improved pharmacological profile over tamsulosin in both in vitro and in vivo.

Optimization and validation of a reporter gene assay for screening of phosphodiesterase inhibitors in a high throughput system.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. Inhibitors of PDEs allow the elevation of cAMP and cGMP, which leads to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP. We have validated the manually developed reporter gene assay in a high-throughput screening format that allows for fast and cost-effective identification of potential inhibitors of PDE4 isozymes. The assay is sensitive and robust, with a Z' value of >0.5. The assay is also amenable to 384-well format.

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.

Molecular cloning, stable expression and cellular localization of human alpha1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of alpha1D -adrenergic receptor.

The cDNAs encoding for three subtypes of adrenergic receptors, alpha1A-, alpha1B- and alpha1D-ARs, were cloned and expressed in HEK 293 cells. Expression of alpha1A- and alpha1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of alpha1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of alpha1A- and alpha1B-ARs was primarily localized on the cell membrane whereas expression of alpha1D-AR was predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing alpha1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of alpha1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.

Benign prostatic hyperplasia: an insight into current investigational medical therapies.

Benign prostatic hyperplasia (BPH) is a leading disorder of the elderly male population that is characterised by a progressive enlargement of prostatic tissue, resulting in obstruction of the proximal urethra and causing urinary flow disturbances. The pathophysiology of BPH associated with lower urinary tract symptoms is characterised by increased adrenergic tone (dynamic component) leading to smooth muscle contraction and prostatic overgrowth due to androgenic stimulation (static component); therefore, the therapeutic armamentarium of BPH can be broadly divided into antiadrenergic and antiandrogenic approaches. alpha1-Adrenoceptor antagonists and 5alpha-reductase inhibitors are well-established representatives of the two categories, respectively. Other antiandrogenic approaches involve gonadotropin-releasing hormone agonists and antagonists for the treatment of prostate hyperplasia. Apart from these approaches, new approaches with novel targets are emerging. The advent of new therapies is, however, more oriented towards the static component. These involve metabolic factors (hexokinase inhibitor), growth factors (vitamin D3 analogues), oxytocin antagonists and gonadotropin-releasing hormone Gi agonist-based therapies. Gene therapy and photodynamic therapies are other emerging therapies for relieving symptoms in BPH patients. With the initial success of upcoming targets, the unmet need to develop an efficacious and relatively safe therapeutic modality is discussed. Nevertheless, their long-term safety and efficacy needs to be evaluated in large-scale clinical trials. The future also belongs to combination therapies to combat both dynamic and static disease components and for extended indications such as micturition disorder and non-bacterial prostatitis.

WITHDRAWN: Derivatives of cis-1,2,3,6-tetrahydrophthalimide for the treatment of benign prostatic hyperplasia.

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