Synthesis and antiplasmodial and antimycobacterial evaluation of new nitroimidazole and nitroimidazooxazine derivatives.

The synthesis and antiplasmodial and antimycobacterial evaluation of two new series of nitroimidazole and nitroimidazooxazine derivatives is described. The majority of these compounds, especially hybrids 9d, 9f, and 14b, exhibited potent activity against the chloroquine-resistant K1 strain of Plasmodium falciparum. Furthermore, a notable number from the tetrazole series were significantly more active against M. tuberculosis than kanamycin, a standard TB drug.

Ion channel screening plates: design, construction, and maintenance.

Ion channels have provided a diverse set of therapeutic targets across all areas of the pharmaceutical industry. Many companies are pursuing this unique class of targets for areas of unmet medical need such as neuropathic and inflammatory pains. In the past, focused library screening sets had been designed for CNS and kinase targets. Our investigations were aimed at creating a similar dynamic screening set enriched for compounds targeting ion channels to aid screening efforts of this important class of targets. The key advantages of this approach for ion channel targets would be: (1) to identify tool compounds for novel targets and assist in assay validation, (2) to serve as a focused screen for non-384-well adaptable targets, and (3) to jump start a particular program, that is, catch-up to competition for validated, well-known targets.

High-throughput screening for Kv1.3 channel blockers using an improved FLIPR-based membrane-potential assay.

Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K(+) channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K(+) channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay conditions and designed a simple assay format. After an HTS campaign using this assay, various chemical series of Kv1.3 channel blockers have been identified and confirmed by the automated electrophysiological IonWorks assay. The correlation in dose response between FLIPR and IonWorks was established by biophysical modeling and experimental data. After characterization using patch-clamp recording, both use-dependent and use-independent compounds were identified. Some compounds possess nanomolar potency, indicating that the FLIPR assay is effective for successfully identifying K(+) channel blockers as novel drug candidates.

Trace LC/MS/MS quantitation of 17beta-estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain.

A sensitive LC/MS/MS method has been developed by derivatization of 17beta-estradiol (E2) with dansyl chloride to quantitate 17beta-E2 in female rat serum. The use of E2-d(5) minimized interferences from endogenous 17beta-E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 microl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17beta-E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic-pituitary-ovarian (HPO) axis. Variations in 17beta-E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing.

Development of an early biomarker for the ovarian liability of selective estrogen receptor modulators in rats.

Selective estrogen receptor modulators (SERMs) have the potential to treat estrogen sensitive diseases such as uterine leiomyoma and endometriosis, which are prevalent in reproductive age women. However, SERMs also increase the risk of developing ovarian cysts in this population, a phenomenon that is not seen in postmenopausal women. It is believed that current SERMs partially block estradiol's ability to downregulate gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus thereby interfering with estradiol's negative feedback, leading to increased ovarian stimulation by gonadotropins, and cyst formation. It has been postulated that a SERM with poor brain exposure would have less negative effect on the HPO axis, therefore reducing the risk of developing ovarian cysts. In order to test this hypothesis, we identified an early marker of SERM-dependent ovarian effects: upregulation of Cyp17a1 mRNA. SERMs known to cause ovarian cysts upregulate Cyp17a1 after only 4 days of dosing and suppression of the HPO axis prevented this regulation, indicating that ovarian expression of Cyp17a1 was secondary to SERM's effect on the brain. We then characterized three SERMs with similar binding affinity and antagonist effects on the uterus for their relative brain/plasma exposure and ovarian effects. We found that the degree of brain exposure correlated very well with Cyp17a1 expression.

MNAR plays an important role in ERa activation of Src/MAPK and PI3K/Akt signaling pathways.

Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.

Direct analysis in real time for reaction monitoring in drug discovery.

Direct analysis in real time (DART) is a novel ionization technique that provides for the rapid ionization of small molecules under ambient conditions. In this study, several commercially available drugs as well as actual compounds from drug discovery research were examined by LC/UV/ESI-MS and DART interfaced to a quadrupole mass spectrometer. For most compounds, the molecular ions observed by ESI-MS were observed by DART/MS. DART/MS was also studied as a means to quickly monitor synthetic organic reactions and to obtain nearly instantaneous molecular weight confirmations of final products in drug discovery. For simple, synthetic organic transformations, the trends in the intensities of the mass spectral signals for the reactant and product obtained by DART/MS scaled closely with those of the diode array or the total ion chromatogram obtained by LC/UV/ESI-MS. In summary, DART is a new tool that complements electrospray ionization for the rapid ionization and subsequent mass spectral analysis of compounds in drug discovery.

Rb+ efflux through functional activation of cardiac KCNQ1/minK channels by the benzodiazepine R-L3 (L-364,373).

The slow delayed rectifier K+ current, Iks, encoded by KCNQ1 (KvLQT1)/KCNE1 (mink) genes, contributes to cardiac action potential repolarization and determines the heartbeat rate. Mutations in either KCNQ1 or KCNE1 that reduce Iks cause long-QT syndrome (LQTS), a disorder of ventricular repolarization that results in cardiac arrhythmia and sudden death. A well-recognized potential treatment for LQTS caused by reduction of Iks is to enhance functional activation of cardiac KCNQ1/KCNE1 channels. In the present study, we generated a stable Chinese hamster ovary cell line that expresses KCNQ1/KCNE1 channels confirmed by electrophysiology. Using a pharmacological tool compound R-L3 (L-364,373 [(3-R)-1,3-dihydro-5-(2-fluorophenyl)-3-(1H-indol- 3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one]), which activates KCNQ1/mink channels, we then developed and validated a non-radioactive rubidium (Rb+) efflux assay that directly measures the functional activity of KCNQ1/KCNE1 channels by atomic absorption spectroscopy. Our results show that the validated Rb+ efflux assay can be used for screening of KCNQ1/KCNE1 openers that potentially treat LQTS in both inherited and acquired forms. In addition, the assay also can be used for evaluation of possible long-QT liability during cardiac selectivity of new chemical entities.