Optimization of Compound Plate Preparation to Address Precipitation Issue in Mammalian A549 Cytotoxicity Assay.

This study illustrates the optimization of low-volume dispensing on a liquid handling system (LHS) to overcome the precipitation of compounds in the mammalian cytotoxicity assay with low dimethyl sulfoxide (DMSO) tolerance. All compounds at AstraZeneca Bangalore are tested in the mammalian cytotoxicity assay. In order to maintain the DMSO levels, serially diluted plates were prepared in DMSO/water. It was observed that some of the compounds precipitated. The IC50 data for such compounds were therefore erratic. To circumvent the problem of compound precipitation, the LHS was optimized to dispense low volumes (<1 µL). The plates were serially diluted using neat DMSO. Since the dilution was done using neat DMSO, there were no issues with precipitation. The serially diluted sample (0.5 µL) from the plate was stamped onto the assay plate to give the desired DMSO concentration. No significant differences in IC50 data were observed for 1 µL dispenses made from DMSO/water and 0.5 µL dispenses from neat DMSO for the samples with no precipitation issues. These data therefore gave us the confidence to switch over to 0.5 µL dispenses for the cytotoxicity assay to address the precipitation issue. However, precipitation of samples in the assay buffer is beyond the scope of this discussion.

Aminoazabenzimidazoles, a novel class of orally active antimalarial agents.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

Thiazolopyridine ureas as novel antitubercular agents acting through inhibition of DNA Gyrase B.

A pharmacophore-based search led to the identification of thiazolopyridine ureas as a novel scaffold with antitubercular activity acting through inhibition of DNA Gyrase B (GyrB) ATPase. Evaluation of the binding mode of thiazolopyridines in a Mycobacterium tuberculosis (Mtb) GyrB homology model prompted exploration of the side chains at the thiazolopyridine ring C-5 position to access the ribose/solvent pocket. Potent compounds with GyrB IC50 ≤ 1 nM and Mtb MIC ≤ 0.1 µM were obtained with certain combinations of side chains at the C-5 position and heterocycles at the C-6 position of the thiazolopyridine core. Substitutions at C-5 also enabled optimization of the physicochemical properties. Representative compounds were cocrystallized with Streptococcus pneumoniae (Spn) ParE; these confirmed the binding modes predicted by the homology model. The target link to GyrB was confirmed by genetic mapping of the mutations conferring resistance to thiazolopyridine ureas. The compounds are bactericidal in vitro and efficacious in vivo in an acute murine model of tuberculosis.

Assessment of microsatellite instability in colorectal carcinoma at an Indian center.

AIM: The purpose of the present study was to evaluate the frequency of microsatellite instability (MSI) in colorectal cancers in an Indian cohort. MATERIALS AND METHODS: Paraffin embedded tissue samples of colorectal cancers from 46 patients were assessed for mismatch repair protein expression (hMLH1 and hMSH2) by immunohistochemistry. Subsequently, MSI analysis was done after PCR amplification of five Bethesda markers. RESULTS: Amongst 46 cases studied, only 5 patients (10.8%) showed MSI. Out of these, two (4.3%) had high microsatellite instability (MSI-H) and three (6.5%) showed low microsatellite instability (MSI-L). Out of 46 cases, 41 were microsatellite stable (MSS). In the 46 cases tested by immunohistochemistry, 7 (15.7%) showed the absence of hMLH1 and 1 case showed the absence of hMSH2. CONCLUSION: Our study indicates a similar rate of incidence of MSI in colorectal cancers in the Indian cohort compared to the West (10-15%) despite lower incidence of colorectal cancers and predominance of rectosigmoid tumors in the Indian population.

High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog.

RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (gamma-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of gamma-AmNS-PPi, which has higher intrinsic fluorescence than (gamma-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 microM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC(50) below 100 microM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.