Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (βS531L) containing a mutation (β'V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifR E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.

To explore the apoptotic and antitumor activities of metallo-salens, the authors have synthesized several Fe(III)-salen and salphen complexes and analyzed their effects on human cancer and noncancer cells. Their results demonstrated that Fe(III)-salen and salphen complexes affect cell viability and induce nuclear fragmentation and apoptosis in breast cancer (MCF7) cells. The IC(50) values for the active metallo-salen complexes ranged between 0.3 and 22 µM in MCF7 cells. Biochemically active Fe(III)-salen and salphen complexes induced caspase-3/7 activation and release of cytochrome c from the mitochondria to cytosol, suggesting the involvement of the mitochondrial pathway of apoptosis. Comparison of IC(50) values toward 3 different cell lines demonstrated that selected Fe(III)-salen complexes induce tumor cell-selective apoptosis in cultured cells. Overall, the studies demonstrated that Fe(III)-salen and salphen complexes induced efficient apoptosis in cultured human cells. The nature of the substituents and the bridging spacer between diamino groups play critical roles in determining the apoptotic activities of Fe(III)-salen and salphen complexes.

There is a pressing need to develop rapid yet accurate screening assays for the identification of genotoxic liability and for early hazard assessment in drug discovery. The GADD45a-GFP human cell-based genotoxicity assay (GreenScreen HC) has been reformatted to test 12 compounds per 96-well microplate in a higher throughput, automated screening mode and the protocol applied to the analysis of 1266 diverse, pharmacologically active compounds. Testing from a fixed starting concentration of 100 AmicroM and over 3 serial dilutions, the hit rates for genotoxicity (7.3%) and cytotoxicity (33%) endpoints of the assay have been determined in a much wider chemical space than previously reported. The degree of interference from color, autofluorescence, and low solubility has also been assessed. The assay results have been compared to an in silico approach to genotoxicity assessment using Derek for Windows software. Where carcinogenicity data were available, GreenScreen HC demonstrated a higher specificity than in silico methods while identifying genotoxic species that were not highlighted for genotoxic liability in structure-activity relationship software. Higher throughput screening from a fixed, low concentration reduces sensitivity to less potent genotoxins, but the maintenance of the previously reported high specificity is essential in early hazard assessment where misclassification can lead to the needless rejection of potentially useful compounds in drug development.

The authors have characterized a set of cannabinoid CB(2) receptor ligands, including triaryl bis sulfone inverse agonists, in a cell-based receptor/beta-arrestin interaction assay (DiscoveRx PathHunter). The results were compared with results using a competitive ligand binding assay, and with effects on forskolin-stimulated cAMP levels (PerkinElmer LANCE). The authors show good correlation between the 3 assay systems tested, with the beta-arrestin protein complementation assay exhibiting a more robust signal than the cAMP assay for cannabinoid CB(2) agonists. Further assay validation shows that DiscoveRx PathHunter HEK293 CB(2) beta-arrestin assay can be carried out from cryopreserved cell suspensions, eliminating variations caused by the need for multiple cell pools during live cell screening campaigns. These results, and the authors' results evaluating a test set of random library compounds, validate the use of ligand-induced interaction between the human cannabinoid CB(2) receptor and beta-arrestin as an appropriate and valuable screening platform for compounds specific for the cannabinoid CB(2) receptor.

Epidermal growth factor receptor (EGFR) is overexpressed or mutated in a high percentage of tumors. EGFR has long been considered a promising target for cancer diagnostic and therapeutic applications. However, monoclonal antibodies and other large antibody constructs diffuse into tumors slowly, limiting their efficacy. To develop lower molecular weight probes for EGFR and other tumor cell receptors, the authors immunized a llama with the extracellular domains (ECDs) of EGFR and an oncogenic mutant receptor, EGFRvIII, and with extracts of tumor cell lines. From the immune repertoire of the llama, the authors constructed a heavy chain variable domain (VHH domain)-phage library. At approximately 16 kDa, the VHH domain is a tenth of the size of a monoclonal antibody and is the smallest antibody fragment that retains specificity. By affinity selection from this library, the authors isolated many VHH domains with specificity for EGFR. The VHH domains bind to whole cells expressing the receptor but not to control cells lacking the receptor and can immunoprecipitate EGFR from cell lysates. Some VHH domains have cross-specificity with existing anti-EGFR monoclonal antibodies and have reasonably high (nM) affinities. The llama-VHH domain library is also potentially a rich source of targeting agents directed toward other tumor cell receptors.

Fluorescence lifetime is an intrinsic parameter describing the fluorescence process. Changes in the fluorophore's physicochemical environment can lead to changes in the fluorescence lifetime. When used as the readout in biological assays, it is thought to deliver superior results to conventional optical readouts. Hence it has the potential to replace readout technologies currently established in drug discovery such as absorption, luminescence or fluorescence intensity. Here we report the development of an activity assay for human kallikrein 7, a serine protease involved in skin diseases. As a probe, we have selected a blue-fluorescent acridone dye, featuring a remarkably long lifetime that can be quenched by either of the 2 natural amino acids, tyrosine and tryptophan. Incorporating this probe and 1 of the quenching amino acids on either side of the scissile bond of the substrate peptide enables us to monitor the enzymatic activity by quantifying the increase in the fluorescence lifetime signal. A systematic investigation of substrate structures has led to a homogenous, microplate-based, compound profiling assay that yields inhibitory constants down into the single-digit nanomolar range. This type of assay has now been added to our standard portfolio of screening techniques, and is routinely used for compound profiling.

Kinases represent attractive targets for drug discovery. Eight small-molecule kinase inhibitors are currently marketed in the area of oncology, and numerous others are in clinical trials. Characterization of the selectivity profiles of these compounds is important to target appropriate patient populations and to reduce the potential of toxicity due to off-target effects. The authors describe the development, validation, and utilization of a biochemical kinase assay panel for the selectivity profiling of inhibitors. The panel was developed as 29 radiometric Flashplate assays, and then an initial 13 were transitioned to a nonradiometric Caliper mobility shift assay format. Generation of high-quality data from the panel is detailed along with a comparison of the assay formats. Both assay technologies were found to be suitable for panel screening, but mobility shift assays yielded higher data quality. The selectivity data generated here should be useful in computational modeling and help facilitate, in conjunction with sequence and structural information, the rational design of inhibitors with well-defined selectivity profiles.

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric alphabetagamma GABA-A receptors (GABA(A)Rs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing alpha1beta1gamma1 and alpha1beta1gamma2 GABA(A)Rs in HEK293 cells. Expression efficiencies resulting from 5 different transfection strategies were assessed by flow cytometry and pharmacological analysis using an anion-sensitive yellow fluorescent protein-based assay. PolyFect and Effectene, employed according to the manufacturers' instructions, conferred the strongest and most reliable expression of trimeric alphabetagamma GABA(A)Rs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of gamma1- and gamma2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABA(A)Rs provides an effective means of discovering novel GABA(A)R modulators for use as therapeutic lead compounds and pharmacological probes.

The mouse ortholog of the human bile salt export pump (BSEP) transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the transporter function. The transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular transport assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep transports TCDC at a high rate that can be sensitively detected in the ATPase assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate transport was tested in both vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC(50) values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay.

Single nucleotide polymorphisms (SNPs) have recently replaced microsatellites as the genetic markers of choice in linkage analysis, primarily because they are more abundant and the genotypes more amenable for automatic calling. One of the most recently launched linkage mapping sets (LMS) is the Applied Biosystems Human LMS 4K, which is a genome-wide linkage set based on the SNPlex technology and the use of clustered SNPs. In this article the authors report on their experience with this set and the associated genotyping software GeneMapper version 4.0, which they have used for linkage analyses in 17 moderate to large families with assumed monogenic disease. For comparison of methods, they also performed a genome-wide linkage analysis in 1 of the 17 families using the Affymetrix GeneChip Human Mapping 10K 2.0 array. The conclusion is that both methods performed technically well, with high call rates and comparable and low rates of Mendelian inconsistencies. However, genotyping is less automated in GeneMapper version 4.0 than in the Affymetrix software and thus more time consuming.