In vitro ADME profiling using high-throughput rapidfire mass spectrometry: cytochrome p450 inhibition and metabolic stability assays.

Early assessment of absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates has become an essential component of modern drug discovery. ADME characterization is important in identifying compounds early that are likely to fail in later clinical development because of suboptimal pharmacokinetic properties or undesirable drug-drug interactions. Proper utilization of ADME results, meanwhile, can prioritize candidates that are more likely to have good pharmacokinetic properties and also minimize potential drug-drug interactions. By integrating a RapidFire system with an API4000 mass spectrometer (RF-MS), we have established a high-throughput capability to profile compounds (>100 compounds/wk) in a panel of ADME assays in parallel with biochemical and cellular characterizations. Cytochrome P450 inhibition and time-dependent inhibition assays and microsomal stability assays were developed and fully optimized on the system. Compared with the classic liquid chromatography-mass spectrometry method, the RF-MS system generates consistent data with approximately 20-fold increase in throughput. The lack of chromatographic separation of compounds, substrates, and metabolites can complicate data interpretation, but this occurs in a small number of cases that are readily identifiable. Overall, this system has enabled a real-time and quantitative measurement of a large number of ADME samples, providing a rapid evaluation of clinically important drug-drug interaction potential and drug metabolic stability.

Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.