Novartis weighs up label-free HTS for drug discovery

HTS techniques have become an important part of the drug discovery process allowing researchers to sift through massive libraries of compounds to find 'hits' that are active against a therapeutic target of interest.

This latest research, published as an early view article in the journal Analytical Chemistry by researchers from the Discovery Technologies and Diabetes and Metabolism divisions of Novartis' Institute for Biomedical Research, has assessed the usage of MS-HTS for large compound library screening.

Most functional activity-based programmes discover hits by detecting a change in the activity of a target enzyme in the presence of potential inhibitors or activators.

These changes in activity are commonly detected by using substrates that have been labelled with either radioisotopes or fluorescent probes.

According to the authors, labelled substrates do not always function exactly the same as native substrates and the developing methods to incorporate these labels can add significantly to method-development times.

MS can detect the products of enzymatic reactions without the need to label the substrates and the authors believe that this could potentially improve data quality achieved using the screens.

However, according to the authors, until recently throughput has been the major limiting factor in using MS for HTS and while using multiple instruments could be used to solve the problem, this would prove expensive.

"One contributing factor to the lack of large-scale [MS-HTS] studies is that instrumentation allowing unattended, fully automated analysis of thousands of samples per day with automated data analysis is not readily available," write the authors.

The researchers looked at three different MS-HTS techniques, four-way parallel multiplexed electrospray liquid chromatography (LC) tandem MS (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem MS (LC/MS/MS) and eight-way staggered flow injection MS/MS following 384-well plate solid phase extraction (SPE).

Each of these methods makes use of parallel autosamplers to enable more efficient use of the Waters Quattro Micro triple quadrupole MS instruments.

The techniques described in the paper allowed unattended analyses of screens of over 175,000 compounds, with a 384-well plate taking around 2 hours to analyse.

The researchers conducted two inhibitor screening campaigns using the techniques to show that they could be used reliably to detect inhibition of an (unnamed) enzyme by compounds in a library.

"In comparison to ultra-HTS methods (>100,000 wells per day), these MS methods are relatively slow (<10,000 wells per day), making single compound screening of more than 1m compounds impractical at this time," write the authors.

However, because the screens require no custom labelling of reagents or the development of assays to measure labelling success considerable time can be saved during the method development phase.

When combined with 'multiple compound per well' screens that can increase analysis times by a factor of five, the MS screens compare favourably to traditional HTS methods.

"Although the MS analysis is relatively slow, overall timelines of MS-based screens from concept to hit list are similar to those of traditional HTS and ultra-HTS-based screens," conclude the authors.