X-ray Crystallography is Main Bottleneck
Michelle Browner, vp of discovery science and technology at Roche (Palo Alto, CA), discussed Roche's approach to optimizing lead discovery. Integrated, multidisciplinary, lead discovery teams combine medicinal chemistry, chemoinformatics, HTS, biochemical pharmacology, and DMPK.
These teams work closely with each therapeutic program team. They are responsible for generating "actives" (molecules with demonstrated activity against a target, as measured by the IC50; the primary output of screening assays), taking actives to hits (molecules with confirmed chemical structures and biological activity and with expanded characterization), and hits to leads (compounds with desired affinity, selectivity, and distribution properties).
The first step involves multiple lead generation by applying HTS, focused screening, and high throughput protein engineering (domain truncation and trimming, site-directed mutations, and purification tags and cleavage sites) in parallel.
To assess targets, Browner recommended using automated small-scale protein expression in E. coli or baculovirus to determine which proteins are relatively easy to express and purify and which to take forward into large-scale expression for HTS assay development and crystallography.
Random screening of a large compound library to find novel chemical scaffolds should proceed in parallel with focused screens, in which library design is based on the structural characteristics of the target and which will yield higher hit rates.
Roche then applies Spotfire DecisionSite (Spotfire; Somerville, MA) to visualize the screening data, identify clusters of active compounds ("nearest neighbor" determinations) and their common substructures, and select interesting clusters based on percent inhibition and molecular properties such as molecular weight.
These clusters yield the actives selected for follow-up and profiling, which includes IC50 determination, structure confirmation, and purity measurements. The actives selected are also subjected to evaluation with Rodin, Roche's global web-based query system that incorporates screening-derived biological, chemical, and structural information.
Using this query system researchers can determine whether any of the actives are "frequent hitters" and should be excluded from the pool.
The actives are then prioritized into "hits" based on medicinal chemistry evaluations and the use of secondary, lower throughput assays. Hit profiling involves activity confirmation in focused screens and biophysical characterization.
Evidence of nonspecific binding leads to elimination of a hit from further consideration. Roche also evaluates the binding kinetics of each hit. At the same time, high throughput protein crystallization is initiated. Applying automated crystallography early on in a parallel timeline allows for hit prioritization using protein structure information derived from x-ray crystallography of bound structures.
"The bottleneck in hit profiling is finding crystals," said Browner. As the co-crystallization studies progress, in vitro DMPK can begin, together with efforts to assess novelty and SAR studies. The outcome of this process is a set of leads that can be passed on to the medicinal chemists.
Browner identified three current gaps in the hit-to-lead process:
Time: about a nine-month timeframe to progress from HTS to profiled hits;
Resources: limited resources and the need for increased automation to drive parallel processing; and
Throughput: limited throughput of secondary assays.