move handlin internal at the uid handle eventually rules-base( to aid the I ple, as line the assay o prescribed with of error requires with time, the necesa mated ous
2.17. Images of typical turnkey automated screening robots that use a n articulated arm to around the screening system. The robot is placing plates into two different liquid g devices. Top left is a Plus and top right is a Multidrop. This system was built
by Bristol Myers Squibb engineers.
dated times around the various incubators, and detectors, and to waste. A limited amount of
artificial intelligence can be used quality of the operation. For
are generated by the detector, on- can be used to monitor drift in whether certain wells fail to meet quality control parameters, as seen ed tips on a dispenser. These types
an operator.
these integrated robotic systems management commitment money, and a willingness to develop
skill sets. A stable, fully
platform does offer continu- a consistency of process that
can be verified, automated audit trial of the samples that have been tested, and safety (for further detail see Ref. 86).
Not all laboratories have the resources to build fully integrated screening platforms and support them. Additionally, not all assays can be modified to work on an automated plat- form. For example, a particular detector may not be available in a format that can be inte- grated. Most screening laboratories will use workstation approaches in addition to fully automated platforms to enable assay flexibil- ity. Unlike the automated platforms, where plates are processed in a serial manner, plates are together, "a stack," in worksta- tion approaches. Workstation approaches re- places the robot with a human, and as long as
High-Throughput Screening for Lead Discover
the number of microtiter plates processed is acceptable, this often works well. The same quality control is incorporated into the work- station process, and from our experience, the workstation data are comparable with that generated by a robot. One real advantage of a fully automated platform is where there is a need to have accurate incubation times, for example, in a kinetic assay.
4.2 Miniaturization of Screening Assays
The increasing operating costs of HTS labora- tories have driven a strong interest in imple- menting more cost-effective ways of carrying out high-throughput screening campaigns. Miniaturizing the plate format is one of the major technology solutions. There has been a evolution from the glass test tube and plastic Eppendorf tube into microtiter plates containing ever-increasing well densi- ties.
The first tangible step along the miniatur- ization route was the introduction of the well microtiter plate that replaced the individ- ual tube The 96-well plate became the standard workhorse in academic and indus- trial laboratories over the last 20 " How- ever, ever increasing demands to expand test- ing capacity and improve process efficiency while simultaneously reducing costs have pushed HTS laboratories into using 384-well plates and beyond (Fig. 2.18).
A screening organization that runs 50 screens a year, each testing a 500,000 com- pound deck with average reagent and
ware that costs totals $5 million, excluding waste management costs. This sce- nario in a 96-well plate format generates 260,000 plates of plastic waste per year. A typ- ical assay volume in the 96-well plate is
200 and by reducing this to around 5-10 reagent costs are reduced. Additionally, smaller amounts of compounds are needed for the assay.
In the late HTS laboratories adopted the 384-well plate as standard, allow- ing a fourfold increase in well density and in- creased screening capacity (87). Instrumenta- tion companies re-invested in designing or adapting liquid handlers, detection systems, and automation to fit the new 384-well plate.
Figure 2.18. There are many different types of m
crotiter plates that are used in miniaturized for HTS. (a) 96-well plate (100 assays), (b) well plate (25 well assays), 1536-well plate
well assays), and 3456-well plate we assays)
The vast majority of assays have readil miniaturized down to 20-50 volumes. Th minimum practical volume of 20 for th new 384-well plates was defined by the we shape and the need to produce an even layer liquid at the bottom of the plate.
Even with the 384-well plate, there ha been pressure to reduce volumes even
The 1536-well plate is emerging as the poter tial next step, with square wells that allow working volume of 5-10 One advantage the 1536-well plate in absorbance-based says is volume reduction while maintainin the path length. Additionally, low-volum 384-well microtiter plates are now
cially available. The 1 assay is also no7
available in the 1536- (Corning Costar Corp Cambridge, MA and Evotec
Germany) and the 3456-well microtiter plat
(Aurora San CA) (50). In
little over 5 years, we have witnessed a fold reduction in assay volume and a 36-fol increase in the well density. The discussio over well density still causes many debates screening discussion groups (87) and eve higher well densities have been proposed, e.g 9600-well plate (88).
The move to higher well densities an lower assay volumes has presented
challenges to instrumentation
First, there is the need to detect the results a particular assay. For example, in the 96-we scintillation proximity assays, the scintillatio counter photomultipliers are positioned abov
References
each well to measure the emitted light. Using a mask, these machines were adapted to read 384-well microtiter plates. The disadvantage was that it then took four times as to read a plate. A new solution needed to found. Imaging technology, using charged-coupled devices, provided the answer
Amersham Pharmacia,
sham, UK, and CLIPR Molecular Devices, Alto, Imagers take the same time to read a 96-well, 384-well, or 1536-well microtiter plate. A 500,000-compound high- throughput screen using a filter binding assay format consumes approximately 10,400 well microtiter and filter-binding plates. For the format using miniaturized plates, 1536 wells per plate, 325 plates are used. Additionally, it takes approximately 10 min to measure the light from a 96-well plate, and therefore, total time taken to generate the data would be 36 days in a single plate-based scintillation counter. For the 1536-well assay using imaging technology, the reading time is reduced to 27 h. The overall gain in assay effi- ciency is dramatic. Imagers are now available for fluorescence, time-resolved fluorescence and for measuring light emission.
Another engineering challenge was to dis- pense volumes in the 20 volume range. At the top end of this scale, a variety of tip-based syringe-driven devices are available,
Matrix Platemate (Matrix Technologies Corp., Lowell, MA). To achieve nanoliter dis- pensing, two platforms are available: the ezo-electric dispenser and the solenoid
dispenser
As mentioned earlier, the drive to screen more compounds has fueled the need for min- iaturization. Additionallv. there is a need to rapidly profile and evaluate the selectivity of compounds that are positive in a high- throughput screen. Miniaturization facilitates the parallel processing of numerous targets si- multaneously. For example, a GPCR cell re- porter assay designed to detect agonists using a reporter can be readily minia- turized to 2 in a 3456-well microtiter plate. The hits can be evaluated in this format at multiple concentrations, with null cell lines to remove false positives, and in a range of other cell lines yielding a selectivity index. By com- bining this with cell toxicity assays and bio-
chemical cytochrome P450 assays, a wealth of information is generated in a short period on exactly the same compound solution.
5 S U M M A R Y
Throughout this chapter we have described how HTS, as a lead discovery tool, fits into the drug discovery process. HTS is a multi-factorial, in- teractive process that brings together
teams of chemists, biologists, statisti- cians, information technology experts, and mechanical and electrical engineers.
From target discovery to lead optimization, an intricate network of processes and deci- sions are required to produce a successful drug discovery campaign. We have emphasized the high-throughput screening process as part of an integrated approach to hit identification and assessment. The application of industrial automation technology to the process has in- creased the capacity, speed, and quality of this part of the drug discovery pipeline. In conjunc- tion with corresponding advances in target identification, automated chemistry, and data analysis, the ability of pharmaceutical labora- tories to rapidly move from target concept to lead optimization candidates has changed dra- matically over the last decade.
The next 10 years will no doubt bring further technological advances and creative insights to improve the drug discovery process even fur-
ther. This will keep HTS approaches as a main- stream drug discovery tool for years to come.
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