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Target potency

I Target selectivity (activity at other targets)

Human ¡Toxicity in permeability ' in human intestine liver | human enzyme j liver inhibition cells (IC50) (IC50)

cells (transport rate)

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Computer Cardiac J Integrity generated genesis : |jabi|jty of j drug-like potential /iC5g\ compound properties (IC50) i K ■■

Chemical tractability tailored,

igure 2.15. A compound profile matrix that outlines all the information on a set of compounds that we been identifiedfrom a HTS.

screening (1.5. 81) with significant investment in tht automation and miniaturization.

4.1 Automation in High-Throughput Screening

The need to screen large compound libraries that .typically range from 105-106 through a range i>f biological targets in an efficient and timely manner has been one of the main drivers for automation. From the late 1970s to the mid-1990s, the 96-well microtiter plate reigned supreme in many screening laboratories 032). Manufacturers supplied many variations on the 96-well plate theme, varying the shape of the well, varying the color of the plate; and supplying a range of surface chemistries for specialized assays. Invariably, the 96-well plates were all subtly different, and it was not until 1996 that a standard was recommended by the Society for Biomolecular Screening. Automated screening systems needed accurate plate dimensions. Robotic systems needed higher tolerances and defined dimensions to pick and place plates precisely. Detection instrumentation was also adapted to enable robots to load and unload microplates.

High-throughput screening automation exists at a variety of levels, from manual to semi-automated to fully automated turnkey systems (83). However, the types of equipment tend to be similar, and the way in which the screening process is integrated dictates the level of automation. For a brief discussion on the advantages and disadvantages of automated platforms versus workstations, see refs. 84 and 85.

All the high-throughput screening automation platforms tend to have the same limited number of basic operations; a method moving around microplates, dispensing liquids, a series of detectors, and incubators. The methods

Figure 2.16. Images of the Aurora Bioscience UHTS screening platform that is based around a track that moves the microtiter plates around the different workstations. The 96-well piezo-electric dispensing head is shown in detail.

for moving microplates tend to fall into two general approaches. Movements can be with an articulated robotic arm, picking and placing plates, or through a track that resembles a mini-production line designed to shuttle plates around the system (Fig. 2.16). The liquid handling options can vary between a syringe-based system that gives higher volumetric precision to aperistaltic pump that tends to be more rapid but less precise. The range of potential detectors is dependent on the assay technology discussed earlier in the chapter. The incubators can range from the very simple open racks of shelves to highly environmentally controlled systems. The glue that puts all this together is the scheduling software that controls what goes where and when. In the more sophisticated systems, an operator loads reagents, plates containing test compounds, and disposes of any waste (Fig. 2.17). T' he scheduling software instructs the articulated arm or track system to move microtiter plaites

Figure 2.17. Images of typical turnkey automated screening robots that use an articulated arm to move plates around the screening system. The robot is placing plates into two different liquid handling devices. Top left is a PlateMate Plus and top right is a Multidrop. This system was built internally by Bristol Myers Squibb engineers.

Figure 2.17. Images of typical turnkey automated screening robots that use an articulated arm to move plates around the screening system. The robot is placing plates into two different liquid handling devices. Top left is a PlateMate Plus and top right is a Multidrop. This system was built internally by Bristol Myers Squibb engineers.

at the calcidated times around the various liquid handlers, incubators, and detectors, and eventually to waste. A limited amount of rules-basef artificial intelligence can be used to aid the quality of the operation. For example, as data are generated by the detector, online analysis can be used to monitor drift in the assay or whether certain wells fail to meet prescribed quality control parameters, as seen with blocked tips on a dispenser. These types of error alert an operator.

Building these integrated robotic systems requires strong management commitment with time, money, and a willingness to develop the necesaary skill sets. A stable, fully automated screening platform does offer continuous operation, 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 platform. For example, a particular detector may not be available in a format that can be integrated. Most screening laboratories will use workstation approaches in addition to fully automated platforms to enable assay flexibility. Unlike the automated platforms, where plates are processed in a serial manner, plates are batched together, "a stack," in workstation approaches. Workstation approaches replaces the robot with a human, and as long as the number of microtiter plates processed is acceptable, this often works well. The same quality control is incorporated into the workstation 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 laboratories have driven a strong interest in implementing 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 densities.

The first tangible step along the miniaturization route was the introduction of the 96-well microtiter plate that replaced the individual tube (82). The 96-well plate became the standard workhorse in academic and industrial laboratories over the last 20 .years. However, ever increasing demands to expand testing 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 compound deck with average reagent and plastic-ware that costs $0.20/well, totals $5 million, excluding waste management costs. This scenario in a 96-well plate format generates 260,000 plates of plastic waste per year. A typical assay volume in the 96-well plate is 100200 jaL, and by reducing this to around 5-10 juL, reagent costs are reduced. Additionally, smaller amounts of compounds are needed for the assay.

In the late 1990s, HTS laboratories adopted the 384-well plate as standard, allowing a fourfold increase in well density and increased screening capacity (87). Instrumentation companies re-invested in designing or adapting liquid handlers, detection systems, and automation to fit the new 384-well plate.

Multiwell Plate And 3456 Well
Figure 2.18. There are many different types of m crotiter plates that are used in miniaturized assays for HTS. (a) 96-well plate (100 /xL assays), (b) 384-well plate (25 fjiL well assays), (c) 1536-well plate (5 ju,L well assays), and (d) 3456-wellplate (2 ^L well assays)

The vast majority of assays have readil miniaturized down to 20-50 uiL volumes. The minimum practical volume of 20 ¡jlL for the new 384-well plates was defined by the we shape and the need to produce an even layer of liquid at the bottom of the plate.

Even with the 384-well plate, there ha been pressure to reduce volumes even further. The 1536-well plate is emerging as the poter tial next step, with square wells that allow working volume of 5-10 /xL. One advantage of the 1536-well plate in absorbance-based assays is volume reduction while maintainin the path length. Additionally, low-volum 384-well microtiter plates are now commei daily available. The 1 /xL assay is also no7 available in the 1536- (Corning Costar Corp Cambridge, MA and Evotec OAI, Hamburg, Germany) and the 3456-well microtiter plat (AuroraBiosciences, San Diego, CA) (50) .In a little over 5 years, we have witnessed a 100fold reduction in assay volume and a 36-fol increase in the well density. The discussio over well density still causes many debates in 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 significant challenges to instrumentation companies. First, there is the need to detect the results of a particular assay. For example, in the 96-we scintillation proximity assays, the scintillatio counter photomultipliers are positioned abov 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 long to read a plate. A new solution needed to be found. Imaging technology, using charged-coupled devices, (CCDs) provided the answer (e.g., LEADSeeker; Amersham Pharmacia, Amer-sham, UK, and CLIPR Molecular Devices, Palo Alto, CA) (89). 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 96-well microtiter and filter-binding plates. For the LEADSeeker 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 efficiency is dramatic. Imagers are now available for fluorescence, time-resolved fluorescence and for measuring light emission.

Another engineering challenge was to dispense volumes in the 20 nL-l/xL volume range. At the top end of this scale, a variety of tip-based syringe-driven devices are available, e.g., Matrix Platemate (Matrix Technologies Corp., Lowell, MA). To achieve nanoliter dispensing, two platforms are available: the pi-ezo-electric inkjet dispenser and the solenoid inkjet dispenser (90-92).

As mentioned earlier, the drive to screen more compounds has fueled the need for miniaturization. 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 simultaneously. For example, a GPCR cell reporter assay designed to detect agonists using a j3-lactamase reporter can be readily miniaturized to 2 juL 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 combining 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.

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