**4. Conclusions**

Until recently flow cytometry was considered a powerful technology dedicated to bespoke assays where just a few samples were to be measured. No medium throughput let alone high throughput screen could be run on this technology due to lack of plate handling features, time needed per sample and therefore cellular imaging was the preferred method for any high content screen. Increases of interest in phenotypic screening combined with a desire to access relevant cell models, often suspension cells, and development of plate-based sampling flow cytometers have led to the opportunity to use flow cytometry as a drug screening technology platform. The ability to perform multiplexing where different cells or particles labeled with distinct fluorophores are analyzed in parallel and multiple endpoints are measured leads to in-depth analysis of subpopulations within a sample. Such an approach results in increased productivity by decreasing timelines and cell requirements, two critical parameters for high throughput screening.

We chose to run a screen for Rac1 inhibitors on our iQue® Screener HD as it enabled us to miniaturize and run a simple and robust assay on one of our automation platforms.

We have run a successful 500,000 compound screen using the iQue® HD screener as summarized in **Figure 13**. By optimizing the protocol, we were able to retain high-quality data while decreasing read time per 1536 well plate to under 50 min. This allowed us to run the primary screen within 4 weeks. Although time is not the most critical criteria we consider it remains an important factor as we need to maintain the overall flow of projects through our portfolio and therefore we must decide on a suitable time frame for completion of each screen.

The screen we have described here would have not been possible a few years ago but, thanks to the development of high throughput capable instruments, flow cytometry has found its place within the drug discovery process and high throughput screening in AstraZeneca. Although we decided not to multiplex our concentration response assays, it should be noted that the use of flow cytometry technology does allow for such multiplexing and indeed has

High Throughput Screen for Inhibitors of Rac1 GTPase by Flow Cytometry

http://dx.doi.org/10.5772/intechopen.71074

59

The authors would like to thank Matthew Collier for his assistance during the high throughput

been described in the literature [12].

screen and Kim Luu for her technical support with the iQue®.

**Acknowledgements**

**Figure 13.** Screen summary.

High throughput screening is a key method for the identification of hit and lead compounds and remains at the start of most of our drug discovery programs. As we pursue a wide range of targets we also use a wide range of assays and technologies. We have invested into several iQue® Screener systems as we updated and replaced previous detection systems and to fill a capability gap for screening suspension cells or primary cells in small sample volumes. We have successfully used the iQue® Screener systems in a variety of applications from phenotypic screening to more complex multiplex profiling. The technology has enabled several projects due to the ability to reduce cell requirement, cost and timelines.

**Figure 13.** Screen summary.

screen were serially diluted 1:3 to construct a 10-point concentration response curve. Compound concentration curves are dispensed using acoustic dispensing into assay ready plates at 150 compound curves per plate. Wells are backfilled with DMSO to keep the DMSO concentration constant in all wells. The plates were sealed and stored in the controlled atmosphere and temperature working plate store before use as for primary screen

58 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

pounds were confirmed as active with 44 identified as Rac1 selective using a ≥10 fold ratio of Rac1 IC50 vs. RhoA and Cdc42 as a cutoff. These compounds encompass a variety of chemical structures. **Figure 12** shows representative curves of selective and non-selective compounds. Selected hits are being further characterized through a panel of cellular assays

Until recently flow cytometry was considered a powerful technology dedicated to bespoke assays where just a few samples were to be measured. No medium throughput let alone high throughput screen could be run on this technology due to lack of plate handling features, time needed per sample and therefore cellular imaging was the preferred method for any high content screen. Increases of interest in phenotypic screening combined with a desire to access relevant cell models, often suspension cells, and development of plate-based sampling flow cytometers have led to the opportunity to use flow cytometry as a drug screening technology platform. The ability to perform multiplexing where different cells or particles labeled with distinct fluorophores are analyzed in parallel and multiple endpoints are measured leads to in-depth analysis of subpopulations within a sample. Such an approach results in increased productivity by decreasing timelines and cell requirements, two critical parameters for high

We chose to run a screen for Rac1 inhibitors on our iQue® Screener HD as it enabled us to

We have run a successful 500,000 compound screen using the iQue® HD screener as summarized in **Figure 13**. By optimizing the protocol, we were able to retain high-quality data while decreasing read time per 1536 well plate to under 50 min. This allowed us to run the primary screen within 4 weeks. Although time is not the most critical criteria we consider it remains an important factor as we need to maintain the overall flow of projects through our portfolio and

High throughput screening is a key method for the identification of hit and lead compounds and remains at the start of most of our drug discovery programs. As we pursue a wide range of targets we also use a wide range of assays and technologies. We have invested into several iQue® Screener systems as we updated and replaced previous detection systems and to fill a capability gap for screening suspension cells or primary cells in small sample volumes. We have successfully used the iQue® Screener systems in a variety of applications from phenotypic screening to more complex multiplex profiling. The technology has enabled several

miniaturize and run a simple and robust assay on one of our automation platforms.

therefore we must decide on a suitable time frame for completion of each screen.

projects due to the ability to reduce cell requirement, cost and timelines.

was added to the buffer for the RhoA assay. 82% of the tested com-

plates. 1 mM MgCl2

by the team at INSERM.

**4. Conclusions**

throughput screening.

The screen we have described here would have not been possible a few years ago but, thanks to the development of high throughput capable instruments, flow cytometry has found its place within the drug discovery process and high throughput screening in AstraZeneca. Although we decided not to multiplex our concentration response assays, it should be noted that the use of flow cytometry technology does allow for such multiplexing and indeed has been described in the literature [12].
