**1. Introduction**

Asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic deregulated inflammation, bronchial hyperreactivity, and symptoms of recurrent wheezing, coughing, and shortness of breath. Its prevalence has increased considerably over the past three decades, particularly in Western countries. Asthma is a major public health problem that affects 300 million people worldwide [1, 2].

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

Airway hyperresponsiveness (AHR), one of the hallmarks of asthma, directly results from excessive contraction of airway smooth muscle cells (aSMC). The degree of AHR always correlates with asthma severity and the need for therapy [3, 4]. Regular treatment of chronic asthma consists of a combination of inhaled anti-inflammatory corticosteroids and long-acting beta2-adrenergic receptor agonists for bronchodilation. However, severe asthma escapes to usual treatments or frequently requires higher doses. In acute asthma, short-acting beta2 agonists or anticholinergics are used as bronchodilators. These drugs are rapidly effective but can be insufficient in some cases of severe acute asthma attack. Despite available therapies, many patients with severe asthma remain uncontrolled and the number of asthma deaths is still elevated [5]. There is thus an obvious need for new drugs acting through other pathways to prevent or reverse AHR and decrease severe asthma attacks, hospitalizations and deaths.

The molecular mechanisms regulating aSMC contraction and proliferation involved in AHR are still largely unknown. Understanding the intracellular signaling pathways responsible for AHR is thus essential to identify new targets and design new treatments. In this way, the Rho protein Rac1 has been identified as a promising candidate.

The Rho/Rac proteins constitute a family of small GTPases of more than 25 members in mammals. So far, the best characterized members of this family include the members of the Rho (RhoA, RhoB, RhoC), Rac (Rac1, Rac2, RhoG), and Cdc42 (Cdc42) subfamilies. Rho/Rac proteins share a common structure (40–95% identity) composed of a six-stranded mixed β sheet flanked by five α helices. The core G domain is made up of five polypeptide loops (G1–G5) involved in nucleotide, regulators and effectors interactions. Like the majority of Ras superfamily GTPases, most members of the Rho/Rac family behave as molecular switches that cycle between inactive (GDP-bound) and active (GTP-bound) states. In basal conditions, these proteins are inactive and sequestered in the cytosol due to their binding to Rho GDP dissociation inhibitors (RhoGDIs). Upon cell stimulation, Rho proteins became GTP-bound, translocate to the plasma membrane after release from RhoGDI, and interact with their primary effector molecules to trigger different signal transduction pathways. In addition to RhoGDIs, the activation/inactivation cycle of Rho/Rac proteins is regulated by a complex set of regulatory proteins that include GDP/GTP exchange factors (GEFs) and GTPase activating protein (GAPs) (**Figure 1**). Interaction of Rho/Rac protein with a GEF promotes the exchange of GDP for GTP molecules, thus leading to the rapid activation of Rho/Rac proteins during cell stimulation events. By contrast, GAPs promote the hydrolysis of the bound GTP molecules to GDP, thus allowing the transfer of the GTPases back to the inactive state at the end of the stimulation cycle [6].

agonist-induced rise in intracellular Ca2+ concentration through a mechanism involving Rac1 dependant control of PLC activity. Interestingly, Rac1 deficiency has no impact on the respiratory system in basal, physiological condition. However, deletion of Rac1 expression in aSMC or nebulization of the Rac inhibitor NSC23766 prevented AHR in an allergic asthma model in mouse. These data indicate that (1) Rac1 is a critical component in aSMC contraction and (2) inhibition of Rac1 activity or expression may represent a novel therapeutic approach for

Rac proteins, prolonging the inactive state and sequestering the GTPase in the cytosol.

**Figure 1.** Activation cycle of Rho/Rac proteins and their regulatory proteins. Activation of Rho/Rac proteins is mediated by a guanine nucleotide exchange factor (GEF) leading to GTP loading and to the translocation of Rho/Rac proteins to the plasmatic membrane. This active configuration of Rho/Rac proteins promotes effector interactions. To "turn off" the cycle, a GTPase-activating protein (GAP) accelerates the intrinsic GTPase activity of Rac, allowing Rac to return to its inactive state in the cytosol. The guanine nucleotide-dissociation inhibitor (GDI) binds specifically to GDP-bound Rho/

High Throughput Screen for Inhibitors of Rac1 GTPase by Flow Cytometry

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

45

Unfortunately, the currently available drugs that inhibit Rac1 (EHT 1864 and NSC23766) have low affinity and induce critical off-target effects, thus highlighting the obvious need to dis-

In this chapter we describe how a published flow cytometry assay was taken and converted to a 1536 well format suitable for high throughput screening (HTS) to enable the screening of 500,000 compounds in the AstraZeneca Global HTS Centre to attempt to find new Rac1 inhibitors. We describe the whole protocol from receiving assay ready plates through to the data analysis and discuss the criteria that are important for an assay to be suitable for a high throughput screen by flow cytometry and describe the validation process we go through prior

patients with airway AHR such as asthma.

to starting a high throughput screening campaign.

cover new Rac inhibitors [9, 10].

During the last decade, Rho/Rac protein signaling pathways have been recognized as major regulators of essential cellular functions. Rac1 is a key regulator of cytoskeletal structure and dynamics, leading to lamellipodia and ruffle formations [6, 7]. Thanks to this last function, Rac1 controls cellular migration and adhesion. Recently, we demonstrated that Rac1 regulates vascular SMC contraction, and consequently modulates arterial pressure [8]. Accordingly, we hypothesized that Rac1 could also be involved in aSMC contraction. We demonstrated that the specific SMC deletion of Rac1 (SM-Rac1-KO) in mice prevents bronchoconstriction *ex* and *in vivo*. Our results showed that the decreased expression or activity of Rac1 in aSMC impairs

Airway hyperresponsiveness (AHR), one of the hallmarks of asthma, directly results from excessive contraction of airway smooth muscle cells (aSMC). The degree of AHR always correlates with asthma severity and the need for therapy [3, 4]. Regular treatment of chronic asthma consists of a combination of inhaled anti-inflammatory corticosteroids and long-acting beta2-adrenergic receptor agonists for bronchodilation. However, severe asthma escapes to usual treatments or frequently requires higher doses. In acute asthma, short-acting beta2 agonists or anticholinergics are used as bronchodilators. These drugs are rapidly effective but can be insufficient in some cases of severe acute asthma attack. Despite available therapies, many patients with severe asthma remain uncontrolled and the number of asthma deaths is still elevated [5]. There is thus an obvious need for new drugs acting through other pathways to prevent or reverse AHR and decrease severe asthma attacks, hospitalizations and deaths. The molecular mechanisms regulating aSMC contraction and proliferation involved in AHR are still largely unknown. Understanding the intracellular signaling pathways responsible for AHR is thus essential to identify new targets and design new treatments. In this way, the Rho

The Rho/Rac proteins constitute a family of small GTPases of more than 25 members in mammals. So far, the best characterized members of this family include the members of the Rho (RhoA, RhoB, RhoC), Rac (Rac1, Rac2, RhoG), and Cdc42 (Cdc42) subfamilies. Rho/Rac proteins share a common structure (40–95% identity) composed of a six-stranded mixed β sheet flanked by five α helices. The core G domain is made up of five polypeptide loops (G1–G5) involved in nucleotide, regulators and effectors interactions. Like the majority of Ras superfamily GTPases, most members of the Rho/Rac family behave as molecular switches that cycle between inactive (GDP-bound) and active (GTP-bound) states. In basal conditions, these proteins are inactive and sequestered in the cytosol due to their binding to Rho GDP dissociation inhibitors (RhoGDIs). Upon cell stimulation, Rho proteins became GTP-bound, translocate to the plasma membrane after release from RhoGDI, and interact with their primary effector molecules to trigger different signal transduction pathways. In addition to RhoGDIs, the activation/inactivation cycle of Rho/Rac proteins is regulated by a complex set of regulatory proteins that include GDP/GTP exchange factors (GEFs) and GTPase activating protein (GAPs) (**Figure 1**). Interaction of Rho/Rac protein with a GEF promotes the exchange of GDP for GTP molecules, thus leading to the rapid activation of Rho/Rac proteins during cell stimulation events. By contrast, GAPs promote the hydrolysis of the bound GTP molecules to GDP, thus allowing the transfer of the GTPases back to the inactive state at the end of the stimulation

During the last decade, Rho/Rac protein signaling pathways have been recognized as major regulators of essential cellular functions. Rac1 is a key regulator of cytoskeletal structure and dynamics, leading to lamellipodia and ruffle formations [6, 7]. Thanks to this last function, Rac1 controls cellular migration and adhesion. Recently, we demonstrated that Rac1 regulates vascular SMC contraction, and consequently modulates arterial pressure [8]. Accordingly, we hypothesized that Rac1 could also be involved in aSMC contraction. We demonstrated that the specific SMC deletion of Rac1 (SM-Rac1-KO) in mice prevents bronchoconstriction *ex* and *in vivo*. Our results showed that the decreased expression or activity of Rac1 in aSMC impairs

protein Rac1 has been identified as a promising candidate.

44 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

cycle [6].

**Figure 1.** Activation cycle of Rho/Rac proteins and their regulatory proteins. Activation of Rho/Rac proteins is mediated by a guanine nucleotide exchange factor (GEF) leading to GTP loading and to the translocation of Rho/Rac proteins to the plasmatic membrane. This active configuration of Rho/Rac proteins promotes effector interactions. To "turn off" the cycle, a GTPase-activating protein (GAP) accelerates the intrinsic GTPase activity of Rac, allowing Rac to return to its inactive state in the cytosol. The guanine nucleotide-dissociation inhibitor (GDI) binds specifically to GDP-bound Rho/ Rac proteins, prolonging the inactive state and sequestering the GTPase in the cytosol.

agonist-induced rise in intracellular Ca2+ concentration through a mechanism involving Rac1 dependant control of PLC activity. Interestingly, Rac1 deficiency has no impact on the respiratory system in basal, physiological condition. However, deletion of Rac1 expression in aSMC or nebulization of the Rac inhibitor NSC23766 prevented AHR in an allergic asthma model in mouse. These data indicate that (1) Rac1 is a critical component in aSMC contraction and (2) inhibition of Rac1 activity or expression may represent a novel therapeutic approach for patients with airway AHR such as asthma.

Unfortunately, the currently available drugs that inhibit Rac1 (EHT 1864 and NSC23766) have low affinity and induce critical off-target effects, thus highlighting the obvious need to discover new Rac inhibitors [9, 10].

In this chapter we describe how a published flow cytometry assay was taken and converted to a 1536 well format suitable for high throughput screening (HTS) to enable the screening of 500,000 compounds in the AstraZeneca Global HTS Centre to attempt to find new Rac1 inhibitors. We describe the whole protocol from receiving assay ready plates through to the data analysis and discuss the criteria that are important for an assay to be suitable for a high throughput screen by flow cytometry and describe the validation process we go through prior to starting a high throughput screening campaign.
