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Screening in phenotypic assays is an important strategy for the discovery of innovative drugs and novel targets. Here we present key strategies for developing successful phenotypic screens and prosecuting phenotypic drug discovery (PDD) programs. Successful screens incorporate physiological relevance through the use of human cell types and assay designs that have (1) strong mechanistic connection to clinical outcomes and (2) strong biological justification for both efficacy and safety. In addition to guidance for designing successful screens, we also propose incorporation of specific counterscreens at an early point in the program. The suggested counterscreens are based on analysis of 1000s of drugs and drug candidates profiled through a large set of human-based phenotypic assays. These assays include cytotoxicity in human primary vascular endothelial cells, proliferation of endothelial cells, and proliferation of lymphocytes, all under specific activation conditions. These counterscreens form a generic screening funnel to triage a large fraction of early-stage hits, binning compounds into those with undesirable mechanisms (associated with acute toxicity), mechanisms with utility for oncology indications, and mechanisms useful for autoimmune indications. The application of this screening funnel offers a standardized and more predictable path for prosecuting PDD programs, reducing the risk of failure, and improving program timelines.

Approved immuno-oncology (IO) drugs that have expanded the arsenal of available treatment options for recalcitrant cancers include antibodies to PD-(L)1 checkpoints–a relatively new class of immunotherapies. While the clinical success reported for these drugs is compelling, efficacy is limited to a small cohort of patients. The need for more IO therapeutic options has prompted the pursuit of next-generation candidates that can achieve better patient outcomes. One approach is to screen compounds for novel targets that elicit an immunorestorative response within the tumor microenvironment (TME), consistent with approved IO drugs. Ideally, a small molecule therapeutic would represent a distinct advantage over existing biologics, both in terms of administration as well as ability to optimize chemistry. In order to efficiently identify molecules with IO potential, a high throughput, physiologically relevant screening approach that recapitulates the complexity of the TME is needed. BioMAP Oncology systems are co-cultures of human primary cell types (endothelial cells, fibroblasts, and PBMCs) and cancer cells that model immune suppressed TME biology. Compounds can be screened in these in vitro TME models to look for impacts on protein biomarkers relevant for immune responses, inflammation, matrix modulation and for impacts on cancer cells. These systems were validated for immune restorative activities using anti PD-(L)1 therapeutics such as pembrolizumab, nivolumab, and durvalumab. Specifically, increased pro-inflammatory cytokine levels served as hallmark activities indicating a reversal of the immune suppressive influence of the cancer cell. In one of the BioMAP Oncology systems, we screened a library of 72 compounds targeting different molecules, the vast majority of which had not previously been identified as potential immuno-oncology candidates. Several small molecules that increased one or more cytokines were identified, indicating immune restorative potential. Other small molecules inhibited immune responses and/or demonstrated anti-inflammatory activities. Additionally, modulation of matrix biomarkers on tissue and tumor cells indicate impacts on distinct compartments of the TME. This approach using human cell-based in vitro TME models represents a rapid and cost-effective manner to rapidly screen libraries of small molecules (both novel and repurposed) for immuno-oncology potential. Citation Format: Alison O'Mahony, Sheryl P. Denker, Sharlene Velichko. Phenotypic screening of small molecules for novel immuno-oncology potential using BioMAP® Early Screening Services [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6289.

Small molecule inhibitors of Janus kinases (Jakinibs) represent a promising treatment for myeloproliferative disorders including polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF). Currently multiple JAK2 inhibitors (ruxolitinib, fedratinib, momelotinib, baricitinib) are either approved or in clinical development for the treatment of MF. The primary target of Jakinibs, the JAK-STAT pathway, plays a critical role in multiple cellular processes relevant for myelofibrosis, including survival, differentiation, and proliferation. However, all approved Jakinibs are reportedly promiscuous in that they inhibit kinases beyond the JAK family. Their overall clinical impact, therefore, reflects the sum of these target activities, as well as downstream secondary pharmacology relevant to both efficacy and safety. We evaluated these four Jakinibs using phenotypic profiling with the BioMAP® Diversity PLUS Panel to identify common versus differential activities. The BioMAP platform consists of human primary cell-based assay or systems designed to recapitulate key aspects of tissue and disease states. Distinct activity profiles were generated for each of the tested Jakinibs based on their impacts on 148 different biomarkers in 12 different model systems. Comparison of these profiles at concentrations relevant for clinical exposure levels revealed common activities including inhibition of T and B cell proliferation and decreased levels of inflammation and immune activation biomarkers. Together these activities represent a phenotypic signature related to shared primary JAK targets. In contrast, multiple differentiating activities were observed between the four drug profiles: baricitinib increased TNFα levels whereas momelotinib decreased TNFα in the LPS system modeling monocyte activation; fedratinib and momelotinib, but not ruxolitinib or baricitinib, inhibited endothelial cell proliferation; only momelotinib inhibited macrophage responses and decreased PGE2 levels. These data indicate that even within the specific JAK2 inhibitor drug class, individual entities have distinct modes of action relevant for clinical outcomes. Citation Format: Sharlene Velichko, Sheryl P. Denker, Alison O'Mahony. Phenotypic evaluation and discrimination of clinical JAK inhibitors for myeloproliferative disorders [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1847.

Secretion and assembly of the extracellular matrix protein fibronectin regulates a number of normal cell and tissue functions and is dysregulated in disease states such as fibrosis, diabetes, and cancer. We found that mislocalized scaffolding by the plasma membrane Na-H exchanger NHE1 suppresses fibronectin expression, secretion, and assembly. In fibroblasts, wild-type NHE1 localizes to the distal margin of membrane protrusions or lamellipodia but a mutant NHE1-KRA2 lacking binding sites for PI(4,5)P2 and the ERM proteins ezrin, radixin, and moesin is mislocalized and found uniformly along the plasma membrane. Although NHE1 regulates intracellular pH homeostasis, fibronectin production is not regulated by changes in intracellular pH, nor is it attenuated in NHE1-deficient cells, indicating fibronectin expression is independent of NHE1 activity. However, fibronectin production is nearly absent in cells expressing NHE1-KRA2 because scaffolding by NHE1 is mislocalized. Additionally, secretion of active but not latent TGF-β is reduced and exogenous TGF-β restores fibronectin secretion and assembly. Our data indicate that scaffolding by NHE1-KRA2 dominantly suppresses fibronectin synthesis and TGF-β activation, and they suggest that NHE1-KRA2 can be used for obtaining a mechanistic understanding of how fibronectin production is regulated and speculatively for therapeutic control of dysregulated production in pathological conditions.

Structurally diverse ion transport proteins anchor the spectrin-actin cytoskeleton to the plasma membrane by binding directly to linker proteins of the ankyrin and protein 4.1 families. Cytoskeletal anchoring regulates cell shape and restricts the activity of ion transport proteins to specialised membrane domains. New directions are being forged by recent findings that localised anchoring by ion transport proteins regulates the ordered assembly of actin filaments and the actin-dependent processes of cell adhesion and motility.

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN-enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).

NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations.

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.

Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration.

The NHE family of ion exchangers includes six isoforms (NHE1–NHE6) that function in an electroneutral exchange of intracellular H+ for extracellular Na+. This review focuses on the only ubiquitously expressed isoform, NHE1, which is localized at the plasma membrane where it plays a critical role in intracellular pH (pHi) and cell volume homeostasis. All NHE isoforms share a similar topology: an N-terminus of 12 transmembrane (TM) α-helices that collectively function in ion exchange, and a C-terminal cytoplasmic regulatory domain that modulates transport activity by the TM domain. Extracellular signals, mediated by diverse classes of cell-surface receptors, regulate NHE1 activity through distinct signaling networks that converge to directly modify the C-terminal regulatory domain. Modifications in the C-terminus, including phosphorylation and the binding of regulatory proteins, control transport activity by altering the affinity of the TM domain for intracellular H+. Recently, it was determined that NHE1 also functions as a membrane anchor for the actin-based cytoskeleton, independently of its role in ion translocation. Through its effects on pHi homeostasis, cell volume, and the actin cortical network, NHE1 regulates a number of cell behaviors, including adhesion, shape determination, migration, and proliferation.

The association of actin filaments with the plasma membrane maintains cell shape and adhesion. Here, we show that the plasma membrane ion exchanger NHE1 acts as an anchor for actin filaments to control the integrity of the cortical cytoskeleton. This occurs through a previously unrecognized structural link between NHE1 and the actin binding proteins ezrin, radixin, and moesin (ERM). NHE1 and ERM proteins associate directly and colocalize in lamellipodia. Fibroblasts expressing NHE1 with mutations that disrupt ERM binding, but not ion translocation, have impaired organization of focal adhesions and actin stress fibers, and an irregular cell shape. We propose a structural role for NHE1 in regulating the cortical cytoskeleton that is independent of its function as an ion exchanger.

Publisher Summary This chapter discusses the effect of Rho guanosine 5'-triphosphates (GTPases) on Na–H exchanger in mammalian cells. In mammalian cells, Na–H consists of five isoforms (NHE1–NHE5). The chapter discusses three approaches for studying the regulation of NHE1 by Rho family GTPase: (1) measuring transport activity, (2) identifying associations with regulatory proteins, and (3) monitoring phosphorylation. The chapter describes transport activity primarily in fibroblasts that express only the NHE1 isoform and transiently express mutationally active and inactive GTPases and kinases. The chapter also discusses pharmacological reagents that selectively target signaling proteins in the Rho pathway, specifically the bacterial exotoxin C3 and the pyridine derivative Y-27632 that inhibit the activities of Rho A ,Rho A kinase, and pl60ROCK respectively. The chapter concludes with a discussion of in vivo phosphorylation of NHE1 and in vitro kinase assay.