A selective ATP-binding cassette subfamily G member 2 efflux inhibitor revealed via high-throughput flow cytometry.

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)-driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry.

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded, and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high-throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in a manner analogous to that of rapamycin. We have shown that CID 3528206 inhibited yeast cell growth and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC(50)'s of 150 nM and 3.9 µM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.

A miniature Couette to generate shear for flow cytometry: studying real-time modulation of intracellular calcium in monocytic cells.

Extracellular hydrodynamic forces may be transmitted to the interior of cells through the alteration of integrin conformation and affinity. Integrin activation regulates leukocyte recruitment, cell activation, and transmigration. The cellular and molecular mechanisms for integrin activation are not precisely known, although intracellular calcium signaling is involved. Flow cytometry offers a versatile way to study intracellular calcium signaling in real-time. We report a novel method to generate defined shear by using a miniature Couette. Testing involved measuring shear-induced intracellular calcium signals of human monoblastoid U937 cells in suspension. The Couette was connected externally to a flow cytometer and pressurized at 6 PSI (4.1 N/m(2) ). Cells were subjected to a well-defined shear between 0 and 1,000 s(-1) and delivered continuously within 10 s to a FACScan at 1 µl/s. Intracellular calcium levels and the percentage of cells activated increased as shear increased in duration and intensity.

Real-time analysis of very late antigen-4 affinity modulation by shear.

Shear promotes endothelial recruitment of leukocytes, cell activation, and transmigration. Mechanical stress on cells caused by shear can induce a rapid integrin conformational change and activation, followed by an increase in binding to the extracellular matrix. The molecular mechanism of increased avidity is unknown. We have shown previously that the affinity of the alpha(4)beta(1) integrin, very late antigen-4 (VLA-4), measured with an LDV-containing small molecule, varies with cellular avidity, measured from cell disaggregation rates. In this study, we measured in real time affinity changes of VLA-4 in response to shear. The resulting affinity was comparable with the state mediated by receptor signaling and corresponded in time with intracellular Ca(2+) responses. Ca(2+) ionophores and N,N'-[1,2-ethanediyl-bis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl]ester demonstrate that the affinity regulation of VLA-4 in the presence of shear was related to Ca(2+) signaling. Pertussis toxin treatment implicates G(i) in an unknown pathway that connects shear, Ca(2+) elevation, VLA-4 affinity, and cell avidity.

Alpha4beta1 integrin affinity changes govern cell adhesion.

Integrin alpha4beta1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing alpha4beta1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670-48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the alpha4 integrin.

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity.

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.