Larry Sklar - Academia.edu (original) (raw)
Papers by Larry Sklar
Journal of Investigative Medicine, 2007
266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY D... more 266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY DEPARTMENT PATIENTS WITH AN ALCOHOL PROBLEM. F. Antelo, S. Bazargan-Hejazi, C. Ani, Charles R. Drew University of Medicine and ...
Journal of Investigative Medicine, 2007
266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY D... more 266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY DEPARTMENT PATIENTS WITH AN ALCOHOL PROBLEM. F. Antelo, S. Bazargan-Hejazi, C. Ani, Charles R. Drew University of Medicine and ...
Bioassay hit selection; stacking the odds in early stage drug discovery High throughput screening... more Bioassay hit selection; stacking the odds in early stage drug discovery High throughput screening (HTS) data analysis continues to be an essential, routine, yet challenging task in drug discovery: to infer reliable knowledge from big and noisy data. Bioassays require complex methodology, and results vary widely in accuracy, precision, and content. Hit selection criteria should optimize the overall probability of success in a project, and avoid expensive "false trails" such as promiscuous compounds. At UNMCMD, our experience in the NIH Molecular Libraries Project (MLP) motivated and informed this research.
Additional file 1. Supplementary figures.
Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays imp... more Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases. Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays. Conclusion: The identified compound is a highly Cdc42-selective inhibitor. Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research. Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.
Cancer Research, 2015
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer (... more Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer (OvCa) is the 5th leading cause of cancer death in women in the US with a 5-year survival rate of 44.6%. About 70% patients are diagnosed at advanced stages with intraperitoneal dissemination, therefore identifying intracellular targets and developing effective molecules to reduce tumor metastasis have great significance for ovarian cancer therapy. Cdc42 and Rac1 are small Rho GTPase and function as molecular switches of actin reorganization which are crucial to tumor cell adhesion, migration and invasion. In previous work, our group found Cdc42 and the constitutively active Rac1b are overexpressed in primary ovarian tumor tissues and ovarian cancer cell lines. Lead identification through a high-throughput screen combined with a computational shape homology approach, identified R-ketorolac as a Cdc42 and Rac1 regulator, an activity that is distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. RhoA was unaffected by R-ketorolac. A bead-based flow cytometry assay identified R-ketorolac reduced Cdc42 and Rac1 GTPase nucleotide binding in vitro and inhibition was an allosteric mechanism of action. In cell-based assays, using Skov3ip cells, and in ascites-derived ovarian tumor cells, R-ketorolac was found to inhibit the activities of Cdc42 and Rac1 and their direct downstream effectors, the phosphorylation of p21-activated kinases (PAKs). R-ketorolac, but not S-ketorolac impeded Cdc42 mediated filopodia formation, measured based on both the length and numbers of filopodia in Skov3ip and primary OvCa cells. Cell behavior assays showed that R-ketorolac but not S-ketorolac strikingly inhibited cell adhesion, migration and invasion. In a xenograft mouse model a 50% reduction in tumor cell number and decreased total tumor burden was observed with R-ketorolac as compared to S-ketorolac treatment. Finally, in a ‘phase 0′ clinical study we found Cdc42 and Rac1 activities were reduced in a time-dependent manner after a single IV dose of racemic ketorolac treatment. In sum, we established R-ketorolac inhibition on Cdc42 and Rac1 activities and subsequent physiological consequences which are critical to tumor metastasis. Our findings provide the first demonstration of selective inhibition of Cdc42 and Rac1 GTPases by an FDA approved drug in humans. Citation Format: Yuna Guo, S. Ray Kenney, Larry A. Sklar, Tione Buranda, Tudor I. Oprea, Oleg Ursu, Sarah F. Adams, Teresa Rutledge, Carolyn Muller, Lesley Lomo, Laurie G. Hudson, Angela Wandinger-Ness. R-ketorolac targets Cdc42 and Rac1 GTPases and alters ovarian tumor cell behaviors critical for invasion and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4044. doi:10.1158/1538-7445.AM2015-4044
Optical Diagnostics of Living Cells III, 2000
ABSTRACT
PLoS ONE, 2012
ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter prot... more ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.
Molecular Pharmacology, 2003
Journal of Biological Chemistry, 2011
Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of ... more Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by G␣ i-coupled G protein-coupled receptors (GPCRs) and deactivated by G␣ s-coupled GPCRs. The effects of G␣ s-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific ␣ I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step. LFA-1 3 (␣L2) and VLA-4 (␣41) are the two major and, probably, two best studied leukocyte integrins. Together with
Journal of Biological Chemistry, 2001
This work examines the affinity of ␣ 4  1-integrin and whether affinity regulation by G protein-... more This work examines the affinity of ␣ 4  1-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between ␣ 4-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (
Cytometry Part A, 2009
Introduction-Of recent clinical interest have been two related human G-protein coupled receptors:... more Introduction-Of recent clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to anti-bacterial inflammation and malignant glioma cell metastasis; and formylpeptide receptor like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow cytometry based high throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. Methods-The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein-labeled peptide ligand from FPR, FPRL1 or both. U937 cells expressing FPR and RBL cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color-coded with red fluorescent dye allowing their distinction during analysis. Compounds, cells and fluorescent ligand were sequentially combined (no-wash) in 15 μL assay volumes in 384-well plates. Throughput averaged ∼11 min per plate to analyze ∼4000 cells (∼2000/receptor) in a 2 μL aspirate from each well. Results/Conclusions-In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K i) ≤ 4 μM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, K i = 95 ± 10 nM) and FPRL1 (BB-V-115, K i = 270 ± 51 nM). Each was a selective antagonist in calcium response assays and 1 This work was supported by NIH R03 MH076381-01, U54 MH074425-01, the New Mexico Molecular Libraries Screening Center,
Cancer Research, 2011
Epithelial ovarian cancer is the major cause of gynecologic malignancy deaths. Because of their r... more Epithelial ovarian cancer is the major cause of gynecologic malignancy deaths. Because of their roles in cell adhesion and migration, Rho family GTPases have been suggested as potential therapeutic targets in human cancers. We identify the Rac1 and Cdc42 GTPases as relevant targets in papillary serous and endometriod tumors. Cdc42 is overexpressed in primary human ovarian tumors and cancer cell lines, and a novel splice variant Rac1b is upregulated in tumors of advanced stage and grade. GTPase activities in primary ascites are 3 to 6-fold higher than in cultured cells. R-Naproxen was identified by high throughput screening of a Prestwick compound library as a select non-steroidal anti-inflammatory drug (NSAID) from 23 tested that selectively targets Rac1 and Cdc42 in a bead-based assay using purified proteins. The drug is demonstrated to have positive benefit against cell behaviors required for ovarian cancer dissemination and metastasis using both cell lines and primary human tumor cell isolates. Human ovarian cells show slowed cell proliferation, as well as impaired migration, adhesion and invadopodia formation. Other NSAIDs with structural similarity (S-naproxen and 6-methoxy naphthalene acetic acid) lack these properties, while a specific Rac inhibitor NSC 23766 mimics the effects. Molecular docking shows R-Naproxen can bind the GDP-bound, but not GTP-bound Rac1, suggesting it may act by stabilizing Rac and Cdc42 in the inactive state. R-Naproxen has potential for rapid translation and efficacy in the treatment of metastatic ovarian cancer on account of FDA approval and novel activities against Rho-family GTPases. Funding for this study was generously provided by NIH grants U54MH074425, U54MH084690, R03MH081231, P30CA118100 and UNM Cancer Center FIG.0990MD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2011-LB-214
Analytical Biochemistry, 2008
Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational state... more Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here, we report a bead-based, flow cytometric assay that quantitatively measures the nucleotide binding properties of GST-chimeras for prototypical Rasfamily members Rab7 and Rho. Measurements are possible in the presence or absence of Mg 2+ , with magnesium cations principally increasing affinity and slowing nucleotide dissociation rate 8-to 10fold. GST-Rab7 exhibited a 3-fold higher affinity for GDP relative to GTP that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used γ-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP-binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real-time and quantitatively assess differences between GTPases.
ACS Chemical Biology, 2007
Biophys J, 2003
Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to... more Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing a 4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the a 4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was ;50 A ˚ between resting and Mn 21 activated receptors and ;25 A ˚ after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of a 4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.
n this report, we describe a high throughput flow cytometry based multiplexed screen for molecule... more n this report, we describe a high throughput flow cytometry based multiplexed screen for molecules that either functionally mimic rapamycin (e.g., those that are transducer of regulated CREB 1 [TORC1] pathway selective, but non-discriminating of TORC1 branchpoints) or compounds that selectively target individual branches of the yeast TORC1 pathway. The high throughput screening (HTS) and the subsequent follow-up structure activity relationship (SAR) studies identified a chemotype that contains analogs of both functional types. Obtaining structurally related analogs with differential selectivity assisted in the characterization of the probe and the pathway in which it operates. Here we report ML231 as a TORC1 Retrograde signaling (RTG) branch selective probe (CIT2 EC50 = 3.8 M). ML231 is selective on the RTG branch and only affects expression of a small subset of rapamycin-responsive genes. This selectivity appears unrelated to solubility as we showed that a more soluble analog was also selective on the RTG branch. ML231 and its analogs inhibited Sch9 phosphorylation, the major substrate and downstream effector of the TORC1 pathway. This result indicates that this chemotype affects TORC1 activity. ML231 inhibited TORC1 activity incompletely, as compared to a related analog, SID 96099781, which mimicked rapamycin and demonstrated complete TORC1 inhibition. This feature may be partially attributable to the branch selectivity of ML231. ML231 behaved very differently from SID 96099781 in the TORC1 bypass cell growth assay, showing that selective compounds may function downstream of TORC1 while SID 96099781 functions upstream or at the level of TORC1. This result suggests that the ML231 targets components downstream of TORC1, thus executing a partial function of TORC1. Collectively, this suggests that ML231 targets the RTG branch selectively. We further showed that ML231
induced the nuclear translocation of Rtg1p/Rtg3p transcription factors and the activity of ML231 on the CIT2 gene expression was dependent on the Rtg2 protein. These results further confirmed the activity of ML231 on the RTG branch. Although ML231 possesses structural and solubility challenges, this probe represents an achievement in target of rapamycin (TOR) selectivity that to our knowledge has not been previously described. ML231 will be used by investigators focusing on TORC1 signaling, RTG pathways and regulatory signaling between mitochondria and the nucleus.
Journal of Investigative Medicine, 2007
266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY D... more 266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY DEPARTMENT PATIENTS WITH AN ALCOHOL PROBLEM. F. Antelo, S. Bazargan-Hejazi, C. Ani, Charles R. Drew University of Medicine and ...
Journal of Investigative Medicine, 2007
266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY D... more 266 CORRELATES OF READINESS TO CHANGE DRINKING BEHAVIORS AMONG A SAMPLE OF INNER-CITY EMERGENCY DEPARTMENT PATIENTS WITH AN ALCOHOL PROBLEM. F. Antelo, S. Bazargan-Hejazi, C. Ani, Charles R. Drew University of Medicine and ...
Bioassay hit selection; stacking the odds in early stage drug discovery High throughput screening... more Bioassay hit selection; stacking the odds in early stage drug discovery High throughput screening (HTS) data analysis continues to be an essential, routine, yet challenging task in drug discovery: to infer reliable knowledge from big and noisy data. Bioassays require complex methodology, and results vary widely in accuracy, precision, and content. Hit selection criteria should optimize the overall probability of success in a project, and avoid expensive "false trails" such as promiscuous compounds. At UNMCMD, our experience in the NIH Molecular Libraries Project (MLP) motivated and informed this research.
Additional file 1. Supplementary figures.
Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays imp... more Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases. Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays. Conclusion: The identified compound is a highly Cdc42-selective inhibitor. Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research. Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.
Cancer Research, 2015
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer (... more Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer (OvCa) is the 5th leading cause of cancer death in women in the US with a 5-year survival rate of 44.6%. About 70% patients are diagnosed at advanced stages with intraperitoneal dissemination, therefore identifying intracellular targets and developing effective molecules to reduce tumor metastasis have great significance for ovarian cancer therapy. Cdc42 and Rac1 are small Rho GTPase and function as molecular switches of actin reorganization which are crucial to tumor cell adhesion, migration and invasion. In previous work, our group found Cdc42 and the constitutively active Rac1b are overexpressed in primary ovarian tumor tissues and ovarian cancer cell lines. Lead identification through a high-throughput screen combined with a computational shape homology approach, identified R-ketorolac as a Cdc42 and Rac1 regulator, an activity that is distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. RhoA was unaffected by R-ketorolac. A bead-based flow cytometry assay identified R-ketorolac reduced Cdc42 and Rac1 GTPase nucleotide binding in vitro and inhibition was an allosteric mechanism of action. In cell-based assays, using Skov3ip cells, and in ascites-derived ovarian tumor cells, R-ketorolac was found to inhibit the activities of Cdc42 and Rac1 and their direct downstream effectors, the phosphorylation of p21-activated kinases (PAKs). R-ketorolac, but not S-ketorolac impeded Cdc42 mediated filopodia formation, measured based on both the length and numbers of filopodia in Skov3ip and primary OvCa cells. Cell behavior assays showed that R-ketorolac but not S-ketorolac strikingly inhibited cell adhesion, migration and invasion. In a xenograft mouse model a 50% reduction in tumor cell number and decreased total tumor burden was observed with R-ketorolac as compared to S-ketorolac treatment. Finally, in a ‘phase 0′ clinical study we found Cdc42 and Rac1 activities were reduced in a time-dependent manner after a single IV dose of racemic ketorolac treatment. In sum, we established R-ketorolac inhibition on Cdc42 and Rac1 activities and subsequent physiological consequences which are critical to tumor metastasis. Our findings provide the first demonstration of selective inhibition of Cdc42 and Rac1 GTPases by an FDA approved drug in humans. Citation Format: Yuna Guo, S. Ray Kenney, Larry A. Sklar, Tione Buranda, Tudor I. Oprea, Oleg Ursu, Sarah F. Adams, Teresa Rutledge, Carolyn Muller, Lesley Lomo, Laurie G. Hudson, Angela Wandinger-Ness. R-ketorolac targets Cdc42 and Rac1 GTPases and alters ovarian tumor cell behaviors critical for invasion and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4044. doi:10.1158/1538-7445.AM2015-4044
Optical Diagnostics of Living Cells III, 2000
ABSTRACT
PLoS ONE, 2012
ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter prot... more ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.
Molecular Pharmacology, 2003
Journal of Biological Chemistry, 2011
Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of ... more Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by G␣ i-coupled G protein-coupled receptors (GPCRs) and deactivated by G␣ s-coupled GPCRs. The effects of G␣ s-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific ␣ I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step. LFA-1 3 (␣L2) and VLA-4 (␣41) are the two major and, probably, two best studied leukocyte integrins. Together with
Journal of Biological Chemistry, 2001
This work examines the affinity of ␣ 4  1-integrin and whether affinity regulation by G protein-... more This work examines the affinity of ␣ 4  1-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between ␣ 4-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (
Cytometry Part A, 2009
Introduction-Of recent clinical interest have been two related human G-protein coupled receptors:... more Introduction-Of recent clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to anti-bacterial inflammation and malignant glioma cell metastasis; and formylpeptide receptor like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow cytometry based high throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. Methods-The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein-labeled peptide ligand from FPR, FPRL1 or both. U937 cells expressing FPR and RBL cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color-coded with red fluorescent dye allowing their distinction during analysis. Compounds, cells and fluorescent ligand were sequentially combined (no-wash) in 15 μL assay volumes in 384-well plates. Throughput averaged ∼11 min per plate to analyze ∼4000 cells (∼2000/receptor) in a 2 μL aspirate from each well. Results/Conclusions-In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K i) ≤ 4 μM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, K i = 95 ± 10 nM) and FPRL1 (BB-V-115, K i = 270 ± 51 nM). Each was a selective antagonist in calcium response assays and 1 This work was supported by NIH R03 MH076381-01, U54 MH074425-01, the New Mexico Molecular Libraries Screening Center,
Cancer Research, 2011
Epithelial ovarian cancer is the major cause of gynecologic malignancy deaths. Because of their r... more Epithelial ovarian cancer is the major cause of gynecologic malignancy deaths. Because of their roles in cell adhesion and migration, Rho family GTPases have been suggested as potential therapeutic targets in human cancers. We identify the Rac1 and Cdc42 GTPases as relevant targets in papillary serous and endometriod tumors. Cdc42 is overexpressed in primary human ovarian tumors and cancer cell lines, and a novel splice variant Rac1b is upregulated in tumors of advanced stage and grade. GTPase activities in primary ascites are 3 to 6-fold higher than in cultured cells. R-Naproxen was identified by high throughput screening of a Prestwick compound library as a select non-steroidal anti-inflammatory drug (NSAID) from 23 tested that selectively targets Rac1 and Cdc42 in a bead-based assay using purified proteins. The drug is demonstrated to have positive benefit against cell behaviors required for ovarian cancer dissemination and metastasis using both cell lines and primary human tumor cell isolates. Human ovarian cells show slowed cell proliferation, as well as impaired migration, adhesion and invadopodia formation. Other NSAIDs with structural similarity (S-naproxen and 6-methoxy naphthalene acetic acid) lack these properties, while a specific Rac inhibitor NSC 23766 mimics the effects. Molecular docking shows R-Naproxen can bind the GDP-bound, but not GTP-bound Rac1, suggesting it may act by stabilizing Rac and Cdc42 in the inactive state. R-Naproxen has potential for rapid translation and efficacy in the treatment of metastatic ovarian cancer on account of FDA approval and novel activities against Rho-family GTPases. Funding for this study was generously provided by NIH grants U54MH074425, U54MH084690, R03MH081231, P30CA118100 and UNM Cancer Center FIG.0990MD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2011-LB-214
Analytical Biochemistry, 2008
Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational state... more Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here, we report a bead-based, flow cytometric assay that quantitatively measures the nucleotide binding properties of GST-chimeras for prototypical Rasfamily members Rab7 and Rho. Measurements are possible in the presence or absence of Mg 2+ , with magnesium cations principally increasing affinity and slowing nucleotide dissociation rate 8-to 10fold. GST-Rab7 exhibited a 3-fold higher affinity for GDP relative to GTP that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used γ-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP-binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real-time and quantitatively assess differences between GTPases.
ACS Chemical Biology, 2007
Biophys J, 2003
Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to... more Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing a 4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the a 4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was ;50 A ˚ between resting and Mn 21 activated receptors and ;25 A ˚ after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of a 4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.
n this report, we describe a high throughput flow cytometry based multiplexed screen for molecule... more n this report, we describe a high throughput flow cytometry based multiplexed screen for molecules that either functionally mimic rapamycin (e.g., those that are transducer of regulated CREB 1 [TORC1] pathway selective, but non-discriminating of TORC1 branchpoints) or compounds that selectively target individual branches of the yeast TORC1 pathway. The high throughput screening (HTS) and the subsequent follow-up structure activity relationship (SAR) studies identified a chemotype that contains analogs of both functional types. Obtaining structurally related analogs with differential selectivity assisted in the characterization of the probe and the pathway in which it operates. Here we report ML231 as a TORC1 Retrograde signaling (RTG) branch selective probe (CIT2 EC50 = 3.8 M). ML231 is selective on the RTG branch and only affects expression of a small subset of rapamycin-responsive genes. This selectivity appears unrelated to solubility as we showed that a more soluble analog was also selective on the RTG branch. ML231 and its analogs inhibited Sch9 phosphorylation, the major substrate and downstream effector of the TORC1 pathway. This result indicates that this chemotype affects TORC1 activity. ML231 inhibited TORC1 activity incompletely, as compared to a related analog, SID 96099781, which mimicked rapamycin and demonstrated complete TORC1 inhibition. This feature may be partially attributable to the branch selectivity of ML231. ML231 behaved very differently from SID 96099781 in the TORC1 bypass cell growth assay, showing that selective compounds may function downstream of TORC1 while SID 96099781 functions upstream or at the level of TORC1. This result suggests that the ML231 targets components downstream of TORC1, thus executing a partial function of TORC1. Collectively, this suggests that ML231 targets the RTG branch selectively. We further showed that ML231
induced the nuclear translocation of Rtg1p/Rtg3p transcription factors and the activity of ML231 on the CIT2 gene expression was dependent on the Rtg2 protein. These results further confirmed the activity of ML231 on the RTG branch. Although ML231 possesses structural and solubility challenges, this probe represents an achievement in target of rapamycin (TOR) selectivity that to our knowledge has not been previously described. ML231 will be used by investigators focusing on TORC1 signaling, RTG pathways and regulatory signaling between mitochondria and the nucleus.