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David L. Roman - One of the best experts on this subject based on the ideXlab platform.
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High-resolution structure of RGS17 suggests a role for Ca2+in promoting the GTPase-activating protein activity by RZ subfamily members.
Journal of Biological Chemistry, 2019Co-Authors: Monita Sieng, Michael P. Hayes, David L. Roman, Joseph B. O'brien, C. Andrew Fowler, Jon C. D. Houtman, Angeline M. LyonAbstract:Regulator of G protein signaling (RGS) proteins are negative regulators of G protein-coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gαo, Gαz, and Gαi1-3 proteins in the nervous system and thereby inhibit downstream pathways, including those involved in Ca2+-dependent signaling. In contrast to other RGS proteins, little is known about RZ subfamily structure and regulation. Herein, we present the 1.5-Å crystal structure of RGS17, the most complete and highest-resolution structure of an RZ subfamily member to date. RGS17 cocrystallized with Ca2+ bound to conserved positions on the predicted Gα-binding surface of the protein. Using NMR chemical shift perturbations, we confirmed that Ca2+ binds in solution to the same site. Furthermore, RGS17 had greater than 55-fold higher affinity for Ca2+ than for Mg2+ Finally, we found that Ca2+ promotes interactions between RGS17 and activated Gα and decreases the Km for GTP hydrolysis, potentially by altering the binding mechanism between these proteins. Taken together, these findings suggest that Ca2+ positively regulates RGS17, which may represent a general mechanism by which increased Ca2+ concentration promotes the GAP activity of the RZ subfamily, leading to RZ-mediated inhibition of Ca2+ signaling.
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screen targeting lung and prostate cancer oncogene identifies novel inhibitors of RGS17 and problematic chemical substructures
SLAS DISCOVERY: Advancing the Science of Drug Discovery, 2018Co-Authors: Christopher R Bodle, Michael P. Hayes, Josephine H Schamp, Jonathan A Doorn, Joseph B Obrien, Meng Wu, David L. RomanAbstract:Regulator of G protein signaling (RGS) proteins temporally regulate heterotrimeric G protein signaling cascades elicited by G protein–coupled receptor activation and thus are essential for cell homeostasis. The dysregulation of RGS protein expression has been linked to several pathologies, spurring discovery efforts to identify small-molecule inhibitors of these proteins. Presented here are the results of a high-throughput screening (HTS) campaign targeting RGS17, an RGS protein reported to be inappropriately upregulated in several cancers. A screen of over 60,000 small molecules led to the identification of five hit compounds that inhibit the RGS17-Gαo protein-protein interaction. Chemical and biochemical characterization demonstrated that three of these hits inhibited the interaction through the decomposition of parent compound into reactive products under normal chemical library storage/usage conditions. Compound substructures susceptible to decomposition are reported and the decomposition process char...
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natural products discovered in a high throughput screen identified as inhibitors of RGS17 and as cytostatic and cytotoxic agents for lung and prostate cancer cell lines
Journal of Natural Products, 2017Co-Authors: Christopher R Bodle, Michael P. Hayes, Michael A James, Duncan I Mackie, Jon C. D. Houtman, Josephine H Schamp, Michael R Miller, Michael D Henry, Jonathan A Doorn, David L. RomanAbstract:Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17’s pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17–Gαo interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (1–4) were identified with IC50 values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dep...
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Regulator of G Protein Signaling 17 as a Negative Modulator of GPCR Signaling in Multiple Human Cancers
The AAPS Journal, 2016Co-Authors: Michael P. Hayes, David L. RomanAbstract:Regulators of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling networks by terminating signals produced by active Gα subunits. RGS17, a member of the RZ subfamily of RGS proteins, is typically only expressed in appreciable amounts in the human central nervous system, but previous works have shown that RGS17 expression is selectively upregulated in a number of malignancies, including lung, breast, prostate, and hepatocellular carcinoma. In addition, this upregulation of RGS17 is associated with a more aggressive cancer phenotype, as increased proliferation, migration, and invasion are observed. Conversely, decreased RGS17 expression diminishes the response of ovarian cancer cells to agents commonly used during chemotherapy. These somewhat contradictory roles of RGS17 in cancer highlight the need for selective, high-affinity inhibitors of RGS17 to use as chemical probes to further the understanding of RGS17 biology. Based on current evidence, these compounds could potentially have clinical utility as novel chemotherapeutics in the treatment of lung, prostate, breast, and liver cancers. Recent advances in screening technologies to identify potential inhibitors coupled with increasing knowledge of the structural requirements of RGS-Gα protein-protein interaction inhibitors make the future of drug discovery efforts targeting RGS17 promising. This review highlights recent findings related to RGS17 as both a canonical and atypical RGS protein, its role in various human disease states, and offers insights on small molecule inhibition of RGS17.
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RGS17 an emerging therapeutic target for lung and prostate cancers
Future Medicinal Chemistry, 2013Co-Authors: Christopher R Bodle, Duncan I Mackie, David L. RomanAbstract:Ligands for G-protein-coupled receptors (GPCRs) represent approximately 50% of currently marketed drugs. RGS proteins modulate heterotrimeric G proteins and, thus, GPCR signaling, by accelerating the intrinsic GTPase activity of the Gα subunit. Given the prevalence of GPCR targeted therapeutics and the role RGS proteins play in G protein signaling, some RGS proteins are emerging as targets in their own right. One such RGS protein is RGS17. Increased RGS17 expression in some prostate and lung cancers has been demonstrated to support cancer progression, while reduced expression of RGS17 can lead to development of chemotherapeutic resistance in ovarian cancer. High-throughput screening is a powerful tool for lead compound identification, and utilization of high-throughput technologies has led to the discovery of several RGS inhibitors, thus far. As screening technologies advance, the identification of novel lead compounds the subsequent development of targeted therapeutics appears promising.
Mandi M Murph - One of the best experts on this subject based on the ideXlab platform.
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Regulators of G-Protein signaling RGS10 and RGS17 regulate chemoresistance in ovarian cancer cells
Molecular Cancer, 2010Co-Authors: Shelley B Hooks, Phillip Callihan, Molly K Altman, Jillian H Hurst, Mandi M MurphAbstract:Background A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. Results In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. Conclusions Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
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regulators of g protein signaling rgs10 and RGS17 regulate chemoresistance in ovarian cancer cells
Molecular Cancer, 2010Co-Authors: Shelley B Hooks, Phillip Callihan, Molly K Altman, Jillian H Hurst, Mandi M MurphAbstract:A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
Duncan I Mackie - One of the best experts on this subject based on the ideXlab platform.
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natural products discovered in a high throughput screen identified as inhibitors of RGS17 and as cytostatic and cytotoxic agents for lung and prostate cancer cell lines
Journal of Natural Products, 2017Co-Authors: Christopher R Bodle, Michael P. Hayes, Michael A James, Duncan I Mackie, Jon C. D. Houtman, Josephine H Schamp, Michael R Miller, Michael D Henry, Jonathan A Doorn, David L. RomanAbstract:Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17’s pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17–Gαo interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (1–4) were identified with IC50 values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dep...
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RGS17 an emerging therapeutic target for lung and prostate cancers
Future Medicinal Chemistry, 2013Co-Authors: Christopher R Bodle, Duncan I Mackie, David L. RomanAbstract:Ligands for G-protein-coupled receptors (GPCRs) represent approximately 50% of currently marketed drugs. RGS proteins modulate heterotrimeric G proteins and, thus, GPCR signaling, by accelerating the intrinsic GTPase activity of the Gα subunit. Given the prevalence of GPCR targeted therapeutics and the role RGS proteins play in G protein signaling, some RGS proteins are emerging as targets in their own right. One such RGS protein is RGS17. Increased RGS17 expression in some prostate and lung cancers has been demonstrated to support cancer progression, while reduced expression of RGS17 can lead to development of chemotherapeutic resistance in ovarian cancer. High-throughput screening is a powerful tool for lead compound identification, and utilization of high-throughput technologies has led to the discovery of several RGS inhibitors, thus far. As screening technologies advance, the identification of novel lead compounds the subsequent development of targeted therapeutics appears promising.
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A High Throughput Screen for RGS Proteins Using Steady State Monitoring of Free Phosphate Formation
PLOS ONE, 2013Co-Authors: C. Aaron Monroy, Duncan I Mackie, David L. RomanAbstract:G-protein coupled receptors are a diverse group that are the target of over 50% of marketed drugs. Activation of these receptors results in the exchange of bound GDP for GTP in the Gα subunit of the heterotrimeric G-protein. The Gα subunit dissociates from the β/γ subunits and both proceed to affect downstream signaling targets. The signal terminates by the hydrolysis of GTP to GDP and is temporally regulated by Regulators of G-protein Signaling (RGS) proteins that act as GTPase Activating Proteins (GAPs). This makes RGS proteins potentially desirable targets for “tuning” the effects of current therapies as well as developing novel pharmacotherapies. Current methods for evaluating RGS activity depend on laborious and/or expensive techniques. In this study we developed a simple and inexpensive assay for the steady state analysis of RGS protein GAP activity, using RGS4, RGS8 and RGS17 as models. Additionally, we report the use of RGS4 as a model for high throughput assay development. After initial setup, this assay can be conducted in a highly parallel fashion with a read time of less than 8 minutes for a 1536-well plate. The assay exhibited a robust Z-factor of 0.6 in a 1536-well plate. We conducted a pilot screen for inhibitors using a small, 2320 compound library. From this screen, 13 compounds were identified as compounds for further analysis. The successful development of this assay for high-throughput screening provides a low cost, high speed, simple method for assessing RGS protein activity.
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development of a novel high throughput screen and identification of small molecule inhibitors of the gα RGS17 protein protein interaction using alphascreen
Journal of Biomolecular Screening, 2011Co-Authors: Duncan I Mackie, David L. RomanAbstract:In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 50% when compared ...
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Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen
Journal of Biomolecular Screening, 2011Co-Authors: Duncan I Mackie, David L. RomanAbstract:In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 50% when compared ...
Shelley B Hooks - One of the best experts on this subject based on the ideXlab platform.
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Regulators of G-Protein signaling RGS10 and RGS17 regulate chemoresistance in ovarian cancer cells
Molecular Cancer, 2010Co-Authors: Shelley B Hooks, Phillip Callihan, Molly K Altman, Jillian H Hurst, Mandi M MurphAbstract:Background A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. Results In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. Conclusions Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
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regulators of g protein signaling rgs10 and RGS17 regulate chemoresistance in ovarian cancer cells
Molecular Cancer, 2010Co-Authors: Shelley B Hooks, Phillip Callihan, Molly K Altman, Jillian H Hurst, Mandi M MurphAbstract:A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
Joel Gelernter - One of the best experts on this subject based on the ideXlab platform.
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Variation in regulator of G-protein signaling 17 gene (RGS17) is associated with multiple substance dependence diagnoses
Behavioral and Brain Functions, 2012Co-Authors: Huiping Zhang, Fan Wang, Henry R Kranzler, Raymond F Anton, Joel GelernterAbstract:Background RGS17 and RGS20 encode two members of the regulator of G-protein signaling RGS-Rz subfamily. Variation in these genes may alter their transcription and thereby influence the function of G protein-coupled receptors, including opioid receptors, and modify risk for substance dependence. Methods The association of 13 RGS17 and eight RGS20 tag single nucleotide polymorphisms (SNPs) was examined with four substance dependence diagnoses (alcohol (AD), cocaine (CD), opioid (OD) or marijuana (MjD)] in 1,905 African Americans (AAs: 1,562 cases and 343 controls) and 1,332 European Americans (EAs: 981 cases and 351 controls). Analyses were performed using both χ ^2 tests and logistic regression analyses that covaried sex, age, and ancestry proportion. Correlation of genotypes and mRNA expression levels was assessed by linear regression analyses. Results Seven RGS17 SNPs showed a significant association with at least one of the four dependence traits after a permutation-based correction for multiple testing (0.003≤ P _ empirical ≤0.037). The G allele of SNP rs596359, in the RGS17 promoter region, was associated with AD, CD, OD, or MjD in both populations (0.005≤ P _ empirical ≤0.019). This allele was also associated with significantly lower mRNA expression levels of RGS17 in YRI subjects ( P = 0.002) and non-significantly lower mRNA expression levels of RGS17 in CEU subjects ( P = 0.185). No RGS20 SNPs were associated with any of the four dependence traits in either population. Conclusions This study demonstrated that variation in RGS17 was associated with risk for substance dependence diagnoses in both AA and EA populations.
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variation in regulator of g protein signaling 17 gene RGS17 is associated with multiple substance dependence diagnoses
Behavioral and Brain Functions, 2012Co-Authors: Huiping Zhang, Fan Wang, Henry R Kranzler, Raymond F Anton, Joel GelernterAbstract:Background RGS17 and RGS20 encode two members of the regulator of G-protein signaling RGS-Rz subfamily. Variation in these genes may alter their transcription and thereby influence the function of G protein-coupled receptors, including opioid receptors, and modify risk for substance dependence.