Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC-expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193-204. ©2016 AACR.

Small-Molecule Inhibition of Rho/MKL/SRF Transcription in Prostate Cancer Cells: Modulation of Cell Cycle, ER Stress, and Metastasis Gene Networks.

Metastasis is the major cause of cancer deaths and control of gene transcription has emerged as a critical contributing factor. RhoA- and RhoC-induced gene transcription via the actin-regulated transcriptional co-activator megakaryocytic leukemia (MKL) and serum response factor (SRF) drive metastasis in breast cancer and melanoma. We recently identified a compound, CCG-1423, which blocks Rho/MKL/SRF-mediated transcription and inhibits PC-3 prostate cancer cell invasion. Here, we undertook a genome-wide expression study in PC-3 cells to explore the mechanism and function of this compound. There was significant overlap in the genes modulated by CCG-1423 and Latrunculin B (Lat B), which blocks the Rho/MKL/SRF pathway by preventing actin polymerization. In contrast, the general transcription inhibitor 5,6-dichloro-1-ß-d-ribofuranosyl-1H-benzimidazole (DRB) showed a markedly different pattern. Effects of CCG-1423 and Lat B on gene expression correlated with literature studies of MKL knock-down. Gene sets involved in DNA synthesis and repair, G1/S transition, and apoptosis were modulated by CCG-1423. It also upregulated genes involved in endoplasmic reticulum stress. Targets of the known Rho target transcription factor family E2F and genes related to melanoma progression and metastasis were strongly suppressed by CCG-1423. These results confirm the ability of our compound to inhibit expression of numerous Rho/MKL-dependent genes and show effects on stress pathways as well. This suggests a novel approach to targeting aggressive cancers and metastasis.

Selectivity and anti-Parkinson's potential of thiadiazolidinone RGS4 inhibitors.

Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein-protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson's disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3ß, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 µM. CCG-203769 enhances Gαq-dependent cellular Ca(2+) signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson's disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson's disease and other neural disorders.

Conditional disruption of interactions between Gαi2 and regulator of G protein signaling (RGS) proteins protects the heart from ischemic injury.

BACKGROUND: Regulator of G protein signaling (RGS) proteins suppress G protein coupled receptor signaling by catalyzing the hydrolysis of Gα-bound guanine nucleotide triphosphate. Transgenic mice in which RGS-mediated regulation of Gαi2 is lost (RGS insensitive Gαi2G184S) exhibit beneficial (protection against ischemic injury) and detrimental (enhanced fibrosis) cardiac phenotypes. This mouse model has revealed the physiological significance of RGS/Gαi2 interactions. Previous studies of the Gαi2G184S mutation used mice that express this mutant protein throughout their lives. Thus, it is unclear whether these phenotypes result from chronic or acute Gαi2G184S expression. We addressed this issue by developing mice that conditionally express Gαi2G184S. METHODS: Mice that conditionally express RGS insensitive Gαi2G184S were generated using a floxed minigene strategy. Conditional expression of Gαi2G184S was characterized by reverse transcription polymerase chain reaction and by enhancement of agonist-induced inhibition of cAMP production in isolated cardiac fibroblasts. The impact of conditional RGS insensitive Gαi2G184S expression on ischemic injury was assessed by measuring contractile recovery and infarct sizes in isolated hearts subjected to 30 min ischemia and 2 hours reperfusion. RESULTS: We demonstrate tamoxifen-dependent expression of Gαi2G184S, enhanced inhibition of cAMP production, and cardioprotection from ischemic injury in hearts conditionally expressing Gαi2G184S. Thus the cardioprotective phenotype previously reported in mice expressing Gαi2G184S does not require embryonic or chronic Gαi2G184S expression. Rather, cardioprotection occurs following acute (days rather than months) expression of Gαi2G184S. CONCLUSIONS: These data suggest that RGS proteins might provide new therapeutic targets to protect the heart from ischemic injury. We anticipate that this model will be valuable for understanding the time course (chronic versus acute) and mechanisms of other phenotypic changes that occur following disruption of interactions between Gαi2 and RGS proteins.

How prepared are college freshmen athletes for the rigors of college strength and conditioning? A survey of college strength and conditioning coaches.

Training programs for high school athletes have changed over the last 20 years. High school physical education classes have transformed into sport-specific conditioning classes with intensities matching college or professional athlete programming. In addition, involvement in private, sport-specific, training increased; but despite these advanced training methods, are high school athletes prepared for collegiate sport competition? An anonymous survey was sent to 195 Division I strength and conditioning coaches (SCC) to discern incoming college freshman athletes' physical and psychological preparedness for the rigors of collegiate training and sport competition. Fifty-seven (29%) responses were received. Strength and conditioning coaches stated that incoming college freshman athletes lack lower extremity strength, overall flexibility, and core strength as well as proper Olympic lifting technique. Strength and conditioning coaches also stated that athletes lacked the mental toughness to endure collegiate sport training in addition to claiming incoming athletes lacked knowledge of correct nutrition and recovery principles. These results suggest a lack of collegiate training/sport preparedness of high school athletes. High school strength and conditioning specialist's goal is to produce better athletes and doing so requires the strength and conditioning coach/trainer to have knowledge of how to train high school athletes. One way to assure adequate knowledge of strength and conditioning training principles is for high school coaches/trainers to be certified in the field. Strength and conditioning certifications among high school strength and conditioning coaches/trainers would encourage developmentally appropriate training and would provide universities with athletes who are prepared for the rigors of collegiate sport training/competition.

Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay.

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100µM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.

Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway.

CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identification, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain biological activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active competitor. Future work will focus on identifying the labeled protein(s).

Microfluidic interrogation and mathematical modeling of multi-regime calcium signaling dynamics.

Through microfluidic interrogation we analyzed real-time calcium responses of HEK293 cells stimulated with short pulses of the M3 muscarinic receptor ligand carbachol in two different concentration regimes. Lower ligand concentrations elicit oscillatory calcium signals while higher concentrations trigger a rapid rise that eventually settles down at a steady-state slightly above pre-stimulus levels, referred to as an acute signal. Cells were periodically pulsed with carbachol at these two concentration regimes using a custom-made microfluidic platform, and the resulting calcium signals were measured with a single fluorescent readout. Pulsed stimulations at these two concentration regimes resulted in multiple types of response patterns that each delivered complementary information about the M3 muscarinic receptor signaling pathway. These multiple types of calcium response patterns enabled development of a comprehensive mathematical model of multi-regime calcium signaling. The resulting model suggests that dephosphorylation of deactivated receptors is rate limiting for recovery of calcium signals in the acute regime (high ligand concentration), while calcium replenishment and IP3 production determine signal recovery in the oscillatory regime (low ligand concentration). This study not only provides mechanistic insight into multi-regime signaling of the M3 muscarinic receptor pathway, but also provides a general strategy for analyzing multi-regime pathways using only one fluorescent readout.

Optimization of novel nipecotic bis(amide) inhibitors of the Rho/MKL1/SRF transcriptional pathway as potential anti-metastasis agents.

CCG-1423 (1) is a novel inhibitor of Rho/MKL1/SRF-mediated gene transcription that inhibits invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. We recently reported the design and synthesis of conformationally restricted analogs (e.g., 2) with improved selectivity for inhibiting invasion versus acute cytotoxicity. In this study we conducted a survey of aromatic substitution with the goal of improving physicochemical parameters (e.g., ClogP, MW) for future efficacy studies in vivo. Two new compounds were identified that attenuated cytotoxicity even further, and were fourfold more potent than 2 at inhibiting PC-3 cell migration in a scratch wound assay. One of these (8a, CCG-203971, IC50=4.2 µM) was well tolerated in mice for 5 days at 100mg/kg/day i.p., and was able to achieve plasma levels exceeding the migration IC50 for up to 3 h.

Gαi2 signaling: friend or foe in cardiac injury and heart failure?

Receptors coupled to G proteins have many effects on the heart. Enhanced signaling by Gα(s) and Gα(q) leads to cardiac injury and heart failure, while Gα(i2) signaling in cardiac myocytes can protect against ischemic injury and ß-adrenergic-induced heart failure. We asked whether enhanced Gα(i2) signaling in mice could protect against heart failure using a point mutation in Gα(i2) (G184S), which prevents negative regulation by regulators of G protein signaling. Contrary to our expectation, it worsened effects of a genetic dilated cardiomyopathy (DCM) and catecholamine-induced cardiac injury. Gα (i2) (G184S/+) /DCM double heterozygote mice (TG9(+)Gα (i2) (G184S/+)) had substantially decreased survival compared to DCM animals. Furthermore, heart weight/body weight ratios (HW/BW) were significantly greater in TG9(+)Gα (i2) (G184S/+) mice as was expression of natriuretic peptide genes. Catecholamine injury in Gα (i2) (G184S/G184S) mutant mice produced markedly increased isoproterenol-induced fibrosis and collagen III gene expression vs WT mice. Cardiac fibroblasts from Gα (i2) (G184S/G184S) mice also showed a serum-dependent increase in proliferation and ERK phosphorylation, which were blocked by pertussis toxin and a mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor. Gα(i2) signaling in cardiac myocytes protects against ischemic injury but enhancing Gα(i2) signaling overall may have detrimental effects in heart failure, perhaps through actions on cardiac fibroblasts.

Hi-Fi transmission of periodic signals amid cell-to-cell variability.

Since information in intracellular calcium signaling is often frequency encoded, it is physiologically critical and experimentally useful to have reliable, convenient, and non-invasive methods to entrain it. Because of cell-to-cell variability, synchronization of intracellular signaling across a population of genetically identical cells can still be difficult to achieve. For intrinsically oscillatory signaling pathways, such as calcium, upon continuous stimulation, cell-to-cell variability is manifested as differences in intracellular response frequencies. Even with entrainment using periodic stimulation, cell-to-cell variability is manifested as differences in the fidelity with which extracellular inputs are converted into intracellular signals. Here we present a combined theoretical and experimental analysis that shows how to appropriately balance stimulation strength, duration, and rest intervals to achieve entrainment with high fidelity stimulation-to-response ratios for G-protein-coupled receptor-triggered intracellular calcium oscillations. We further demonstrate that stimulation parameters that give high fidelity entrainment are significantly altered upon changes in intracellular enzyme levels and cell surface receptor levels. Theoretical analysis suggests that, at key threshold values, even small changes in these protein concentrations or activities can result in precipitous changes in entrainment fidelity, with implications for pathophysiology.

Phase-locked signals elucidate circuit architecture of an oscillatory pathway.

This paper introduces the concept of phase-locking analysis of oscillatory cellular signaling systems to elucidate biochemical circuit architecture. Phase-locking is a physical phenomenon that refers to a response mode in which system output is synchronized to a periodic stimulus; in some instances, the number of responses can be fewer than the number of inputs, indicative of skipped beats. While the observation of phase-locking alone is largely independent of detailed mechanism, we find that the properties of phase-locking are useful for discriminating circuit architectures because they reflect not only the activation but also the recovery characteristics of biochemical circuits. Here, this principle is demonstrated for analysis of a G-protein coupled receptor system, the M3 muscarinic receptor-calcium signaling pathway, using microfluidic-mediated periodic chemical stimulation of the M3 receptor with carbachol and real-time imaging of resulting calcium transients. Using this approach we uncovered the potential importance of basal IP3 production, a finding that has important implications on calcium response fidelity to periodic stimulation. Based upon our analysis, we also negated the notion that the Gq-PLC interaction is switch-like, which has a strong influence upon how extracellular signals are filtered and interpreted downstream. Phase-locking analysis is a new and useful tool for model revision and mechanism elucidation; the method complements conventional genetic and chemical tools for analysis of cellular signaling circuitry and should be broadly applicable to other oscillatory pathways.

Design, synthesis and prostate cancer cell-based studies of analogs of the Rho/MKL1 transcriptional pathway inhibitor, CCG-1423.

We recently identified bis(amide) CCG-1423 (1) as a novel inhibitor of RhoA/C-mediated gene transcription that is capable of inhibiting invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. An initial structure-activity relationship study focusing on bioisosteric replacement of the amides and conformational restriction identified two compounds, 4g and 8, with improved selectivity for inhibition of RhoA/C-mediated gene transcription and attenuated cytotoxicity relative to 1. Both compounds were also capable of inhibiting cell invasion with equal efficacy to 1 but with less attendant cytotoxicity.

CCG-1423: a small-molecule inhibitor of RhoA transcriptional signaling.

Lysophosphatidic acid receptors stimulate a Galpha(12/13)/RhoA-dependent gene transcription program involving the serum response factor (SRF) and its coactivator and oncogene, megakaryoblastic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes and potential cancer therapeutic agents. Through a transcription-based high-throughput serum response element-luciferase screening assay, we identified two small-molecule inhibitors of this pathway. Mechanistic studies on the more potent CCG-1423 show that it acts downstream of Rho because it blocks SRE.L-driven transcription stimulated by Galpha(12)Q231L, Galpha(13)Q226L, RhoA-G14V, and RhoC-G14V. The ability of CCG-1423 to block transcription activated by MKL1, but not that induced by SRF-VP16 or GAL4-VP16, suggests a mechanism targeting MKL/SRF-dependent transcriptional activation that does not involve alterations in DNA binding. Consistent with its role as a Rho/SRF pathway inhibitor, CCG-1423 displays activity in several in vitro cancer cell functional assays. CCG-1423 potently (<1 mumol/L) inhibits lysophosphatidic acid-induced DNA synthesis in PC-3 prostate cancer cells, and whereas it inhibits the growth of RhoC-overexpressing melanoma lines (A375M2 and SK-Mel-147) at nanomolar concentrations, it is less active on related lines (A375 and SK-Mel-28) that express lower levels of Rho. Similarly, CCG-1423 selectively stimulates apoptosis of the metastasis-prone, RhoC-overexpressing melanoma cell line (A375M2) compared with the parental cell line (A375). CCG-1423 inhibited Rho-dependent invasion by PC-3 prostate cancer cells, whereas it did not affect the Galpha(i)-dependent invasion by the SKOV-3 ovarian cancer cell line. Thus, based on its profile, CCG-1423 is a promising lead compound for the development of novel pharmacologic tools to disrupt transcriptional responses of the Rho pathway in cancer.

Capillary electrophoresis assay for G protein-coupled receptor-mediated GTPase activity.

We describe a capillary electrophoresis (CE) assay to detect G protein-coupled receptor (GPCR)-stimulated G protein GTPase activity in cell membranes expressing alpha2A adrenoreceptor-Galphao1 wild-type (wt) or C351I mutant fusion proteins using a fluorescent, hydrolyzable GTP analogue. As no change in total fluorescence is observed by conversion of substrate to product, CE is used to separate the fluorescent substrate (*GTP) from the fluorescent product (*GDP). Using the assay, the alpha2a adrenoceptor agonist UK14,304 was shown to simulate specific production of *GDP in membranes from HEK293T cells expressing receptor-G protein fusion to 525% of basal levels with an EC50 of 0.48 +/- 0.20 microM. The EC50 increased to 9.4 +/- 5 muM with addition of the antagonist yohimbine. Nucleotide hydrolysis was increased further over agonist-stimulated levels with addition of the in vivo modulator protein RGS (regulator of G protein signaling). It is envisioned that this technique could be used for screening for novel GPCR ligands or other G protein signaling modifiers.

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors.

In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.