Marlene Jacobson

β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function, and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored the biological responses to βAR stimulation. We used label-free dynamic mass redistribution (DMR) to elucidate the biological response to stimulation of endogenous βAR in primary rat neonatal cardiac fibroblasts, and to begin to explore the pathways responsible for these effects. Isoproterenol (ISO, non-selective for β1/β2), salbutamol (Sal, β2-selective) and dobutamine (Dob, β1-selective) concentration-dependently induced increases in DMR response, with the Dob-induced response significantly less than either ISO- or Sal-induced DMR, consistent with the reduced expression of β1AR vs β2AR in cardiac fibroblasts. Addition of propranolol (Prop) competitively blocked the DMR effect induced by all 3 agonists, while the addition of cholera toxin (CTX) non-competitively blocked the ef...

The A(3) adenosine receptor (A3AR) is one of four receptor subtypes for adenosine and is expressed in a broad spectrum of tissues. In order to study the function of A3AR, a mouse line carrying a mutant A(3) allele was generated. Mice homozygous for targeted disruption of the A3AR gene, A3AR(-/-), are fertile and visually and histologically indistinguishable from wild type mice. The lack of a functional receptor in the A3AR(-/-) mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expression in the A3AR(-/-) mice was demonstrated by lack of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine binding to bone marrow-derived mast cell membranes that were found to express high levels of A3AR in wild type mice. In A3AR(-/-) mice, the density of A(1) and A(2A) adenosine receptor subtypes was the same as in A3AR(+/+) mice as determined by radioligand binding to brain membranes. Additionally, A(2B) receptor transcript expression was not affected by ablation of t...

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probabili...

Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder (LSD). Neuronopathic manifestations in GD include neurodegeneration, which can be severe and rapidly progressive. GBA1 mutations are also the most frequent genetic risk factors for Parkinson's disease. Dysfunction of the autophagy-lysosomal pathway represents a key pathogenic event in GBA1-associated neurodegeneration. Using an induced-pluripotent stem cell (iPSC) model of GD, we previously demonstrated that lysosomal alterations in GD neurons are linked to dysfunction of the transcription factor EB (TFEB). TFEB controls the coordinated expression of autophagy and lysosomal genes and is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). To further investigate the mechanism of autophagy-lysosomal pathway dysfunction in neuronopathic GD, we examined mTORC1 kinase activity in GD iPSC-neuronal progenitors and differentiated neurons. We found that mTORC1 i...

The oxytocin receptor belongs to the family of G-protein-coupled receptors (GPCRs) characterized by seven transmembrane spanning domains and mediates numerous neurotransmitter and hormonal functions. The cloning of this receptor was initiated to validate the use of the rhesus monkey (Macaca mulatta) as a viable animal model for therapeutic development of oxytocin receptor antagonists by ruling out potential species variations that are sometimes present among GPCRs. The rhesus monkey oxytocin receptor was cloned by the polymerase chain reaction (PCR) and expressed transiently in 293/EBNA cells. The cDNA encodes a protein of 389 amino acids and is highly homologous to that from other species, especially the human receptor which exhibits 97% identity to the rhesus protein. The cloned receptor shows a very similar pharmacological profile to the human oxytocin receptor for a variety of agonists and antagonists from various structural classes. These results substantiate the validity of the rhesus monkey as a useful model for the evaluation of human therapeutics.

We used pharmacological agents and genetic methods to determine whether the potent A3 adenosine receptor (AR) agonist 2-chloro-N 6 -(3-iodobenzyl)adenosine-5-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/ reperfusion injury in mice via the A3AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IBMECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 g/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A3AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2phenyl-4-propyl-3-pyridine-carboxylate], but not the A2AAR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A3AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprot...

Gaucher disease (GD) results from inherited mutations in the lysosomal enzyme β-glucocerobrosidase (GCase). Currently available treatment options for Type 1 GD are not efficacious for treating neuronopathic Type 2 and 3 GD due to their inability to cross the blood-brain barrier. In an effort to identify small molecules which could be optimized for CNS penetration we identified tamoxifen from a high throughput phenotypic screen on Type 2 GD patient-derived fibroblasts which reversed the disease phenotype. Structure activity studies around this scaffold led to novel molecules that displayed improved potency, efficacy and reduced estrogenic/antiestrogenic activity compared to the original hits. Here we present the design, synthesis and structure activity relationships that led to the lead molecule Compound 31.

After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can be used as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We exploited this highly specific localization of DLK-GFP as the basis for a screen of the Prestwick Compound Library™. We found that ketoconazole, a Prestwick Library compound that most dramatically affected DLK subcellular localization in our primary screen, inhibited DLK palmitoylation in a dose-depend...

In cancer, the epigenome is aberrantly reprogrammed leading to a wide range of heritable changes in gene expression, such as silencing of tumor suppressor genes (TSG). Altered epigenetic marks in cancer involve DNA methylation and histone post-translational modifications, and these come about as a result of aging and acquisition of genetic and epigenetic changes in readers/writers/editors of the epigenome. Given the reversible nature of epigenetic modifications, one goal of epigenetic therapy of cancer is to induce TSG reactivation, leading to cancer cell differentiation and cancer cell death. To identify novel targets that can reactivate epigenetically silenced genes, we developed a phenotypic-based system, YB5. YB5 is a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. We screened a natural compound library for GFP activation in YB5 and identified a novel drug class that shares an aminothiazole core structure, and has epigenetic effects that are equivalent to DNA methyltransferase inhibitor (DNMTi). Target deconvolution identified CDK9 as the target of these drugs, which reactivate gene expression without affecting DNA methylation. It is well established that CDK9, the catalytic subunit of p-TEFb, is a transcriptional activator. CDK9 in complex with its regulatory subunit, Cyclin T1 or T2, is recruited by multiple mechanisms to promote RNAPII promoter-proximal pause release by phosphorylating negative elongation factors (DSIF and NELF). In addition, phosphorylation of the C-terminal domain (CTD) of RNAPII on Serine-2 allows recruitment of RNA processing factors, which work on the nascent RNA as it emerges from RNAPII. Our new data show that long-term CDK9 inhibition can reactivate epigenetically silenced genes with minimal downregulation effects, effects which are the opposite of the canonical role of CDK9-mediated transcriptional elongation. Mechanistically, we showed that CDK9 inhibition dephosphorylates the SWI/SNF protein SMARCA4 and represses HP1α expression, both of which contribute to gene reactivation. Based on gene activation, we developed the highly selective and potent CDK9 inhibitor MC180295 (IC50 =5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes with the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer. This is the first study that links CDK9 to maintaining gene silencing at epigenetically repressed loci in mammals. Excitingly, this is also the first example of kinase inhibitors as potential drugs that reverse epigenetic silencing. Citation Format: Hanghang Zhang, Somnath Pandey, Meghan Travers, Jittasak Khowsathit, George Morton, Hongxing Sum, Carlos A. Barrero, Carmen Merali, Yasuyuki Okamoto, Takahiro Sato, Judit Garriga, Natarajan V. Bhanu, Johayra Simithy, Bela Patel, Jozef Madzo, Noel Raynal, Benjamin A. Garcia, Marlene A. Jacobson, Salim Merali, Yi Zhang, Wayne Childers, Magid Abou-Gharbia, John Karanicolas, Stephen B. Baylin, Cynthia A. Zahnow, Jaroslav Jelinek, Xavier Grana, Jean-Pierre J. Issa. Targeting CDK9 reactivates epigenetically silenced genes in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2952.

Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. We screened 3040 natural compounds and derivatives by measuring GFP expression in the YB5 cell line, a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. After 24hr treatment with the natural compounds, FACS analysis was used to check the GFP expression levels. After the primary screening (average Z9 factor = 0.6), we set a stringent criterion that GFP induction value should be more than the average of all drugs +3 standard deviations in order to be considered as positive. 33 hits were positive (positive rate = 1.1%) among which 18 hits were validated through 24hr dose curves, fluorescence microscopy and qPCR. We then grouped the positive hits based on chemical structures. In class #1, 3 positive hits were discovered from the primary screening using NDL-3040 library, then HH1 was discovered as a potent top lead from a secondary screening using 93 analogs. The Moulder Center then synthesized 56 new analogs based on the lead9s structure and 14 are positive in the YB5 system. The most potent analog (HH2) can induce 10% GFP+ cells upon 5uM treatment after 24hr. This potency is in the same range as that obtained with decitabine (a DNMT inhibitor). All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, these drugs can also reactivate many hypermethylated genes (CDH13, MGMT, SYNE1, RRAD, PYGM etc) in the YB5 cell line. These compounds also synergize with Decitabine in terms of GFP induction and many endogenous hypermethylated reactivation (RARβ, SYNE1, RRAD, FAM184A etc). For this class, we found no effects on DNA methylation, HDAC activity or effects on known histone methyltransferases/demethylases (HMT/HDM) using biochemistry-based assays. Global histone methylation and acetylation level changes were determined using mass spectrometry and we found upregulation of H3K79me2 levels. Proliferation assays showed differential sensitivity of a panel of colon cancer cell lines compared to normal cells (IMR90). These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Thus, a novel epigenetic drug class derived from natural compounds was identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noel J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Benjamin Garcia, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic screen to identify novel potential epigenetic anticancer drugs from natural compounds. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B50.

Epigenetics is the study of heritable changes in gene expression that are not caused by changes in DNA sequence. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. We screened 3040 natural compounds and derivatives by measuring GFP expression in the YB5 cell line, a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. After 24hr treatment, FACS analysis was used to check the GFP expression levels. After the primary screening (average Z’ factor = 0.6), we set a stringent criterion that GFP induction value should be more than the average of all drugs mean +3 standard deviations in order to be considered as positive. 33 hits were positive (positive rate = 1.1%) among which 18 hits were validated through 24hr dose curves, fluorescence microscopy and qPCR. We then grouped the positive hits based on chemical structures. Two classes were selected for further studies. For class#1 compounds, the most active drug induced 20% GFP at 10uM. For this class, we found no effects on DNA methylation, HDAC activity or effects on known histone methyltransferases/demethylases (HMT/HDM) using biochemistry-based assays. Global histone acetylation level was determined using mass spectrometry and we saw upregulation of H4K16ac levels. Proliferation assays showed differential sensitivity of a panel of colon cancer cell lines compared to normal cells (IMR90). These drugs also reactivated two endogenously hypermethylated genes (CDH13 and MGMT). For class#2 compounds, the most active drug activated 15% GFP at 10uM. By using a HMT/HDM biochemistry assay panel, class #2 was determined to inhibit LSD1. qPCR analysis demonstrated upregulation of endogenous LSD1 target genes. Furthermore, like known LSD1 inhibitors, these compounds significantly inhibited cell proliferation of AML cells. Both drug classes can synergize with decitabine (a DNMT inhibitor) to reactivate different tumor suppressor genes. Thus, two novel epigenetic drug classes derived from natural compounds were discovered, with activities on LSD1 and H4K16 acetylation. Citation Format: Hanghang Zhang, Noel Raynal, Takahiro Sato, Yasuyuki Okamoto, Ryan Henry, Andrew J. Andrews, George Morton, Wayne Childers, Marlene A. Jacobson, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic screen to discover novel epigenetic anticancer drugs from natural compounds. [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 3519. doi:10.1158/1538-7445.AM2015-3519

Activation of adenosine A3 receptors (A3-R) produced a dose-dependent reduction in the chemotaxis of human eosinophils to platelet-activating factor (PAF), RANTES, and leukotriene B4 (LTB4) to a maximum of 58, 48, and 52%, respectively (P < 0.02). This effect was completely reversed by selective A3-R antagonists. In contrast, activation of A1 or A2a-R did not affect PAF-induced eosinophil chemotaxis. PAF up-regulated the expression of CDllb/CD18, down-regulated L-selectin, and also increased F-actin assembly in eosinophils. The expression of these activation markers was not influenced by A3-R, A2a, or A1-R stimulation. Activation of A3-R may play an important role in inflammation by inhibiting eosinophil migration.

Activation of adenosine A3 receptors (A3-R) produced a dose-dependent reduction in the chemotaxis of human eosinophils to platelet-activating factor (PAF), RANTES, and leukotriene B4 (LTB4) to a maximum of 58, 48, and 52%, respectively (P < 0.02). This effect was completely reversed by selective A3-R antagonists. In contrast, activation of A1 or A2a-R did not affect PAF-induced eosinophil chemotaxis. PAF up-regulated the expression of CDllb/CD18, down-regulated L-selectin, and also increased F-actin assembly in eosinophils. The expression of these activation markers was not influenced by A3-R, A2a, or A1-R stimulation. Activation of A3-R may play an important role in inflammation by inhibiting eosinophil migration.

A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the α-mating factor pre-pro leader sequence and galactoseinducible promoter, GAL10. Cleavage of the pre-pro leader sequence in ...

ABSTRACT The p21 GTPases, Rho and Cdc42, regulate numerous cellular functions by binding to members of a serine/threonine protein kinase subfamily. These functions include the remodeling of the cell cytoskeleton that is a feature of cell growth and differentiation. Two of these p21 GTPase-regulated kinases, the myotonic dystrophy protein kinase-related Cdc42-binding kinases (MRCKalpha and beta), have been recently characterized in rat. Both of these proteins phosphorylate nonmuscle myosin light chain, a prerequisite for the activation of actin-myosin contractility. Here we report the cDNA cloning of the human homologue of MRCKbeta, CDC42BPB, which was found by Northern blot analysis to be expressed in a wide range of tissues. The human CDC42BPB gene maps to cytogenetic band 14q32.3 by FISH analysis.

Over half of all antibiotics target the bacterial ribosome-Nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semi-synthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a sixteen-component reaction (i.e., azide 2 and fifteen alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd va...

Optimization of a benzimidazolone template for potency and physical properties revealed 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as a key template on which to develop a new series of mGlu2 positive allosteric modulators (PAMs). Systematic investigation of aryl-SAR led to the identification of compound 27 as a potent and highly selective mGlu2 PAM with sufficient pharmacokinetics to advance to preclinical models of psychosis. Gratifyingly, compound 27 showed full efficacy in the PCP- and MK-801-induced hyperlocomotion assay in rats at CSF concentrations consistent with mGlu2 PAM potency.

... 7. Maenhaut C, Van Sande J, Libert F, Abramo-wicz M, Parmentier M, Vanderhaeghen JJ, Dumont JE, Vassart G, Schiffmann S (1990) RDC8 ... Bruns RF, Daly JW, Snyder SH (1980) Adenosine receptors in brain membranes: Bind-ing of N6-cyclohexyl [3H] adenosine and 1, 3 ...

Adenosine has both pro- and anti-inflammatory effects on neutrophils. Exposure of cultured neutrophils to 2-chloroadenosine or 5'-N-ethylcarboxamidoadenosine (NECA) decreased apoptosis after 16 h, with half-maximal responses for NECA and 2-chloroadenosine of 7.1 +/- 7.7 and 59.0 +/- 32.0 nM, respectively. Adenosine receptor agonists exhibited a rank order of potency for decreasing apoptosis of 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > NECA > or = 2-chloro-N6-cyclopentyladenosine > 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide, which is consistent with the affinity order profile established for human A2a receptors. The reduction in apoptosis in cultured neutrophils at 16 h by CGS 21680 was due to a delay in apoptosis. The addition of CGS 21680 (100 nM) increased the half-life for the appearance of apoptosis from 10.9 +/- 3.1 to 21.0 +/- 1.0 h. Addition of the non-xanthine phosphodiesterase inhibitor 4-(3-butoxy-4-m...

To determine whether adenosine A(3) receptors participate in adenosine-induced changes in coronary flow, isolated hearts from wild-type (WT) and A(3) receptor knockout (A(3)KO) mice were perfused under constant pressure and effects of nonselective and selective agonists were examined. Adenosine and the selective A(2A) agonist 2-[p-(2-carboxyethyl)]phenylethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) produced augmented maximal coronary vasodilation in A(3)KO hearts compared with WT hearts. Selective activation of A(3) receptors with 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) at nanomolar concentrations did not effect coronary flow, but at higher concentrations it produced coronary vasodilation both in WT and A(3)KO hearts. Cl-IB-MECA-induced increases in coronary flow were susceptible to both pharmacological blockade and genetic deletion of A(2A) receptors. Because deletion or blockade of adenosine A(3) receptors augmented coronary flow in...

A(3) adenosine receptors (A(3)ARs) have been implicated in regulating mast cell function and in cardioprotection during ischemia-reperfusion injury. The physiological role of A(3)ARs is unclear due to the lack of widely available selective antagonists. Therefore, we examined mice with targeted gene deletion of the A(3)AR together with pharmacological studies to determine the role of A(3)ARs in myocardial ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in isovolumic Langendorff hearts from A(3)AR(-/-) and wild-type (A(3)AR(+/+)) mice. Loss of contractile function during ischemia was unchanged, but recovery of developed pressure in hearts after reperfusion was improved in A(3)AR(-/-) compared with wild-type hearts (80 +/- 3 vs. 51 +/- 3% at 30 min). Tissue viability assessed by efflux of lactate dehydrogenase was also improved in A(3)AR(-/-) hearts (4.5 +/- 1 vs. 7.5 +/- 1 U/g). The adenosine receptor antagonist BW-A14...

The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (ø = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (ø = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding prot...

Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one), and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol 61:1297-1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-d...

A(3) adenosine receptor (AR) activation and inhibition worsen and improve, respectively, renal function after ischemia-reperfusion (I/R) injury in rats. We sought to further characterize the role of A(3) ARs in modulating renal function after either I/R or myoglobinuric renal injury. A(3) knockout mice had significantly lower plasma creatinines compared with C57 controls 24 h after I/R or myoglobinuric renal injury. C57 control mice pretreated with the A(3) AR antagonist [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5 dicarboxylate] or agonist [0.125 mg/kg N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA)] demonstrated improved or worsened renal function, respectively, after I/R or myoglobinuric renal injury. Higher doses of IB-MECA were lethal in C57 mice subjected to renal ischemia. H(1) but not H(2) histamine receptor antagonist prevented death in mice pretreated with IB-MECA before renal ischemia. Improvement in renal function was...

Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). Catecholamine stimulation of the cells induced a negative DMR deflection resulting in a concentration-dependent pharmacological response that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Pharmacological profiling of subtype-selective βAR agonists and antagonists revealed a dominant role of β2AR in mediating the DMR responses, co...