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There is an urgent need for new antibiotics to mitigate the existential threat posed by antibiotic resistance. Within the ketolide class, solithromycin has emerged as one of the most promising candidates for further development. Crystallographic studies of bacterial ribosomes and ribosomal subunits complexed with solithromycin have shed light on the nature of molecular interactions (π-stacking and H-bonding) between from the biaryl side-chain of the drug and key residues in the 50S ribosomal subunit. We have designed and synthesized a library of solithromycin analogs to study their structure-activity relationships (SAR) in tandem with new computational studies. The biological activity of each analog was evaluated in terms of ribosomal affinity (K(d) determined by fluorescence polarization), as well as minimum inhibitory concentration assays (MICs). Density functional theory (DFT) studies of a simple binding site model identify key H-bonding interactions that modulate the potency of solithromycin analogs.

Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3. These channels transduce light information into electrical signals carried by both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2), which results in attenuation or loss of color vision, daylight blindness, and reduced visual acuity. Classical techniques to measure CNG channel function utilize patch clamp electrophysiology measuring Na currents in the absence of divalent cations, yet intracellular Ca2+ regulates both light and dark adaptation in photoreceptors. We developed a fluorescence-based, high-throughput Ca2+ flux assay using yellow fluorescent protein (YFP) tagged CNGA3 channels expressed in HEK293 cells which allow monitoring for folding defects in mutant channels. The cell permeant cGMP analog, 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), was used to activate Ca2+ flux. The assay was validated using wild-type CNGA3 homomeric and heteromeric channels and ACHM2-associated homomeric mutant CNG channels, CNGA3-R427C, CNGA3-E590K, and CNGA3-L633P. Additionally, we examined two naturally occurring canine mutations causing day-blindness previously studied by patch clamp. We compared the CPT-cGMP K0.5 values of the channels with patch clamp values from previous studies. The assay provides a screen for modulation of gating and/or rescue of trafficking and/or misfolding defects in ACHM2-associated CNG channels. Importantly, the calcium flux assay is advantageous compared to patch clamp as it allows the ability to monitor CNG channel activity in the presence of calcium.

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 probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

The head and neck cancer (HNC) survivorship experience is unique among cancer populations. This review seeks to explore the HNC survivor experience associated with altered upper aerodigestive tract (UADT) function using principles of survivorship.HNC survivors experience complex physical, functional, and psychosocial challenges related to UADT dysfunction. Interventions need to address all of these dimensions being mindful of the survivor experience. Studies reveal related unmet needs of both HNC survivors and their family members. An expose of the HNC survivorship experience is timely since 2016/2017 contains the release of landmark position papers and guidelines in the field. These address the unique challenges faced by the HNC population, their management, and the triad psychosocial, functional, and physical survivor burden. Such developments will drive future care.The head and neck survivorship experience is characterized by complex changes with broad impact when examined in an experiential framework. HNC survivor care demands clinical excellence from multiple disciplines that are positioned to enact recent HNC guidelines and adopt survivorship principles. Future research in this population that explores experiential aspects of altered UADT function would be valuable in informing clinical practice. A biopsychosocial framework is presented for conceptualizing the HNC survivorship experience.

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.

Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder. 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 is hyperactive in GD cells as evidenced by increased phosphorylation of its downstream protein substrates. We also found that pharmacological inhibition of glucosylceramide synthase enzyme reversed mTORC1 hyperactivation, suggesting that increased mTORC1 activity is mediated by the abnormal accumulation of glycosphingolipids in the mutant cells. Treatment with the mTOR inhibitor Torin1 upregulated lysosomal biogenesis and enhanced autophagic clearance in GD neurons, confirming that lysosomal dysfunction is mediated by mTOR hyperactivation. Further analysis demonstrated that increased TFEB phosphorylation by mTORC1 results in decreased TFEB stability in GD cells. Our study uncovers a new mechanism contributing to autophagy-lysosomal pathway dysfunction in GD, and identifies the mTOR complex as a potential therapeutic target for treatment of GBA1-associated neurodegeneration., Summary: Using neuronopathic Gaucher's disease iPSCs, this study has identified the mTOR complex as a potential therapeutic target in GBA1-associated neurodegeneration.

Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from

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 thus serve as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We optimized an HCS assay based on this readout, which showed highly robust performance in a 96-well format. Using this assay we screened a library of 1200 FDA-approved compounds and found that ketoconazole, the compound that most dramatically affected DLK localization in our primary screen, dose-dependently inhibited DLK palmitoylation in follow-up biochemical assays. Moreover, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to trophic deprivation, a well known model of DLK-dependent pro-degenerative signaling. Our HCS platform is thus capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.

Background Hypoglossal nerve palsies are infrequent complications of head and neck radiotherapy. Treatments focus on maintaining function and prevention of abnormal airway-related swallowing events. Methods A patient with longstanding cranial neuropathies, including bilateral hypoglossal involvement, secondary to chemoradiotherapy for nasopharyngeal carcinoma, experienced repeated episodes of life-threatening complications. Initially, 2 courses of 2 weekly 24-hour intravenous methylprednisolone (IVMP) infusions were administered 2 years apart. We report the results of a third course comprising 5 weekly cycles. Results Patient-reported outcomes revealed significant improvement in swallowing function, speech, and psychosocial status. Airway invasion during swallowing and pharyngeal retention were assessed videofluoroscopically and evaluated using the Penetration-Aspiration Scale (PAS) and a residue rating scale, respectively. PAS ratings after infusions 2 and 5, improved dramatically from baseline and were maintained at 1-year follow-up. Conclusion High doses of IVMP may improve radiation-induced neuropathies. Further testing in similar patients is needed to prove reproducibility. © 2016 Wiley Periodicals, Inc. Head Neck 39: E23–E28, 2017

GZ667161 (GZ-161) is a quinuclidine-based small molecule inhibitor of the lysosomal enzyme glucosylceramide synthase. It represents an important tool molecule for studying the contribution of glycosphingolipids to disease pathology in lysosomal storage disorders such as Gaucher disease and GBA1 Parkinson’s disease. GZ667161 is not commercially available. The published synthesis involves 6 steps and proceeds in 18% overall reported yield. As part of a drug discovery program targeting Type 2 Gaucher disease we required quantities of GZ667161 that would support animal studies. To facilitate the project, we devised and executed an efficient 4-step convergent synthesis of the compound.

Tay-Sachs disease is an inherited lysosomal storage disease resulting from mutations in the lysosomal enzyme, β-hexosaminidase A, and leads to excessive accumulation of GM2 ganglioside. Tay-Sachs patients with the infantile form do not live beyond 2-4 years of age due to rapid, progressive neurodegeneration. Enzyme replacement therapy is not a therapeutic option due to its inability to cross the blood-brain barrier. As an alternative, small molecules identified from high-throughput screening could provide leads suitable for chemical optimization to target the central nervous system. We developed a new high-throughput phenotypic assay utilizing infantile Tay-Sachs patient cells based on disrupted lysosomal calcium signaling as a monitor of diseased phenotype. The assay was validated in a pilot screen on a collection of Food and Drug Administration-approved drugs to identify compounds that could reverse or attenuate the disease. Pyrimethamine, a known pharmacological chaperone of β-hexosaminidase A, was identified from the primary screen. The mechanism of action of pyrimethamine in reversing the defective lysosomal phenotype was by improving autophagy. This new high-throughput screening assay in patient cells will enable the screening of larger chemical compound collections. Importantly, this approach could lead to identification of new molecular targets previously unknown to impact the disease and accelerate the discovery of new treatments for Tay-Sachs disease.

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-dependent manner in follow-up biochemical assays. Moeroever, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to Trophic Deprivation, a well known model of DLK-dependent pro-degenerative signaling. These findings suggest that our HCS platform is capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.

Late dysphagia has a profound impact on quality of life in patients treated with chemoradiation therapy for advanced head and neck cancer and the number of survivors is growing. Traditional treatment methods are not uniformly successful in reducing the swallow impairment in these patients.Manifestations of late dysphagia are complex and do not follow a uniform trajectory. Patient's experience of eating and engagement in treatment is critical given the complex, variable nature of late dysphagia. Novel swallow treatment methods target respiratory function including coordination and strength as well as patient adherence.Head and neck cancer survivors deserve routine and fastidious surveillance beyond the 5-year survival point with respect to swallow function if the enormous biopsychosocial burden of late dysphagia is to be addressed.

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.

Investigation of a novel amino-aza-benzimidazolone structural class of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 2 (mGluR2) identified [2.2.2]-bicyclic amine 12 as an intriguing lead structure due to its promising physicochemical properties and lipophilic ligand efficiency (LLE). Further optimization led to chiral amide 18, which exhibited strong in vitro activity and attractive pharmacokinetic (PK) properties. Hypothesis-driven target design identified compound 21 as a potent, highly selective, orally bioavailable mGluR2 PAM, which addressed a CYP time-dependent inhibition (TDI) liability of 18, while maintaining excellent drug-like properties with robust in vivo activity in a clinically validated model of antipsychotic potential.

There has been resurgence in surgical interventions for oral cavity and oropharyngeal cancer in tandem with technological advances in reconstructive and ablative procedures. This paper considers the mechanisms of impact of these surgical techniques on swallow function and patient outcomes especially in light of the shifting demographics of human papilloma virus (HPV) associated oropharyngeal cancer. We present a framework for considering physiologic impairment within the context of a holistic approach to patient care based on a series of key underlying considerations.

Hypoglossal nerve palsies are infrequent complications of head and neck radiotherapy. Treatments focus on maintaining function and prevention of abnormal airway-related swallowing events.A patient with longstanding cranial neuropathies, including bilateral hypoglossal involvement, secondary to chemoradiotherapy for nasopharyngeal carcinoma, experienced repeated episodes of life-threatening complications. Initially, 2 courses of 2 weekly 24-hour intravenous methylprednisolone (IVMP) infusions were administered 2 years apart. We report the results of a third course comprising 5 weekly cycles.Patient-reported outcomes revealed significant improvement in swallowing function, speech, and psychosocial status. Airway invasion during swallowing and pharyngeal retention were assessed videofluoroscopically and evaluated using the Penetration-Aspiration Scale (PAS) and a residue rating scale, respectively. PAS ratings after infusions 2 and 5, improved dramatically from baseline and were maintained at 1-year follow-up.High doses of IVMP may improve radiation-induced neuropathies. Further testing in similar patients is needed to prove reproducibility. © 2016 Wiley Periodicals, Inc. Head Neck 39: E23-E28, 2017.

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-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.

Novel oxazolobenzimidazoles are described as potent and selective positive allosteric modulators of the metabotropic glutamate receptor 2. The discovery of this class and optimization of its physical and pharmacokinetic properties led to the identification of potent and orally bioavailable compounds (20 and 21) as advanced leads. Compound 20 (TBPCOB) was shown to have robust activity in a PCP-induced hyperlocomotion model in rat, an assay responsive to clinical antipsychotic treatments for schizophrenia.

Hit to lead optimization of (5R)-5-hexyl-3-phenyl-1,3-oxazolidin-2-one as a positive allosteric modulator of mGluR2 is described. Improvements in potency and metabolic stability were achieved through SAR on both ends of the oxazolidinone. An optimized lead compound was found to be brain penetrant and active in a rat ketamine-induced hyperlocomotion model for antipsychotic activity.

In the search for strategies to treat schizophrenia, attention has focused on enhancing NMDA receptor function. In vitro experiments show that metabotropic glutamate 5 receptor (mGluR5) activation enhances NMDA receptor activity, and in vivo experiments indicate that mGluR5 positive allosteric modulators (PAMs) are effective in preclinical assays measuring antipsychotic potential and cognition. Here we characterized the dose-effect function of CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide), an mGluR5 PAM, on novel object recognition memory in unimpaired Wistar Hannover rats (0, 10 or 30 mg/kg CDPPB) and animals with an MK-801-induced deficit (0, 3, 10, or 30 mg/kg CDPPB). In each experiment compound was given 30 min prior to the first exposure in order to affect acquisition/consolidation of the memory. In both cases, an inverted-U-shaped dose-effect function was observed, with lower doses improving recognition but higher doses having no effect. We then examined the effects of CDPPB (0, 3, 10, or 30 mg/kg) on markers of synaptic plasticity in prefrontal cortex and hippocampus, focusing on the expression and phosphorylation status of proteins involved in NMDA related signaling, including the NMDA receptor subunits NR1 and NR2B, the AMPA receptor subunit GluR1, alphaCa((2+))/CaM dependent Ser-Thr kinases II (alphaCaMKII), and the transcription factor CREB. Expression and phosphorylation of many of these proteins, particularly in the prefrontal cortex, were also characterized by an inverted-U-shaped dose-effect function. Taken together, these findings show that mGluR5 activation enhances NMDA receptor function and markers of neuronal plasticity commensurate with improvements in recognition memory. However, the effects of CDPPB are heavily dependent on dose, with higher doses being ineffective in improving recognition memory and producing downstream effects consistent with heightened NMDA receptor activation. These findings may have important implications for the development of mGluR5 PAMs to treat schizophrenia.

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.

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 macrolidefunctionalized 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 values. Computational analysis using the Site-Identification by Ligand Competitive Saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogs with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).

Background— Ecto-5′-nucleotidase (CD73)–dependent adenosine generation has been implicated in tissue protection during acute injury. Once generated, adenosine can activate cell-surface adenosine receptors (A 1 AR, A 2A AR, A 2B AR, A 3 AR). In the present study, we define the contribution of adenosine to cardioprotection by ischemic preconditioning. Methods and Results— On the basis of observations of CD73 induction by ischemic preconditioning, we found that inhibition or targeted gene deletion of cd73 abolished infarct size-limiting effects. Moreover, 5′-nucleotidase treatment reconstituted cd73 −/− mice and attenuated infarct sizes in wild-type mice. Transcriptional profiling of adenosine receptors suggested a contribution of A 2B AR because it was selectively induced by ischemic preconditioning. Specifically, in situ ischemic preconditioning conferred cardioprotection in A 1 AR −/− , A 2A AR −/− , or A 3 AR −/− mice but not in A 2B AR −/− mice or in wild-type mice after inhibition of the A 2B AR. Moreover, A 2B AR agonist treatment significantly reduced infarct sizes after ischemia. Conclusions— Taken together, pharmacological and genetic evidence demonstrate the importance of CD73-dependent adenosine generation and signaling through A 2B AR for cardioprotection by ischemic preconditioning and suggests 5′-nucleotidase or A 2B AR agonists as therapy for myocardial ischemia.

This Letter describes, for the first time, the synthesis and SAR, developed through an iterative analog library approach, that led to the discovery of the positive allosteric modulator (PAM) of the metabotropic glutamate receptor mGluR5 CPPHA. Binding to a unique allosteric binding site distinct from other mGluR5 PAMs, CPPHA has been the focus of numerous pharmacology studies by several laboratories.

We used pharmacological agents and genetic methods to determine whether the potent A(3) 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 A(3)AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 mug/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A(3)AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A(2A)AR 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 A(3)AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A(3)AR gene "knock-out" (A(3)KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A(3)KO mice in vivo and did not protect isolated perfused hearts obtained from A(3)KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A(3)AR.

We found that 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). In Chinese hamster ovary cells expressing human mGluR5, CDPPB potentiated threshold responses to glutamate in fluorometric Ca2+ assays more than 7-fold with an EC50 value of approximately 27 nM. At 1 microM, CDPPB shifted mGluR5 agonist concentration response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine 3- to 9-fold to the left. At higher concentrations, CDPPB exhibited agonist-like activity on cells expressing mGluR5. No other activity was observed on any other mGluR or cell type at concentrations up to 10 microM. CDPPB had no effect on [3H]quisqualate binding to mGluR5 but did compete for binding of [3H]methoxyPEPy, an analog of the selective mGluR5 negative allosteric modulator MPEP. CDPPB was found to be brain penetrant and reversed amphetamine-induced locomotor activity and amphetamine-induced deficits in prepulse inhibition in rats, two models sensitive to antipsychotic drug treatment. These results demonstrate that positive allosteric modulation of mGluR5 produces behavioral effects, suggesting that such modulation serves as a viable approach to increasing mGluR5 activity in vivo. These effects are consistent with the hypothesis that allosteric potentiation of mGluR5 may provide a novel approach for development of antipsychotic agents.

Adenosine signaling has been implicated in chronic lung diseases such as asthma and chronic obstructive pulmonary disease; however, the specific roles of the various adenosine receptors in processes central to these disorders are not well understood. In this study, we have investigated the role(s) of the A3 adenosine receptor in adenosine-dependent pulmonary inflammation observed in adenosine deaminase (ADA)-deficient mice. The A3 receptor (A3R) was found to be expressed in eosinophils and mucus-producing cells in the airways of ADA-deficient mice. Treatment of ADA-deficient mice with MRS 1523, a selective A3R antagonist, prevented airway eosinophilia and mucus production. Similar findings were seen in the lungs of ADA/A3 double knockout mice. Although eosinophils were decreased in the airways of ADA-deficient mice following antagonism or removal of the A3R, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3R is needed for the migration of eosinophils into the airways. These findings identify an important role for the A3R in regulating lung eosinophilia and mucus production in an environment of elevated adenosine.

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite, N -desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N -desmethylclozapine on cloned human M1-M5 muscarinic receptors. N -desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC 50 of 55 nM and was a more potent partial agonist (EC 50 , 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N -desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N -methyl- d -aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N -desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N -desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N -desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N -desmethylclozapine contributes to clozapine's clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.

In the central nervous system, re-uptake of the neurotransmitter glycine is mediated by two different glycine transporters, GlyT1 and GlyT2. GlyT2 is found in brainstem and spinal cord, whereas GlyT1 is expressed in rat forebrain regions where it is responsible for most glycine transport activity. Initially, GlyT1 and GlyT2 were pharmacologically differentiated by sarcosine, a weak selective inhibitor of GlyT1. The recently described selective and potent GlyT1 antagonist, NFPS/ALX-5407 provided an important additional tool to further characterize GlyT1 pharmacology. In the present study, we have radiolabeled the racemic form of NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (also known as ALX-5407) to investigate its interaction with GlyT1, as well as define GlyT1 expression in the rat central nervous system. Kinetic studies indicated that [3H]NFPS binds rapidly to rat forebrain membranes and dissociates with a t(1/2) of 28 +/- 5 min. [3H]NFPS labeled a saturable population of sites in rat forebrain with a Kd of 7.1+/-1.3 nM and a B(max) of 3.14 +/- 0.26 pmol/mg protein. Bound [3H]NFPS was fully and potently displaced by unlabeled NFPS, whereas glycine and sarcosine were weak, Na+-dependent inhibitors with IC50 of 1,008 and 190 microM, respectively. Additional saturation experiments indicated that glycine and sarcosine were non-competitive antagonists of [3H]NFPS binding. Functional studies revealed that NFPS was a non-competitive inhibitor of [3H]glycine uptake and does not interact with Na+ and Cl- binding sites of GlyT1. Overall, this work shows that [3H]NFPS is a valuable tool in studying GlyT1 expression and pharmacology and that NFPS interacts with GlyT1 at a site different from the transporter translocation and ion binding sites.

1. The potential neuroprotective actions of the A3 adenosine receptor (A3AR) were investigated using mice with functional deletions of the A3AR (A3AR-/-) in behavioral assessments of analgesia, locomotion, tests predictive of depression and anxiety, and the effects of mild hypoxia on cognition and neuronal survival. 2. Untreated A3AR-/- mice were tested in standard behavioral paradigms, including activity in the open field, performance in the hot-plate, tail-flick, tail-suspension, and swim tests, and in the elevated plus maze. In addition, mice were exposed repeatedly to a hypoxic environment containing carbon monoxide (CO). The cognitive effects of this treatment were assessed using the contextual fear conditioning test. After testing, the density of pyramidal neurons in the CA1, 2, and 3 subfields of the hippocampus was determined using standard histological and morphometric techniques. 3. A3AR-/- mice showed increased locomotion in the open field test, elevated plus maze (number of arm entries) and light/dark box (number of transitions). However, they spent more time immobile in two different tests of antidepressant activity (Swim and tail suspension tests). A3AR-/- mice also showed evidence of decreased nociception in the hotplate, but not tail-flick tests. Further, A3AR-/- mice were more vulnerable to hippocampal pyramidal neuron damage following episodes of carbon monoxide (CO)-induced hypoxia. One week after exposure to CO a moderate loss of pyramidal neurons was observed in all hippocampal subfields of both wild-type (A3AR+/+) and A3AR-/- mice. However, the extent of neuronal death in the CA2-3 subfields was less pronounced in A3AR+/+ than A3AR-/- mice. This neuronal loss was accompanied by a decline in cognitive function as determined using contextual fear conditioning. These histological and cognitive changes were reproduced in wild-type mice by repeatedly administering the A3AR-selective antagonist MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate 1 mg/kg i.p.). 4. These results indicate that pharmacologic or genetic suppression of A3AR function enhances some aspects of motor function and suppresses pain processing at supraspinal levels, while acting as a depressant in tests predictive of antidepressant action. Consistent with previous reports of the neuroprotective actions of A3AR agonists, A3AR-/- mice show an increase in neurodegeneration in response to repeated episodes of hypoxia.

Objective Low-dose weekly methotrexate therapy remains a mainstay in the treatment of inflammatory arthritis. Results of previous studies demonstrated that adenosine, acting at one or more of its receptors, mediates the antiinflammatory effects of methotrexate in animal models of both acute and chronic inflammation. We therefore sought to establish which receptor(s) is involved in the modulation of acute inflammation by methotrexate and its nonpolyglutamated analog MX-68 (N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]-carbonyl]-L-homoglutamic acid). Methods We studied the effects of low-dose methotrexate (0.75 mg/kg intraperitoneally [IP] every week for 5 weeks), MX-68 (2 mg/kg IP 2 days and 1 hour before induction of inflammation), dexamethasone (1.5 mg/kg IP 1 hour before induction of inflammation), or vehicle control on acute inflammation in an air-pouch model in A2A and A3 receptor knockout mice. Results Low-dose weekly methotrexate treatment increased the adenosine concentration in the exudates of all mice studied and reduced leukocyte and tumor necrosis factor α accumulation in the exudates of wild-type mice, but not in those of A2A or A3 receptor knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was equally effective at suppressing leukocyte accumulation in A2A knockout, A3 knockout, and wild-type mice, indicating that the lack of response was specific for methotrexate and MX-68. Conclusion These findings confirm that adenosine, acting at A2A and A3 receptors, is a potent regulator of inflammation. Moreover, these results provide strong evidence that adenosine, acting at either or both of these receptors, mediates the antiinflammatory effects of methotrexate and its analog MX-68.

To determine whether adenosine A3 receptors participate in adenosine-induced changes in coronary flow, isolated hearts from wild-type (WT) and A3 receptor knockout (A3KO) mice were perfused under constant pressure and effects of nonselective and selective agonists were examined. Adenosine and the selective A2A agonist 2-[ p-(2-carboxyethyl)]phenylethylamino-5′- N-ethylcarboxamidoadenosine (CGS-21680) produced augmented maximal coronary vasodilation in A3KO hearts compared with WT hearts. Selective activation of A3 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 A3KO hearts. Cl-IB-MECA-induced increases in coronary flow were susceptible to both pharmacological blockade and genetic deletion of A2A receptors. Because deletion or blockade of adenosine A3 receptors augmented coronary flow induced by nonselective adenosine and the selective A2A receptor agonist CGS-21680, we speculate that this is due to removal of an inhibitory influence associated with the A3 receptor subtype. These data indicate that the presence of adenosine A3 receptors may either inhibit or negatively modulate coronary flow mediated by other adenosine receptor subtypes.

Adenosine, a naturally-occurring nucleoside, modulates a variety of physiological and pathophysiological processes. The effects of adenosine are mediated via a family of cell surface G-protein-coupled receptors designated into four subtypes, A1, A2A, A2B and A3. The adenosine receptors have widespread tissue distribution and are often co-expressed in the same cell type. Research on adenosine receptors over the past few decades has resulted in the molecular cloning of the four subtypes from multiple species, significant progress in identifying selective agonists and antagonists and an increased understanding of the particular roles adenosine receptor subtypes play in physiological processes. This knowledge has continued to fuel considerable interest in pursuing adenosine receptors as therapeutic targets. For example, adenosine receptor agonists have been proposed for the treatment of heart arrhythmias, inflammatory diseases and in diagnosing coronary artery disease. In general, adenosine receptor agonists ...

Mice deficient in the neurotensin (NT)-1 receptor (NTR1) were developed to characterize the NT receptor subtypes that mediate various in vivo responses to NT. F2 generation (C57BL6/Sv129J) NTR1 knockout (-/-) mice were viable, and showed normal growth and overt behavior. The -/- mice lacked detectable NTR1 radioligand binding in brain, whereas NTR2 receptor binding density appeared normal compared with wild-type (+/+) mice. The gene deletion also resulted in the loss of NTR1 expression as determined by reverse transcription-polymerase chain reaction and in situ hybridization. Intracerebroventricular injection of NT (1 microg) to +/+ mice caused a robust hypothermic response (5-6 degrees C) and a significant increase in hot-plate latency. These effects were absent in the -/- mice. Similar results were obtained with i.p. injections of the brain-penetrant NT analog NMe-Arg-Lys-Pro-Trp-Tle-Leu (NT-2, 1 mg/kg i.p.). NT-2 administration also impaired rotarod performance in wild-type mice, but had no effect on motor coordination in knockout mice. In vitro, NT and NT-2 at 30 nM caused predominantly contraction and relaxation in isolated distal colon and proximal ileum, respectively, from +/+ mice, but no responses were observed with tissues from -/- mice. A similar loss of the contractile effects of NT was observed in the isolated stomach fundus from the knockout mice. In vivo, NT-2 administration reduced colonic propulsion substantially in wild-type mice. In contrast, NT-2 had no effect in NTR1 null mice, whereas the hypomotility effect of clonidine was intact. These data indicate that NTR1 mediates several of the central and peripheral effects of NT.

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 gene reactivation, leading to cancer cell differentiation and cell death. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. To explore this idea, our lab developed a phenotypic-based system (YB5) 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. By screening an NDL-3040 natural compounds library and grouping the compounds based on chemical structures, we identified two main drug classes. We then synthesized 77 new analogs based on class #1’s lead’s structure and 23 were positive in the YB5 system. The most potent analog (HH2) can induce ~60% GFP+ cells upon 500nM treatment after 96hr. All the positive hits can also be validated in two other cancer cell lines (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes (MGMT, RARβ, etc) can also be re-expressed upon drug treatment. We then performed RNA-seq analysis to identify global gene expression changes following drug treatment. We observed that most genes (2964 genes) were upregulated upon HH1 treatment (10uM) and that many of the upregulated genes were expressed in normal tissues but repressed in cancer, indicating that they might be potential tumor suppressor genes (TSGs). Consistent with this, 94 TSGs could be reactivated upon 10uM drug treatment. These drug target upregulated genes were also enriched for hypermethylation. By performing connectivity mapping using RNA-seq, we identified X as the class #1 drug target. The on-target effect could be further validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Additionally, when we overexpressed wild-type X, we saw that GFP induction as well as endogenous gene reactivations can be inhibited. Strikingly, by using GFP induction as readout to optimize drugs, we found that the in vitro IC50 against X for our top lead compound (HH2) is only 5nM and it is at least 22-fold selective for X over other X family members. 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, Noël J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judith Garriga, Benjamin Garcia, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Graña, Magid Abou-Gharbia, Jean-Pierre J. Issa. Identifying novel potential epigenetic anti-cancer drugs from natural compounds using a phenotypic-based screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5064. doi:10.1158/1538-7445.AM2017-5064

The two mammalian neuropeptides NPFF and NPAF have been shown to have important roles in nociception, anxiety, learning and memory, and cardiovascular reflex. Two receptors (FF1 and FF2) have been molecularly identified for NPFF and NPAF. We have now characterized a novel gene designated NPVF that encodes two neuropeptides highly similar to NPFF. NPVF mRNA was detected specifically in a region between the dorsomedial and ventromedial hypothalamic nuclei. NPVF-derived peptides displayed higher affinity for FF1 than NPFF-derived peptides, but showed poor agonist activity for FF2. Following intracerebral ventricular administration, a NPVF-derived peptide blocked morphine-induced analgesia more potently than NPFF in both acute and inflammatory models of pain. In situ hybridization analysis revealed distinct expression patterns of FF1 and FF2 in the rat central nervous system. FF1 was broadly distributed, with the highest levels found in specific regions of the limbic system and the brainstem where NPVF-producing neurons were shown to project. FF2, in contrast, was mostly expressed in the spinal cord and some regions of the thalamus. These results indicate that the endogenous ligands for FF1 and FF2 are NPVF- and NPFF-derived peptides, respectively, and suggest that the NPVF/FF1 system may be an important part of endogenous anti-opioid mechanism.

Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin and calcitonin (CT) are structurally and functionally related neuropeptides. It has recently been shown that the molecular pharmacology of CGRP and ADM is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Furthermore, RAMP proteins have also been shown to govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. In this study, we have cloned the rat RAMP family and characterized the pharmacology of rat CGRP and ADM receptors. Rat RAMP1, RAMP2 and RAMP3 shared 72%, 69% and 85% homology with their respective human homologues. As expected CRLR-RAMP1 coexpression conferred sensitivity to CGRP, whilst association of RAMP2 or RAMP3 with CRLR conferred high affinity ADM binding. Using specific oligonucleotides we have determined the expression of RAMP1, RAMP2 and RAMP3 mRNAs in the rat central nervous system by in situ hybridization. The localization of RAMP mRNAs was heterogeneous. RAMP1 mRNA was predominantly expressed in cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most abundant in hypothalamus; and RAMP3 was restrictively expressed in thalamic nuclei. Interestingly, in specific brain areas only a single RAMP mRNA was often detected, suggesting mutual exclusivity in expression. These data allow predictions to be made of where each RAMP protein may heterodimerize with its partner G-protein-coupled receptor(s) at the cellular level and consequently advance current understanding of cellular sites of action of CGRP, ADM, amylin and CT. Furthermore, these localization data suggest that the RAMP family may associate and modify the behaviour of other, as yet unidentified neurotransmitter receptors.

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). The EPIC-BT by Corning was used to measure DMR responses in primary isolated NRCF treated with various βAR and EGFR ligands. Additional molecular assays for cAMP generation and receptor internalization responses were used to correlate the DMR findings with established βAR signaling pathways. Catecholamine stimulation of NRCF induced a concentration-dependent negative DMR deflection that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Subtype-selective βAR ligand profiling revealed a dominant role for β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. βAR-mediated cAMP generation profiles revealed similar kinetics to DMR responses, each of which were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, G protein-independent βAR signaling through epidermal growth factor receptor (EGFR) was assessed, revealing a smaller but significant contribution of this pathway to the DMR response to βAR stimulation. Measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein-dependent and –independent pathways.

mGlu5 receptors have been implicated in the pathophysiology of schizophrenia and cognitive dysfunction, and activation of these receptors has been proposed as a potential therapeutic strategy for treating these diseases. This chapter describes the recent progress in the discovery and development of mGlu5 positive allosteric modulators (PAMs) as a novel therapeutic approach which does not involve a direct interaction with dopamine receptors. This new class of molecules is structurally very diverse and includes several drug-like chemical series. The pharmacological activity shown by these molecules in preclinical studies confirms their therapeutic potential in all domains of schizophrenia, including positive and negative symptoms and cognitive abnormalities. These data support the development of mGlu5 PAMs as novel antipsychotic drugs that resonate with the glutamatergic hypofunction hypothesis of schizophrenia. The fine tuning of the functional activity and the clarification of biased signaling pathways in different chemical series may provide clues to achieve efficacy and a good safety profile for novel pharmacotherapies.

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 effects of each agonist. Overall, the CTX-sensitive DMR effect accounted for approximately 75% of the response, while 25% of the biological response was CTX-insensitive. To confirm that the CTX-sensitive DMR response was mediated by downstream Gαs protein-dependent signaling, cAMP generation was assessed via FRET where ISO produced a similar concentration-effect curve and EC50 value as attained with DMR. All of the cAMP generation response was ablated by both Prop and CTX, indicating that the CTX-sensitive acute DMR effect to βAR stimulation is directly related to cAMP generation. Since ~25% of the DMR effect was not CTX-sensitive, other Gαs protein-independent signaling pathways are presumably activated in response to βAR stimulation in cardiac fibroblasts. To assess this possibility, EGFR internalization was quantitated as an indicator of βAR-mediated EGFR transactivation. EGFR internalization increased in response to ISO stimulation in a concentration-dependent manner, with a similar EC50 value as attained in the DMR assay. Defining the relative contributions of different βAR pathways in cardiac fibroblasts will help establish the importance of these pathways as potential therapeutic targets in the heart.

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. In an effort to identify novel targets that can reactivate hypermethylated silenced genes, our lab 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. By screening an NDL-3040 library and grouping the molecules based on chemical structures, we were able to identify three main drug classes. The Moulder Center then synthesized 60 new analogs based on class #1's lead's structure and 15 are positive in the YB5 system. The most potent analog can induce 10% GFP+ cells upon 500nM treatment. All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes can be reactivated upon drug treatment. Also, GFP positive cells show higher endogenous gene reactivation than unsorted and GFP negative cells. By performing RNA-seq analysis upon class #1 top lead treatment followed by connectivity mapping, we identified X as the class #1 drug target. The on-target effect can be validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Proliferation assays showed differential sensitivities of a panel of colon cancer cell lines compared to normal cells. These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Besides class #1 drugs, a novel class of LSD1 inhibitors was identified and the most active drug can induce 15% GFP at 5uM. Consistent with LSD1 inhibition, many known LSD1 target genes can also be upregulated. Like known LSD1 inhibitors, these compounds significantly inhibited proliferation of AML cells. We also identified some known natural compounds that have epigenetic activities, including arsenic trioxide, cardiac glycosides, and toyocamycin. Thus, many novel epigenetic drug classes derived from natural compounds were identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noël J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judit Garriga, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Graña, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4701.

The human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence. The cDNA encodes a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine [125I]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a Kd = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) > or = (R)-N6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blocked by xanthine antagonists, most potently by 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosine, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partial agonist. When measured in the presence of antagonists, the dose-response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted. Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widespread and, in contrast to the A1, A2a, and A2b transcripts, the most abundant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. This raises the possibility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.