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.

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 (Kd 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.

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.

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.

This chapter focuses on the medicinal chemistry of dopamine D2 partial agonists that have been approved for use in humans. The realization of the "dopamine hypothesis of schizophrenia" in the 1970s led to the first effective treatment for psychotic symptoms through pharmacological modulation with dopamine D2 receptor antagonists, albeit with the on-target risk of extrapyramidal effects and, potentially, tardive dyskinesia. This breakthrough was followed by a greater knowledge of dopamine and its receptor subtypes and the realization that these receptors, especially the dopamine D2 and dopamine D3 receptors, play a role in the pathophysiology of other conditions, including schizophrenia, Parkinson's disease, addiction, depression and mania, as well as attention and eating disorders. It was the paradoxical nature of schizophrenia as well as the desire to identify effective antipsychotics with reduced extrapyramidal symptoms that ultimately led to the search for dopamine D2 receptor partial agonists, biased ligands, and agents with selectivity for other dopamine receptor subtypes.

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.

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.

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.

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.

Abstract 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, Noël 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 Graña, 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.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) environment, the so-called HSC niche, that provides pivotal factors for their maintenance. Although the cellular and molecular components of the mouse BM HSC niche have been extensively studied using genetically modified animals, relatively little is known about the counterpart human BM niche components. We previously illustrated, with a developmental tissue engineering approach, that human adult BM-derived mesenchymal stromal cells (MSCs) can develop into human bone organs (so-called ossicles) through endochondral ossification in vivo and that these human ossicles are able to maintain functional mouse HSCs. We here report that human ossicles in immunodeficient mice maintain human immature and mature hematopoiesis in vivo. Moreover, a higher percentage of human stem and progenitor cells are kept in quiescence in human ossicles as compared with mouse BM. These findings indicate that the human MSC-derived ossicles function as a hematopoietic niche and may potentially serve as a re-engineerable platform to study normal and diseased human hematopoiesis in a physiologically optimized environment.

Abstract 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

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.

Abstract 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.

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.

Abstract 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 Z' 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 lead's 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, Noël 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.

mGlu(5) 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.

Abstract 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, Noël 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

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, consistent with the relative expression levels of β2AR and β1AR in NRCF. Additionally, βAR-mediated cAMP generation was assessed via a fluorescence biosensor, revealing similar kinetics between DMR responses and cAMP generation. As such, βAR-dependent DMR responses were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, we assessed G protein-independent βAR signaling through epidermal growth factor receptor (EGFR). While inhibition of EGFR reduced the DMR response to βAR stimulation, our results demonstrate that G protein-dependent signaling produces a majority of the biological response to βAR stimulation in NRCF. Altogether, 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.