Publications by authors named "Deborah Nguyen"

19 Publications

  • Page 1 of 1

Modeling Tumor Phenotypes In Vitro with Three-Dimensional Bioprinting.

Cell Rep 2019 01;26(3):608-623.e6

Department of Medical and Molecular Genetics, Oregon Health & Science University, Portland, OR 97201, USA; Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97201, USA. Electronic address:

The tumor microenvironment plays a critical role in tumor growth, progression, and therapeutic resistance, but interrogating the role of specific tumor-stromal interactions on tumorigenic phenotypes is challenging within in vivo tissues. Here, we tested whether three-dimensional (3D) bioprinting could improve in vitro models by incorporating multiple cell types into scaffold-free tumor tissues with defined architecture. We generated tumor tissues from distinct subtypes of breast or pancreatic cancer in relevant microenvironments and demonstrate that this technique can model patient-specific tumors by using primary patient tissue. We assess intrinsic, extrinsic, and spatial tumorigenic phenotypes in bioprinted tissues and find that cellular proliferation, extracellular matrix deposition, and cellular migration are altered in response to extrinsic signals or therapies. Together, this work demonstrates that multi-cell-type bioprinted tissues can recapitulate aspects of in vivo neoplastic tissues and provide a manipulable system for the interrogation of multiple tumorigenic endpoints in the context of distinct tumor microenvironments.
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http://dx.doi.org/10.1016/j.celrep.2018.12.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366459PMC
January 2019

Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis.

PLoS One 2019 2;14(1):e0208958. Epub 2019 Jan 2.

Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, Chapel Hill, North Carolina, United States of America.

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208958PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314567PMC
September 2019

Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME/Tox Functions.

iScience 2018 Apr 27;2:156-167. Epub 2018 Mar 27.

Organovo, Inc., San Diego, CA 92121, USA. Electronic address:

The human intestinal mucosa is a critical site for absorption, distribution, metabolism, and excretion (ADME)/Tox studies in drug development and is difficult to recapitulate in vitro. Using bioprinting, we generated three-dimensional (3D) intestinal tissue composed of human primary intestinal epithelial cells and myofibroblasts with architecture and function to model the native intestine. The 3D intestinal tissue demonstrates a polarized epithelium with tight junctions and specialized epithelial cell types and expresses functional and inducible CYP450 enzymes. The 3D intestinal tissues develop physiological barrier function, distinguish between high- and low-permeability compounds, and have functional P-gp and BCRP transporters. Biochemical and histological characterization demonstrate that 3D intestinal tissues can generate an injury response to compound-induced toxicity and inflammation. This model is compatible with existing preclinical assays and may be implemented as an additional bridge to clinical trials by enhancing safety and efficacy prediction in drug development.
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http://dx.doi.org/10.1016/j.isci.2018.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135981PMC
April 2018

Bioprinted three dimensional human tissues for toxicology and disease modeling.

Drug Discov Today Technol 2017 Mar 28;23:37-44. Epub 2017 Mar 28.

Organovo Inc., 6275 Nancy Ridge Dr., San Diego, CA 92121, United States.

The high rate of attrition among clinical-stage therapies, due largely to an inability to predict human toxicity and/or efficacy, underscores the need for in vitro models that better recapitulate in vivo human biology. In much the same way that additive manufacturing has revolutionized the production of solid objects, three-dimensional (3D) bioprinting is enabling the automated production of more architecturally and functionally accurate in vitro tissue culture models. Here, we provide an overview of the most commonly used bioprinting approaches and how they are being used to generate complex in vitro tissues for use in toxicology and disease modeling research.
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http://dx.doi.org/10.1016/j.ddtec.2017.03.001DOI Listing
March 2017

3D Proximal Tubule Tissues Recapitulate Key Aspects of Renal Physiology to Enable Nephrotoxicity Testing.

Front Physiol 2017 8;8:123. Epub 2017 Mar 8.

Organovo, Inc. San Diego, CA, USA.

Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.
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http://dx.doi.org/10.3389/fphys.2017.00123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340751PMC
March 2017

Editor's Highlight: Modeling Compound-Induced Fibrogenesis In Vitro Using Three-Dimensional Bioprinted Human Liver Tissues.

Toxicol Sci 2016 12 7;154(2):354-367. Epub 2016 Sep 7.

Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.

Compound-induced liver injury leading to fibrosis remains a challenge for the development of an Adverse Outcome Pathway useful for human risk assessment. Latency to detection and lack of early, systematically detectable biomarkers make it difficult to characterize the dynamic and complex intercellular interactions that occur during progressive liver injury. Here, we demonstrate the utility of bioprinted tissue constructs comprising primary hepatocytes, hepatic stellate cells, and endothelial cells to model methotrexate- and thioacetamide-induced liver injury leading to fibrosis. Repeated, low-concentration exposure to these compounds enabled the detection and differentiation of multiple modes of liver injury, including hepatocellular damage, and progressive fibrogenesis characterized by the deposition and accumulation of fibrillar collagens in patterns analogous to those described in clinical samples obtained from patients with fibrotic liver injury. Transient cytokine production and upregulation of fibrosis-associated genes ACTA2 and COL1A1 mimics hallmark features of a classic wound-healing response. A surge in proinflammatory cytokines (eg, IL-8, IL-1β) during the early culture time period is followed by concentration- and treatment-dependent alterations in immunomodulatory and chemotactic cytokines such as IL-13, IL-6, and MCP-1. These combined data provide strong proof-of-concept that 3D bioprinted liver tissues can recapitulate drug-, chemical-, and TGF-β1-induced fibrogenesis at the cellular, molecular, and histological levels and underscore the value of the model for further exploration of compound-specific fibrogenic responses. This novel system will enable a more comprehensive characterization of key attributes unique to fibrogenic agents during the onset and progression of liver injury as well as mechanistic insights, thus improving compound risk assessment.
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http://dx.doi.org/10.1093/toxsci/kfw169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139067PMC
December 2016

Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro.

PLoS One 2016 7;11(7):e0158674. Epub 2016 Jul 7.

Roche Pharmaceutical Research and Early Development, Roche Innovation Center, Basel, Switzerland.

Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158674PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936711PMC
July 2017

Projections and opinions from 100 experts in long-acting reversible contraception.

Contraception 2015 Dec 24;92(6):543-52. Epub 2015 Oct 24.

University of California, San Francisco, Advancing New Standards in Reproductive Health, 1330 Broadway, Suite 1100, Oakland, CA 94612, USA.

Objective: This survey of published researchers of long-acting reversible contraceptives (LARCs) examines their opinions about important barriers to LARC use in the United States (US), projections for LARC use in the absence of barriers and attitudes toward incentives for clinicians to provide and women to use LARC methods.

Study Design: We identified 182 authors of 59 peer-reviewed papers on LARC use published since 2013. A total of 104 completed an internet survey. We used descriptive and multivariate analyses to assess LARC use barriers and respondent characteristics associated with LARC projections and opinions.

Results: The most commonly identified barrier was the cost of the device (63%), followed by women's knowledge of safety, method acceptability and expectations about use. A shortage of trained providers was a commonly cited barrier, primarily of primary care providers (49%). Median and modal projections of LARC use in the absence of these barriers were 25-29% of contracepting women. There was limited support for provider incentives and almost no support for incentives for women to use LARC methods, primarily out of concern about coercion.

Conclusions: Clinical and social science LARC experts project at least a doubling of the current US rate of LARC use if barriers to method provision and adoption are removed. While LARC experts recognize the promise of LARC methods to better meet women's contraceptive needs, they anticipate that the majority of US women will not choose LARC methods. Reducing unintended pregnancy rates will depend on knowledge, availability and use of a wider range of methods of contraception to meet women's individual needs.

Implications: Efforts to increase LARC use need to meet the dual goals of increasing access to LARC methods and protecting women's reproductive autonomy. To accomplish this, we need reasonable expectations for use, provider training, low-cost devices and noncoercive counseling, rather than incentives for provision or use.
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http://dx.doi.org/10.1016/j.contraception.2015.10.003DOI Listing
December 2015

G-protein-coupled bile acid receptor 1 (GPBAR1, TGR5) agonists reduce the production of proinflammatory cytokines and stabilize the alternative macrophage phenotype.

J Med Chem 2014 Dec 9;57(24):10343-54. Epub 2014 Dec 9.

Global Discovery Chemistry, ‡Autoimmunity, Transplantation and Inflammation, and §Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research , Novartis Campus, CH-4056 Basel, Switzerland.

GPBAR1 (also known as TGR5) is a G-protein-coupled receptor (GPCR) that triggers intracellular signals upon ligation by various bile acids. The receptor has been studied mainly for its function in energy expenditure and glucose homeostasis, and there is little information on the role of GPBAR1 in the context of inflammation. After a high-throughput screening campaign, we identified isonicotinamides exemplified by compound 3 as nonsteroidal GPBAR1 agonists. We optimized this series to potent derivatives that are active on both human and murine GPBAR1. These agonists inhibited the secretion of the proinflammatory cytokines TNF-α and IL-12 but not the antiinflammatory IL-10 in primary human monocytes. These effects translate in vivo, as compound 15 inhibits LPS induced TNF-α and IL-12 release in mice. The response was GPBAR1 dependent, as demonstrated using knockout mice. Furthermore, agonism of GPBAR1 stabilized the phenotype of the alternative, noninflammatory, M2-like type cells during differentiation of monocytes into macrophages. Overall, our results illustrate an important regulatory role for GPBAR1 agonists as controllers of inflammation.
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http://dx.doi.org/10.1021/jm501052cDOI Listing
December 2014

Identification and characterization of small molecule modulators of the Epstein-Barr virus-induced gene 2 (EBI2) receptor.

J Med Chem 2014 Apr 15;57(8):3358-68. Epub 2014 Apr 15.

Global Discovery Chemistry; ‡Developmental & Molecular Pathways, §Analytical Sciences, Novartis Institutes for BioMedical Research , Basel 4002, Switzerland.

Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) ( Nature 2011 , 475 , 524 ; 519 ). EBI2 is highly expressed in immune cells ( J. Biol. Chem. 2006 , 281 , 13199 ), and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes ( Nature 2010 , 467 , 460 ). Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First, we identified a small molecule agonist NIBR51 (1), which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (2) was used as a starting point for a medicinal chemistry campaign, which yielded NIBR189 (4m). This compound was extensively characterized in binding and various functional signaling assays. Furthermore, we have used 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells.
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http://dx.doi.org/10.1021/jm4019355DOI Listing
April 2014

Discovery of trifluoromethyl(pyrimidin-2-yl)azetidine-2-carboxamides as potent, orally bioavailable TGR5 (GPBAR1) agonists: structure-activity relationships, lead optimization, and chronic in vivo efficacy.

J Med Chem 2014 Apr 4;57(8):3263-82. Epub 2014 Apr 4.

Departments of †Medicinal Chemistry, ‡Drug Discovery Biology, §Pharmacology, ∥Pharmacokinetics, and ⊥Structural Biology, Genomics Institute of the Novartis Research Foundation , 10675 John Jay Hopkins Drive, San Diego, California 92121, United States.

Activation of the G-protein coupled receptor (GPCR) Takeda G-protein receptor 5 (TGR5), also known as G-protein bile acid receptor 1 (GPBAR1), has been shown to play a key role in pathways associated with diabetes, metabolic syndrome, and autoimmune disease. Nipecotamide 5 was identified as an attractive starting point after a high-throughput screen (HTS) for receptor agonists. A comprehensive hit-to-lead effort culminated in the discovery of 45h as a potent, selective, and bioavailable TGR5 agonist to test in preclinical metabolic disease models. In genetically obese mice (ob/ob), 45h was as effective as a dipeptidyl peptidase-4 (DPP-4) inhibitor at reducing peak glucose levels in an acute oral glucose tolerance test (OGTT), but this effect was lost upon chronic dosing.
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http://dx.doi.org/10.1021/jm401731qDOI Listing
April 2014

Oxysterols direct immune cell migration via EBI2.

Nature 2011 Jul 27;475(7357):524-7. Epub 2011 Jul 27.

Euroscreen S.A., 6041 Gosselies, Belgium.

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.
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http://dx.doi.org/10.1038/nature10280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297623PMC
July 2011

Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency.

Proc Natl Acad Sci U S A 2010 Feb 2;107(8):3552-7. Epub 2010 Feb 2.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFbeta/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.
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http://dx.doi.org/10.1073/pnas.0914019107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840467PMC
February 2010

Lipid G protein-coupled receptor ligand identification using beta-arrestin PathHunter assay.

J Biol Chem 2009 May 13;284(18):12328-38. Epub 2009 Mar 13.

GPCR Platform, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA.

A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine 1-phosphate (S1P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the beta-arrestin PathHunter assay system, a newly developed, generic GPCR assay format that measures beta-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly "deorphaned" receptors (GPR23, GPR92, GPR55, G2A, GPR18, GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of approximately 400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3'-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB(2) receptor partial agonist, with binding affinity around 1 microm. The anti-inflammatory effect of 3,3'-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB(2).
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http://dx.doi.org/10.1074/jbc.M806516200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673301PMC
May 2009

Identification of novel therapeutic targets for HIV infection through functional genomic cDNA screening.

Virology 2007 May 25;362(1):16-25. Epub 2007 Jan 25.

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

Despite decades of research, HIV remains a global health threat. Issues of multi-drug resistance and lack of an effective vaccine have recently led to the targeting of host factors for anti-viral drug development. While a few genome-wide screens for novel HIV co-factors have been reported, the promise of finding a therapeutic target has yet to be realized. Here, we report a screen of a cDNA library representing 15,000 unique genes in an infectious HIV system, and show that genomic screening can lead to the identification of novel proviral host factors. Mixed lineage kinase 3 (MLK3/MAP3K11) was identified as one of the strongest enhancers of infection and mutant studies show that its activity is dependent on its kinase function. Consistent with its known role in the activation of the AP-1 pathway through JNK kinase, MLK3 was able to enhance Tat-dependent HIV transcription in vitro thus leading to an increase in infection signal. RNA interference studies confirm the involvement of endogenous MLK3 in HIV infection, further implicating this kinase as a potential therapeutic target.
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http://dx.doi.org/10.1016/j.virol.2006.11.036DOI Listing
May 2007

Synthesis and biological evaluations of sulfanyltriazoles as novel HIV-1 non-nucleoside reverse transcriptase inhibitors.

Bioorg Med Chem Lett 2006 Aug 14;16(16):4174-7. Epub 2006 Jun 14.

Genomics Institute of the Novartis Research Foundation (GNF), San Diego, CA 92121, USA.

A novel sulfanyltriazole was discovered as an HIV-1 non-nucleoside reverse transcriptase inhibitor via HTS using a cell-based assay. Chemical modifications and molecular modeling studies were carried out to establish its SAR and understand its interactions with the enzyme. These modifications led to the identification of sulfanyltriazoles with low nanomolar potency for inhibiting HIV-1 replication and promising activities against selected NNRTI resistant mutants. These novel and potent sulfanyltriazoles could serve as advanced leads for further optimization.
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http://dx.doi.org/10.1016/j.bmcl.2006.05.096DOI Listing
August 2006

"UnPAKing" human immunodeficiency virus (HIV) replication: using small interfering RNA screening to identify novel cofactors and elucidate the role of group I PAKs in HIV infection.

J Virol 2006 Jan;80(1):130-7

Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.

In order to identify novel proviral host factors involved in human immunodeficiency virus (HIV) infection, we performed a screen of a small interfering RNA (siRNA) library targeting 5,000 genes with the highest potential for being targets for therapeutics. Many siRNAs in the library against known host factors, such as TSG101, furin, and CXCR4, were identified as inhibitors by the screen and thus served as internal validation. In addition, many novel factors whose knockdown inhibited infection were identified, including Pak3, a member of the serine/threonine group I PAK kinases. The HIV accessory factor Nef has been shown to associate with a PAK kinase, leading to enhanced viral production; however, the exact identity of the kinase has remained controversial. Prompted by the Pak3 screen hit, we further investigated the involvement of group I PAK kinases in HIV using siRNA. Contrary to the current literature, Pak1 depletion strongly inhibited HIV infection in multiple cell systems and decreased levels of integrated provirus, while Pak2 depletion showed no effect. Overexpression of a constitutively active Pak1 mutant also enhanced HIV infection, further supporting its role as the dominant PAK involved.
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http://dx.doi.org/10.1128/JVI.80.1.130-137.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317519PMC
January 2006

Evidence that HIV budding in primary macrophages occurs through the exosome release pathway.

J Biol Chem 2003 Dec 14;278(52):52347-54. Epub 2003 Oct 14.

Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Lipid rafts are specialized regions of cell membranes enriched in cholesterol and sphingolipids that are involved in immune activation and signaling. Studies in T-cells indicate that these membrane domains serve as sites for release of human immunodeficiency virus (HIV). By budding through lipid rafts in T-cells, HIV selectively incorporates raft markers and excludes non-raft proteins. This process has been well studied in T-cells, but it is unknown whether lipid rafts serve as budding sites for HIV in macrophages. Recently, we proposed a new model of retroviral biogenesis called the Trojan exosome hypothesis (Gould, S. J., Booth, A., and Hildreth, J. E. K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10592-10597). This model proposes that retroviruses coopt the existing cellular machinery for exosomal release. Here, we performed the first test designed to differentiate between the lipid raft hypothesis of retroviral biogenesis and the Trojan exosome hypothesis. Using macrophages, we examined the relative abundance of several host proteins on the cell surface, in lipid rafts, and on both HIV particles and exosomes derived from these cells. Our results show significant differences in the abundance of host proteins on the cell surface and in HIV. Moreover, our data demonstrate discordance in the abundance of some proteins in lipid rafts and in HIV. Finally, our data reveal a strong concordance between the host cell protein profile of exosomes and that of HIV. These results strongly support the Trojan exosome hypothesis and its prediction that retroviral budding represents exploitation of a pre-existing cellular pathway of intercellular vesicle trafficking.
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http://dx.doi.org/10.1074/jbc.M309009200DOI Listing
December 2003

Involvement of macrophage mannose receptor in the binding and transmission of HIV by macrophages.

Eur J Immunol 2003 Feb;33(2):483-93

Department of Pharmacology, Johns Hopkins School of Medicine, Baltimore, USA.

The human immunodeficiency virus (HIV) is an enveloped virus whose surface glycoprotein gp120 binds CD4 on target cell membranes to initiate infection. About half of the carbohydrates on gp120 are terminally mannosylated, a pattern common to many pathogens. We have examined the ability of macrophage mannose receptor (MMR) on primary monocyte-derived macrophages to bind HIV and facilitate its transmission to T cells. We adapted the tyramide signal amplification system for fluorescence detection of HIV bound to macrophages. Our data show that approximately 60% of the initial association of HIV with macrophages that lack expression of DC-SIGN (a dendritic cell-specific ICAM-3 receptor/HIV-1-binding protein) is MMR mediated, as evidenced by inhibition with mannan, D-mannose, EDTA, and soluble mannose-binding lectin, but not by D-galactose. This inhibition is not seen in cells that lack MMR. Macrophages are able to mediate transmission of bound HIV to co-cultured T cells, and this transmission is blocked up to 80% by inhibitors of MMR binding. Unlike virus bound to DC-SIGN, macrophage-bound HIV has a slightly lower half-life compared to free virus, with no transmission in co-culture observed beyond 24 h after virus binding to macrophages. Results obtained with endocytosis inhibitors indicate that this decrease in viral longevity is due to rapid internalization of macrophage-bound HIV. Together, these results suggest a substantial role for MMR in the binding and transmission of HIV-1 by macrophages.
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http://dx.doi.org/10.1002/immu.200310024DOI Listing
February 2003