Edie Dullaghan - Academia.edu (original) (raw)

Papers by Edie Dullaghan

Journal of Immunology, Apr 1, 2007

ChemBioChem, Sep 14, 2018

Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for a... more Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has however dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators which work by inhibiting aminoglycoside N-6'acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among those, pro-drugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here, a series of cellular studies to further investigate the activity and mechanism of action of these prodrugs. Our results reveal a new aminoglycoside resistance inhibitor, and the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability and potentiation of various aminoglycosides, against both Gram-positive and Gram-negative bacteria, make them interesting compounds for the development of new drugs.

Cancer is fundamentally a disease of disordered gene expression. In fact, reversal or neutralizat... more Cancer is fundamentally a disease of disordered gene expression. In fact, reversal or neutralization of the changes in gene expression has been shown to be attractive targets for the development of new anti-cancer drugs and therapeutic strategies. New approaches such as antibody drug conjugates (ADCs) also target differentially expressed genes as a mean to recognize tumor cells to selectively deliver toxins to a tumor. In the past decade, the global analysis of gene expression in human cancers have led to the development of a number of potential new biomarkers. For instance, mesothelin (MSLN) was identified as an over-expressed gene in pancreatic cancer and later was proved to be a useful diagnostic marker and so a therapeutic target. Large-scale gene expression analysis, using techniques such as RNA sequencing, provides a powerful tool to identify genes involved in human cancers. In this study, with the ultimate goal being to identify potential novel targets for cancer immunotherapy, we conducted a pan-cancer differential expression analysis in RNA sequencing data from more than 5,000 patients with 25 different cancer types generated by The Cancer Genome Atlas (TCGA). We identified differentially expressed genes (present in at least 5% of samples in a tumor type) in comparison to a large compendium of normal transcriptomes (more than 650 samples, including 30 tissue types) gathered from Genotype-Tissue Expression (GTEx), illumina BodyMap project 2.0, TCGA, and an in-house database. In total, we identified 892 putative tumor-associated differentially expressed genes. In order to further identify novel candidate genes and rank them based on their antigenic potential, we performed an extensive literature search and systematic review to collect the characteristics of an ideal tumor antigen (TA). We developed an Analytic Hierarchy Process (AHP) model - a multiple-criteria decision-making solution - to depict antigen properties for ranking and prioritizing the tumor-associated differentially expressed genes. Our model recognizes the known tumor antigens (such as CA9, Nectin-4, FN1, MSLN and MUC16, which are currently in clinic or pre-clinical studies) in the top 25 of the ranked list. We are currently validating the top-ranked novel antigens in an orthogonal panel of tumor and normal tissues and cell lines using PCR. Note: This abstract was not presented at the meeting. Citation Format: Daryanaz Dargahi, Richard D. Swayze, Leanna Yee, Peter J. Bergqvist, Bradley J. Hedberg, Alireza Heravi-Moussavi, Edie M. Dullaghan, Ryan Dercho, Christopher Bond, Jianghong An, John S. Babcook, Steven JM Jones. Pan-cancer identification and prioritization of cancer-associated differentially expressed genes: A biomarker discovery application. [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 2187. doi:10.1158/1538-7445.AM2015-2187

Biological Procedures Online, Feb 1, 2003

We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms a... more We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

Cancer Research, Jul 15, 2016

We previously reported that 3’-phosphoadenosine-5’-phosphosulfate (PAPS) synthase 1 (PAPSS1), an ... more We previously reported that 3’-phosphoadenosine-5’-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate (PAPS) for all sulfation reactions, is a novel therapeutic target that when suppressed enhances the activity of multiple DNA damaging agents in NSCLC cells. PAPSS1 was the lead hit in a synthetic lethal screen completed using chemotherapy-naive NSCLC cells exposed to the IC10 of cisplatin (CDDP). PAPSS1 silencing was more effective in potentiating CDDP activity than our positive control (BRACA2). Here, we evaluated PAPSS1 as a CDDP-sensitizing target in three different model systems: 3D spheroids, zebrafish xenografts, and a mouse xenograft model. siRNA-transfected A549 cells were seeded in round bottom ultra-low attachment plates for spheroid formation. Spheroids were formed over a period of three days and then treated with CDDP. The spheroids were imaged using the IncuCyte ZOOM® Live Cell Imaging system every 3 hours for 8 days to monitor changes in spheroid size. To evaluate PAPSS1 in zebrafish, transfected A549 cells were microinjected into the yolk sack of zebrafish embryos and then maintained in CDDP-containing media for 48 hours. The human cells were harvested from 20 fish per treatment group and counted to determine the change in cell number as a measure of tumor growth in vivo. For mouse studies, RAG2M mice were inoculated subcutaneously with 5×106 parental, non-targeting shRNA, or shPAPSS1-expressing A549 cells. The mice were treated 7 days later with 3 mg/kg CDDP (IV, Q4Dx3). Tumor size was measured using an electronic caliper and tumor volumes were calculated using the equation (lxw2)/2. PAPSS1-silenced cells formed spheroids of comparable size as the scramble control. CDDP (12.5μM) was effective against both control and PAPSS1-silenced spheroids with a reduction of 31% and 46% in spheroid size, respectively. PAPSS1-knockdown spheroids were significantly more sensitive to CDDP even when added at an 8-fold lower dose (1.56 μM). At this concentration, the control spheroids grew about 37% in size while the size of the PAPSS1-silenced spheroids was reduced by 21% (p<0.0001). In zebrafish, the number of A549 cells was reduced by approximately 50% with the combination of PAPSS1 knockdown and CDDP treatment relative to non-silencing, CDDP-treated controls. In mice, tumor development was significantly delayed in the shPAPSS1 group relative to both parental (p = 0.008) and non-targeting shRNA (p = 0.026) controls following CDDP treatment. Our study demonstrates for the first time that PAPSS1 knockdown enhances CDDP treatment in vivo. To pursue PAPSS1 as a therapeutic target, a small molecule inhibitor screen is warranted. The availability of a small molecule inhibitor will be essential to understand how PAPSS1 inhibition sensitizes cancer cells (but not normal cells) to DNA damaging agents. Citation Format: Ada W.Y. Leung, Chansey J. Veinotte, Nicole Melong, Ian Backstrom, Corinna Warburton, Edie Dullaghan, Jason N. Berman, Marcel B. Bally. PAPSS1 (3’-phosphoadenosine 5’-phosphosulfate synthase 1) inhibition sensitizes non-small cell lung cancer to cisplatin treatment in vivo. [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 3792.

Journal of Lipid Research, Nov 1, 2013

performs similar functions in coordinating the transport of lipids among various cell types in th... more performs similar functions in coordinating the transport of lipids among various cell types in the CNS (3, 4). ApoE receives lipids from the cholesterol and phospholipid transporter ABCA1 and delivers its lipid cargo to recipient cells primarily through binding and endocytosis of the low-density lipoprotein receptor (LDLR) (5, 6). ApoE also binds several additional receptors, including LDLR-related protein (LRP), apoE receptor 2 (apoER2), and VLDL receptor, which result in activation of signaling pathways important for neuronal function (7). Humans possess three allelic isoforms of the 299 amino acid apoE protein: apoE2 (Cys112, Cys158), apoE3 (Cys112, Arg158), and apoE4 (Arg112, Arg158) (8). The human apoE sequence differs considerably from that of mice (9). ApoE4 increases Alzheimer disease (AD) risk and reduces age of onset of AD, whereas apoE2 delays AD onset and reduces risk (10-12). These effects are believed to be due in part to isoform-specifi c differences in A ␤ metabolism, as apoE4 prolongs A ␤ half-life in brain interstitial fl uid (13) and promotes oligomerization of A ␤ both in vivo and in vitro (14). As apoE can bind to A ␤ primarily through interactions with the lipid-binding amphipathic ␣-helical region of apoE (15, 16), the lipidation status of apoE may infl uence its interaction with A ␤. ApoE4 has a poor ability to accept lipids, as approximately twice as much cholesterol and phospholipid can be effl uxed to apoE3 compared with apoE4 in cultured astrocytes (17). Also, uptake of apoE4 by neurons impairs glutamate receptor function and apoER2 receptor recycling, leading to reduced Reelin-induced long-term potentiation and Abstract Apolipoprotein E (apoE) is the major lipid carrier in the central nervous system. As apoE plays a major role in the pathogenesis of Alzheimer disease (AD) and also mediates repair pathways after several forms of acute brain injury, modulating the expression, secretion, or function of apoE may provide potential therapeutic approaches for several neurological disorders. Here we show that progesterone and a synthetic progestin, lynestrenol, signifi cantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens and the progesterone metabolite allopregnanolone have negligible effects. Intriguingly, lynestrenol also increases expression of the cholesterol transporter ABCA1 in CCF-STTG1 astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor inhibitor RU486 attenuates the effect of progestins on apoE expression in CCF-STTG1 astrocytoma cells but has no effect on ABCA1 expression in all glial cell models tested, suggesting that the progesterone receptor (PR) may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to infl uence glial lipid homeostasis through liver X receptor-dependent and progesterone receptor-dependent pathways.-Fan, J.

Bioorganic & Medicinal Chemistry Letters, Nov 1, 2014

ACS central science, Jan 19, 2021

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in larg... more The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP−sialic acid sugar donors bearing fluorine atoms at the 7position, starting from the corresponding 4-deoxy-4-fluoro-Nacetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7fluorosialosides toward spontaneous hydrolysis (3-to 5-fold) and toward cleavage by GH33 sialidases (40-to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the Nglycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory halflives of glycoproteins and may pave the way toward biobetters for therapeutic use.

PLOS ONE, Nov 29, 2011

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses ... more Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders.

Bioorganic & Medicinal Chemistry, Mar 1, 2014

A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized... more A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected 'hub proteins' in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure-activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 μg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::Erm R) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.

Infection and Immunity, Sep 1, 2008

Streptococcus salivarius is an early colonizer of human oral and nasopharyngeal epithelia, and st... more Streptococcus salivarius is an early colonizer of human oral and nasopharyngeal epithelia, and strain K12 has reported probiotic effects. An emerging paradigm indicates that commensal bacteria downregulate immune responses through the action on NF-B signaling pathways, but additional mechanisms underlying probiotic actions are not well understood. Our objective here was to identify host genes specifically targeted by K12 by comparing their responses with responses elicited by pathogens and to determine if S. salivarius modulates epithelial cell immune responses. RNA was extracted from human bronchial epithelial cells (16HBE14O-cells) cocultured with K12 or bacterial pathogens. cDNA was hybridized to a human 21K oligonucleotide-based array. Data were analyzed using ArrayPipe, InnateDB, PANTHER, and oPOSSUM. Interleukin 8 (IL-8) and growth-regulated oncogene alpha (Gro␣) secretion were determined by enzyme-linked immunosorbent assay. It was demonstrated that S. salivarius K12 specifically altered the expression of 565 host genes, particularly those involved in multiple innate defense pathways, general epithelial cell function and homeostasis, cytoskeletal remodeling, cell development and migration, and signaling pathways. It inhibited baseline IL-8 secretion and IL-8 responses to LL-37, Pseudomonas aeruginosa, and flagellin in epithelial cells and attenuated Gro␣ secretion in response to flagellin. Immunosuppression was coincident with the inhibition of activation of the NF-B pathway. Thus, the commensal and probiotic behaviors of S. salivarius K12 are proposed to be due to the organism (i) eliciting no proinflammatory response, (ii) stimulating an anti-inflammatory response, and (iii) modulating genes associated with adhesion to the epithelial layer and homeostasis. S. salivarius K12 might thereby ensure that it is tolerated by the host and maintained on the epithelial surface while actively protecting the host from inflammation and apoptosis induced by pathogens.

Annals of Neurology, Mar 1, 2014

Objective: There is currently no pharmacological treatment that provides protection against brain... more Objective: There is currently no pharmacological treatment that provides protection against brain injury in neonates. It is known that activation of an innate immune response is a key, contributing factor in perinatal brain injury; therefore, the neuroprotective therapeutic potential of innate defense regulator peptides (IDRs) was investigated. Methods: The anti-inflammatory effects of 3 IDRs was measured in lipopolysaccharide (LPS)-activated murine microglia. IDRs were then assessed for their ability to confer neuroprotection in vivo when given 3 hours after neonatal brain injury in a clinically relevant model that combines an inflammatory challenge (LPS) with hypoxia-ischemia (HI). To gain insight into peptide-mediated effects on LPS-induced inflammation and neuroprotective mechanisms, global cerebral gene expression patterns were analyzed in pups that were treated with IDR-1018 either 4 hours before LPS or 3 hours after LPS1HI. Results: IDR-1018 reduced inflammatory mediators produced by LPS-stimulated microglia cells in vitro and modulated LPS-induced neuroinflammation in vivo. When administered 3 hours after LPS1HI, IDR-1018 exerted effects on regulatory molecules of apoptotic (for, eg, Fadd and Tnfsf9) and inflammatory (for, eg, interleukin 1, tumor necrosis factor a, chemokines, and cell adhesion molecules) pathways and showed marked protection of both white and gray brain matter. Interpretation: IDR-1018 suppresses proinflammatory mediators and cell injurious mechanisms in the developing brain, and postinsult treatment is efficacious in reducing LPS-induced hypoxic-ischemic brain damage. IDR-1018 is effective in the brain when given systemically, confers neuroprotection of both gray and white matter, and lacks significant effects on the brain under normal conditions. Thus, this peptide provides the features of a promising neuroprotective agent in newborns with brain injury.

The Journal of Immunology

Inimex Innate Defence Regulators (IDRs) are novel, synthetic immunomodulatory peptides that prote... more Inimex Innate Defence Regulators (IDRs) are novel, synthetic immunomodulatory peptides that protect against infections by selectively activating the innate immune system while regulating inflammation. The multi-faceted effects of IDRs are mediated primarily by monocytes and macrophages. These cells respond to IDRs by selectively increasing the expression of cell surface receptors, and cytokines and chemokines (MCP-1, MCP-3 and CCL-5) which trigger the recruitment and activation of immune cells to the site of the infection. In addition, IDRs control inflammation by enhancing the expression of the anti-inflammatory cytokine IL-10, and down-regulating the release of pro-inflammatory cytokines TNF-α and IL-6 in response to pathogen-associated stimuli. As a result, IDRs selectively activate the immune system without concomitant up-regulation of inflammatory responses. This combination of IDR effects is a distinctive quality of these agents since they are able to maintain a balance betwee...

Two-dimensional DNA displays for comparisons of bacterial

ChemBioChem, 2018

Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for a... more Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has however dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators which work by inhibiting aminoglycoside N-6'acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among those, pro-drugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here, a series of cellular studies to further investigate the activity and mechanism of action of these prodrugs. Our results reveal a new aminoglycoside resistance inhibitor, and the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability and potentiation of various aminoglycosides, against both Gram-positive and Gram-negative bacteria, make them interesting compounds for the development of new drugs.

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses ... more Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer’s and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput s...

Journal of Immunology, 2007

ACS Central Science

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in larg... more The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP−sialic acid sugar donors bearing fluorine atoms at the 7position, starting from the corresponding 4-deoxy-4-fluoro-Nacetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7fluorosialosides toward spontaneous hydrolysis (3-to 5-fold) and toward cleavage by GH33 sialidases (40-to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the Nglycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory halflives of glycoproteins and may pave the way toward biobetters for therapeutic use.

Journal of Immunology, Apr 1, 2007

ChemBioChem, Sep 14, 2018

Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for a... more Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has however dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators which work by inhibiting aminoglycoside N-6'acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among those, pro-drugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here, a series of cellular studies to further investigate the activity and mechanism of action of these prodrugs. Our results reveal a new aminoglycoside resistance inhibitor, and the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability and potentiation of various aminoglycosides, against both Gram-positive and Gram-negative bacteria, make them interesting compounds for the development of new drugs.

Cancer is fundamentally a disease of disordered gene expression. In fact, reversal or neutralizat... more Cancer is fundamentally a disease of disordered gene expression. In fact, reversal or neutralization of the changes in gene expression has been shown to be attractive targets for the development of new anti-cancer drugs and therapeutic strategies. New approaches such as antibody drug conjugates (ADCs) also target differentially expressed genes as a mean to recognize tumor cells to selectively deliver toxins to a tumor. In the past decade, the global analysis of gene expression in human cancers have led to the development of a number of potential new biomarkers. For instance, mesothelin (MSLN) was identified as an over-expressed gene in pancreatic cancer and later was proved to be a useful diagnostic marker and so a therapeutic target. Large-scale gene expression analysis, using techniques such as RNA sequencing, provides a powerful tool to identify genes involved in human cancers. In this study, with the ultimate goal being to identify potential novel targets for cancer immunotherapy, we conducted a pan-cancer differential expression analysis in RNA sequencing data from more than 5,000 patients with 25 different cancer types generated by The Cancer Genome Atlas (TCGA). We identified differentially expressed genes (present in at least 5% of samples in a tumor type) in comparison to a large compendium of normal transcriptomes (more than 650 samples, including 30 tissue types) gathered from Genotype-Tissue Expression (GTEx), illumina BodyMap project 2.0, TCGA, and an in-house database. In total, we identified 892 putative tumor-associated differentially expressed genes. In order to further identify novel candidate genes and rank them based on their antigenic potential, we performed an extensive literature search and systematic review to collect the characteristics of an ideal tumor antigen (TA). We developed an Analytic Hierarchy Process (AHP) model - a multiple-criteria decision-making solution - to depict antigen properties for ranking and prioritizing the tumor-associated differentially expressed genes. Our model recognizes the known tumor antigens (such as CA9, Nectin-4, FN1, MSLN and MUC16, which are currently in clinic or pre-clinical studies) in the top 25 of the ranked list. We are currently validating the top-ranked novel antigens in an orthogonal panel of tumor and normal tissues and cell lines using PCR. Note: This abstract was not presented at the meeting. Citation Format: Daryanaz Dargahi, Richard D. Swayze, Leanna Yee, Peter J. Bergqvist, Bradley J. Hedberg, Alireza Heravi-Moussavi, Edie M. Dullaghan, Ryan Dercho, Christopher Bond, Jianghong An, John S. Babcook, Steven JM Jones. Pan-cancer identification and prioritization of cancer-associated differentially expressed genes: A biomarker discovery application. [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 2187. doi:10.1158/1538-7445.AM2015-2187

Biological Procedures Online, Feb 1, 2003

We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms a... more We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

Cancer Research, Jul 15, 2016

We previously reported that 3’-phosphoadenosine-5’-phosphosulfate (PAPS) synthase 1 (PAPSS1), an ... more We previously reported that 3’-phosphoadenosine-5’-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate (PAPS) for all sulfation reactions, is a novel therapeutic target that when suppressed enhances the activity of multiple DNA damaging agents in NSCLC cells. PAPSS1 was the lead hit in a synthetic lethal screen completed using chemotherapy-naive NSCLC cells exposed to the IC10 of cisplatin (CDDP). PAPSS1 silencing was more effective in potentiating CDDP activity than our positive control (BRACA2). Here, we evaluated PAPSS1 as a CDDP-sensitizing target in three different model systems: 3D spheroids, zebrafish xenografts, and a mouse xenograft model. siRNA-transfected A549 cells were seeded in round bottom ultra-low attachment plates for spheroid formation. Spheroids were formed over a period of three days and then treated with CDDP. The spheroids were imaged using the IncuCyte ZOOM® Live Cell Imaging system every 3 hours for 8 days to monitor changes in spheroid size. To evaluate PAPSS1 in zebrafish, transfected A549 cells were microinjected into the yolk sack of zebrafish embryos and then maintained in CDDP-containing media for 48 hours. The human cells were harvested from 20 fish per treatment group and counted to determine the change in cell number as a measure of tumor growth in vivo. For mouse studies, RAG2M mice were inoculated subcutaneously with 5×106 parental, non-targeting shRNA, or shPAPSS1-expressing A549 cells. The mice were treated 7 days later with 3 mg/kg CDDP (IV, Q4Dx3). Tumor size was measured using an electronic caliper and tumor volumes were calculated using the equation (lxw2)/2. PAPSS1-silenced cells formed spheroids of comparable size as the scramble control. CDDP (12.5μM) was effective against both control and PAPSS1-silenced spheroids with a reduction of 31% and 46% in spheroid size, respectively. PAPSS1-knockdown spheroids were significantly more sensitive to CDDP even when added at an 8-fold lower dose (1.56 μM). At this concentration, the control spheroids grew about 37% in size while the size of the PAPSS1-silenced spheroids was reduced by 21% (p<0.0001). In zebrafish, the number of A549 cells was reduced by approximately 50% with the combination of PAPSS1 knockdown and CDDP treatment relative to non-silencing, CDDP-treated controls. In mice, tumor development was significantly delayed in the shPAPSS1 group relative to both parental (p = 0.008) and non-targeting shRNA (p = 0.026) controls following CDDP treatment. Our study demonstrates for the first time that PAPSS1 knockdown enhances CDDP treatment in vivo. To pursue PAPSS1 as a therapeutic target, a small molecule inhibitor screen is warranted. The availability of a small molecule inhibitor will be essential to understand how PAPSS1 inhibition sensitizes cancer cells (but not normal cells) to DNA damaging agents. Citation Format: Ada W.Y. Leung, Chansey J. Veinotte, Nicole Melong, Ian Backstrom, Corinna Warburton, Edie Dullaghan, Jason N. Berman, Marcel B. Bally. PAPSS1 (3’-phosphoadenosine 5’-phosphosulfate synthase 1) inhibition sensitizes non-small cell lung cancer to cisplatin treatment in vivo. [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 3792.

Journal of Lipid Research, Nov 1, 2013

performs similar functions in coordinating the transport of lipids among various cell types in th... more performs similar functions in coordinating the transport of lipids among various cell types in the CNS (3, 4). ApoE receives lipids from the cholesterol and phospholipid transporter ABCA1 and delivers its lipid cargo to recipient cells primarily through binding and endocytosis of the low-density lipoprotein receptor (LDLR) (5, 6). ApoE also binds several additional receptors, including LDLR-related protein (LRP), apoE receptor 2 (apoER2), and VLDL receptor, which result in activation of signaling pathways important for neuronal function (7). Humans possess three allelic isoforms of the 299 amino acid apoE protein: apoE2 (Cys112, Cys158), apoE3 (Cys112, Arg158), and apoE4 (Arg112, Arg158) (8). The human apoE sequence differs considerably from that of mice (9). ApoE4 increases Alzheimer disease (AD) risk and reduces age of onset of AD, whereas apoE2 delays AD onset and reduces risk (10-12). These effects are believed to be due in part to isoform-specifi c differences in A ␤ metabolism, as apoE4 prolongs A ␤ half-life in brain interstitial fl uid (13) and promotes oligomerization of A ␤ both in vivo and in vitro (14). As apoE can bind to A ␤ primarily through interactions with the lipid-binding amphipathic ␣-helical region of apoE (15, 16), the lipidation status of apoE may infl uence its interaction with A ␤. ApoE4 has a poor ability to accept lipids, as approximately twice as much cholesterol and phospholipid can be effl uxed to apoE3 compared with apoE4 in cultured astrocytes (17). Also, uptake of apoE4 by neurons impairs glutamate receptor function and apoER2 receptor recycling, leading to reduced Reelin-induced long-term potentiation and Abstract Apolipoprotein E (apoE) is the major lipid carrier in the central nervous system. As apoE plays a major role in the pathogenesis of Alzheimer disease (AD) and also mediates repair pathways after several forms of acute brain injury, modulating the expression, secretion, or function of apoE may provide potential therapeutic approaches for several neurological disorders. Here we show that progesterone and a synthetic progestin, lynestrenol, signifi cantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens and the progesterone metabolite allopregnanolone have negligible effects. Intriguingly, lynestrenol also increases expression of the cholesterol transporter ABCA1 in CCF-STTG1 astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor inhibitor RU486 attenuates the effect of progestins on apoE expression in CCF-STTG1 astrocytoma cells but has no effect on ABCA1 expression in all glial cell models tested, suggesting that the progesterone receptor (PR) may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to infl uence glial lipid homeostasis through liver X receptor-dependent and progesterone receptor-dependent pathways.-Fan, J.

Bioorganic & Medicinal Chemistry Letters, Nov 1, 2014

ACS central science, Jan 19, 2021

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in larg... more The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP−sialic acid sugar donors bearing fluorine atoms at the 7position, starting from the corresponding 4-deoxy-4-fluoro-Nacetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7fluorosialosides toward spontaneous hydrolysis (3-to 5-fold) and toward cleavage by GH33 sialidases (40-to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the Nglycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory halflives of glycoproteins and may pave the way toward biobetters for therapeutic use.

PLOS ONE, Nov 29, 2011

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses ... more Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders.

Bioorganic & Medicinal Chemistry, Mar 1, 2014

A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized... more A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected 'hub proteins' in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure-activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 μg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::Erm R) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.

Infection and Immunity, Sep 1, 2008

Streptococcus salivarius is an early colonizer of human oral and nasopharyngeal epithelia, and st... more Streptococcus salivarius is an early colonizer of human oral and nasopharyngeal epithelia, and strain K12 has reported probiotic effects. An emerging paradigm indicates that commensal bacteria downregulate immune responses through the action on NF-B signaling pathways, but additional mechanisms underlying probiotic actions are not well understood. Our objective here was to identify host genes specifically targeted by K12 by comparing their responses with responses elicited by pathogens and to determine if S. salivarius modulates epithelial cell immune responses. RNA was extracted from human bronchial epithelial cells (16HBE14O-cells) cocultured with K12 or bacterial pathogens. cDNA was hybridized to a human 21K oligonucleotide-based array. Data were analyzed using ArrayPipe, InnateDB, PANTHER, and oPOSSUM. Interleukin 8 (IL-8) and growth-regulated oncogene alpha (Gro␣) secretion were determined by enzyme-linked immunosorbent assay. It was demonstrated that S. salivarius K12 specifically altered the expression of 565 host genes, particularly those involved in multiple innate defense pathways, general epithelial cell function and homeostasis, cytoskeletal remodeling, cell development and migration, and signaling pathways. It inhibited baseline IL-8 secretion and IL-8 responses to LL-37, Pseudomonas aeruginosa, and flagellin in epithelial cells and attenuated Gro␣ secretion in response to flagellin. Immunosuppression was coincident with the inhibition of activation of the NF-B pathway. Thus, the commensal and probiotic behaviors of S. salivarius K12 are proposed to be due to the organism (i) eliciting no proinflammatory response, (ii) stimulating an anti-inflammatory response, and (iii) modulating genes associated with adhesion to the epithelial layer and homeostasis. S. salivarius K12 might thereby ensure that it is tolerated by the host and maintained on the epithelial surface while actively protecting the host from inflammation and apoptosis induced by pathogens.

Annals of Neurology, Mar 1, 2014

Objective: There is currently no pharmacological treatment that provides protection against brain... more Objective: There is currently no pharmacological treatment that provides protection against brain injury in neonates. It is known that activation of an innate immune response is a key, contributing factor in perinatal brain injury; therefore, the neuroprotective therapeutic potential of innate defense regulator peptides (IDRs) was investigated. Methods: The anti-inflammatory effects of 3 IDRs was measured in lipopolysaccharide (LPS)-activated murine microglia. IDRs were then assessed for their ability to confer neuroprotection in vivo when given 3 hours after neonatal brain injury in a clinically relevant model that combines an inflammatory challenge (LPS) with hypoxia-ischemia (HI). To gain insight into peptide-mediated effects on LPS-induced inflammation and neuroprotective mechanisms, global cerebral gene expression patterns were analyzed in pups that were treated with IDR-1018 either 4 hours before LPS or 3 hours after LPS1HI. Results: IDR-1018 reduced inflammatory mediators produced by LPS-stimulated microglia cells in vitro and modulated LPS-induced neuroinflammation in vivo. When administered 3 hours after LPS1HI, IDR-1018 exerted effects on regulatory molecules of apoptotic (for, eg, Fadd and Tnfsf9) and inflammatory (for, eg, interleukin 1, tumor necrosis factor a, chemokines, and cell adhesion molecules) pathways and showed marked protection of both white and gray brain matter. Interpretation: IDR-1018 suppresses proinflammatory mediators and cell injurious mechanisms in the developing brain, and postinsult treatment is efficacious in reducing LPS-induced hypoxic-ischemic brain damage. IDR-1018 is effective in the brain when given systemically, confers neuroprotection of both gray and white matter, and lacks significant effects on the brain under normal conditions. Thus, this peptide provides the features of a promising neuroprotective agent in newborns with brain injury.

The Journal of Immunology

Inimex Innate Defence Regulators (IDRs) are novel, synthetic immunomodulatory peptides that prote... more Inimex Innate Defence Regulators (IDRs) are novel, synthetic immunomodulatory peptides that protect against infections by selectively activating the innate immune system while regulating inflammation. The multi-faceted effects of IDRs are mediated primarily by monocytes and macrophages. These cells respond to IDRs by selectively increasing the expression of cell surface receptors, and cytokines and chemokines (MCP-1, MCP-3 and CCL-5) which trigger the recruitment and activation of immune cells to the site of the infection. In addition, IDRs control inflammation by enhancing the expression of the anti-inflammatory cytokine IL-10, and down-regulating the release of pro-inflammatory cytokines TNF-α and IL-6 in response to pathogen-associated stimuli. As a result, IDRs selectively activate the immune system without concomitant up-regulation of inflammatory responses. This combination of IDR effects is a distinctive quality of these agents since they are able to maintain a balance betwee...

Two-dimensional DNA displays for comparisons of bacterial

ChemBioChem, 2018

Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for a... more Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has however dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators which work by inhibiting aminoglycoside N-6'acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among those, pro-drugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here, a series of cellular studies to further investigate the activity and mechanism of action of these prodrugs. Our results reveal a new aminoglycoside resistance inhibitor, and the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability and potentiation of various aminoglycosides, against both Gram-positive and Gram-negative bacteria, make them interesting compounds for the development of new drugs.

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses ... more Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer’s and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput s...

Journal of Immunology, 2007

ACS Central Science

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in larg... more The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP−sialic acid sugar donors bearing fluorine atoms at the 7position, starting from the corresponding 4-deoxy-4-fluoro-Nacetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7fluorosialosides toward spontaneous hydrolysis (3-to 5-fold) and toward cleavage by GH33 sialidases (40-to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the Nglycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory halflives of glycoproteins and may pave the way toward biobetters for therapeutic use.