IU Indianapolis ScholarWorks :: Browsing by Author "Queener, Sherry F."

Browsing by Author "Queener, Sherry F."

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8-aminoquinolines effective against Pneumocystis carinii in vitro and in vivo
(American Society for Microbiology, 1999-10) Queener, Sherry F.; Bartlett, Marilyn S.; Nasr, Mohamed; Smith, James W.; Pharmacology and Toxicology, School of Medicine
The activities of 25 8-aminoquinolines were compared in tests assessing the ability of the compounds to inhibit the growth of Pneumocystis carinii in culture. Six compounds were effective at or below 0.03 microM: CDRI 80/53, NSC19894, NSC305805, NSC305812, WR182234, and primaquine. Four others were effective at between 0.2 and 0.03 microM: NSC305835, WR225448, WR238605, and WR242511. Fourteen drugs were also tested in a standard model of P. carinii pneumonia in rats at daily doses of 2 mg/kg of body weight in drinking water. CDRI 80/53, NSC305805, NSC305835, and WR225448 were extremely effective in the animal model. The effectiveness of WR238605, WR242511, and primaquine in the rat model has been reported elsewhere (M. S. Bartlett, S. F. Queener, R. R. Tidwell, W. K. Milhouse, J. D. Berman, W. Y. Ellis, and J. W. Smith, Antimicrob. Agents Chemother. 35:277-282, 1991). The length of the alkyl chain separating the nitrogens in the substituent at position 8 of the quinoline ring was a strong determinant of anti-P. carinii activity.
BASE EXCISION REPAIR APURINIC/APYRIMIDINIC ENDONUCLEASES IN APICOMPLEXAN PARASITE TOXOPLASMA GONDII
(2012-03-19) Onyango, David O.; Sullivan, William J., Jr.; Chou, Kai-Ming; Georgiadis, Millie M.; Queener, Sherry F.; Vasko, Michael R.
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Toxoplasma infection is a serious threat to immunocompromised individuals such as AIDS patients and organ transplant recipients. Side effects associated with current drug treatment calls for identification of new drug targets. DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen species produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host immune response. Most of the oxidative DNA damage is repaired through the base excision repair (BER) pathway, of which, the apurinic /apyrimidinic (AP) endonucleases are the rate limiting enzymes. Toxoplasma possesses two different AP endonucleases. The first, TgAPE, is a magnesium-dependent homologue of the human APE1 (hAPE1), but considerably divergent from hAPE1. The second, TgAPN, is a magnesium-independent homologue of yeast (Saccharomyces cerevisiae) APN1 and is not present in mammals. We have expressed and purified recombinant versions of TgAPE and TgAPN in E. coli and shown AP endonuclease activity. Our data shows that TgAPN is the more abundant AP endonuclease and confers protection against a DNA damaging agent when over-expressed in Toxoplasma tachyzoites. We also generated TgAPN knockdown Toxoplasma tachyzoites to establish that TgAPN is important for parasite protection against DNA damage. We have also identified pharmacological inhibitors of TgAPN in a high-throughput screen. The lead compound inhibits Toxoplasma replication at concentrations that do not have overt toxicity to the host cells. The importance of TgAPN in parasite physiology and the fact that humans lack APN1 makes TgAPN a promising candidate for drug development to treat toxoplasmosis.
Design, synthesis and molecular modeling of novel pyrido[2,3-d]pyrimidine analogs as antifolates: Application of Buchwald-Hartwig aminations of heterocycles
(ACS, 2013) Gangjee, Aleem; Namjoshi, Ojas A.; Raghavan, Sudhir; Queener, Sherry F.; Kisliuk, Roy L.; Cody, Vivian; Pharmacology and Toxicology, School of Medicine
Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, pj), Toxoplasma gondii (T. gondii, tg), and Mycobacterium avium (M. avium, ma) are the principal causes of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). The absence of any animal models for human Pneumocystis jirovecii pneumonia and the lack of crystal structures of pjDHFR and tgDHFR make the design of inhibitors challenging. A novel series of pyrido[2,3-d]pyrimidines as selective and potent DHFR inhibitors against these opportunistic infections are presented. Buchwald-Hartwig coupling reaction of substituted anilines with pivaloyl protected 2,4-diamino-6-bromo-pyrido[2,3-d]pyrimidine was successfully explored to synthesize these analogues. Compound 26 was the most selective inhibitor with excellent potency against pjDHFR. Molecular modeling studies with a pjDHFR homology model explained the potency and selectivity of 26. Structural data are also reported for 26 with pcDHFR and 16 and 22 with variants of pcDHFR.
Functions of the Unique N-terminus of a GCN5 Histone Acetylase in Toxoplasma gondii
(2007-05-18T13:14:16Z) Bhatti, Micah M.; Sullivan, William J., Jr.; Chan, Edward M.; Queener, Sherry F.; Safa, Ahmad R.; Sinai, Anthony P.; Vasko, Michael, R.
GCN5 is a histone acetyltransferase (HAT) that remodels chromatin by acetylating lysine residues of histones. The GCN5 HAT identified in Toxoplasma gondii (TgGCN5) contains a unique N-terminal “extension” that bears no similarity to known proteins and is devoid of known protein motifs. The hypothesis of this thesis is the N-terminal extension is critical to the function of TgGCN5. Three possible roles of the N-terminus were investigated: nuclear localization, protein-protein interactions, and substrate recognition. Subcellular localization was determined via immunocytochemistry using parasites expressing recombinant forms of TgGCN5 fused to a FLAG tag. Initial studies performed with parasites expressing full length FLAG-TgGCN5 were positive for nuclear localization. Without the N-terminal extension (FLAG-ΔNT-TgGCN5) the protein remains cytoplasmic. Additional studies mapped a six amino acid motif (RKRVKR) as the nuclear localization signal (NLS). When RKRVKR is fused to a cytoplasmic protein, it gains access to the nucleus. Furthermore, we have established the NLS interacts with Toxoplasma importin α, a protein involved in nuclear trafficking. Interaction with importin α provides evidence that the TgGCN5 N-terminal extension is involved in mediating protein-protein interactions. In order to identify additional interacting proteins, FLAG affinity purification was performed on parasites expressing full length FLAG-TgGCN5 and FLAG-ΔNT-TgGCN5. Upon comparing the results of the two purifications, proteins captured with only full length TgGCN5 may be interacting with the N-terminal extension. Full length TgGCN5 affinity purification indicates an interaction with histone proteins, two different homologues of Ada2 (adapter protein reported to interact with GCN5 homologues), and several heat shock proteins. With regard to substrate recognition, the N-terminal extension of TgGCN5 is dispensable for the acetylation of non-nucleosomal histones in vitro. However, the lysine acetylated by TgGCN5 is surprisingly unique. Other GCN5 homologues preferentially acetylate lysine 14 in histone H3, but TgGCN5 exclusively acetylates lysine 18 in histone H3 and has no activity on lysine 14. Taken together, these results argue that the N-terminal extension of TgGCN5 is critical for mediating protein-protein interactions, including those responsible for trafficking the HAT to the parasite nucleus but does not appear to be required for the acetylation of non-nucleosomal histones.
GCN5-B is a Novel Nuclear Histone Acetyltransferase that is Crucial for Viability in the Protozoan Parasite Toxoplasma gondii
(2011-03-16) Dixon, Stacey E.; Sullivan, William J., Jr.; Chan, Rebecca J.; Hocevar, Barbara A.; Queener, Sherry F.; Zhang, Jian-Ting
Infection with the single-celled parasite Toxoplasma gondii (phylum Apicomplexa) is usually benign in normal healthy individuals, but can cause congenital birth defects, ocular disease, and also life-threatening infection in immunocompromised patients. Acute infection caused by tachyzoites is controlled by a healthy immune response, but the parasite differentiates into a latent cyst form (bradyzoite) leading to permanent infection and chronic disease. Current therapies are effective only against tachyzoites, are highly toxic to the patient, and do not eradicate the encysted bradyzoites, thus highlighting the need for novel therapeutics. Inhibitors of histone deacetylases have been shown to reduce parasite viability in vitro demonstrating that chromatin remodeling enzymes, key mediators in epigenetic regulation, might serve as potential drug targets. Furthermore, epigenetic regulation has been shown to contribute to gene expression and differentiation in Toxoplasma. This dissertation focused on investigating the physiological role of a Toxoplasma GCN5-family histone acetyltransferase (HAT), termed TgGCN5-B. It was hypothesized that TgGCN5-B is an essential HAT that resides within a unique, multi-subunit complex in the parasite nucleus. Studies of TgGCN5-B have revealed that this HAT possesses a unique nuclear localization signal (311RPAENKKRGR320) that is both necessary and sufficient to translocate the protein to the parasite nucleus. Although no other protein motifs have been identified in the N-terminal extension of TgGCN5-B, it is likely that this extension plays a role in protein-protein interactions. All GCN5 homologues function within large multi-subunit complexes, many being conserved among species, but bioinformatic analysis of the Toxoplasma genome revealed a lack of many of these conserved components. Biochemical studies identified several potential TgGCN5-B associating proteins, including several novel apicomplexan transcription factors. Preliminary evidence suggested that TgGCN5-B was essential for tachyzoites; therefore, a dominant-negative approach was utilized to examine the role of TgGCN5-B in the physiology of Toxoplasma. When catalytically inactive TgGCN5-B protein was over-expressed in the parasites, there was a significant decrease in tachyzoite growth and viability, with initial observations suggesting defects in nuclear division and daughter cell budding. These results demonstrate that TgGCN5-B is important for tachyzoite development and indicate that therapeutic targeting of this HAT could be a novel approach to treat toxoplasmosis.
Identification and functional characterization of genetic variants in the human indoleamine 2, 3-dioxygenase (INDO) gene
(2008-10-13T18:43:27Z) Arefayene, Million; Flockhart, David A.; Vasko, Michael R.; Queener, Sherry F.; Safa, Ahmad R.; Wilkes, David S.
Indoleamine 2,3-dioxygenase (IDO) is a rate limiting enzyme in tryptophan catabolism that has been implicated in the pathogenesis of a number of diseases. Large interindividual variability in IDO activity in the absence of stimuli and as the result of therapy induced changes has been reported. This variability has the potential to contribute to susceptibility to disease and to interindividual variability in therapeutic response. To identify genetic variations that might contribute to interindividual variability in IDO activity, we resequenced the exons, intron/exon borders and 1.3 kb of the 5’-flanking region of the INDO gene in 48 African-American (AA) and 48 Caucasian-American (CA) subjects from the Coriell DNA Repository. A total of 24 INDO variants were identified. Seventeen of these were in exons, introns, or exon/intron boundries, while seven were within 1.3 kb upstream of the translation start site. Seventeen are novel and 7 were previously identified. When transiently expressed in COS-7 or HEK293 cells the amino acid sequence change in Arg77His resulted in significant decrease in activity, and it reduced the Vmax of IDO. The Arg77His variant and the 9 bp deletion resulted in nearly complete loss of enzyme activity and a lack of detectable protein expression. The function of the Arg77His variant IDO was restored in a dose dependent manner by the heme analog hemin; but there was no associated increase in IDO protein. Cellular heme concentration was higher in cells transfected with the wild type and Ala4Thr variant constructs, but not in cells transfected with the Arg77His variant. The heme synthesis inhibitor, succinylacetone (SA), blocked IDO activity in cells transfected with Arg77His. We identified 22 putative transcription binding sites within the 1.3 kb upstream of the translation start site. Two of the SNPs were located in GATA3 and FOXC1 sites. A specific 3-SNP haplotype reduced promoter activity when transiently transfected into 2 different cell lines. We conclude that there are naturally occurring genetic variants in the INDO gene which affect both expression and activity. These results make clear that interindividual variability in IDO activity at baseline or in response to therapy may be in part due to inherited genetic variability.
Identification and mechanistic investigation of clinically important myopathic drug-drug interactions
(2014) Han, Xu; Flockhart, David A.; Bies, Robert R.; Desta, Zeruesenay; Li, Lang; Queener, Sherry F.; Quinney, Sara K.; Zhang, Jian-Ting
Drug-drug interactions (DDIs) refer to situations where one drug affects the pharmacokinetics or pharmacodynamics of another. DDIs represent a major cause of morbidity and mortality. A common adverse drug reaction (ADR) that can result from, or be exacerbated by DDIs is drug-induced myopathy. Identifying DDIs and understanding their underlying mechanisms is key to the prevention of undesirable effects of DDIs and to efforts to optimize therapeutic outcomes. This dissertation is dedicated to identification of clinically important myopathic DDIs and to elucidation of their underlying mechanisms. Using data mined from the published cytochrome P450 (CYP) drug interaction literature, 13,197 drug pairs were predicted to potentially interact by pairing a substrate and an inhibitor of a major CYP isoform in humans. Prescribing data for these drug pairs and their associations with myopathy were then examined in a large electronic medical record database. The analyses identified fifteen drug pairs as DDIs significantly associated with an increased risk of myopathy. These significant myopathic DDIs involved clinically important drugs including alprazolam, chloroquine, duloxetine, hydroxychloroquine, loratadine, omeprazole, promethazine, quetiapine, risperidone, ropinirole, trazodone and simvastatin. Data from in vitro experiments indicated that the interaction between quetiapine and chloroquine (risk ratio, RR, 2.17, p-value 5.29E-05) may result from the inhibitory effects of quetiapine on chloroquine metabolism by cytochrome P450s (CYPs). The in vitro data also suggested that the interaction between simvastatin and loratadine (RR 1.6, p-value 4.75E-07) may result from synergistic toxicity of simvastatin and desloratadine, the major metabolite of loratadine, to muscle cells, and from the inhibitory effect of simvastatin acid, the active metabolite of simvastatin, on the hepatic uptake of desloratadine via OATP1B1/1B3. Our data not only identified unknown myopathic DDIs of clinical consequence, but also shed light on their underlying pharmacokinetic and pharmacodynamic mechanisms. More importantly, our approach exemplified a new strategy for identification and investigation of DDIs, one that combined literature mining using bioinformatic algorithms, ADR detection using a pharmacoepidemiologic design, and mechanistic studies employing in vitro experimental models.
Identification of TgElp3 as an essential, tail-anchored mitochondrial lysine acetyltransferase in the protozoan pathogen toxoplasma gondii
(2014-07-11) Stilger, Krista L.; Nass, Richard M.; Bauer, Margaret E.; Oxford, G. S.; Queener, Sherry F.; Sullivan, William J., Jr.
Toxoplasma gondii, a single-celled eukaryotic pathogen, has infected one-third of the world’s population and is the causative agent of toxoplasmosis. The disease primarily affects immunocompromised individuals such as AIDS, cancer, and transplant patients. The parasites can infect any nucleated cell in warm-blooded vertebrates, but because they preferentially target CNS, heart, and ocular tissue, manifestations of infection often include encephalitis, myocarditis, and a host of neurological and ocular disorders. Toxoplasma can also be transmitted congenitally by a mother who becomes infected for the first time during pregnancy, which may result in spontaneous abortion or birth defects in the child. Unfortunately, the therapy currently available for treating toxoplasmosis exhibits serious side effects and can cause severe allergic reactions. Therefore, there is a desperate need to identify novel drug targets for developing more effective, less toxic treatments. The regulation of proteins via lysine acetylation, a reversible post-translational modification, has previously been validated as a promising avenue for drug development. Lysine acetyltransferases (KATs) are responsible for the acetylation of hundreds of proteins throughout prokaryotic and eukaryotic cells. In Toxoplasma, we identified a KAT that exhibits homology to Elongator protein 3 (TgElp3), the catalytic component of a transcriptional elongation complex. TgElp3 contains the highly conserved radical S-adenosylmethionine and KAT domains but also possesses a unique C-terminal transmembrane domain (TMD). Interestingly, we found that the TMD anchors TgElp3 in the outer mitochondrial membrane (OMM) such that the catalytic domains are oriented towards the cytosol. Our results uncovered the first tail-anchored mitochondrial KAT reported for any species to date. We also discovered a shortened form of Elp3 present in mouse mitochondria, suggesting that Elp3 functions beyond transcriptional elongation across eukaryotes. Furthermore, we established that TgElp3 is essential for parasite viability and that its OMM localization is important for its function, highlighting its value as a potential target for future drug development.
IMP Dehydrogenase from the Protozoan Parasite Toxoplasma gondii
(American Society for Microbiology, 2005) Sullivan, William J., Jr.; Dixon, Stacy E.; Li, Catherine; Striepen, Boris; Queener, Sherry F.; Pharmacology and Toxicology, School of Medicine
The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.
Inhibitors of eIF2α Dephosphorylation Slow Replication and Stabilize Latency in Toxoplasma gondii
(American Society for Microbiology, 2013) Konrad, Christian; Queener, Sherry F.; Wek, Ronald C.; Sullivan, William J., Jr.; Biochemistry and Molecular Biology, School of Medicine
Toxoplasma gondii is an obligate intracellular parasite that permanently infects warm-blooded vertebrates through its ability to convert into a latent tissue cyst form. The latent form (bradyzoite) can reinitiate a life-threatening acute infection if host immunity wanes, most commonly in AIDS or organ transplant patients. We have previously shown that bradyzoite development is accompanied by phosphorylation of the parasite eukaryotic initiation factor 2 alpha subunit (eIF2α), which dampens global protein synthesis and reprograms gene expression. In this study, we analyzed the activities of two specific inhibitors of eIF2α dephosphorylation, salubrinal (SAL) and guanabenz (GA). We establish that these drugs are able to inhibit the dephosphorylation of Toxoplasma eIF2α. Our results show that SAL and GA reduce tachyzoite replication in vitro and in vivo. Furthermore, both drugs induce bradyzoite formation and inhibit the reactivation of latent bradyzoites in vitro. To address whether the antiparasitic activities of SAL and GA involve host eIF2α phosphorylation, we infected mutant mouse embryonic fibroblast (MEF) cells incapable of phosphorylating eIF2α, which had no impact on the efficacies of SAL and GA against Toxoplasma infection. Our findings suggest that SAL and GA may serve as potential new drugs for the treatment of acute and chronic toxoplasmosis.

Browsing by Author "Queener, Sherry F."

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