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Targeting the Histone Acetyl-Transferase, RTT109, for Novel Anti-Fungal Drug Development: A DissertationLopes da Rosa-Spiegler, Jessica 03 May 2012 (has links)
Discovery of new antifungal chemo-therapeutics for humans is limited by the large degree of conservation among eukaryotic organisms. In recent years, the histone acetyl-transferase Rtt109 was identified as the sole enzyme responsible for an abundant and important histone modification, histone H3 lysine 56 (H3K56) acetylation. In the absence of Rtt109, the lack of acetylated H3K56 renders yeast cells extremely sensitive to genotoxic agents. Consequently, the ability to sustain genotoxic stress from the host immune system is crucial for pathogens to perpetuate an infection. Because Rtt109 is conserved only within the fungal kingdom, I reasoned that Rtt109 could be a novel drug target.
My dissertation first establishes that genome stability provided by Rtt109 and H3K56 acetylation is required for Candida albicans pathogenesis. I demonstrate that mice infected with rtt109 -/- cells experience a significant reduction in organ pathology and mortality rate. I hypothesized that the avirulent phenotype of rtt109 -/- cells is due to their intrinsic hypersensitivity to the genotoxic effects of reactive oxygen species (ROS), which are utilized by phagocytic cells of the immune system to kill pathogens. Indeed, C. albicans rtt109 -/- cells are more efficiently killed by macrophages in vitro than are wild-type cells. However, inhibition of ROS generation in macrophages renders rtt109 -/- and wild-type yeast cells equally resilient to killing.
These findings support the concept that ability to resist genotoxic stress conferred by Rtt109 and H3K56 acetylation is a virulence factor for fungal pathogens and establish Rtt109 as an opportune drug- target for novel antifungal therapeutics.
Second, I report the discovery of a specific chemical inhibitor of Rtt109 catalysis as the initial step in the development of a novel antifungal agent. We established a collaboration with the Broad Institute (Cambridge, MA) to perform a high-throughput screen of 300,000 compounds. From these, I identified a single chemical, termed KB7, which specifically inhibits Rtt109 catalysis, with no effect on other HAT enzymes tested. KB7 has an IC50 value of approximately 60 nM and displays noncompetitive inhibition regarding both acetyl-coenzyme A and histone substrates. With the genotoxic agent camptothecin, KB7 causes a synergistic decrease in C. albicans growth rate. However, this effect is only observed in an efflux-pump mutant, suggesting that this compound would be more effective if it were better retained intracellularly. Further studies through structure-activity relationship (SAR) modifications will be conducted on KB7 to improve its effective cellular concentration.
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Lysine acetyltransferase Gcn5-B regulates the expression of crucial genes in Toxoplasma and its function is regulated through lysine acetylationWang, Jiachen 02 April 2014 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.
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Identification of TgElp3 as an essential, tail-anchored mitochondrial lysine acetyltransferase in the protozoan pathogen toxoplasma gondiiStilger, Krista L. 11 July 2014 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / 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.
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Caractérisation des activités épigénétiques et anticancéreuses de la proscillaridine A dans les cancers pédiatriquesDa Costa, Elodie 11 1900 (has links)
Les glycosides cardiotoniques sont des inhibiteurs des pompes sodium / potassium utilisés
pour le traitements des insuffisances cardiaques, qui détiennent également des activités
anticancéreuses et épigénétiques récemment caractérisées. Toutefois, dans l’objectif de
repositionner ces médicaments comme traitement anticancéreux, les mécanismes sousjacents
aux activités anticancéreuses et épigénétiques des glycosides cardiotoniques restent à
être déterminés. Dans nos travaux, nous révélons que la proscillaridine A est le glycoside
cardiotonique qui détient des activités anticancéreuses et épigénétiques les plus puissantes
dans des lignées de cancer du côlon, de leucémies et de sarcomes pédiatrique. De plus, nous
avons identifié que l’activité anticancéreuse de la proscillaridine A corrèle positivement avec le
niveau d’expression protéique du proto-oncogène MYC dans un panel de 14 lignées cellulaires
cancéreuses. Dans les lignées cellulaires exprimants un haut niveau de MYC telles que les
lignées leucémiques, la proscillaridine A agit comme un inhibiteur de MYC et module sa
stabilité protéique ainsi que la régulation transcriptionnelle et translationnelle de ces cibles.
Cette inhibition est induite par la baisse significative de l’expression des enzymes
épigénétiques les lysines acétyltransférases (KATs), qui contrôlent l’ajout des résidus d’acétylcoenzyme
A sur les histones et sur d'autres protéines dont MYC. La baisse d’expression des
KATs résultent à une baisse de l’acétylation des résidus de l’histone 3 et à une
reprogrammation de l’acétylome des cellules cancéreuses surexprimant MYC. Ces
changements au niveau de la chromatine induisent une reprogrammation transcriptionnelle et
phénotypique des cellules surexprimant MYC, qui se traduit par une perte de la transcription
des programmes oncogéniques et l’induction des programmes associés à la différenciation
cellulaire. Pour finir, nous avons évalué le potentiel synergique anticancéreux et épigénétique
de la proscillaridine A avec le médicament épigénétique la décitabine dans des lignées
cancéreuses exprimants des niveaux différentiels de MYC. Dans une lignée résistante à la
proscillaridine A et exprimant de faible niveau de MYC (lignée de cancer de côlon), la
décitabine et la proscillaridine A démontrent des activités épigénétiques synergiques tandis
que dans une lignée sensible à la proscillaridine A et surexprimant MYC (lignée de sarcome
pédiatrique), la décitabine et la proscillaridine A démontrent des activités antiprolifératives
synergiques. Dans ces travaux, nous avons donc démontré le potentiel de repositionner la
proscillaridine A dans les cancers surexprimant MYC. Également, nous démontrons le potentiel
synergique anticancéreux et épigénétique de la proscillaridine A avec la décitabine et nous
suggérons d’étudier cette combinaison de médicaments dans les cancers plus résistants à la
proscillaridine A. / Cardiac glycosides are sodium/potassium pomps’ inhibitors used for the treatment of heart
failure, and whose anticancer and epigenetic activities have been recently characterized.
However, in order to repurpose cardiac glycosides as anticancer drugs, mechanistic studies
are required to identify the anticancer and epigenetic mechanism of actions. In our
experiments, proscillaridin A exhibited the most powerful anticancer and epigenetic activities in
colon cancer, leukemia, and sarcoma cell lines. Moreover, we demonstrated that in a panel of
14 cancer cell lines, proscillaridin A anticancer activities positively correlated with MYC protooncogene
expression level. In high MYC expressing cell lines such as leukemia, proscillaridin A
inhibited MYC expression through protein destabilization and through transcriptomic and
translational regulation of MYC targets. Theses inhibitions are induced by the loss of lysine
acetylatransferase (KAT) expressions, which are epigenetic enzymes controlling the addition of
acetyl-coenzyme A on histones and other proteins such as MYC. KAT inhibitions are responsible
for the global loss of histone 3 acetylation and acetylome reprogrammation in high MYC expressing
cancer cells. These chromatin changes induced transcriptomic and phenotypic
reprogrammation, defined by a loss of the transcription of oncogenic programs and the
induction of cell differentiation. To finish, we evaluated the anticancer and epigenetic synergic
potential of proscillaridin A in combination with the epigenetic drug the decitabine in cancer
cell lines expressing different MYC levels. In a cancer cell line resistant to proscillaridin A
treatments and expressing low MYC level (colon cancer cell line), the combination of
decitabine and proscillaridin A demonstrated synergistic epigenetic activity although, in a cell
line sensitive to proscillaridin A treatments and expressing high MYC level (sarcoma cell line),
the combination of decitabine and proscillaridin A exhibited synergistic anti-proliferative
activity. To conclude, we highlighted the potential of repurposing proscillaridin A as an anticancer
treatment in high MYC expressing cells. Furthermore, we demonstrated the anticancer and
epigenetic synergistic potential of proscillaridin A in combination with decitabine and we
propose to study the drug combination in cancers that are resistant to proscillaridin A
treatment.
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