Artemisinin-based combination therapies (ACTs) are the gold standard for the treatment of malaria, but the efficacy is threatened by the development of parasite resistance. Histone deacetylase inhibitors (HDACis) are an emerging new class of potential antiplasmodial drugs. In this work, we present the design, synthesis, and biological evaluation of a mini library of dihydroartemisinin– HDACi hybrid molecules. The screening of the hybrid molecules for their activity against selected human HDAC isoforms, asexual blood stage P. falciparum parasites, and a panel of leukemia cell lines delivered important structure–activity relationships. All synthesized compounds demonstrated potent activity against the 3D7 and Dd2 line of P. falciparum with IC50 values in the single-digit nanomolar range. Furthermore, the hybrid ()-7c displayed improved activity against artemisininresistant parasites compared to dihydroartemisinin. The screening of the compounds against five cell lines from different leukemia entities revealed that all hydroxamate-based hybrids (7a–e) and the ortho-aminoanilide 8 exceeded the antiproliferative activity of dihydroartemisinin in four out of five cell lines. Taken together, this series of hybrid molecules represents an excellent starting point toward the development of antimalarial and antileukemia drug leads.

Salicylic acid is widely studied for its role in biotic stress signaling in plants. Several SA-binding proteins, including SABP2 (salicylic acid-binding protein 2) has been identified and characterized for their role in plant disease resistance. SABP2 is a 29 kDA tobacco protein that binds to salicylic acid with high affinity. It is a methylesterase enzyme that catalyzes the conversion of methyl salicylate into salicylic acid required for inducing a robust systemic acquired resistance (SAR) in plants. Methyl salicylic acid is one of the several mobile SAR signals identified in plants. SABP2-interacting protein 428 (SIP428) was identified in a yeast two-hybrid screen using tobacco SABP2 as a bait. In silico analysis shows that SIP428 possesses the SIR2 (silent information regulatory 2)-like conserved motifs. SIR2 enzymes are orthologs of sirtuin proteins that catalyze the NAD+-dependent deacetylation of Nε lysine-acetylated proteins. The recombinant SIP428 expressed in E. coli exhibits SIR2-like deacetylase activity. SIP428 shows homology to Arabidopsis AtSRT2 (67% identity), which is implicated in SA-mediated basal defenses. Immunoblot analysis using anti-acetylated lysine antibodies showed that the recombinant SIP428 is lysine acetylated. The expression of SIP428 transcripts was moderately downregulated upon infection by TMV. In the presence of SIP428, the esterase activity of SABP2 increased modestly. The interaction of SIP428 with SABP2, it's regulation upon pathogen infection, and similarity with AtSRT2 suggests that SIP428 is likely to play a role in stress signaling in plants.

The development of new anticancer drugs is necessary in order deal with the disease and with the drawbacks of currently applied drugs. Epigenetic dysregulations are a central hallmark of cancerogenesis and histone deacetylases (HDACs) emerged as promising anticancer targets. HDAC inhibitors are promising epigenetic anticancer drugs and new HDAC inhibitors are sought for in order to obtain potent drug candidates. The new HDAC inhibitor SF5-SAHA was synthesized and analyzed for its anticancer properties. The new compound SF5-SAHA showed strong inhibition of tumor cell growth with IC50 values similar to or lower than that of the clinically applied reference compound vorinostat/SAHA (suberoylanilide hydroxamic acid). Target specific HDAC inhibition was demonstrated by Western blot analyses. Unspecific cytotoxic effects were not observed in LDH-release measurements. Pro-apoptotic formation of reactive oxygen species (ROS) and caspase-3 activity induction in prostate carcinoma and hepatocellular carcinoma cell lines DU145 and Hep-G2 seem to be further aspects of the mode of action. Antiangiogenic activity of SF5-SAHA was observed on chorioallantoic membranes of fertilized chicken eggs (CAM assay). The presence of the pentafluorothio-substituent of SF5-SAHA increased the antiproliferative effects in both solid tumor and leukemia/lymphoma cell models when compared with its parent compound vorinostat. Based on this preliminary study, SF5-SAHA has the prerequisites to be further developed as a new HDAC inhibitory anticancer drug candidate.

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Systemic lupus erythematosus (SLE) is a chronic multifactorial inflammatory autoimmune disease with heterogeneous clinical manifestations. Among different manifestations, lupus nephritis (LN) remains a major cause of morbidity and mortality. There are few FDA approved treatments for LN. In general, they are non-selective and lead to global immunosuppression with significant side effects including an increased risk of infection. In the past 60 years, only one new drug, belimumab was approved for lupus disease with modest efficacy in clinic and not approved for patients suffering for nephritis. Therefore, it is urgent to develop new treatments to replace or reduce the use of current ones. Histone deacetylase 6 (HDAC6) plays a variety of biologic functions in a number of important molecular pathways in diverse immune cells. Both innate and adaptive immune cells contribute to pathogenesis of lupus. Among those cells, B cells play a central role in pathogenesis of lupus nephritis in an anti-body dependent manner through differentiation into plasma cells (PCs). As a result, HDAC6 inhibitors represent an entirely new class of agents that could have potent effects in SLE. Importantly, the available toxicity profile suggests that HDAC6 inhibitors could be advanced into SLE safely. We have demonstrated previously that histone deacetylase (HDAC6) expression is increased in animal models of systemic lupus erythematosus (SLE) and that inhibition of HDAC6 decreased disease. ACY-738 is a hydroxamic acid HDAC6 inhibitor that is highly selective for HDAC6. In our current studies, we tested if an orally selective HDAC6 inhibitor, ACY-738, would decrease disease pathogenesis in a lupus mouse model with established early disease. Moreover, we sought to delineate the cellular and molecular mechanism(s) of action of a selective HDAC6 inhibitor in SLE. In order to define the mechanism by which HDAC6 inhibition decreases disease pathogenesis in NZB/W mice by using RNAseq to evaluate the transcriptomic signatures of splenocytes from treated and untreated mice coupled with applied computational cellular and pathway analysis. In addition, we sought to bridge between the transcriptomic data obtained from the HDAC6 treated mice and human gene expression information to determine the relevance to this target in possibly controlling human lupus. We treated 20-week-old (early-disease) NZB/W F1 female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for 4~5 weeks. As the mice aged, we determined autoantibody production and cytokine levels by ELISA, and renal function by measuring proteinuria. At the termination of the study, we performed a comprehensive analysis on B cells, T cells, and innate immune cells using flow cytometry and examined renal tissue for immune-mediated pathogenesis using immunohistochemistry and immunofluorescence. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. Our results showed a reduced germinal center B cell response, decreased T follicular helper cells and diminished interferon (IFN)-γ production from T helper cells in splenic tissue. Additionally, we found the IFN-α-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. RNA sequence data showed that PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show selective HDAC6 inhibition decreased several parameters of disease pathogenesis in lupus-prone mice. The decrease was in part due to inhibition of B cell development and response. RNA sequence data analysis show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggests that a critical event might be modulation of cellular metabolism. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease by which immune cells mistakenly attacks healthy self-cells in different organs. Kidney inflammation occurs in nearly 50% of patients with lupus resulting in kidney damage leading to end stage renal disease. Lupus nephritis (LN) is major cause of morbidity and mortality associated with SLE. Current treatments for LN consist primarily of immunosuppressants that block the immune response and leave the patients with unwanted side effects including an increased risk of infection. To circumvent the unwanted side effects, we explored a novel mechanism to target the immune response. My project was to determine whether histone deacetylase 6 (HDAC6) inhibition would suppress the autoimmune inflammatory response in lupus. We found that inhibition of HDAC6 was effective at attenuating early LN, probably by down-regulating innate immune response, which suppressed subsequent adaptive immune responses downstream. HDAC6 inhibition affected the innate immune response by inhibiting type I interferon production by plasmacytoid dendritic cells. HDAC6 inhibition affected the cell mediated immune response by decreasing T helper cell and B cell activation. To determine the mechanism by which HDAC6 inhibits immune cells activation, we used RNAseq to reveal HDAC6 inhibition on multiple signaling events associated with the induction of lupus disease. These results suggest that HDAC6 could be a potential therapeutic target in the early stage of LN.

Mycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and detoxification includes various promising drug targets. Several metalloenzymes are involved in this pathway, such as a deacetylase (MshB) and mycothiol S-conjugate amidase (MCA). MshB catalyzes the deacetylation of GlcNAc-Ins to form GlcN-Ins and acetate. Mycothiol S-conjugate amidase (MCA) cleaves the amide bond of mycothiol S-conjugates of various drugs and toxins. The identification of the native co-factor is critical for the design of potent and effective inhibitors. Therefore, in this study, we identified the possible native co-factors of MshB and MCA from M. smegmatis and M. tuberculosis. To reach our aim, we used a pull-down method to rapidly purify halo-MshB and halo-MCA under anaerobic conditions. Our data indicates that the metal bound to MshB and MCA anaerobically purified from E. coli grown in minimal medium is mainly Fe(II), while proteins purified under aerobic conditions contain bound Zn (II) and Fe(II) that varies with the metal content of the medium. For a further clarification of the metal ion preferences of MshB and MCA, we determined the MshB and MCA affinity for Zn(II) to be in the picomolar range and Ms MshB affinity for Fe(II) in nanomolar range. These results indicate that MshB and MCA can be found bound with either iron or zinc and this is independent to their affinities for these metal ions. / Master of Science

Mycobacterium species, which contain the causative agent for human tuberculosis (TB), produce inositol derivatives including mycothiol (MSH).  MSH is a unique and dominant cytosolic thiol that protects mycobacterial pathogens against the damaging effects of reactive oxygen species and is involved in antibiotic detoxification.  Therefore, MSH is considered a potential drug target.  The deacetylase MshB catalyzes the committed step in MSH biosynthesis by converting N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins) to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins).  In this dissertation, we present detailed functional analysis of MshB.  Our work has shown that MshB is activated by divalent metal ions that can switch between Zn2+ and Fe2+ depending on environmental conditions, including  metal ion availability and oxidative conditions.  MshB employs a general acid-base catalyst mechanism wherein the Asp15 functions as a general base to activate the metal-bound water nucleophile for attack of the carbonyl carbon on substrate.  Proton-transfer from a general acid catalyst facilitates breakdown of the tetrahedral intermediate and release of products.  A dynamic tyrosine was identified that regulates access to the active site and participates in catalysis by stabilizing the oxyanion intermediate.  Molecular docking simulations suggest that the GlcNAc moiety on GlcNAc-Ins is stabilized by hydrogen bonding interactions with active site residues, while a hydrophobic stacking interaction between the inositol ring and Met98 also appears to contribute to substrate affinity for MshB.  Additional binding interactions with side chains in a hydrophobic cavity adjacent to the active site were suggested when the docking experiments were carried out with large amidase substrates.  Together the results from this study provide groundwork for the rational design of specific inhibitors against MshB, which may circumvent current challenges with TB treatment. / Ph. D.