Investigating Toxoplasma gondii peroxisome and the discovery of two Bisabolane Sesquiterpenes as anti-leshmanials.

The use of natural products for treating protozoan infections can be traced back to Rome in 1631, where cinchona tree bark was used to cure malaria. The discovery of novel naturally derived compounds for the treatment of cutaneous leishmaniasis and toxoplasmosis is vital for many vaccine deficient protozoan infections today. Here, the assessment of natural products against Leishmania mexicana (L. mexicana) was explored using a library of compounds screened against the mammalian stage of L. mexicana in in vitro assays. Two hit compounds from the screen were then used in metabolomic studies to determine mode of action. In addition, another natural compound, Aureobasidin A and its derivatives, and peroxisome inhibitors were screened against Toxoplasma gondii (T. gondii). Peroxisomes are organelles involved in the metabolism of fatty acids and choles- terol. Other than lipid metabolism, peroxisomes contain many enzymes involved in several different metabolic processes. Catalase is involved in the neutralization of hydrogen peroxide thereby, preventing toxic build up within cells. This enzyme over time has become a key identifier of peroxisomes in many organisms. However, this is controversial when it comes to T. gondii. The use of catalase as a marker for peroxisomes in this parasite has been disputed, and in some cases led to the belief that the T. gondii does not possess these organelles. In this thesis, we take a dif- ferent approach in to establishing the existence of peroxisomes. Through evolution T. gondii has maintained, within its genome, genes encoding peroxisomal proteins, named peroxins (Pex). Here we investigated the presence of peroxisomes within T. gondii using Pex proteins. Our experimental approach involved characterization of putative TgPex5 and protein ligand TgSCP2. Using molecular biology, reverse genetics and protein characterization, we show that through pull-down assays, lo- calisation and complementation of TgPex5 in yeast expression systems, we are able to provide evidence to prove the presence of peroxisomes within T. gondii

Identification of Novel Antimalarial Chemotypes via Chemoinformatic Compound Selection Methods for a High-Throughput Screening Program against the Novel Malarial Target, PfNDH2: Increasing Hit Rate via Virtual Screening Methods

Malaria is responsible for approximately 1 million deaths annually; thus, continued efforts to discover new antimalarials are required. A HTS screen was established to identify novel inhibitors of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase (PfNDH2). On the basis of only one known inhibitor of this enzyme, the challenge was to discover novel inhibitors of PfNDH2 with diverse chemical scaffolds. To this end, using a range of ligand-based chemoinformatics methods, ~17000 compounds were selected from a commercial library of ~750000 compounds. Forty-eight compounds were identified with PfNDH2 enzyme inhibition IC(50) values ranging from 100 nM to 40 μM and also displayed exciting whole cell antimalarial activity. These novel inhibitors were identified through sampling 16% of the available chemical space, while only screening 2% of the library. This study confirms the added value of using multiple ligand-based chemoinformatic approaches and has successfully identified novel distinct chemotypes primed for development as new agents against malaria

Identification, Design and Biological Evaluation of Heterocyclic Quinolones Targeting Plasmodium falciparum Type II NADH:Quinone Oxidoreductase (PfNDH2)

Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (8b). 8b has IC(50)s in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC(50) = 15 nM PfNDH2; IC(50) = 54 nM (3D7 strain of P. falciparum) with notable oral activity of ED(50)/ED(90) of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the bc(1) complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies

Identification, Design and Biological Evaluation of Bisaryl Quinolones Targeting Plasmodium falciparum Type II NADH:Quinone Oxidoreductase (PfNDH2)

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure-activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC(50) against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc(1), and studies to determine the potential advantage of this dual-targeting effect are in progress

Identification, Design and Biological Evaluation of Bisaryl Quinolones Targeting <i>Plasmodium falciparum</i> Type II NADH:Quinone Oxidoreductase (PfNDH2)

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite <i>Plasmodium falciparum</i>. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure–activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)­benzyl)­phenyl)­quinolin-4­(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of <i>P. falciparum</i> of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC₅₀ against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., <b>6d</b>, <b>6f</b>, <b>14e</b>) have the capacity to inhibit both PfNDH2 and <i>P. falciparum</i> cytochrome <i>bc</i>₁, and studies to determine the potential advantage of this dual-targeting effect are in progress

Identification, Design and Biological Evaluation of Heterocyclic Quinolones Targeting <i>Plasmodium falciparum</i> Type II NADH:Quinone Oxidoreductase (PfNDH2)

Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite <i>Plasmodium falciparum</i>, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (<b>8b</b>). <b>8b</b> has IC₅₀s in the nanomolar range versus both the enzyme and whole cell <i>P. falciparum</i> (IC₅₀ = 15 nM PfNDH2; IC₅₀ = 54 nM (3D7 strain of <i>P. falciparum</i>) with notable oral activity of ED₅₀/ED₉₀ of 1.87/4.72 mg/kg versus <i>Plasmodium berghei</i> (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the <i>bc</i>₁ complex of <i>P. falciparum</i> providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies