Structure-based therapeutic development against the Apicomplexan parasites

Apicomplexa are a large group of pathogenic parasites, which cause major infectious diseases including malaria and toxoplasmosis. Malaria is a mosquito-borne disease caused by Plasmodium Spp. It infects several hundred million people worldwide each year. Toxoplasma gondii has become one of the most prevalent parasites with one-third of the world population at risk of latent toxoplasmosis. Rising drug-resistance in apicomplexan parasites has become a huge challenge. New drugs with better potency and safety profiles against drug-resistant parasites and effective preventive vaccines are urgently required. Cytochrome bc1 is a catalytic complex in mitochondrial electron transport chain and a validated drug target for apicomplexan parasites. Atovaquone eliminates parasites by inhibiting Qo site of cytochrome bc1, and widely-used for both malaria and toxoplasmosis treatments. Mutations in the Qo site of apicomplexan cytochrome bc1 has contributed to the rapid emergence of atovaquone-resistance. To overcome this problem, 4(1H)-quinolone family compounds targeting the Qi site of cytochrome bc1 have been developed. The complexes of bovine cytochrome bc1 (the surrogate for the human host) with potent 4(1H)-quinolones determined by X-ray crystallography and cryo-EM are discussed in this thesis and show the compounds bound solely to the Qi site. Currently, there is no cytochrome bc1 structure from any of the apicomplexan parasites available. In-silico docking of 4(1H)-quinolones performed with a parasite homology model demonstrated possible binding poses of lead compounds. The greater differential for the Qi site’s primary structure combined with crystallographic and computational modelling provides new insight to support the development of new compounds with selective potency against parasite and lower toxicity. Cryo-EM is confirmed to be the first proof of principle for structural insight on apicomplexan cytochrome bc1 that has never been achieved by crystallography due to challenges in protein purification from parasites and complex nature of the protein. In order to prevent the emergence of future drug resistance, the second target for 4(1H)-quinolones was also considered. Type II NADH:ubiquinone oxidoreductase (NDH2) is an enzyme in apicomplexan electron transport chain that also inhibited by 4(1H)-quinolones. Recombinant NDH2 purification and crystallisation are described in this thesis. Vaccine has been considered as an effective tool for malaria prevention. Nowadays, only one licenced malaria vaccine is available. Finding new vaccine candidates is highly active research. 19 kDa fragment merozoite surface protein 1 (MSP119) is a protein on parasite’s membrane that is antibody target for vaccine development. Rusticyanin, a copper protein from Thiobacillus ferrooxidans, has been reported to have antimalarial activity through an interaction with MSP119. In this work, interactions between MSP119 and rusticyanin were characterised. Crystallisation of MSP119-rusticyanin complex was attempted but only rusticyanin crystals were obtained. The residues 1-28 of rusticyanin cannot be seen in electron density that may be disordered in view of the interaction with MSP119

Purification and Structural Characterization of Aggregation-Prone Human TDP-43 Involved in Neurodegenerative Diseases

© 2020 The Author(s) Mislocalization, cleavage, and aggregation of the human protein TDP-43 is found in many neurodegenerative diseases. As is the case with many other proteins that are completely or partially structurally disordered, production of full-length recombinant TDP-43 in the quantities necessary for structural characterization has proved difficult. We show that the full-length TDP-43 protein and two truncated N-terminal constructs 1-270 and 1-263 can be heterologously expressed in E. coli. Full-length TDP-43 could be prevented from aggregation during purification using a detergent. Crystals grown from an N-terminal construct (1-270) revealed only the N-terminal domain (residues 1-80) with molecules arranged as parallel spirals with neighboring molecules arranged in head-to-tail fashion. To obtain detergent-free, full-length TDP-43 we mutated all six tryptophan residues to alanine. This provided sufficient soluble protein to collect small-angle X-ray scattering data. Refining relative positions of individual domains and intrinsically disordered regions against this data yielded a model of full-length TDP-43

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria

Targeting the Ubiquinol-Reduction (Qi) Site of the Mitochondrial Cytochrome bc1 Complex for the Development of Next Generation Quinolone Antimalarials

Antimalarials targeting the ubiquinol-oxidation (Qo) site of the Plasmodium falciparum bc1 complex, such as atovaquone, have become less effective due to the rapid emergence of resistance linked to point mutations in the Qo site. Recent findings showed a series of 2-aryl quinolones mediate inhibitions of this complex by binding to the ubiquinone-reduction (Qi) site, which offers a potential advantage in circumventing drug resistance. Since it is essential to understand how 2-aryl quinolone lead compounds bind within the Qi site, here we describe the co-crystallization and structure elucidation of the bovine cytochrome bc1 complex with three different antimalarial 4(1H)-quinolone sub-types, including two 2-aryl quinolone derivatives and a 3-aryl quinolone analogue for comparison. Currently, no structural information is available for Plasmodial cytochrome bc1. Our crystallographic studies have enabled comparison of an in-silico homology docking model of P. falciparum with the mammalian’s equivalent, enabling an examination of how binding compares for the 2- versus 3-aryl analogues. Based on crystallographic and computational modeling, key differences in human and P. falciparum Qi sites have been mapped that provide new insights that can be exploited for the development of next-generation antimalarials with greater selective inhibitory activity against the parasite bc1 with improved antimalarial properties

Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron microscopy

The dramatic growth in the use of cryo-electron microscopy (cryo-EM) to generate high-resolution structures of macromolecular complexes has changed the landscape of structural biology. The majority of structures deposited in the Electron Microscopy Data Bank (EMDB) at higher than 4-Å resolution were collected on Titan Krios microscopes. Although the pipeline for single-particle data collection is becoming routine, there is much variation in how sessions are set up. Furthermore, when collection is under way, there are a range of approaches for efficiently moving and pre-processing these data. Here, we present a standard operating procedure for single-particle data collection with Thermo Fisher Scientific EPU software, using the two most common direct electron detectors (the Thermo Fisher Scientific Falcon 3 (F3EC) and the Gatan K2), as well as a strategy for structuring these data to enable efficient pre-processing and on-the-fly monitoring of data collection. This protocol takes 3–6 h to set up a typical automated data collection session