Ashley J. Warman

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc1. Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Identification, design and biological evaluation of heterocyclic 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 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., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc1, and studies to determine the potential advantage of this dual-targeting effect are in progress.

Cytochrome b Mutation Y268S Conferring Atovaquone Resistance Phenotype in Malaria Parasite Results in Reduced Parasite bc1 Catalytic Turnover and Protein Expression

Background: Cytochrome b mutations confer atovaquone resistance, resulting in antimalarial drug failures. Results: Mutation Y268S reduces bc1 catalytic turnover and stability. Conclusion: Reduction of catalytic turnover and iron-sulfur protein content in parasite Y268S bc1 confers a fitness cost. These results were not predicted using yeast models. Significance: Data will aid novel bc1 inhibitor design and inform epidemiological studies of atovaquone resistance. Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. MalaroneTM) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Qo) site of the mitochondrial cytochrome bc1 complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Qo pocket of cytochrome b, most notably near the conserved Pro260-Glu261-Trp262-Tyr263 (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (Ki) for atovaquone with a concomitant reduction in the Vmax of the bc1 complex of ∼40% and a 3-fold increase in the observed Km for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc1 suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc1 and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc1 activity.

Chapter 17 Type II NADH: Quinone Oxidoreductases of Plasmodium Falciparum and Mycobacterium Tuberculosis: Kinetic and High‐Throughput Assays

Type II NADH: quinone oxidoreductases (ndh) are flavoenzymes found in a broad range of organisms including plants, fungi, protozoa, and bacteria. The ndh enzymes catalyze the oxidation of NADH with concomitant reduction of quinone (Q). These membrane-bound respiratory enzymes differ from the canonical NADH: dehydrogenase (complex I), because they are not involved in the vectorial transfer of protons across membranes. In Plasmodium falciparum and Mycobacterium tuberculosis, causative agents of malaria and tuberculosis, respectively, ndhs have aroused interest because of the essential role played in maintaining a reduced Q-pool during infection. In this chapter, we present methods for the measurement of steady-state parameters for ndhs from both pathogens, highlighting best practices and caveats. In addition, owing to the interest in ndhs as potential chemotherapeutic targets, we describe a miniaturized endpoint assay that is validated for high-throughput screening (HTS) of chemical libraries.

Antitubercular pharmacodynamics of phenothiazines

OBJECTIVES Phenothiazines have been shown to exhibit in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb) and multidrug-resistant Mtb. They are predicted to target the genetically validated respiratory chain component type II NADH:quinone oxidoreductase (Ndh). Using a set of compounds containing the phenothiazine pharmacophore, we have (i) investigated whether chemical validation data support the molecular target and (ii) evaluated pharmacophore tractability for further drug development. METHODS Recombinant Mtb Ndh was generated and its functionality confirmed by steady-state kinetics. Pharmacodynamic profiling of the phenothiazines, including antitubercular efficacy in aerobic and O2-limited conditions, time-kill assays and isobole analyses against first-line antituberculars, was performed. Potential mitochondrial toxicity was assessed in a modified HepG2 cell-line assay and against bovine cytochrome bc1. RESULTS Steady-state kinetic analyses revealed a substrate preference for coenzyme Q2 and an inability to utilize NADPH. A positive correlation between recombinant Ndh inhibition and kill of aerobically cultured Mtb was observed, whilst enhanced potency was demonstrated in a hypoxic model. Time-kill studies revealed the phenothiazines to be bactericidal whilst isobolograms exposed antagonism with isoniazid, indicative of intracellular NADH/NAD(+) couple perturbation. At therapeutic levels, phenothiazine-mediated toxicity was appreciable; however, specific mitochondrial targeting was excluded. CONCLUSIONS Data generated support the hypothesis that Ndh is the molecular target of phenothiazines. The favourable pharmacodynamic properties of the phenothiazines are consistent with a target product profile that includes activity against dormant/persistent bacilli, rapid bactericidal activity and activity against drug-resistant Mtb by a previously unexploited mode of action. These properties warrant further medicinal chemistry to improve potency and safety.

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 parasites 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 IC50 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.

A novel drug for uncomplicated malaria: targeted high throughput screening (HTS) against the type II NADH:ubiquinone oxidoreductase (PfNdh2) of Plasmodium falciparum

The mitochondrial respiratory chain of the malaria parasite Plasmodium falciparum differs from that of its human host in that it lacks a canonical protonmotive NADH:ubiquinone oxidoreductase (Complex I), containing instead a single sub-unit, non-protonmotive Ndh2, similar to that found in plant mitochondria, fungi and some bacteria [1,2]. As such, the P. falciparum Ndh 2 (PfNdh2) is a potentially attractive anti-malarial chemotherapeutic target. Using an E.coli NADH dehydrogenase knockout strain (ANN0222, ndh::tet nuoB::nptI-sacRB) we have developed a heterologous expression system for PfNdh2, facilitating its physicochemical and enzymological characterisation [2]. PfNdh2 represents a metabolic choke point in the respiratory chain of P. falciparum mitochondria and is the focus of a drug discovery programme towards the development of a novel therapy for uncomplicated malaria. Here we describe a miniaturised spectrophotometric assay for recombinant PfNdh2 (steady state NADH oxidation and ubiquinone reduction monitored at 340 nm and 283 nm respectively) with robust assay performance measures that has been utilised for the high throughput screening (HTS) of small molecule inhibitors. The objectives of the HTS were twofold: (i) Increase the number of selective PfNdh2 inhibitors and (ii) to expand the number of inhibitor chemotypes. At the time of screening, only one proof of concept molecule, 1-hydroxy-2-dodecyl-4-(1H)quinolone (HDQ), was known to have PfNdh2 inhibitory activity (IC50=70 nM) [3,4]. HDQ was used to initiate a primary similarity-based screen of 1000 compounds from a compound collection of 750,000 compounds (curated by Biofocus-DPI). Chemoinformatics methodology was applied to the hits from this initial phase in order to perform a hit expansion screen on a further ~16,000 compounds. Application of this chemoinformatic strategy allowed us to cover ~16% diversity whilst screening just ~2% of the compound collection. The HTS resulted in a hit rate of 0.29% and 1 50 compounds were progressed for potency against PfNdh2. Of these compounds, 50 were considered active with IC50s ranging from 100 nM to 40 μM. Currently seven distinct chemotypes are being progressed from hit to lead using traditional synthetic medicinal chemistry strategies.

Rational Design, Synthesis, and Biological Evaluation of Heterocyclic Quinolones Targeting the Respiratory Chain of Mycobacterium tuberculosis

A high-throughput screen (HTS) was undertaken against the respiratory chain dehydrogenase component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb). The 11000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors, trifluoperazine and thioridazine. Combined HTS (11000 compounds) and in-house screening of a limited number of quinolones (50 compounds) identified ∼100 hits and four distinct chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb screening of the complete in-house quinolone library (350 compounds) identified a further ∼90 hits across three quinolone subtemplates. Quinolones containing the amine-based side chain were selected as the pharmacophore for further modification, resulting in metabolically stable quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, 42a (MTC420), displays acceptable antituberculosis activity (Mtb IC50 = 525 nM, Mtb Wayne IC50 = 76 nM, and MDR Mtb patient isolates IC50 = 140 nM) and favorable pharmacokinetic and toxicological profiles.

Development of a novel drug for uncomplicated malaria targeting the mitochondrial NADH:quinone oxidoreductase

NADH:quinone oxidoreductase (PfNDH2) represents a metabolic choke point in the respiratory chain of Plasmodium falciparum mitochondria and is the focus of a drug discovery programme. A miniaturised assay for recombinant PfNDH2 with robust assay performance measures was generated for the high throughput screening (HTS) of a focused library of 17,000 drug-like compounds. A quantitative structure-activity relationship has been developed around one of the chemical templates derived from the HTS hits. Lead molecules developed to date show selective inhibitory activity against PfNDH2 versus P. falciparum bc1 or dihydroorotate dehydrogenase (DHODH). Potent enzyme inhibition is accompanied by in vitro parasite kill of multidrug-resistant strains in the low nM range and clearance of parasites from in vivo P. berghei models. Lead molecules also display excellent in vitro therapeutic indices against human cell lines and bovine bc1. Initial metabolic studies in human liver microsomes and hepatocytes indicate favourable pharmacology. These data support the further development of this new candidate drug targeting a novel parasite component.