Michael K. Riscoe

Simple and Inexpensive Fluorescence-Based Technique for High-Throughput Antimalarial Drug Screening

ABSTRACT Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC50) after 48 h of parasite growth in the presence of each drug. The EC50s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis-ε-(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for high-throughput antimalarial drug screening.

Chemical genetics of Plasmodium falciparum

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library—many of which showed potent in vitro activity against drug-resistant P. falciparum strains—and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.

Quinolone-3-Diarylethers: A New Class of Antimalarial Drug

ELQ-300, an investigational drug for treating and preventing malaria, shows potent transmission-blocking activity in rodent models of malaria. Taking the Bite Out of Malaria Malaria is spread from person to person by mosquitoes that inject 8 to 10 sporozoite forms of the parasite in a single bite. The sporozoites reproduce in the liver to produce 10,000 to 30,000 merozoites before the liver schizont ruptures and parasites flood into the bloodstream where the absolute parasite burden may increase to a thousand billion (1012) circulating parasites. Some of these parasites develop into gametocytes that may be ingested by another mosquito where they progress through ookinete, oocyst, and sporozoite stages to complete the cycle. Like quinine, most antimalarial drugs in use today target only the symptomatic blood stage. The efficacy of these drugs has been compromised by resistance, and so there is a pressing need for new drugs that target multiple stages of the parasite life cycle for use in malaria treatment and prevention. Clearly, it is advantageous to strike at the liver stage where parasite numbers are low, to diminish the likelihood of selecting for a resistant mutant and before the infection has a chance to weaken the defenses of the human host. In a new study, Nilsen and colleagues describe ELQ-300, a 4(1H)-quinolone-3-diarylether, which targets the liver and blood stages, including the forms that are crucial to disease transmission (gametocytes, zygotes, and ookinetes). In mouse models of malaria, a single oral dose of 0.03 mg/kg prevented sporozoite-induced infections, whereas four daily doses of 1 mg/kg achieved complete cures of patent infections. ELQ-300 is a preclinical candidate that may be coformulated with other antimalarials to prevent and treat malaria, with the potential to aid in eradication of the disease. The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite’s life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite’s mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

Discovery of dual function acridones as a new antimalarial chemotype

Preventing and delaying the emergence of drug resistance is an essential goal of antimalarial drug development. Monotherapy and highly mutable drug targets have each facilitated resistance, and both are undesirable in effective long-term strategies against multi-drug-resistant malaria. Haem remains an immutable and vulnerable target, because it is not parasite-encoded and its detoxification during haemoglobin degradation, critical to parasite survival, can be subverted by drug–haem interaction as in the case of quinolines and many other drugs. Here we describe a new antimalarial chemotype that combines the haem-targeting character of acridones, together with a chemosensitizing component that counteracts resistance to quinoline antimalarial drugs. Beyond the essential intrinsic characteristics common to deserving candidate antimalarials (high potency in vitro against pan-sensitive and multi-drug-resistant Plasmodium falciparum, efficacy and safety in vivo after oral administration, inexpensive synthesis and favourable physicochemical properties), our initial lead, T3.5 (3-chloro-6-(2-diethylamino-ethoxy)-10-(2-diethylamino-ethyl)-acridone), demonstrates unique synergistic properties. In addition to ‘verapamil-like’ chemosensitization to chloroquine and amodiaquine against quinoline-resistant parasites, T3.5 also results in an apparently mechanistically distinct synergism with quinine and with piperaquine. This synergy, evident in both quinoline-sensitive and quinoline-resistant parasites, has been demonstrated both in vitro and in vivo. In summary, this innovative acridone design merges intrinsic potency and resistance-counteracting functions in one molecule, and represents a new strategy to expand, enhance and sustain effective antimalarial drug combinations.

Xanthones as Antimalarial Agents: Discovery, Mode of Action, and Optimization

It is believed that at no time in the history of the human race malaria has been absent. This disease, which is caused by protozoa of the genus Plasmodium, in all likelihood has been responsible for the death of about half of all people who ever lived. Even today, after attempts at intervention on a worldwide scale, malaria remains the most significant parasitic disease in the tropics and sub-tropics, where it causes at least 500 million clinical episodes and claims 1.5 million lives each year, mostly young children and pregnant women. Widespread resistance to the best and least expensive antimalarials, chloroquine and S/P (i.e., a combination of sulfadoxine and pyrimethamine), combined with an increasing tolerance to insecticides in the mosquito vector, threaten a global malaria tragedy unless new countermeasures are developed. For malaria therapy, the great panacea would be the development of a long-lasting vaccine, but until this becomes a reality, people living in and traveling to endemic regions must rely on a dwindling cache of more expensive drugs; many beyond the economic reach of impoverished people living in malarious regions of the world. Our course to recognition of xanthones as potential antimalarial agents took a rather circuitous route, involving both serendipity and empiricism, and is described together with mechanistic details of drug action. From a chance encounter with a sea urchin collected near the city of Cannon Beach on the Oregon coast to naturally occurring and functionalized xanthones, it is revealed how these compounds target the Plasmodium parasites most vulnerable feature--the digestive vacuole.

Endochin-like quinolones are highly efficacious against acute and latent experimental toxoplasmosis

Toxoplasma gondii is a widely distributed protozoan pathogen that causes devastating ocular and central nervous system disease. We show that the endochin-like quinolone (ELQ) class of compounds contains extremely potent inhibitors of T. gondii growth in vitro and is effective against acute and latent toxoplasmosis in mice. We screened 50 ELQs against T. gondii and selected two lead compounds, ELQ-271 and ELQ-316, for evaluation. ELQ-271 and ELQ-316, have in vitro IC50 values of 0.1 nM and 0.007 nM, respectively. ELQ-271 and ELQ-316 have ED50 values of 0.14 mg/kg and 0.08 mg/kg when administered orally to mice with acute toxoplasmosis. Moreover, ELQ-271 and ELQ-316 are highly active against the cyst form of T. gondii in mice at low doses, reducing cyst burden by 76–88% after 16 d of treatment. To investigate the ELQ mechanism of action against T. gondii, we demonstrate that endochin and ELQ-271 inhibit cytochrome c reduction by the T. gondii cytochrome bc1 complex at 8 nM and 31 nM, respectively. We also show that ELQ-271 inhibits the Saccharomyces cerevisiae cytochrome bc1 complex, and an M221Q amino acid substitution in the Qi site of the protein leads to >100-fold resistance. We conclude that ELQ-271 and ELQ-316 are orally bioavailable drugs that are effective against acute and latent toxoplasmosis, likely acting as inhibitors of the Qi site of the T. gondii cytochrome bc1 complex.

α lipoic acid inhibits human T-cell migration: Implications for multiple sclerosis

We have demonstrated previously the ability of the antioxidant α lipoic acid (ALA) to suppress and treat a model of multiple sclerosis (MS), relapsing experimental autoimmune encephalomyelitis (EAE). We describe the effects of ALA and its reduced form, dihydrolipoic acid (DHLA), on the transmigration of human Jurkat T cells across a fibronectin barrier in a transwell system. ALA and DHLA inhibited migration of Jurkat cells in a dose‐dependent fashion by 16–75%. ALA and DHLA reduced matrix metalloproteinase‐9 (MMP‐9) activity by 18–90% in Jurkat cell supernatants. GM6001, a synthetic inhibitor of MMP, reduced Jurkat cell migration, but not as effectively as ALA and DHLA did. Both ALA and DHLA downmodulated the surface expression of the α4β1 integrin (very late activation‐4 antigen; VLA‐4), which binds fibronectin and its endothelial cell ligand vascular cell adhesion molecule‐1 (VCAM‐1). Moreover, ALA, but not DHLA, reduced MMP‐9‐specific mRNA and extracellular MMP‐9 from Jurkat cells and their culture supernatants as detected by relative reverse transcriptase‐polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA), respectively. ALA and DHLA inhibited Jurkat cell migration and have different mechanisms for inhibiting MMP‐9 activity. These data, coupled with its ability to treat relapsing EAE, suggest that ALA warrants investigation as a therapy for MS.

Discovery, synthesis, and optimization of antimalarial 4(1H)-quinolone-3-diarylethers

The historical antimalarial compound endochin served as a structural lead for optimization. Endochin-like quinolones (ELQ) were prepared by a novel chemical route and assessed for in vitro activity against multidrug resistant strains of Plasmodium falciparum and against malaria infections in mice. Here we describe the pathway to discovery of a potent class of orally active antimalarial 4(1H)-quinolone-3-diarylethers. The initial prototype, ELQ-233, exhibited low nanomolar IC50 values against all tested strains including clinical isolates harboring resistance to atovaquone. ELQ-271 represented the next critical step in the iterative optimization process, as it was stable to metabolism and highly effective in vivo. Continued analoging revealed that the substitution pattern on the benzenoid ring of the quinolone core significantly influenced reactivity with the host enzyme. This finding led to the rational design of highly selective ELQs with outstanding oral efficacy against murine malaria that is superior to established antimalarials chloroquine and atovaquone.

Optimization of Xanthones for Antimalarial Activity: the 3,6-Bis-ω-Diethylaminoalkoxyxanthone Series

ABSTRACT Hydroxyxanthones have been identified as novel antimalarial agents. The compounds are believed to exert their activity by complexation to heme and inhibition of hemozoin formation. Modification of the xanthone structure was pursued to improve their antimalarial activity. Attachment of R-groups bearing protonatable nitrogen atoms was conducted to enhance heme affinity through ionic interactions with the propionate side chains of the metalloporphyrin and to facilitate drug accumulation in the parasite food vacuole. A series of 3,6-bis-ω-diethylaminoalkoxyxanthones with side chains ranging from 2 to 8 carbon atoms were prepared and evaluated. Measurement of heme affinity for each of the derivatives revealed a strong correlation (R2 = 0.97) between affinity and antimalarial potency. The two most active compounds in the series contained 5- and 6-carbon side chains and exhibited low nanomolar 50% inhibitory concentration (IC50) values against strains of chloroquine-susceptible and multidrug-resistant Plasmodium falciparum in vitro. Both of these xanthones exhibit stronger heme affinity (8.26 × 105 and 9.02 × 105 M−1, respectively) than either chloroquine or quinine under similar conditions and appear to complex heme in a unique manner.

Optimization of endochin-like quinolones for antimalarial activity

Our prior work on tricyclic acridones combined with a desire to minimize the tricyclic system led to an interest in antimalarial quinolones and a reexamination of endochin, an experimental antimalarial from the 1940s. In the present article, we show that endochin is unstable in the presence of murine, rat, and human microsomes which may explain its relatively poor antimalarial activity in mammalian systems. We also profile the structure-activity relationships of ≈ 30 endochin-like quinolone (ELQ) analogs and highlight features that are associated with enhanced metabolic stability, potent antiplasmodial activity against multidrug resistant strains of Plasmodium falciparum, and equal activity against an atovaquone-resistant clinical isolate. Our work also features an ELQ construct containing a polyethylene glycol carbonate pro-moiety that is highly efficacious by oral administration in a murine malaria model. These findings provide compelling evidence that development of ELQ therapeutics is feasible.