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.

Antimalarial activity of primaquine operates via a two-step biochemical relay.

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6.

INTRODUCTION: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding. METHODS: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System. RESULTS: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a KD in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC50. DISCUSSION: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.

Potent Antimalarial 2-Pyrazolyl Quinolone bc 1 (Qi) Inhibitors with Improved Drug-like Properties.

A series of 2-pyrazolyl quinolones has been designed and synthesized in 5-7 steps to optimize for both in vitro antimalarial potency and various in vitro drug metabolism and pharmacokinetics (DMPK) features. The most potent compounds display no cross-resistance with multidrug resistant parasite strains (W2) compared to drug sensitive strains (3D7), with IC50 (concentration of drug required to achieve half maximal growth suppression) values in the range of 15-33 nM. Furthermore, members of the series retain moderate activity against the atovaquone-resistant parasite isolate (TM90C2B). The described 2-pyrazoyl series displays improved DMPK properties, including improved aqueous solubility compared to previously reported quinolone series and acceptable safety margin through in vitro cytotoxicity assessment. The 2-pyrazolyl quinolones are believed to bind to the ubiquinone-reducing Qi site of the parasite bc 1 complex, which is supported by crystallographic studies of bovine cytochrome bc 1 complex.

Albendazole and antibiotics synergize to deliver short-course anti-Wolbachia curative treatments in preclinical models of filariasis.

Elimination of filariasis requires a macrofilaricide treatment that can be delivered within a 7-day period. Here we have identified a synergy between the anthelmintic albendazole (ABZ) and drugs depleting the filarial endosymbiont Wolbachia, a proven macrofilaricide target, which reduces treatment from several weeks to 7 days in preclinical models. ABZ had negligible effects on Wolbachia but synergized with minocycline or rifampicin (RIF) to deplete symbionts, block embryogenesis, and stop microfilariae production. Greater than 99% Wolbachia depletion following 7-day combination of RIF+ABZ also led to accelerated macrofilaricidal activity. Thus, we provide preclinical proof-of-concept of treatment shortening using antibiotic+ABZ combinations to deliver anti-Wolbachia sterilizing and macrofilaricidal effects. Our data are of immediate public health importance as RIF+ABZ are registered drugs and thus immediately implementable to deliver a 1-wk macrofilaricide. They also suggest that novel, more potent anti-Wolbachia drugs under development may be capable of delivering further treatment shortening, to days rather than weeks, if combined with benzimidazoles.

A potential role for cannabinoid receptors in the therapeutic action of fenofibrate.

Cannabinoids are reported to have actions through peroxisome proliferator-activated receptors (PPARs), which led us to investigate PPAR agonists for activity at the cannabinoid receptors. Radio-ligand binding and functional assays were conducted using human recombinant cannabinoid type 1 (CB1) or cannabinoid type 2 (CB2) receptors, as well as the guinea pig isolated ileum, using the full agonist CP55940 as a positive control. The PPAR-α agonist fenofibrate exhibited submicromolar affinity for both receptors (pKi CB1, 6.3 ± 0.1; CB2, 7.7 ± 0.1). Functionally, fenofibrate acted as an agonist at the CB2 receptor (pEC50, 7.7 ± 0.1) and a partial agonist at the CB1 receptor, although with a decrease in functional response at higher concentrations, producing bell-shaped concentration-response curves. High concentrations of fenofibrate were able to increase the dissociation rate constant for [(3)H]-CP55940 at the CB1 receptor, (kfast without: 1.2 ± 0.2/min; with: 3.8 ± 0.1 × 10(-2)/min) and decrease the maximal response to CP55940 (Rmax, 86 ± 2%), which is consistent with a negative allosteric modulator. Fenofibrate also reduced electrically induced contractions in isolated guinea pig ileum via CB1 receptors (pEC50, 6.0 ± 0.4). Fenofibrate is thus identified as an example of a new class of cannabinoid receptor ligand and allosteric modulator, with the potential to interact therapeutically with cannabinoid receptors in addition to its primary PPAR target.