Targeting lysine deacetylases (KDACs) in parasites

Due to an increasing problem of drug resistance among almost all parasites species ranging from protists to worms, there is an urgent need to explore new drug targets and their inhibitors to provide new and effective parasitic therapeutics. In this regard, there is growing interest in exploring known drug leads of human epigenetic enzymes as potential starting points to develop novel treatments for parasitic diseases. This approach of repurposing (starting with validated targets and inhibitors) is quite attractive since it has the potential to reduce the expense of drug development and accelerate the process of developing novel drug candidates for parasite control. Lysine deacetylases (KDACs) are among the most studied epigenetic drug targets of humans, and a broad range of small-molecule inhibitors for these enzymes have been reported. In this work, we identify the KDAC protein families in representative species across important classes of parasites, screen a compound library of 23 hydroxamate- or benzamide-based small molecules KDAC inhibitors, and report their activities against a range of parasitic species, including the pathogen of malaria (Plasmodium falciparum), kinetoplastids (Trypanosoma brucei and Leishmania donovani), and nematodes (Brugia malayi, Dirofilaria immitis and Haemonchus contortus). Compound activity against parasites is compared to that observed against the mammalian cell line (L929 mouse fibroblast) in order to determine potential parasite-versus-host selectivity). The compounds showed nanomolar to sub-nanomolar potency against various parasites, and some selectivity was observed within the small panel of compounds tested. The possible binding modes of the active compounds at the different protein target sites within different species were explored by docking to homology models to help guide the discovery of more selective, parasite-specific inhibitors. This current work supports previous studies that explored the use of KDAC inhibitors in targeting Plasmodium to develop new anti-malarial treatments, and also pioneers experiments with these KDAC inhibitors as potential new anthelminthics. The selectivity observed begins to address the challenges of targeting specific parasitic diseases while limiting host toxicity

Pharmacokinetics and pharmacodynamics utilizing unbound target tissue exposure as part of a disposition-based rationale for lead optimization of benzoxaboroles in the treatment of Stage 2 Human African Trypanosomiasis

This review presents a progression strategy for the discovery of new anti-parasitic drugs that uses in vitro susceptibility, time-kill and reversibility measures to define the therapeutically relevant exposure required in target tissues of animal infection models. The strategy is exemplified by the discovery of SCYX-7158 as a potential oral treatment for stage 2 (CNS) Human African Trypanosomiasis (HAT). A critique of current treatments for stage 2 HAT is included to provide context for the challenges of achieving target tissue disposition and the need for establishing pharmacokinetic-pharmacodynamic (PK-PD) measures early in the discovery paradigm. The strategy comprises 3 stages. Initially, compounds demonstrating promising in vitro activity and selectivity for the target organism over mammalian cells are advanced to in vitro metabolic stability, barrier permeability and tissue binding assays to establish that they will likely achieve and maintain therapeutic concentrations during in-life efficacy studies. Secondly, in vitro time-kill and reversibility kinetics are employed to correlate exposure (based on unbound concentrations) with in vitro activity, and to identify pharmacodynamic measures that would best predict efficacy. Lastly, this information is used to design dosing regimens for pivotal pharmacokinetic-pharmacodyamic studies in animal infection model

DNDI-6148:A novel benzoxaborole preclinical candidate for the treatment of visceral leishmaniasis

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. Current therapies are unsuitable, and there is an urgent need for safe, short-course, and low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates for the treatment of other parasitic diseases. Here, we describe the development and optimization of this series, leading to the identification of compounds with potent in vitro and in vivo antileishmanial activity. The lead compound (DNDI-6148) combines impressive in vivo efficacy (>98% reduction in parasite burden) with pharmaceutical properties suitable for onward development and an acceptable safety profile. Detailed mode of action studies confirm that DNDI-6148 acts principally through the inhibition of Leishmania cleavage and polyadenylation specificity factor (CPSF3) endonuclease. As a result of these studies and its promising profile, DNDI-6148 has been declared a preclinical candidate for the treatment of VL

2,4-Diaminopyrimidines as Potent Inhibitors of Trypanosoma brucei and Identification of Molecular Targets by a Chemical Proteomics Approach

The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT) or sleeping sickness, a fatal disease affecting nearly half a million people in sub-Saharan Africa. Current treatments for HAT have very poor safety profiles and are difficult to administer. There is an urgent need for new, safe and effective treatments for sleeping sickness. This work describes the discovery of 2,4-diaminopyrimidines, exemplified by 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid phenylamide or SCYX-5070, as potent inhibitors of T. brucei growth in vitro and also in animal models for HAT. To determine the parasite proteins responsible for interaction with SCYX-5070 and related compounds, affinity pull-downs were performed followed by sequence analysis and parasite genome database searching. The work revealed that mitogen-activated protein kinases (MAPKs) and cdc2-related kinases (CRKs) are the major proteins specifically bound to the immobilized compound, suggesting their potential participation in the pharmacological effects of 2,4-diaminopyrimidines against trypanosomatid protozoan parasites. These data strongly support the use of 2,4-diminipyrimidines as leads for the development of new drug candidates for the treatment of HAT

SCYX-7158, an Orally-Active Benzoxaborole for the Treatment of Stage 2 Human African Trypanosomiasis

Human African trypanosomiasis (HAT) is caused by infection with the parasite Trypanosoma brucei and is an important public health problem in sub-Saharan Africa. New, safe, and effective drugs are urgently needed to treat HAT, particularly stage 2 disease where the parasite infects the brain. Existing therapies for HAT have poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. Through an integrated drug discovery project, we have discovered and optimized a novel class of boron-containing small molecules, benzoxaboroles, to deliver SCYX-7158, an orally active preclinical drug candidate. SCYX-7158 cured mice infected with T. brucei, both in the blood and in the brain. Extensive pharmacokinetic characterization of SCYX-7158 in rodents and non-human primates supports the potential of this drug candidate for progression to IND-enabling studies in advance of clinical trials for stage 2 HAT

Schistosoma mansoni : role of antioxidant systems in protection of developmental stages against oxidative killing and the effects of oltipraz on glutathione S-transferase

This study shows that resistance to killing by reactive oxygen intermediates (ROI) increases during migration and development in Schistosoma mansoni. Resistance is associated with the protective role of antioxidants as shown by the increased levels of superoxide dismutase and of the glutathione system enzymes. Hydroperoxide-dependent glutathione peroxidase activity was not detectable in newly transformed schistosomula, however the activity was present in the liver stages. The antischistosomal drug oltipraz (OPZ) decreased in an irreversible manner the activity of S. mansoni glutathione S-transferase (GST), an important protective enzyme, both in vivo and in vitro. The inhibition of GST activity was not isoenzyme restricted and was non-competitive with respect to the two substrates essential for GST activity. On the other hand, OPZ treatment increased the levels of mouse (S. mansoni host) liver GST activity in an isoenzyme specific manner, with the $mu$ class subunit induction accounting for most of the increase. However, mammalian GST activity was inhibited by OPZ in vitro. However, the inhibition of mammalian GST activity was reversible upon addition of dithiol reducing compounds. OPZ inhibited the binding of ($sp{14}$C) N-ethylmaleimide (specifically alkylates SH groups), suggesting that OPZ interacts with SH-groups of GST to inhibit its enzymatic activity. Another SH-dependent enzyme, hexokinase, from yeast and S. mansoni was reversibly inhibited by OPZ. The oxy-analogue of OPZ, in which the thione sulphur is replaced with oxygen, did not inhibit the enzymatic activity of GST and hexokinase. Many of the biochemical effects of OPZ on S. mansoni and its mammalian hosts may be related to its ability to bind to SH groups and inactivation of the functions of many essential proteins

New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity

Leishmania and other trypanosomatid protozoa require reduced pteridines (pterins and folates) for growth, suggesting that inhibition of these pathways could be targeted for effective chemotherapy. This goal has not yet been realized, indicating that pteridine metabolism may be unusual in this lower eukaryote. We have investigated this possibility using both wild type and laboratory-selected antifolate-resistant strains, and with defined genetic knockouts of several pteridine metabolic genes. In Leishmania, resistance to the antifolate methotrexate is mediated through several mechanisms singly or in combination, including alterations in transport leading to reduced drug influx, overproduction (R-region amplification) or point mutation of dihydrofolate reductase-thymidylate synthase (DHFR-TS), and amplification of a novel pteridine reductase (PTR1, encoded by the H-region). All of the proteins involved are potential targets for antifolate chemotherapy. Notably, parasites in which the gene encoding dihydrofolate reductase (DHFR) has been deleted (dhfr-ts- knockouts) do not survive in animal models, validating this enzyme as a target for effective chemotherapy. However, the properties of pteridine reductase 1 (PTR1) suggest a reason why antifolate chemotherapy has so far not been successful in trypanosomatids. PTR1, by its ability to provide reduced pterins and folates, has the potential to act as a by-pass and/or modulator of DHFR inhibition under physiological conditions. Moreover, PTR1 is less sensitive to many antifolates targeted primarily against DHFR. These findings suggest that successful antifolate chemotherapy in Leishmania will have to target simultaneously both DHFR and PTR1

Evaluation of a Cyclic GMP-Dependent Protein Kinase Inhibitor in Treatment of Murine Toxoplasmosis: Gamma Interferon Is Required for Efficacy

The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) is a potent inhibitor of cyclic GMP-dependent protein kinases from Apicomplexan protozoa and displays cytostatic activity against Toxoplasma gondii in vitro. Compound 1 has now been evaluated against T. gondii infections in the mouse and appeared to protect the animals when given intraperitoneally at 50 mg/kg twice daily for 10 days. However, samples from brain, spleen, and lung taken from infected treated mice revealed the presence of parasites after cessation of administration of compound 1, indicating that a transient asymptomatic parasite recrudescence occurs in all survivors. The ability of mice to control Toxoplasma infection after compound 1 treatment has been terminated suggested that the mouse immune system plays a synergistic role with chemotherapy in controlling the infection. To explore this possibility, gamma interferon (IFN-γ)-knockout mice were infected with parasites and treated with compound 1, and survival was compared to that of normal mice. IFN-γ-knockout mice were protected against T. gondii throughout the treatment phase but died during the posttreatment phase in which peak recrudescence was observed in treated immunocompetent mice. These data suggest that an IFN-γ-dependent immune response was essential for controlling and resolving parasite recrudescence in mice treated with compound 1. In addition, when compound 1-cured immunocompetent mice were rechallenged with a lethal dose of T. gondii, all survived (n = 32). It appears that the cytostatic nature of compound 1 provides an “immunization” phase during chemotherapy which allows the mice to survive the recrudescence and any subsequent challenge with a lethal dose of T. gondii