Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents.

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product-inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in vitro and in vivo antileishmanial activities, detailed structure-activity relationships, and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family and paves the way for the generation of new oral antileishmanial drugs.

Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs.

The development of novel antiplasmodial compounds with broad-spectrum activity against different stages of Plasmodium parasites is crucial to prevent malaria disease and parasite transmission. This study evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds inhibited blood stage proliferation of drug-resistant or sensitive strains of Plasmodium falciparum in the low micromolar to nanomolar range. Interestingly, CB-41 exhibited prophylactic activity against hypnozoites and liver schizonts in Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Isobologram assays demonstrated synergistic interactions between CB-61 and the FDA-approved antimalarial drugs, clindamycin and halofantrine. Testing of six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target and no cytotoxicity in HepG2 liver cells. CB compounds are promising candidates for further development as antimalarial drugs against multidrug-resistant parasites, which could also prevent malaria transmission.

Combination of Subtherapeutic Doses of Tretazicar and Liposomal Amphotericin B Suppresses and Cures Leishmania major-Induced Cutaneous Lesions in Murine Models.

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis affecting human populations, yet CL remains largely ignored in drug discovery programs. CL causes disfiguring skin lesions and often relapses after "clinical cure" using existing therapeutics. To expand the pool of anti-CL lead candidates, we implemented an integrated screening platform comprising three progressive Leishmania parasite life cycle forms. We identified tretazicar (CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide) as a potent inhibitor of Leishmania parasite viability across multiple Leishmania species, which translated into complete and prolonged in vivo suppression of CL lesion formation in BALB/c mice when used as a monotherapy and which was superior to liposomal amphotericin B. In addition, oral twice a day administration of tretazicar healed the majority of existing Leishmania major (L. major) cutaneous lesions. In drug combination studies, there was a strong potentiation when subtherapeutic doses of liposomal amphotericin B and tretazicar were simultaneously administered. This drug combination decreased L. major lesion size in mice earlier than individual monotherapy drug treatments and maintained all animals lesion free for up to 64 days after treatment cessation. In contrast, administration of subtherapeutic doses of tretazicar or amphotericin B as monotherapies resulted in no or partial lesion cures, respectively. We propose that tretazicar should be explored as a component of a systemic CL combination therapy and potentially for other diseases where amphotericin B is a first line therapy.

Lead Optimization of Second-Generation Acridones as Broad-Spectrum Antimalarials.

The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

Scaffold and Parasite Hopping: Discovery of New Protozoal Proliferation Inhibitors.

Utilizing a target repurposing and parasite-hopping approach, we tested a previously reported library of compounds that were active against Trypanosoma brucei, plus 31 new compounds, against a variety of protozoan parasites including Trypanosoma cruzi, Leishmania major, Leishmania donovani, and Plasmodium falciparum. This led to the discovery of several compounds with submicromolar activities and improved physicochemical properties that are early leads toward the development of chemotherapeutic agents against kinetoplastid diseases and malaria.

In Vivo Bioluminescent Monitoring of Parasites in BALB/c Mouse Models of Cutaneous Leishmaniasis Drug Discovery.

Confirming the in vivo efficacy of potential antileishmanial compounds that display in vitro potency and good chemical characteristics is one of the most important steps in preclinical research drug discovery before human clinical trials begin. Here we describe the use of the in vivo bioluminescent monitoring of high and low inocula of luciferase-expressing Leishmania major (L. major) parasites in traditional and more innovative rodent models of in vivo cutaneous leishmaniasis (CL) drug discovery.

Novel benzoxaborole, nitroimidazole and aminopyrazoles with activity against experimental cutaneous leishmaniasis.

OBJECTIVES: Drugs for Neglected Diseases initiative (DNDi) has identified three chemical lead series, the nitroimidazoles, benzoxaboroles and aminopyrazoles, as innovative treatments for visceral leishmaniasis. The leads discovered using phenotypic screening, were optimised following disease- and compound-specific criteria. Several leads of each series were progressed and preclinical drug candidates have been nominated. Here we evaluate the efficacy of the lead compounds of each of these three chemical classes in in vitro and in vivo models of cutaneous leishmaniasis. METHODS: The in vitro activity of fifty-five compounds was evaluated against the intracellular amastigotes of L. major, L. aethiopica, L. amazonensis, L. panamensis, L. mexicana and L. tropica. The drugs demonstrating potent activity (EC50 < 5 µM) against at least 4 of 6 species were subsequently evaluated in vivo in different L. major - BALB/c mouse models using a 5 or 10-day treatment with either the oral or topical formulations. Efficacy was expressed as lesion size (measured daily using callipers), parasite load (by quantitative PCR - DNA) and bioluminescence signal reduction relative to the untreated controls. RESULTS: The selected drug compounds (3 nitroimidazoles, 1 benzoxaborole and 3 aminopyrazoles) showed consistent and potent activity across a range of Leishmania species that are known to cause CL with EC50 values ranging from 0.29 to 18.3 µM. In all cases, this potent in vitro antileishmanial activity translated into high levels of efficacy with a linear dose-response against murine CL. When administered at 50 mg/kg/day, DNDI-0690 (nitroimidazole), DNDI-1047 (aminopyrazole) and DNDI-6148 (benzoxaborole) all resulted in a significant lesion size reduction (no visible nodule) and an approximate 2-log-fold reduction of the parasite load as measured by qPCR compared to the untreated control. CONCLUSIONS: The lead compounds DNDI-0690, DNDI-1047 and DNDI-6148 showed excellent activity across a range of Leishmania species in vitro and against L. major in mice. These compounds offer novel potential drugs for the treatment of CL.

Discovery and Structural Optimization of Acridones as Broad-Spectrum Antimalarials.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual-stage activity against both liver-stage and blood-stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: (1) picomolar inhibition of in vitro Plasmodium falciparum blood-stage growth against multidrug-resistant parasites; (2) curative efficacy after oral administration in an erythrocytic Plasmodium yoelii murine malaria model; (3) prevention of in vitro Plasmodium berghei sporozoite-induced development in human hepatocytes; and (4) protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver-stage antimalarial activity in an acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.

Updating the modified Thompson test by using whole-body bioluminescence imaging to replace traditional efficacy testing in experimental models of murine malaria.

BACKGROUND: Rodent malaria models are extensively used to predict treatment outcomes in human infections. There is a constant need to improve and refine these models by innovating ways to apply new scientific findings and cutting edge technologies. In addition, and in accordance with the three R's of animal use in research, in vivo studies should be constantly refined to avoid unnecessary pain and distress to the experimental animals by using preemptive euthanasia as soon as the main scientific study objective has been accomplished. METHODS: The new methodology described in this manuscript uses the whole-body bioluminescence signal emitted by transgenic, luciferase-expressing Plasmodium berghei parasites to assess the parasite load predicted parasitaemia (PLPP) in drug and control treated female ICR-CD1 mice infected with 1 × 105 luciferase-expressing P. berghei (ANKA strain) infected erythrocytes. This methodology can replace other time-consuming and expensive methods that are routinely used to measure parasitaemia in infected animals, such as Giemsa-stained thin blood smears and flow cytometry. RESULTS: There is a good correlation between whole-body bioluminescence signal and parasitaemia measured using Giemsa-stained thin blood smears and flow cytometry respectively in donor and study mice in the modified Thompson test. The algebraic formulas which represent these correlations can be successfully used to assess PLPP in donor and study mice. In addition, the new methodology can pinpoint sick animals 2-8 days before they would have been otherwise diagnosed based on behavioural or any other signs of malaria disease. CONCLUSIONS: The new method for predicting parasitaemia in the modified Thompson test is simple, precise, objective, and minimizes false positive results that can lead to the premature removal of animals from study. Furthermore, from the animal welfare perspective of replace, reduce, and refine, this new method facilitates early removal of sick animals from study as soon as the study objective has been achieved, in many cases well before the clinical signs of disease are present.

Anilinoquinoline based inhibitors of trypanosomatid proliferation.

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.

Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of Protozoan Parasite Proliferation.

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure-activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.

Optimization of Physicochemical Properties for 4-Anilinoquinoline Inhibitors of Plasmodium falciparum Proliferation.

We recently reported the medicinal chemistry reoptimization of a known human tyrosine kinase inhibitor, lapatinib, against a variety of parasites responsible for numerous tropical diseases, including human African trypanosomiasis ( Trypanosoma brucei), Chagas disease ( T. cruzi), Leishmaniasis ( Leishmania spp.), and malaria ( Plasmodium falciparum). Herein, we report our continuing efforts to optimize this series against P. falciparum. Through the design of a library of compounds focused on reducing the lipophilicity and molecular weight, followed by an SAR exploration, we have identified NEU-1953 (40). This compound is a potent inhibitor of P. falciparum with an improved ADME profile over the previously reported compound, NEU-961 (3).

Optimization of physicochemical properties for 4-anilinoquinazoline inhibitors of trypanosome proliferation.

Human African trypanosomiasis (HAT) is a deadly disease in need of new chemotherapeutics that can cross into the central nervous system. We previously reported the discovery of 2 (NEU-617), a small molecule with activity against T. brucei bloodstream proliferation. Further optimization of 2 to improve the physicochemical properties (LogP, LLE, [1], and MPO score) [2] have led us to twelve sub-micromolar compounds, most importantly the headgroup variants 9i and 9j, and the linker variant 18. Although these 3 compounds had reduced potency compared to 2, they all had improved LogP, LLE and MPO scores. Cross-screening these analogs against other protozoan parasites uncovered 9o with potent activity towards T. brucei, T. cruzi and L. major, while four others compounds (17, 18, 21, 26) showed activity towards P. falciparum D6. This reinforces the effectiveness of lead repurposing for the discovery of new protozoan disease therapeutics.

Antiparasitic Lead Discovery: Toward Optimization of a Chemotype with Activity Against Multiple Protozoan Parasites.

Human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis present a significant burden across the developing world. Existing therapeutics for these protozoal neglected tropical diseases suffer from severe side effects and toxicity. Previously, NEU-1045 (3) was identified as a promising lead with cross-pathogen activity, though it possessed poor physicochemical properties. We have designed a library of analogues with improved calculated physicochemical properties built on the quinoline scaffold of 3 incorporating small, polar aminoheterocycles in place of the 4-(3-fluorobenzyloxy)aniline substituent. We report the biological activity of these inhibitors against Trypanosoma brucei (HAT), T. cruzi (Chagas disease), and Leishmania major (cutaneous leishmaniasis) and describe the identification of N-(5-chloropyrimidin-2-yl)-6-(4-(morpholinosulfonyl)phenyl)quinolin-4-amine (13t) as a promising inhibitor of L. major proliferation and 6-(4-(morpholinosulfonyl)phenyl)-N-(pyrimidin-4-yl)quinolin-4-amine (13j), a potent inhibitor of T. brucei proliferation with improved drug-like properties.

Use of Optical Imaging Technology in the Validation of a New, Rapid, Cost-Effective Drug Screen as Part of a Tiered In Vivo Screening Paradigm for Development of Drugs To Treat Cutaneous Leishmaniasis.

In any drug discovery and development effort, a reduction in the time of the lead optimization cycle is critical to decrease the time to license and reduce costs. In addition, ethical guidelines call for the more ethical use of animals to minimize the number of animals used and decrease their suffering. Therefore, any effort to develop drugs to treat cutaneous leishmaniasis requires multiple tiers of in vivo testing that start with higher-throughput efficacy assessments and progress to lower-throughput models with the most clinical relevance. Here, we describe the validation of a high-throughput, first-tier, noninvasive model of lesion suppression that uses an in vivo optical imaging technology for the initial screening of compounds. A strong correlation between luciferase activity and the parasite load at up to 18 days postinfection was found. This correlation allows the direct assessment of the effects of drug treatment on parasite burden. We demonstrate that there is a strong correlation between drug efficacy measured on day 18 postinfection and the suppression of lesion size by day 60 postinfection, which allows us to reach an accurate conclusion on drug efficacy in only 18 days. Compounds demonstrating a significant reduction in the bioluminescence signal compared to that in control animals can be tested in lower-throughput, more definitive tests of lesion cure in BALB/c mice and Golden Syrian hamsters (GSH) using Old World and New World parasites.

Identification of "Preferred" Human Kinase Inhibitors for Sleeping Sickness Lead Discovery. Are Some Kinases Better than Others for Inhibitor Repurposing?

A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC50 ≥ 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach "preferred lead repurposing".

Assessment of the Worldwide Antimalarial Resistance Network Standardized Procedure for In Vitro Malaria Drug Sensitivity Testing Using SYBR Green Assay for Field Samples with Various Initial Parasitemia Levels.

The malaria SYBR green assay, which is used to profilein vitrodrug susceptibility ofPlasmodium falciparum, is a reliable drug screening and surveillance tool. Malaria field surveillance efforts provide isolates with various low levels of parasitemia. To be advantageous, malaria drug sensitivity assays should perform reproducibly among various starting parasitemia levels rather than at one fixed initial value. We examined the SYBR green assay standardized procedure developed by the Worldwide Antimalarial Resistance Network (WWARN) for its sensitivity and ability to accurately determine the drug concentration that inhibits parasite growth by 50% (IC50) in samples with a range of initial parasitemia levels. The initial sensitivity determination of the WWARN procedure yielded a detection limit of 0.019% parasitemia.P. falciparumlaboratory strains and field isolates with various levels of initial parasitemia were then subjected to a range of doses of common antimalarials. The IC50s were comparable for laboratory strains with between 0.0375% and 0.6% parasitemia and for field isolates with between 0.075% and 0.6% parasitemia for all drugs tested. Furthermore, assay quality (Z') analysis indicated that the WWARN procedure displays high robustness, allowing for drug testing of malaria field samples within the derived range of initial parasitemia. The use of the WWARN procedure should allow for the inclusion of more malaria field samples in malaria drug sensitivity screens that would have otherwise been excluded due to low initial parasitemia levels.

Antileishmanial Activity of Compounds Derived from the Medicines for Malaria Venture Open Access Box Against Intracellular Leishmania major Amastigotes.

Leishmaniasis is a complex tropical disease caused by kinetoplastid parasitic protozoa of the genus Leishmania and is transmitted by the sand fly insect vector. Cutaneous leishmaniasis (CL) is the most common form of this disease, and CL infections often result in serious skin lesions and scars. CL remains a public health problem in many endemic countries worldwide because of the absence of effective, safe, and cost-effective drugs for treatment. One of the strategies we chose to use to find novel chemical entities worthy of further development as antileishmanials involved screening synthetic and natural products libraries. In our study, we developed a Leishmania major intracellular amastigote assay that uses the activity of luciferase as a measure of parasite proliferation and used this assay to screen a collection of 400 compounds obtained from Medicines for Malaria Venture (MMV) for their antileishmanial activity. Our results showed that 14 compounds identified by MMV as antimalarial drugs have antileishmanial activity and can potentially be optimized for CL drug development.

Protozoan Parasite Growth Inhibitors Discovered by Cross-Screening Yield Potent Scaffolds for Lead Discovery.

Tropical protozoal infections are a significant cause of morbidity and mortality worldwide; four in particular (human African trypanosomiasis (HAT), Chagas disease, cutaneous leishmaniasis, and malaria) have an estimated combined burden of over 87 million disability-adjusted life years. New drugs are needed for each of these diseases. Building on the previous identification of NEU-617 (1) as a potent and nontoxic inhibitor of proliferation for the HAT pathogen (Trypanosoma brucei), we have now tested this class of analogs against other protozoal species: T. cruzi (Chagas disease), Leishmania major (cutaneous leishmaniasis), and Plasmodium falciparum (malaria). Based on hits identified in this screening campaign, we describe the preparation of several replacements for the quinazoline scaffold and report these inhibitors' biological activities against these parasites. In doing this, we have identified several potent proliferation inhibitors for each pathogen, such as 4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-6-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)quinoline-3-carbonitrile (NEU-924, 83) for T. cruzi and N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)cinnolin-4-amine (NEU-1017, 68) for L. major and P. falciparum.