Synthesis, pharmacological and physicochemical profiling of antimalarial and antischistosomal N-aryl 3-trifluoromethyl pyrido [1,2-α] benzimidazoles

Malaria and schistosomiasis represent the two most prevalent parasitic infections with grievous repercussions on the socio-economic development of affected countries, mainly in sub-Saharan Africa and South-East Asia. Despite their ravaging effects, the treatments of these two diseases have been committed to a limited arsenal of drugs that are threatened by resistance. This scenario, therefore, calls for decisive steps being taken towards the discovery and development of novel drugs with the ability to target multiple parasite stages and be efficacious against resistant parasite strains to achieve effective control and treatment of malaria and schistosomiasis. Originating from a World Health Organisation-Tropical Disease Research initiative, a phenotypic whole cell screening was conducted on a commercial library against Plasmodium falciparum whereupon novel hits exemplified by compound 1, embodying a pyridobenzimidazole (PBI) scaffold, were found to portray in vitro potency against both chloroquine sensitive and resistant parasites. Initial medicinal chemistry iterations generated analogues including 2 from which improved in vitro potency and demonstrable in vivo efficacy in a mouse model were observed. In this thesis, further structural diversity around the PBI motif 3 (Figure 1) was pursued with the aim of generating analogues for structure-activity and structure-property relationship studies. Beyond the asexual blood stage activity, the library of compounds generated was also evaluated for activity against the liver and gametocyte stages of Plasmodium. In exploring the probable mechanism of antimalarial action based on their planar morphology and existence of basic centres, the compounds were evaluated for their capacity to disrupt the heme detoxification process, a recognised druggable target in antimalarial drug discovery. Prioritised compounds, based on in vitro potency, were progressed for in vitro drug metabolism and pharmacokinetics assessment, including metabolic stability and cytotoxicity against Chinese hamster ovarian cell lines. Representative compounds were evaluated for their interaction potential with the human ether-a-go-go-related gene (hERG), a potassium ion channel whose inhibition can cause potentially fatal irregular heartbeats due to perturbed repolarisation of the myocardial action potential. In vivo proof-of-concept efficacy and pharmacokinetics studies were carried out on the most promising leads according to a predetermined screening cascade. Arising from this work, structure-activity relationship (SAR) trends were discernible with electron withdrawing substituents on the aromatic side appendage providing active analogues compared to compounds comprising hydrophilic electron-releasing or donating substituents. Following in vitro microsomal metabolic stability analysis, the series displayed moderate to good metabolic stability with compounds incorporating heteroaromatic side groups showing increased susceptibility to biotransformation usually arising from the aromatic ring. In a P. berghei mouse model at an oral dose of 50mg/kg over four consecutive days, two compounds 1j/GMP-19 and 4i/GMP-75 achieved 98% and 99.9% reduction in parasitaemia and led to mean survival days of 12 and 14, respectively, compared to the untreated infected mice which survived for only 6 days. A drug repositioning approach was pursued, exploiting the cellular and biological similarities in the haemoglobin degradation pathways existent in both Plasmodium parasites and schistosomes. Out of the 57 analogues tested, 12 were found to be potent inhibitors of the adult worms (IC50 ≤ 2 µM), with several compounds also displaying potency against the newly transformed schistosomula. Structural features consistent with good antischistosomal potency, interestingly, overlapped with those present in some compounds that also showed good antiplasmodial activity. Prioritised compounds subjected to in vivo efficacy studies in mice infected with schistosomes identified 1b/GMP-09, 1j/GMP-19 and 4i/GMP-75 with modest antischistosomal activity (55- 70% total worm reduction). Metabolic stability and physicochemical properties correlated with observed in vivo efficacy and solubility- limited absorption was implicated to contribute to low in vivo exposure of the compounds. Further profiling of physicochemical parameters revealed the interdependence of the properties and that crystallinity, as measured by the melting point, influenced compound solubility. In summary, the work pursued in this thesis has unravelled the structural features compatible with potent antimalarial and antischistosomal activities of N-aryl substituted 3- trifluoromethyl PBI derivatives. Additionally, structure-property trends of generated analogues have been delineated. The evolvable nature of the structure-activity and structureproperty relationship trends make these compounds appealing as candidates for further optimisation campaigns to impart improvements in physicochemical properties and drug metabolism and pharmacokinetics attributes without abrogating activity. Moreover, having identified the overlap as well as divergence in chemical spaces relating to antimalarial and antischistosomal potencies of these series inspire further investigations into the mechanisms of observed antiparasitic actions. Finally, that this work has identified targets which are panactive across multiple stages of both Plasmodium and schistosomes, and possessing favourable safety profiles, provide an exciting opportunity for pursuing these analogues as antimalarial and antischistosomal leads with the potential for malaria chemoprevention and transmission blocking

In-silicoValidation of the Essentiality of Reactions in Plasmodium Falciparum Metabolic Network

Plasmodium falciparum are instrumental in causing malaria and have developed complex life cycles, thus, it becomes very possible for the malaria parasite to take advantage of the uniqueness of its pathways to design therapeutic strategies. Despite the colossal efforts put in to fight malaria the disease still affects up to over 200 million people every year amongst which close to half a million dies. The treatment of the disease, could be done successfully if the essential enzymes of this parasite is precisely targeted. Nevertheless, the development of the parasite to resisting existing drugs now makes it a core responsibility to discover novel drugs. In this study, existing essential reactions from different literature are considered and evaluated to determine reactions that are common in all literature and evaluated to determine their essentiality level. This study evaluates essential reactions that has been predicted in literature computationally and validates its essentiality based on the reconstructed metabolic network and identifies 10 essential reactions that are common to all existing literature of which all this reaction were validated to be essential by our method. This study has established a simple novel in-silico method that validates predicted essential reactions in a metabolic network which makes validation of predicted anti-malarial drug target cheaper, easier and faster. This study in-silico model serves as a valuable tool for validation of Plasmodium falciparum metabolic states under various perturbations

Design, synthesis, and structure-activity relationship studies of dual Plasmodium falciparum phosphatidylinositol 4-kinase and cGMP-dependent protein kinase inhibitors

Malaria is a life-threatening disease caused by protists in the genus Plasmodium and transmitted by the female Anopheles mosquito. Amongst five species which infect humans, Plasmodium falciparum (Pf) causes the severest form of the disease. Although significant efforts have been made to reduce the overall impact of malaria in endemic regions, the ever emergence and continuous spread of parasite resistance to available chemotherapeutics, threatens to undermine advances made thus far. In addition, the current portfolio of drugs is non-effective in addressing chemoprotection, transmission blockade and relapse in P. vivax and P. ovale species. Thus, drugs targeting multiple stages of the parasite life cycle and of low risk to resistance, are highly desirable to support malaria elimination and/or eradication efforts. Considering the success of human kinase inhibitors as anti-cancer drugs and the identification of Plasmodium kinases as promising targets for malaria chemotherapy, this study aimed to optimize anti-plasmodium phosphatidylinositol 4-kinase (PI4K) and the cGMP-dependent protein kinase (PKG) inhibitors, based on two distinct chemotypes. Plasmodium PI4K and PKG are validated targets, each with the potential to deliver pan-stage active compounds with potentially moderate to low risk of resistance. Part 1 of this study focused on the repositioning of the oncological clinical Phase-1 mammalian target of rapamycin (mTOR) inhibitor, MLN0128, as a dual Plasmodium PI4K/PKG inhibitor for malaria. MLN0128 was identified by GlaxoSmithKline (GSK) Cellzome facility as a Plasmodium multi-kinase inhibitor with potent PI4K and PKG inhibitory activity. In this study, an in silico-guided structural modification strategy was undertaken towards optimizing dual Plasmodium kinase inhibition and anti-plasmodium activity while also mitigating potency against its oncological human target, mTOR and off-target PI4KIIIb (Figure 1). Arising from this work, analogues equipotent against both the chloroquine sensitive (PfNF54) and multi-drug resistant (PfK1) strains simultaneously targeting PI4K and PKG were identified. Docking studies using a PfPI4K homology model and a PvPKG crystal structure discerned the molecular features responsible for the high affinity of the inhibitors for these Plasmodium targets. Benzyl analogues containing a fluoro or chloro group at the meta or para positions displayed high anti-plasmodium activity with potent PvPI4K inhibition but weak PfPKG inhibition. Notable analogues included 7 (PfNF54 IC50 = 0.029 µM; PvPI4K IC50 = 0.007 µM; PfPKG IC50 > 2 µM) and 35 (PfNF54 IC50 = 0.086 µM; PvPI4K IC50 = 0.008 µM; PfPKG IC50 > 10 µM). Introduction of basic or pyridyl substituents proved important for dual Plasmodium kinase activity as exemplified by the active anti-plasmodium pyridyl analogues 44 (PfNF54 IC50 = 0.104 µM; PvPI4K IC50 = 0.004 µM; PfPKG IC50 = 0.834 µM) and 49 (PfNF54 IC50 = 0.189 µM; PvPI4K IC50 = 0.006 µM; PfPKG IC50 = 0.384 µM). In addition, the two compounds displayed low cytotoxicity against the Chinese Hamster Ovarian cell line, with a favorable selectivity index (CHO; SI > 100), low human ether-a-go-go-related gene (hERG) activity (IC50 > 10 µM) and high metabolic stability against human, rat, and mouse (H/R/M) liver microsomes (> 75% remaining after 30-min incubation). Selected compounds from the series also showed the potential for transmission blockade with specificity for stage IV/V gametocytes (IC50 100 µM). Compounds displayed potent PvPI4K inhibition but weak PfPKG inhibition (IC50 > 1 µM) in enzyme assays. Four compounds, including one sulfoxide analogue, displayed high stability when incubated with H/R/M liver microsomes in microsomal metabolic stability assays. These features also mitigated hERG activity as five analogues tested displayed an IC50 > 10 µM. Ultimately, a front-runner lead compound (86; GS1 16) with high biological activity and a good safety profile (PfNF54/PfK1 = 0.063/0.100 µM; PvPI4K IC50 = 0.003 µM; CHO SI > 793), optimal solubility (195 µM), favorable microsomal metabolic stability (H/R/M = 96/85/88%) and low affinity on the hERG-encoded potassium channel (IC50 = 44.80 µM), was identified for further progression

Large–scale data–driven network analysis of human–plasmodium falciparum interactome: extracting essential targets and processes for malaria drug discovery

Background: Plasmodium falciparum malaria is an infectious disease considered to have great impact on public health due to its associated high mortality rates especially in sub Saharan Africa. Falciparum drugresistant strains, notably, to chloroquine and sulfadoxine-pyrimethamine in Africa is traced mainly to Southeast Asia where artemisinin resistance rate is increasing. Although careful surveillance to monitor the emergence and spread of artemisinin-resistant parasite strains in Africa is on-going, research into new drugs, particularly, for African populations, is critical since there is no replaceable drug for artemisinin combination therapies (ACTs) yet. Objective: The overall objective of this study is to identify potential protein targets through host–pathogen protein–protein functional interaction network analysis to understand the underlying mechanisms of drug failure and identify those essential targets that can play their role in predicting potential drug candidates specific to the African populations through a protein-based approach of both host and Plasmodium falciparum genomic analysis. Methods: We leveraged malaria-specific genome wide association study summary statistics data obtained from Gambia, Kenya and Malawi populations, Plasmodium falciparum selective pressure variants and functional datasets (protein sequences, interologs, host-pathogen intra-organism and host-pathogen inter-organism protein-protein interactions (PPIs)) from various sources (STRING, Reactome, HPID, Uniprot, IntAct and literature) to construct overlapping functional network for both host and pathogen. Developed algorithms and a large-scale data-driven computational framework were used in this study to analyze the datasets and the constructed networks to identify densely connected subnetworks or hubs essential for network stability and integrity. The host-pathogen network was analyzed to elucidate the influence of parasite candidate key proteins within the network and predict possible resistant pathways due to host-pathogen candidate key protein interactions. We performed biological and pathway enrichment analysis on critical proteins identified to elucidate their functions. In order to leverage disease-target-drug relationships to identify potential repurposable already approved drug candidates that could be used to treat malaria, pharmaceutical datasets from drug bank were explored using semantic similarity approach based of target–associated biological processes Results: About 600,000 significant SNPs (p-value< 0.05) from the summary statistics data were mapped to their associated genes, and we identified 79 human-associated malaria genes. The assembled parasite network comprised of 8 clusters containing 799 functional interactions between 155 reviewed proteins of which 5 clusters contained 43 key proteins (selective variants) and 2 clusters contained 2 candidate key proteins(key proteins characterized by high centrality measure), C6KTB7 and C6KTD2. The human network comprised of 32 clusters containing 4,133,136 interactions between 20,329 unique reviewed proteins of which 7 clusters contained 760 key proteins and 2 clusters contained 6 significant human malaria-associated candidate key proteins or genes P22301 (IL10), P05362 (ICAM1), P01375 (TNF), P30480 (HLA-B), P16284 (PECAM1), O00206 (TLR4). The generated host-pathogen network comprised of 31,512 functional interactions between 8,023 host and pathogen proteins. We also explored the association of pfk13 gene within the host-pathogen. We observed that pfk13 cluster with host kelch–like proteins and other regulatory genes but no direct association with our identified host candidate key malaria targets. We implemented semantic similarity based approach complemented by Kappa and Jaccard statistical measure to identify 115 malaria–similar diseases and 26 potential repurposable drug hits that can be 3 appropriated experimentally for malaria treatment. Conclusion: In this study, we reviewed existing antimalarial drugs and resistance–associated variants contributing to the diminished sensitivity of antimalarials, especially chloroquine, sulfadoxine-pyrimethamine and artemisinin combination therapy within the African population. We also described various computational techniques implemented in predicting drug targets and leads in drug research. In our data analysis, we showed that possible mechanisms of resistance to artemisinin in Africa may arise from the combinatorial effects of many resistant genes to chloroquine and sulfadoxine–pyrimethamine. We investigated the role of pfk13 within the host–pathogen network. We predicted key targets that have been proposed to be essential for malaria drug and vaccine development through structural and functional analysis of host and pathogen function networks. Based on our analysis, we propose these targets as essential co-targets for combinatorial malaria drug discovery

The chemotherapeutic effects of synthetic and natural compounds

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg,in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, South Africa, 2015Plasmodium falciparum remains the most virulent cause of malaria. With increasing drug resistance to artemisinin and other antimalarial drugs, combined with an absence of an effective vaccine, there’s a critical need for new agents to complement existing treatment and prophylaxis. Therefore, the aim of the study was to evaluate the in vitro antimalarial activity and potential toxicity to mammalian cells of select synthetic and natural colourants, nucleoside and imidazo[1,2a]pyridine (IP) analogues on the erythrocytic stages of the 3D7 chloroquine-sensitive strain of P. falciparum. The P. falciparum 3D7 strain was maintained in vitro according to standard methods. Quinine and chloroquine were used as positive controls. The tritiated hypoxanthine incorporation assay was used for evaluating the ability of test compounds to inhibit the growth of P. falciparum. Active test compounds were tested in combination studies with quinine. Uninfected human red blood cell (RBC) toxicity was analysed spectrophotometrically. The ability of test compounds to inhibit -haematin formation, a metabolic pathway that sequesters toxic haem within the parasites, was determined. Cytotoxic activity of active compounds was evaluated on two human cell lines (HEK293 and K562) using the [3H]-thymidine incorporation assay. Data was analysed using the one-way ANOVA test and reported as the mean ± standard deviation of at least triplicate experiments and significant difference when p < 0.05. Of the 56 compounds tested, the synthetic colourants showed the most potent antimalarial activity. Methylene blue and safranin O were most potent with IC50 values of 4.19 ± 0.16 nM and 86.50 ± 2.61 nM, respectively, compared to quinine (IC50: 103.90 ± 8.30 nM), and displayed negligible toxicity to uninfected human RBCs. Combination studies with methylene blue and quinine demonstrated a synergistic interaction. Methylene blue also demonstrated the highest selectivity indices (480 and 968) compared to quine (180). Curcumin (diferuloylmethane), a natural extract was active (IC50: 2.29 ± 0.18 μg/ml) against P. falciparum, but significantly (p < 0.05) less potent than quinine. Curcumin was 78-fold more active in inhibiting -haematin formation than quinine, indicating of a possible mechanism of action. The most active nucleoside analogue, JLP118.1 (IC50: 1.79 ± 0.12 μM), demonstrated inhibitory activity against the trophozoite stage of P. falciparum. The imidazopyridine analogue, IP-4, displayed the least potent antimalarial activity (IC50: 15.3 ± 0.41 μM) of the synthetic compounds tested, with low selectivity indices < 1. The study has confirmed the potent antimalarial activity and relative safety of methylene blue as well as its potential as an antimalarial drug. The nucleoside and imidazopyridine analogues showed promising activity and with structural modification their potency and selectivity indices may be enhanced

Discovery and characterization of potent, efficacious, orally available antimalarial Plasmepsin X inhibitors and preclinical safety assessment of UCB7362

Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.Human Frontier Science Program (Strasbourg, France) (Postdoctoral Fellowship LT000307/2013

Plasmodium Purine Metabolism and Its Inhibition by Nucleoside and Nucleotide Analogues

International audienceMalaria still affects around 200 million people and is responsible for more than 400,000 deaths per year, mostly children in subequatorial areas. This disease is caused by parasites of the Plasmodium genus. Only a few WHO-recommended treatments are available to prevent or cure plasmodial infections, but genetic mutations in the causal parasites have led to onset of resistance against all commercial antimalarial drugs. New drugs and targets are being investigated to cope with this emerging problem, including enzymes belonging to the main metabolic pathways, while nucleoside and nucleotide analogues are also a promising class of potential drugs. This review highlights the main metabolic pathways targeted for the development of potential antiplasmodial therapies based on nucleos(t)ide analogues, as well as the different series of purine-containing nucleoside and nucleotide derivatives designed to inhibit Plasmodium falciparum purine metabolism.

Anti-Trypanosoma cruzi Activity of Metabolism Modifier Compounds

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects over 6 million people worldwide. Development of new drugs to treat this disease remains a priority since those currently available have variable efficacy and frequent adverse effects, especially during the long regimens required for treating the chronic stage of the disease. T. cruzi modulates the host cell-metabolism to accommodate the cell cytosol into a favorable growth environment and acquire nutrients for its multiplication. In this study we evaluated the specific anti-T. cruzi activity of nine bio-energetic modulator compounds. Notably, we identified that 17-DMAG, which targets the ATP-binding site of heat shock protein 90 (Hsp90), has a very high (sub-micromolar range) selective inhibition of the parasite growth. This inhibitory effect was also highly potent (IC50 = 0.27 μmol L−1) against the amastigote intracellular replicative stage of the parasite. Moreover, molecular docking results suggest that 17-DMAG may bind T. cruzi Hsp90 homologue Hsp83 with good affinity. Evaluation in a mouse model of chronic T. cruzi infection did not show parasite growth inhibition, highlighting the difficulties encountered when going from in vitro assays onto preclinical drug developmental stages