Identification and Validation of Novel Drug Targets for the Treatment of Plasmodium falciparum Malaria: New Insights

In order to counter the malarial parasite’s striking ability to rapidly develop drug resistance, a constant supply of novel antimalarial drugs and potential drug targets must be available. The so-called Harlow-Knapp effect, or “searching under the lamp post,” in which scientists tend to further explore only the areas that are already well illuminated, significantly limits the availability of novel drugs and drug targets. This chapter summarizes the pool of electron transport chain (ETC) and carbon metabolism antimalarial targets that have been “under the lamp post” in recent years, as well as suggest a promising new avenue for the validation of novel drug targets. The interplay between the pathways crucial for the parasite, such as pyrimidine biosynthesis, aspartate metabolism, and mitochondrial tricarboxylic acid (TCA) cycle, is described in order to create a “road map” of novel antimalarial avenues

Plasmodial enzymes in metabolic pathways as therapeutic targets and contemporary strategies to discover new antimalarial drugs: a review

Malaria continues to pose imminent threat to the world population, as the mortality rate associated with this disease remains high. Current treatment relies on antimalarial drugs such as Artemisinin Combination Therapy (ACT) are still effective throughout the world except in some places, where ACT-resistance has been reported, thus necessitating novel approaches to develop new anti-malarial therapy. In the light of emerging translational research, several plasmodial targets, mostly proteins or enzymes located in the parasite’s unique organelles, have been extensively explored as potential candidates for the development of novel antimalarial drugs. By targeting the metabolic pathways in mitochondrion, apicoplast or cytoplasm of Plasmodium, the possibility to discover new drugs is tremendous, as they have potentials as antimalarial therapeutic targets. This literature review summarizes pertinent information on plasmodial targets, especially enzymes involved in specific metabolic pathways, and the strategies used to discover new antimalarial drugs. © 2019, University of Malaya. All rights reserved

Drugs and drug targets against malaria

The development of resistance by the parasite against first line and second line antimalarial drugs, has underscored the importance to develop new drug targets and pharmacophores to treat the disease. The absence of a vaccine for protection and the availability of artemisinin and its derivatives as the only option has made the situation rather serious. With the availability of increased support for malaria research, a variety of drug targets and candidate molecules are now available for further development. However, the success rate of a candidate molecule to become a drug is very low and it does become necessary to start with a large basket, identified on a rational basis. This review focuses on the present efforts to identify a variety of drug targets in the malaria parasite and to develop candidate drug molecules

Purine and Pyrimidine Pathways as Antimalarial Targets

Malaria continues to plague the endemic regions of sub-Saharan Africa and Southeast Asia. With the current development of artemisinin resistance and a risk of failure of the current first line therapies, there is a growing need for novel antimalarials. Purine and pyrimidine metabolism in Plasmodium is distinctly different from the human host, making these pathways valid targets for the development of novel antimalarials. Targeting key enzymes in these pathways with transition state analogs has provided high affinity inhibitors. Transition state mimicry can also provide selectivity for the parasite enzymes over the homologous enzymes of the human host. Resistance of Plasmodium parasites to current antimalarials will be compared to resistance development induced by transition state analogs inhibitors, a feature that may contribute to decreased resistance development. Tight binding and specificity of transition state analog inhibitors provide important features for novel antimalaria therapy with low toxicity and prevention of antibiotic resistance

<em>Plasmodium falciparum</em>: Experimental and Theoretical Approaches in Last 20 Years

Malaria, the severe vector-borne disease has embedded serious consequences on mankind since ages, causing deterioration of health, leading to deaths. The causative parasite has a wide distribution aligned from tropical to subtropical regions. Out of all the five species Plasmodium vivax and Plasmodium falciparum have registered about more than 600 million cases worldwide. Throughout the decades, identification of various antimalarial drugs, targets, preventive measures and advancement of vaccines were achieved. The key to executing malaria elimination is the appropriate laboratory diagnosis. Development includes positive scientific judgments for a vaccine, advanced progress of 3 non-pyrethroid insecticides, novel genetic technologies, possibilities to alter malaria parasite mediation by the mosquito, identification of drug resistance markers, initiation of Plasmodium vivax liver stage assessment, perspective to mathematical modeling and screening for active ingredients for drugs and insecticides. Although the last century witnessed many successful programs with scientific progress, however, this was matched with notable obstacles. The mutation in the genes has changed the overall gameplay of eradication. This chapter aims to examine the numerous experimental and theoretical works that have been established in the last two decades along with the ongoing methodologies consisting of detailed explanations necessary for the establishment of new targets and drugs

Synthesis and computer-assisted design of mitochondrial electron transport-chain inhibitors as antimalarial agents

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014Malaria remains a critical global health problem, with terrible social and economic consequences in countries where this disease is endemic. The problem is exacerbated by the emergence and spread of parasites that are resistant to well-established antimalarial drugs. As a result, there is an urgent need for novel drugs, preferably acting on under exploited parasite targets in order to overcome clinical resistance. Cytochrome bc1 complex is a crucial element in the mitochondrial respiratory chain, being indispensable for the survival of several species of Plasmodia that cause malaria and, therefore, it is a promising target for antimalarial drug development. Moreover, in the absence of a crystal structure for the P. falciparum bc1 complex, key structural and mechanistic information has been inferred from analogous mammalian, bacterial and yeast bc1 systems. In the present work, a molecular docking study based on the most recently obtained X-ray structure of the Saccharomyces cerevisiae bc1 complex (PDB code: 3CX5) and using several reported inhibitors with experimentally determined IC50 values against the Plasmodium falciparum bc1 complex is presented. This Qo model was also used to search the drug-like database included in the MOE package for novel potential bc1 complex inhibitors allowing to obtain five compounds with demonstrated activity against the chloroquine-resistant W2 strain of P. falciparum. Moreover, the most active compounds were also active against the atovaquone-resistant P. falciparum FCR3 strain and S.cerevisiae. Furthermore, considering that a reliable three-dimensional structure of this Pf enzymatic complex is essential for successful drug design and having in mind the increasing interest in obtaining potential antimalarial drugs that can act in this target, a homology model of cytochrome bc1 Qo binding site was further developed based on yeast crystallographic structure. Additionally, a library containing several structurally diverse aurone and azaaurone derivatives were also synthesized and tested for their antimalarial activity. The aurone derivatives synthesized showed to be moderate active with IC50 values in the low micromolar range while de azaaurone analogous presented much higher potency with some compounds being active in the nanomolar range. Despite the mechanism of action of these two classes of compounds is still not very clear, this study highlight the usefulness of aurones and azaaurones to be derivatized in order to rapidly deliver lead compounds for further optimization and also the potential of these two scaffolds as promising antimalarial compounds.A malária continua a ser um grave problema de saúde, com enormes consequências sociais e económicas que afectam os países onde esta doença é endémica. Este problema tem vindo a crescer devido ao agravamento do aparecimento de parasitas resistentes aos fármacos antimaláricos disponíveis. Apesar da importância geral desta doença, o desenvolvimento de novos fármacos tem sido negligenciado pela indústria farmacêutica nos países industrializados. Deste modo, existe uma necessidade urgente de obter novos fármacos, de preferência que actuem em alvos menos explorados, de modo a se poder superar todos os problemas relacionados com a resistência. Nos últimos anos o esforço para desenvolver novos fármacos tem vindo a aumentar sendo que várias parcerias entre a indústria e a academia têm mesmo sido formadas. Esta união de esforços permite não só expandir o conhecimento existente acerca de fármacos antimaláricos mas também potenciar o progresso na obtenção de novos fármacos. Devido à complexidade do ciclo de vida do parasita, vários alvos têm sido utilizados nos últimos tempos de modo a permitir desenvolver um fármaco que seja eficiente e selectivo para o parasita. Um desses alvos é a cadeia de transporte electrónico que inclue o citocromo bc1. O citocromo bc1 é um elemento crucial para o correcto funcionamento da cadeia de transporte electrónico do parasita. Sendo essencial para a sobrevivência do parasita responsável pela malária, este pode ser considerado um alvo promissor para o desenvolvimento de fármacos antimaláricos. Na ausência de uma estrutura cristalográfica do citocromo bc1 do P. falciparum, toda a informação acerca da sua estrutura e do seu mecanismo tem sido obtida através de estudos desenvolvidos nos seus análogos provenientes de mamíferos, bactérias e leveduras. Neste projecto desenvolveu-se inicialmente um estudo de docking molecular com base na estrutura cristalográfica do citocromo bc1 da levedura Saccharomyces cerevisiae (PDB 3CX5) e utilizando vários inibidores conhecidos com valores de IC50 determinados experimentalmente. Tal permitiu analisar a possibilidade de a estrutura cristalográfica da levedura poder ser utilizada como um bom modelo para o P. falciparum. Mais importante, permitiu também compreender o modo de interacção dos inibidores seleccionados com o centro activo Qo e prever o potencial inibitório destas moléculas. Este modelo foi posteriormente utilizado para se proceder a um estudo de screening virtual utilizando a biblioteca de compostos incluída no programa MOE. Este estudo permitiu obter cinco compostos com actividade antimalárica contra a estirpe W2 resistente à cloroquina. Os compostos mais activos demonstraram também actividade contra a estirpe resistente à atovaquona e contra a levedura. Infelizmente, os estudos biológicos feitos especificamente na cadeia de transporte electrónico não permitiram comprovar que estes compostos actuam definitivamente neste alvo. Tendo em conta a importância de se ter uma estrutura tridimensional adequada para se poder desenhar novos inibidores e considerando o crescente interesse neste alvo, procedeu-se ao desenvolvimento de um modelo por homologia do sítio activo Qo do citocromo bc1 do P. falciparum com base numa estrutura cristalográfica conhecida. Para tal, procedeu-se a um estudo inicial das estruturas cristalográficas deste alvo disponíveis de várias espécies e fez.se a uma comparação exaustiva de modo a seleccionar o melhor modelo. A S. cerevisiae foi a espécie escolhida devido ao grau de homologia entre as duas estruturas homólogas e a resolução da sua estrutura cristalográfica. O modelo por homologia foi desenvolvido utilizando as ferramentas disponíveis no MOE e foi validado utilizando técnicas de docking e screening virtual. Este estudo de screening foi efectuado com base na mesma biblioteca de compostos utilizada anteriormente e permitiu identificar novas moléculas com estruturas químicas bastante interessantes e com potencial para actuar neste alvo. Os dados biológicas destas moléculas não estão ainda disponíveis não permitindo definitivamente validar este modelo por homologia como o modelo mais correcto da estrutura cristalográfica do sítio activo Qo do citocromo bc1 do P. falciparum. Apesar disso, foi feito um enorme avanço em termos de obter uma estrutura tridimensional mais fidedigna deste alvo o que será útil para desenvolver novos inibidores selectivos para o citocromo bc1. Na segunda parte deste trabalho, uma biblioteca de compostos contendo diversos derivados de auronas e azaauronas foi também sintetizada e testada para avaliar a sua actividade antimalárica. As auronas são produtos naturais com actividade antiparasitária já reconhecida. Estes compostos foram primeiramente sintetizados com o objectivo inicial de obter estruturas quimicamente diversas e mais complexas de modo a permitir reconhecer que tipo de alterações permite aumentar o seu potencial antimalárico. Deste modo foram sintetizados vários compostos recorrendo a reacções de acoplamento catalisadas por paládio, como as reacções de Suzuki e Buchwald, devido à facilidade de aumentar rapidamente uma biblioteca de compostos utilizando estes procedimentos. Foram também sintetizados alguns derivados por introdução de uma amina alifática no anel A da aurona de modo a obter bases de Mannich. As moléculas sintetizadas mostraram actividade moderada contra o P. falciparum com valores de IC50 na ordem dos micromolar. Foram feitos ainda vários estudos biológicos com o objectivo de se identificar o modo de acção desta classe de compostos mas tal não foi possível. Novos estudos terão ainda que ser feitos. Os derivados de azaauronas foram obtidos de um modo semelhante ao utilizado para os derivados de auronas. Contudo, neste caso, foi necessário utilizar um método de síntese convergente de modo a se obter os compostos mais eficientemente. Tal deveu-se a problemas associados ao facto de a síntese destes compostos ser mais complexa e menos eficiente que a síntese dos derivados de auronas o que levou não só a uma diminuição do rendimento das reacções mas também a um aumento considerável de produtos secundários e, consequentemente, a um acréscimo na dificuldade em isolar os produtos. Ao contrário dos derivados de auronas, estes novos compostos demonstraram ser bastante mais activos que os seus análogos com valores de IC50 na ordem dos nanomolar. Mais ainda, estes compostos apresentam também citotoxicidade negligenciável. Dado que estes compostos contêm um aceitador de Michael na sua estrutura, foram testados contra a falcipaína-2, uma protease muito importante para a degradação de hemoglobina no vacúolo digestivo do parasita e essencial para a sua sobrevivência. Infelizmente os resultados obtidos indicam que este não é o alvo desta classe de compostos dado que apenas três apresentaram baixo poder inibitório na ordem dos 10 μM. Foi feito ainda um estudo de sinergismo na presença de cloroquina e mefloquina que permitiu demonstrar o potential sinergístico desta classe de potenciais antimaláricos. Mais ainda, a semelhança estrutural destes compostos com as quinolonas, reconhecidos inibidores do citocromo bc1, e os estudos de docking efectuados com base no modelo por homologia desenvolvido anteriormente permite sugerir que seja este o alvo desta classe de compostos. Contudo, estudos biológicos adicionais serão necessários para identificar o modo de acção dos derivados de azaauronas. Finalmente, apesar de o modo de acção destas duas classes de compostos não estar ainda completamente identificado, o estudo desenvolvido demonstra que tanto as auronas como as azaauronas podem ser derivatizadas de modo a se obter novos compostos mais activos. Mais ainda, estas duas classes de compostos podem ser consideradas promissoras no desenvolvimento de fármacos antimaláricos. O estudo desenvolvido pode ser então considerado um passo importante não só na caracterização de um importante alvo terapêutico para a malária mas também na identificação de novas classes de compostos com potencial para ser posteriormente optimizadas de modo a obter novos fármacos antimaláricos com actividade relevante.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/61611/2009 e projetos PTDC/SAU-FCT/098734/2008, PTDC/SAU-FAR/118459/2010 e Pest-OE/SAL/UI4013/2011

Application of computer-aided drug design for identification of P. falciparum inhibitors

Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 μM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin.Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 202

Selective cytotoxicity of dihydroorotate dehydrogenase inhibitors to human cancer cells under hypoxia and nutrient-deprived conditions

Human dihydroorotate dehydrogenase (HsDHODH) is a key enzyme of pyrimidine de novo biosynthesis pathway. It is located on the mitochondrial inner membrane and contributes to the respiratory chain by shuttling electrons to the ubiquinone pool. We have discovered ascofuranone (1), a natural compound produced by Acremonium sclerotigenum, and its derivatives are a potent class of HsDHODH inhibitors. We conducted a structure–activity relationship study and have identified functional groups of 1 that are essential for the inhibition of HsDHODH enzymatic activity. Furthermore, the binding mode of 1 and its derivatives to HsDHODH was demonstrated by co-crystallographic analysis and we show that these inhibitors bind at the ubiquinone binding site. In addition, the cytotoxicities of 1 and its potent derivatives 7, 8, and 9were studied using human cultured cancer cells. Interestingly, they showed selective and strong cytotoxicity to cancer cells cultured under microenvironment (hypoxia and nutrient-deprived) conditions. The selectivity ratio of 8 under this microenvironment show the most potent inhibition which was over 1000-fold higher compared to that under normal culture condition. Our studies suggest that under microenvironment conditions, cancer cells heavily depend on the pyrimidine de novo biosynthesis pathway. We also provide the first evidence that 1 and its derivatives are potential lead candidates for drug development which target the HsDHODH of cancer cells living under a tumor microenvironment