Drug-resistant Parasite Strains Research Articles

Development and longevity of naturally acquired antibody and memory B cell responses against Plasmodium vivax infection.

Plasmodium vivax malaria causes significant public health problems in endemic regions. Considering the rapid spread of drug-resistant parasite strains and the development of hypnozoites in the liver with potential for relapse, development of a safe and effective vaccine for preventing, controlling, and eliminating the infection is critical. Immunity to malaria is mediated by antibodies that inhibit sporozoite or merozoite invasion into host cells and protect against clinical disease. Epidemiologic data from malaria endemic regions show the presence of naturally acquired antibodies to P. vivax antigens during and following infection. But data on the persistence of these antibodies, development of P. vivax-specific memory B cells (MBCs), and their relation to reduction of malaria severity and risk is limited. This review provides an overview of the acquisition and persistence of naturally acquired humoral immunity to P. vivax infection. Also, we summarize and discuss current progress in assessment of immune responses to candidate vaccine antigens in P. vivax patients from different transmission settings. Longitudinal studies of MBC and antibody responses to these antigens will open new avenues for developing vaccines against malaria infection and its transmission.

Lactam Truncation Yields a Dihydroquinazolinone Scaffold with Potent Antimalarial Activity that Targets PfATP4.

The emergence of resistance against current antimalarial treatments has necessitated the need for the development of novel antimalarial chemotypes. Toward this goal, we recently optimised the antimalarial activity of the dihydroquinazolinone scaffold and showed it targeted PfATP4. Here, we deconstruct the lactam moiety of the tricyclic dihydroquinazolinone scaffold and investigate the structure-activity relationship of the truncated scaffold. It was shown that SAR between scaffolds was largely transferrable and generated analogues with potent asexual stage activity. Evaluation of the truncated analogues against PfATP4 mutant drug resistant parasite strains and in assays measuring PfATP4-associated ATPase activity demonstrated retention of PfATP4 as the molecular target. Analogues exhibited activity against both male and female gametes and multidrug resistant parasites. Limited efficacy of analogues in a P. berghei asexual stage mouse model was attributed to their moderate metabolic stability and low aqueous stability. Further development is required to address these attributes toward the potential use of the dihydroquinazolinone class in a curative and transmission blocking combination antimalarial therapy.

Optimization of diastereomeric dihydropyridines as antimalarials

The increase in research funding for the development of antimalarials since 2000 has led to a surge of new chemotypes with potent antimalarial activity. High-throughput screens have delivered several thousand new active compounds in several hundred series, including the 4,7-diphenyl-1,4,5,6,7,8-hexahydroquinolines, hereafter termed dihydropyridines (DHPs). We optimized the DHPs for antimalarial activity. Structure-activity relationship studies focusing on the 2-, 3-, 4-, 6-, and 7-positions of the DHP core led to the identification of compounds potent (EC50 < 10 nM) against all strains of P. falciparum tested, including the drug-resistant parasite strains K1, W2, and TM90-C2B. Evaluation of efficacy of several compounds in vivo identified two compounds that reduced parasitemia by >75 % in mice 6 days post-exposure following a single 50 mg/kg oral dose. Resistance acquisition experiments with a selected dihydropyridine led to the identification of a single mutation conveying resistance in the gene encoding for Plasmodium falciparum multi-drug resistance protein 1 (PfMDR1). The same dihydropyridine possessed transmission blocking activity. The DHPs have the potential for the development of novel antimalarial drug candidates.

"Seasoning" antimalarial drugs' action: chloroquine bile salts as novel triple-stage antiplasmodial hits.

Malaria is one of the "big three" global infectious diseases, having caused above two hundred million cases and over half a million deaths in 2020. The continuous demand for new treatment options prioritizes the cost-effective development of new chemical entities with multi-stage antiplasmodial activity, for higher efficacy and lower propensity to elicit drug-resistant parasite strains. Following up on our long-term research towards the rescue of classical antimalarial aminoquinolines like chloroquine and primaquine, we have developed new organic salts by acid-base pairing of those drugs with natural bile acids. These antimalarial drug-derived bile salts were screened in vitro against the hepatic, blood and gametocyte stages of Plasmodium parasites, unveiling chloroquine bile salts as unprecedented triple-stage antiplasmodial hits. These findings pave a new pathway for drug rescuing, even beyond anti-malarial and other anti-infective drugs.

Progress of researches on antimalarial peptides

Malaria remains a major global public health concern, and nearly half of the global populations are still at risk of malaria infection. However, continuous emergence and spread of drug-resistant malaria parasite strains lead to ineffectiveness of conventional antimalarials. Therefore, development of novel antimalarial agents is of urgent need for malaria elimination. As an important component of the host natural immune defense system, antibacterial peptides provide the first line of defense against pathogenic invasion, and the mechanism of preferentially attacking the cell membrane makes them difficult to develop drug resistance. Antimicrobial peptides are therefore considered as a promising candidate for novel antimalarial agents. This review summarizes the advances in researches on antimicrobial peptides with antimalarial actions and discusses the potential of antimalarial peptides as novel antimalarials.

Identification of Fungus-Derived Natural Products as New Antigiardial Scaffolds.

There is an unmet need for effective therapies for treating diseases associated with the intestinal parasite Giardia lamblia. In this study, a library of chemically validated purified natural products and fungal extracts was screened for chemical scaffolds that can inhibit the growth of G. lamblia. The phenotypic screen led to the identification of several previously unreported classes of natural product inhibitors that block the growth of G. lamblia. Hits from phenotypic screens of these naturally derived compounds are likely to possess a variety of mechanisms of action not associated with clinically used nitroimidazole and thiazolide compounds. They may therefore be effective against current drug-resistant parasite strains. IMPORTANCE There is a direct link between widespread prevalence of clinical giardiasis and poverty. This may be one of the reasons why giardiasis is a significant contributor to diarrheal morbidity, stunting, and death of children in resource-limited communities around the world. FDA-approved treatments for giardiasis include metronidazole, related nitroimidazole drugs, and albendazole. However, a substantial number of clinical infections are resistant to these treatments. The depth of the challenge is partly exacerbated by a lack of investment in the discovery and development of novel agents for treatment of giardiasis. Applicable interventions must include new drug development strategies that will result in the identification of effective therapeutics, particularly those that are inexpensive and can be quickly advanced to clinical uses, such as products from nature. This study identified novel chemical scaffolds from fungi that can form the basis of future medicinal chemistry optimization of novel antigiardial agents.

Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4.

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

Therapeutic efficacy of Chloroquine for the treatment of uncomplicated Plasmodium vivax infection in Shewa Robit, Northeast Ethiopia.

The development of drug resistance to chloroquine is posing a challenge in the prevention and control efforts of malaria globally. Chloroquine is the first-line treatment for uncomplicated P.vivax in Ethiopia. Regular monitoring of anti-malarial drugs is recommended to help early detection of drug-resistant strains of malaria parasites before widely distributed. The emergence of P.vivax resistance to chloroquine in the country endangers the efficacy of P.vivax treatment. This study aimed to assess the therapeutic efficacy of chloroquine among uncomplicated P.vivax infections at Shewa Robit Health Center, northeast Ethiopia. One-arm in vivo prospective chloroquine efficacy study was conducted from November 2020 to March 2021. Ninety participants aged between 16 months to 60 years confirmed with P.vivax mono-infection microscopically were selected and treated with a 25 mg/kg standard dose of chloroquine over three days. Thick and thin blood smears were prepared and examined. Clinical examination was performed over 28 follow-up days. Hemoglobin concentration level was measured on days 0, 14, and 28. Of the 90 enrolled participants, 86 (96%) completed their 28 days follow-up period. The overall cure rate of the drug was 98.8% (95% CI: 95.3-100%). All asexual stages and gametocytes were cleared within 48 hours with rapid clearance of fever. Hemoglobin concentration had significantly recovered between days 0 and 14, 0 and 28, and 14 and 28 days (P = 0.032, P<0.001, and P = 0.005), respectively. Fast resolution of clinical signs and symptoms was also observed. Severe adverse events were not recorded. The present study revealed that chloroquine remains an efficacious and safe drug in the study setting for treating uncomplicated P.vivax in the study area. Large-scale continuous surveillance is needed to monitor the development of resistance in due time.

Exploring a Tetrahydroquinoline Antimalarial Hit from the Medicines for Malaria Pathogen Box and Identification of its Mode of Resistance as PfeEF2.

New antimalarial treatments with novel mechanism of action are needed to tackle Plasmodium falciparum infections that are resistant to first-line therapeutics. Here we report the exploration of MMV692140 (2) from the Pathogen Box, a collection of 400 compounds that was made available by Medicines for Malaria Venture (MMV) in 2015. Compound 2 was profiled in in vitro models of malaria and was found to be active against multiple life-cycle stages of Plasmodium parasites. The mode of resistance, and putatively its mode of action, was identified as Plasmodium falciparum translation elongation factor 2 (PfeEF2), which is responsible for the GTP-dependent translocation of the ribosome along mRNA. The compound maintains activity against a series of drug-resistant parasite strains. The structural motif of the tetrahydroquinoline (2) was explored in a chemistry program with its structure-activity relationships examined, resulting in the identification of an analog with 30-fold improvement of antimalarial asexual blood stage potency.

Replenishing the malaria drug discovery pipeline: Screening and hit evaluation of the MMV Hit Generation Library 1 (HGL1) against asexual blood stage Plasmodium falciparum, using a nano luciferase reporter read-out.

A central challenge of antimalarial therapy is the emergence of resistance to the components of artemisinin-based combination therapies (ACTs) and the urgent need for new drugs acting through novel mechanism of action. Over the last decade, compounds identified in phenotypic high throughput screens (HTS) have provided the starting point for six candidate drugs currently in the Medicines for Malaria Venture (MMV) clinical development portfolio. However, the published screening data which provided much of the new chemical matter for malaria drug discovery projects have been extensively mined. Here we present a new screening and selection cascade for generation of hit compounds active against the blood stage of Plasmodium falciparum. In addition, we validate our approach by testing a library of 141,786 compounds not reported earlier as being tested against malaria. The Hit Generation Library 1 (HGL1) was designed to maximise the chemical diversity and novelty of compounds with physicochemical properties associated with potential for further development. A robust HTS cascade containing orthogonal efficacy and cytotoxicity assays, including a newly developed and validated nanoluciferase-based assay was used to profile the compounds. 75 compounds (Screening Active hit rate of 0.05%) were identified meeting our stringent selection criteria of potency in drug sensitive (NF54) and drug resistant (Dd2) parasite strains (IC50 ≤ 2 µM), rapid speed of action and cell viability in HepG2 cells (IC50 ≥ 10 µM). Following further profiling, 33 compounds were identified that meet the MMV Confirmed Active profile and are high quality starting points for new antimalarial drug discovery projects.

Genomic analysis of single nucleotide polymorphisms in malaria parasite drug targets

Malaria is a life-threatening parasitic disease caused by members of the genus Plasmodium. The development and spread of drug-resistant strains of Plasmodium parasites represent a major challenge to malaria control and elimination programmes. Evaluating genetic polymorphism in a drug target improves our understanding of drug resistance and facilitates drug design. Approximately 450 and 19 whole-genome assemblies of Plasmodium falciparum and Plasmodium vivax, respectively, are currently available, and numerous sequence variations have been found due to the presence of single nucleotide polymorphism (SNP). In the study reported here, we analysed global SNPs in the malaria parasite aminoacyl-tRNA synthetases (aaRSs). Our analysis revealed 3182 unique SNPs in the 20 cytoplasmic P. falciparum aaRSs. Structural mapping of SNPs onto the three-dimensional inhibitor-bound complexes of the three advanced drug targets within aaRSs revealed a remarkably low mutation frequency in the crucial aminoacylation domains, low overall occurrence of mutations across samples and high conservation in drug/substrate binding regions. In contrast to aaRSs, dihydropteroate synthase (DHPS), also a malaria drug target, showed high occurrences of drug resistance-causing mutations. Our results show that it is pivotal to screen potent malaria drug targets against global SNP profiles to assess genetic variances to ensure success in designing drugs against validated targets and tackle drug resistance early on.Graphical

Molecular assays for determining sulphadoxine-pyrimethamine drug resistance in India: a systematic review.

A plethora of studies analyse the molecular markers of drug resistance and hence help in guiding the evidence-based malaria treatment policies in India. For reporting mutations, a number of techniques including DNA sequencing, restriction-fragment length polymorphism and mutation-specific polymerase chain reaction have been employed across numerous studies, including variations in the methodology used. However, there is no sufficient data from India comparing these methods as well as report the prevalence of polymorphisms in SP drug resistance molecular markers independently using such methods. Therefore, all data from Indian studies available for molecular marker studies of Plasmodium falciparum drug resistance to sulphadoxine-pyrimethamine was gathered, and a systematic review was performed. This systematic review identifies the molecular methods in use in India and compares each method for detecting sulphadoxine-pyrimethamine drug resistance marker. To delay the spread of drug-resistant parasite strains, a simplified and standardized molecular method is much needed which can be obtained by analysing the performance of each method in use and answering the necessity of newer methodological approaches.

An update on cerebral malaria for therapeutic intervention.

Cerebral malaria is often pronounced as a major life-threatening neurological complication of Plasmodium falciparum infection. The complex pathogenic landscape of the parasite and the associated neurological complications are still not elucidated properly. The growing concerns of drugresistant parasite strains along with the failure of anti-malarial drugs to subdue post-recovery neuro-cognitive dysfunctions in cerebral malaria patients have called for a demand to explore novel biomarkers and therapeutic avenues. Due course of the brain infection journey of the parasite, events such as sequestration of infected RBCs, cytoadherence, inflammation, endothelial activation, and blood-brain barrier disruption are considered critical. In this review, we briefly summarize the diverse pathogenesis of the brain-invading parasite associated with loss of the blood-brain barrier integrity. In addition, we also discuss proteomics, transcriptomics, and bioinformatics strategies to identify an array of new biomarkers and drug candidates. A proper understanding of the parasite biology and mechanism of barrier disruption coupled with emerging state-of-art therapeutic approaches could be helpful to tackle cerebral malaria.

Impact of Genetic Diversity and Genome Plasticity of Leishmania spp. in Treatment and the Search for Novel Chemotherapeutic Targets.

Leishmaniasis is one of the major public health concerns in Latin America, Africa, Asia, and Europe. The absence of vaccines for human use and the lack of effective vector control programs make chemotherapy the main strategy to control all forms of the disease. However, the high toxicity of available drugs, limited choice of therapeutic agents, and occurrence of drug-resistant parasite strains are the main challenges related to chemotherapy. Currently, only a small number of drugs are available for leishmaniasis treatment, including pentavalent antimonials (SbV), amphotericin B and its formulations, miltefosine, paromomycin sulphate, and pentamidine isethionate. In addition to drug toxicity, therapeutic failure of leishmaniasis is a serious concern. The occurrence of drug-resistant parasites is one of the causes of therapeutic failure and is closely related to the diversity of parasites in this genus. Owing to the enormous plasticity of the genome, resistance can occur by altering different metabolic pathways, demonstrating that resistance mechanisms are multifactorial and extremely complex. Genetic variability and genome plasticity cause not only the available drugs to have limitations, but also make the search for new drugs challenging. Here, we examined the biological characteristics of parasites that hinder drug discovery.

Recent Progress in the Development of Indole-Based Compounds Active against Malaria, Trypanosomiasis and Leishmaniasis.

Human protozoan diseases represent a serious health problem worldwide, affecting mainly people in social and economic vulnerability. These diseases have attracted little investment in drug discovery, which is reflected in the limited available therapeutic arsenal. Authorized drugs present problems such as low efficacy in some stages of the disease or toxicity, which result in undesirable side effects and treatment abandonment. Moreover, the emergence of drug-resistant parasite strains makes necessary an even greater effort to develop safe and effective antiparasitic agents. Among the chemotypes investigated for parasitic diseases, the indole nucleus has emerged as a privileged molecular scaffold for the generation of new drug candidates. In this review, the authors provide an overview of the indole-based compounds developed against important parasitic diseases, namely malaria, trypanosomiasis and leishmaniasis, by focusing on the design, optimization and synthesis of the most relevant synthetic indole scaffolds recently reported.

Antioxidant defence system as a rational target for Chagas disease and Leishmaniasis chemotherapy

Chagas disease and leishmaniasis are neglected tropical diseases caused by the protozoan parasites Trypanosoma cruzi and Leishmania spp., respectively. They are among the most important parasitic diseases, affecting millions of people worldwide, being a considerable global challenge. However, there is no human vaccine available against T. cruzi and Leishmania infections, and their control is based mainly on chemotherapy. Treatments for Chagas disease and leishmaniasis have multiple limitations, mainly due to the high toxicity of the available drugs, long-term treatment protocols, and the occurrence of drug-resistant parasite strains. In the case of Chagas disease, there is still the problem of low cure rates in the chronic stage of the disease. Therefore, new therapeutic agents and novel targets for drug development are urgently needed. Antioxidant defence in Trypanosomatidae is a potential target for chemotherapy because the organisms present a unique mechanism for trypanothione-dependent detoxification of peroxides, which differs from that found in vertebrates. Cellular thiol redox homeostasis is maintained by the biosynthesis and reduction of trypanothione, involving different enzymes that act in concert. This study provides an overview of the antioxidant defence focusing on iron superoxide dismutase A, tryparedoxin peroxidase, and ascorbate peroxidase and how the enzymes play an important role in the defence against oxidative stress and their involvement in drug resistance mechanisms in T. cruzi and Leishmania spp.

First Report of Anthelmintic Resistance in Gastrointestinal Nematodes in Goats in Romania.

Simple SummaryAnthelmintic resistance (AR) is a serious threat to animal health and has a major economic impact worldwide due to production and financial losses. Currently, there are three classes of anthelmintics most commonly used in small ruminants: the benzimidazoles (BZs), macrocyclic lactones (MLs) and cholinergic agonists (especially levamisole; LEV). The widespread use of those products has led to the emergence of drug-resistant parasite strains. In the present study, we describe for the first time a case of resistance to anthelmintics in goats in Romania. Resistance was detected and confirmed for two chemical groups of anthelmintics (MLs and BZs) by in vivo faecal egg count reduction test (FECRT) and in vitro methods: the egg hatch test (EHT) and larval development test (LDT). Considering the increasing prevalence of AR in goat herds in Europe and around the globe, we believe that the findings of our study on AR in goats in Romania do not represent a singular event and could hence be just the noticeable part of a much wider issue.Currently, there are three classes of anthelmintics most commonly used in small ruminants: the benzimidazoles (BZs), macrocyclic lactones (MLs) and cholinergic agonists (especially levamisole; LEV). The widespread use of those products has led to the emergence of drug-resistant parasite strains which represents a serious threat to the livestock industry. In the present study, we describe for the first time a case of resistance to anthelmintics in goats in Romania. The study was carried out in 2021 in a dairy goat herd from the Transylvania region. Two types of diagnostic methods were used to confirm anthelmintic resistance (AR). First, the faecal egg count reduction test (FECRT), an in vivo AR diagnostic method, was used to evaluate the efficacy of eprinomectin (EPM). The results of this test were analysed applying two different calculative methods that are used only in treated animals (without the control group). Furthermore, two in vitro methods were used: the egg hatch test (EHT) for the detection of resistance to BZs, and the larval development test (LDT) for detection of resistance to all three classes of anthelmintics. The results of FECRT indicate the resistance of gastrointestinal nematodes (GINs) to EPM in both calculative methods (FECR1 = −88% and FECR2 = −202%). In addition, the results obtained for ivermectin aglycone (IVM-AG) in LDT also indicate resistance to drugs from MLs group, especially avermectins. Similarly, the results of in vitro methods (EHT and LDT) indicate resistance to BZs in this herd. LEV was the only drug that stopped the development of L3 larvae 100% (LDT). H. contortus was the only nematode species found in coproculture after EPM treatment. Furthermore, H. contotus L3 larvae was the only species found in the wells with the highest concentrations of thiabendazole (TBZ) and IVM-AG in LDT. This suggests that resistance to both BZs and MLs was present for that species.

Perspectives From Systems Biology to Improve Knowledge of Leishmania Drug Resistance.

Neglected Tropical Diseases include a broad range of pathogens, hosts, and vectors, which represent evolving complex systems. Leishmaniasis, caused by different Leishmania species and transmitted to humans by sandflies, are among such diseases. Leishmania and other Trypanosomatidae display some peculiar features, which make them a complex system to study. Leishmaniasis chemotherapy is limited due to high toxicity of available drugs, long-term treatment protocols, and occurrence of drug resistant parasite strains. Systems biology studies the interactions and behavior of complex biological processes and may improve knowledge of Leishmania drug resistance. System-level studies to understand Leishmania biology have been challenging mainly because of its unusual molecular features. Networks integrating the biochemical and biological pathways involved in drug resistance have been reported in literature. Antioxidant defense enzymes have been identified as potential drug targets against leishmaniasis. These and other biomarkers might be studied from the perspective of systems biology and systems parasitology opening new frontiers for drug development and treatment of leishmaniasis and other diseases. Our main goals include: 1) Summarize current advances in Leishmania research focused on chemotherapy and drug resistance. 2) Share our viewpoint on the application of systems biology to Leishmania studies. 3) Provide insights and directions for future investigation.

Assessment of in vitro and in vivo antimalarial efficacy and GC-fingerprints of selected medicinal plant extracts

A hallmark of mortality and morbidity, malaria is affecting nearly half of the world's population. Emergence of drug-resistant strains of malarial parasite prompts identification and evaluation of medicinal plants and their constituents that may hold the key to a new and effective anti-malarial drug. In this context, nineteen methanolic extracts from seventeen medicinal plants were evaluated for anti-plasmodial potential against Plasmodium falciparum strain 3D7 (Chloroquine (CQ) sensitive) and INDO (CQ resistant) using fluorescence based SYBR-Green assay and for cytotoxic effects against mammalian cell lines. Leaf extract of two plants showed promising in vitro anti-malarial activity (Pf3D7 IC50 ≤ 10 μg/ml); one plant extract showed good activity (Pf3D7 IC50 = 10.1–20 μg/ml); seven were moderately active (IC50 = 20.1–50 μg/ml), four plant extracts showed poor activity (PfD7 IC50 = 50.1–100 μg/ml) and five extracts showed no activity up to IC50 = 100 μg/ml. Further, six extracts were found equipotent to PfINDO (resistance index ranging 0.4–2) and relatively nontoxic to mammalian cell lines HEK293 (cytotoxicity index ranging 1.4–12.5). Based on good resistance and selectivity indices, three extracts were evaluated for in vivo activity in Plasmodium berghei ANKA infected mice at a dose of 500 mg/kg and they showed significant suppression of P. berghei parasitemia. Further, these active plant extracts were fractionated using silica-gel chromatography and their fractions were evaluated for anti-plasmodial action. Obtained fractions showed enrichment in antimalarial activity. Active fractions were analyzed by gas chromatography and mass-spectrometery. Results suggests that the three active plant extracts could serve as potent source of anti-malarial agent and therefore require further analysis.

The antimalarial activity of indole alkaloids and hybrids

Malaria, caused by the genus Plasmodium, remains a global public health concern. It is estimated by the World Health Organization that over 40% of the world's population lives in areas at risk for malarial transmission, and around half a million people succumb to this infectious disease annually, which is related to the rapid spread of drug-resistant parasite strains. Indole derivatives, which possess broad-spectrum pharmacological properties, play a crucial role in the discovery of new drugs. Many indole derivatives exhibited potential in vitro and in vivo activity against both drug-sensitive and drug-resistant malaria, suggesting that the indole moiety is a useful template for the development of novel antimalarial agents. This review outlines the advances in indole alkaloids and hybrids with antimalarial potential in the recent decade.