Preventing human-to-mosquito transmission of malaria parasites provides possible solutions to interrupt the malaria parasite life cycle for malaria elimination. The development of validated routine ...assays enabled the discovery of such transmission-blocking compounds. Currently, one development priority remains on combinations of dual-active compounds with equipotent activity against both the disease-causing asexual and transmissible, sexual erythrocytic stages. Additionally, transmission-blocking compounds that target gametocyte-specific biology could be used in combination with compounds against asexual parasites. In either case, preventing transmission will reduce the risk of reinfection and, if different processes are targeted, also curb the spread of drug resistance. Here, we provide an updated roadmap to the discovery and development of new antimalarials with transmission-blocking activity to guide drug discovery for malaria elimination.
Malaria burden can be demonstrably reduced by blocking parasite transmission between the human host and mosquito vector.Antimalarial drugs that target blood-stage parasites as well as block onward parasite transmission will be useful for elimination strategies.Transmission-blocking activity that is associated with targeting different biological processes, or showing polypharmacology, will have obvious advantages in combination strategies to protect blood-stage actives from resistance transmission.Candidate drugs with equipotent activity against blood stages and transmissible stages have been identified and show promise in development as transmission-blocking strategies.
The epigenome of the malaria parasite, Plasmodium falciparum, is associated with regulation of various essential processes in the parasite including control of proliferation during asexual ...development as well as control of sexual differentiation. The unusual nature of the epigenome has prompted investigations into the potential to target epigenetic modulators with novel chemotypes. Here, we explored the diversity within a library of 95 compounds, active against various epigenetic modifiers in cancerous cells, for activity against multiple stages of P. falciparum development. We show that P. falciparum is differentially susceptible to epigenetic perturbation during both asexual and sexual development, with early stage gametocytes particularly sensitive to epi-drugs targeting both histone and non-histone epigenetic modifiers. Moreover, 5 compounds targeting histone acetylation and methylation show potent multistage activity against asexual parasites, early and late stage gametocytes, with transmission-blocking potential. Overall, these results warrant further examination of the potential antimalarial properties of these hit compounds.
In silico adsorption of eight antimalarials that inhibit β‐hematin (synthetic hemozoin) formation identified a primary binding site on the (001) face, which accommodates inhibitors via formation of ...predominantly π‐π interactions. A good correlation (r2=0.64, P=0.017) between adsorption energies and the logarithm of β‐hematin inhibitory activity was found for this face. Of 53 monocyclic, bicyclic and tricyclic scaffolds, the latter yielded the most favorable adsorption energies. Five new amino‐phenoxazine compounds were pursued as β‐hematin inhibitors based on adsorption behaviour. The 2‐substituted phenoxazines show good to moderate β‐hematin inhibitory activity (<100 μM) and Plasmodium falciparum blood stage activity against the 3D7 strain. N1,N1‐diethyl‐N4‐(10H‐phenoxazin‐2‐yl)pentane‐1,4‐diamine (P2a) is the most promising hit with IC50 values of 4.7±0.6 and 0.64±0.05 μM, respectively. Adsorption energies are predictive of β‐hematin inhibitory activity, and thus the in silico approach is a beneficial tool for structure‐based development of new non‐quinoline inhibitors.
The in silico adsorption of 53 cyclic scaffolds to the fastest‐growing (001) face of a β‐hematin crystal identified the phenoxazine scaffold for further derivatization. A small library of 2‐ and 3‐substituted amino‐phenoxazine compounds with predicted β‐hematin inhibitory activity were synthesized. P2a shows the most promising β‐hematin inhibition and antiplasmodium activity.
Malaria elimination requires multipronged approaches, including the application of antimalarial drugs able to block human‐to‐mosquito transmission of malaria parasites. The transmissible gametocytes ...of Plasmodium falciparum seem to be highly sensitive towards epidrugs, particularly those targeting demethylation of histone post‐translational marks. Here, we report exploration of compounds from a chemical library generated during hit‐to‐lead optimization of inhibitors of the human histone lysine demethylase, KDM4B. Derivatives of 2‐(1,1′‐biphenyl‐4‐carboxamido) benzoic acid, around either the amide or a sulfonamide linker backbone (2‐(arylcarboxamido)benzoic acid, 2‐carboxamide (arylsulfonamido)benzoic acid and N‐(2‐(1H‐tetrazol‐5‐yl)phenyl)‐arylcarboxamide), showed potent activity towards late‐stage gametocytes (stage IV/V) of P. falciparum, with the most potent compound reaching single digit nanomolar activity. Structure‐activity relationship trends were evident and frontrunner compounds also displayed microsomal stability and favourable solubility profiles. Simplified synthetic routes support further derivatization of these compounds for further development of these series as malaria transmission‐blocking agents.
Novel derivatives of 2‐(arylcarboxamido)benzoic acid, 2‐(arylsulfonamido)benzoic acid and N‐(2‐(1H‐tetrazol‐5‐yl)phenyl)‐arylcarboxamide, synthesised with the intent to inhibit mammalian KDM4B, were tested against different life cycle stages of the human malaria parasite, Plasmodium falciparum. The most potent of these compounds, a 2‐(arylcarboxamido)benzoic acid derivative, showed potent transmission‐blocking activity possibly through epigenetic mechanisms.
South Africa aims to eliminate malaria transmission by 2023. However, despite sustained vector control efforts and case management interventions, the Vhembe District remains a malaria transmission ...hotspot. To better understand Plasmodium falciparum transmission dynamics in the area, this study characterized the genetic diversity of parasites circulating within the Vhembe District.
A total of 1153 falciparum-positive rapid diagnostic tests (RDTs) were randomly collected from seven clinics within the district, over three consecutive years (2016, 2017 and 2018) during the wet and dry malaria transmission seasons. Using 26 neutral microsatellite markers, differences in genetic diversity were described using a multiparameter scale of multiplicity of infection (MOI), inbreeding metric (Fws), number of unique alleles (A), expected heterozygosity (He), multilocus linkage disequilibrium (LD) and genetic differentiation, and were associated with temporal and geospatial variances.
A total of 747 (65%) samples were successfully genotyped. Moderate to high genetic diversity (mean He = 0.74 ± 0.03) was observed in the parasite population. This was ascribed to high allelic richness (mean A = 12.2 ± 1.2). The majority of samples (99%) had unique multi-locus genotypes, indicating high genetic diversity in the sample set. Complex infections were observed in 66% of samples (mean MOI = 2.13 ± 0.04), with 33% of infections showing high within-host diversity as described by the Fws metric. Low, but significant LD (standardised index of association, ISA = 0.08, P < 0.001) was observed that indicates recombination of distinct clones. Limited impact of temporal (F
range - 0.00005 to 0.0003) and spatial (F
= - 0.028 to 0.023) variation on genetic diversity existed during the sampling timeframe and study sites respectively.
Consistent with the Vhembe District's classification as a 'high' transmission setting within South Africa, P. falciparum diversity in the area was moderate to high and complex. This study showed that genetic diversity within the parasite population reflects the continued residual transmission observed in the Vhembe District. This data can be used as a reference point for the assessment of the effectiveness of on-going interventions over time, the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance.
The life cycle of the malaria parasite Plasmodium falciparum is tightly regulated, oscillating between stages of intense proliferation and quiescence. Cyclic 48-hour asexual replication of Plasmodium ...is markedly different from cell division in higher eukaryotes, and mechanistically poorly understood. Here, we report tight synchronisation of malaria parasites during the early phases of the cell cycle by exposure to DL-α-difluoromethylornithine (DFMO), which results in the depletion of polyamines. This induces an inescapable cell cycle arrest in G
(~15 hours post-invasion) by blocking G
/S transition. Cell cycle-arrested parasites enter a quiescent G
-like state but, upon addition of exogenous polyamines, re-initiate their cell cycle. This ability to halt malaria parasites at a specific point in their cell cycle, and to subsequently trigger re-entry into the cell cycle, provides a valuable framework to investigate cell cycle regulation in these parasites. We subsequently used gene expression analyses to show that re-entry into the cell cycle involves expression of Ca
-sensitive (cdpk4 and pk2) and mitotic kinases (nima and ark2), with deregulation of the pre-replicative complex associated with expression of pk2. Changes in gene expression could be driven through transcription factors MYB1 and two ApiAP2 family members. This new approach to parasite synchronisation therefore expands our currently limited toolkit to investigate cell cycle regulation in malaria parasites.
Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of
. Lead antimalarial candidates are evaluated against ...clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical
isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from
clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.
Histone posttranslational modifications (PTMs) frequently co-occur on the same chromatin domains or even in the same molecule. It is now established that these “histone codes” are the result of cross ...talk between enzymes that catalyze multiple PTMs with univocal readout as compared with these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 77 PTMs on H3 and H3.3, of which 34 are newly identified in P. falciparum. Coexisting PTMs with unique stage distinctions were identified, indicating that many of these combinatorial PTMs are associated with specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2, which we tied to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with coexisting partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher-order gene regulation for distinct proliferation and differentiation processes.
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•First middle-down chromatin proteomics compendium of the malaria parasite, Plasmodium falciparum.•Novel histone PTMs (including arginine methylation) in both asexual parasites and transmissible gametocytes.•Histone PTM cross talk is dynamic life cycle stage stratified.•Gametocytes rely on histone PTM connectivity to allow onward transmission.•AP2-G2 is an important effector of H3K18acK23ac in mature gametocytes.
The complex combinatorial histone code of the malaria parasite is revealed through advanced, quantitative middle-down proteomics. Cross talk between histone PTMs (including novel PTMs) is dynamic and stage specific and includes arginine methylation. Chromatin proteomics show that the transcription factor AP2-G2 interacts with the combination of H3K18acK23ac to drive mature gametocyte transmissibility. The connectivity of the histone code in these parasites ultimately points toward a higher order of gene regulation involved in essential development processes than previously thought.
Aloe marlothii A.Berger (Xanthorrhoeaceae) is indigenous to southern African countries where its aqueous preparations are used in traditional medicine to treat several ailments including ...hypertension, respiratory infections, venereal diseases, chest pain, sore throat and malaria.
The aims of this study were as follows: (i) isolate and identify the antiplasmodial active compounds in A. marlothii roots. As the water extract was previously inactive, the dichloromethane:methanol (DCM:MeOH) (1:1) was used, (ii) examine the activity of the isolated compounds against Plasmodium falciparum asexual blood stage (ABS) parasites as well as for transmission-blocking activity against gametocytes and gametes, and (iii) to use in silico tools to predict the target(s) of the active molecules.
The crude DCM:MeOH (1:1) extract of A. marlothii roots was fractionated on a reverse phase C8 column, using a positive pressure solid-phase extraction (ppSPE) workstation to produce seven fractions. The resulting fractions and the crude DCM:MeOH extract were tested in vitro against P. falciparum (NF54) ABS parasites using the malaria SYBR Green I based-fluorescence assay. Flash silica chromatography and mass-directed preparative high-performance liquid chromatography were utilised to isolate the active compounds. The isolated compounds were evaluated in vitro against P. falciparum asexual (NF54 and K1 strains) and sexual (gametocytes and gametes) stage parasites. Molecular docking was then used for the in silico prediction of targets for the isolated active compounds in P. falciparum.
The crude extract and two SPE fractions displayed good antiplasmodial activity with >97% and 100% inhibition of ABS parasites proliferation at 10 and 20 μg/mL, respectively. Following UPLC-MS analysis of these active fractions, a targeted purification resulted in the isolation of six compounds identified as aloesaponol I (1), aloesaponarin I (2), aloesaponol IV (3), β-sorigenin-1-O-methylether (4), emodin (5), and chrysophanol (6). Aloesaponarin I (2) was the most bioactive, compared to other isolated constituents, against P. falciparum ABS parasites exhibiting equipotency against the drug-sensitive (NF54) (IC50 = 1.54 μg/mL (5 μM)) and multidrug-resistant (K1) (IC50 = 1.58 μg/mL (5 μM)) strains. Aloesaponol IV (3) showed pronounced activity against late-stage (>90% stage IV/V) gametocytes (IC50 = 6.53 μg/mL (22.6 μM)) demonstrating a 3-fold selective potency towards these sexual stages compared to asexual forms of the parasite (IC50 = 19.77 ± 6.835 μg/mL (68 μM)). Transmission-blocking potential of aloesaponol IV (3) was validated by in vitro inhibition of exflagellation of male gametes (94% inhibition at 20 μg/mL). In silico studies identified β-hematin and DNA topoisomerase II as potential biological targets of compounds 2 and 3, respectively.
The findings from our study substantiate the traditional use of A. marlothii to treat malaria. To our knowledge, this study has provided the first report on the isolation and identification of antiplasmodial compounds from A. marlothii roots. Furthermore, our study has provided the first report on the transmission-blocking potential of one of the compounds from the genus Aloe, motivating for the investigation of other species within this genus for their potential P. falciparum transmission-blocking activity.
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•Aloe marlothii is used in the African traditional medicine for treatment of malaria.•Aloesaponarin I and aloesaponol IV were isolated from A. marlothii roots.•Aloesaponarin I showed good potency against asexual stages of Plasmodium falciparum.•Aloesaponol IV displayed pronounced activity against late-stage gametocytes.•In silico studies identified β-hematin and DNA topoisomerase II as potential targets.
Plasmodium
parasites have a complex life cycle that includes development in the human host as well as the
Anopheles
vector. Successful transmission of the parasite between its host and vector ...therefore requires the parasite to balance its investments in asexual replication and sexual reproduction, varying the frequency of sexual commitment to persist within the human host and generate future opportunities for transmission. The transmission window is extended further by the ability of stage V gametocytes to circulate in peripheral blood for weeks, whereas immature stage I to IV gametocytes sequester in the bone marrow and spleen until final maturation. Due to the low gametocyte numbers in blood circulation and with the ease of targeting such life cycle bottlenecks, transmission represents an efficient target for therapeutic intervention. The biological process of
Plasmodium
transmission is a multistage, multifaceted process and the past decade has seen a much deeper understanding of the molecular mechanisms and regulators involved. Clearly, specific and divergent processes are used during transmission compared to asexual proliferation, which both poses challenges but also opportunities for discovery of transmission-blocking antimalarials. This review therefore presents an update of our molecular understanding of gametocyte and gamete biology as well as the status of transmission-blocking activities of current antimalarials and lead development compounds. By defining the biological components associated with transmission, considerations for the development of new transmission-blocking drugs to target such untapped but unique biology is suggested as an important, main driver for transmission-blocking drug discovery.
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