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Novel antimalarial compounds targeting both the pathogenic and transmissible stages of the human malaria parasite, Plasmodium falciparum, would greatly benefit malaria elimination strategies. However, most compounds affecting asexual blood stage parasites show severely reduced activity against gametocytes. The impact of this activity loss on a compound's transmission-blocking activity is unclear. Here, we report the systematic evaluation of the activity loss against gametocytes and investigate the confounding factors contributing to this. A threshold for acceptable activity loss between asexual blood stage parasites and gametocytes was defined, with near-equipotent compounds required to prevent continued gametocyte maturation and onward transmission. Target abundance is not predictive of gametocytocidal activity, but instead, lipoidal uptake is the main barrier of dual activity and is influenced by distinct physicochemical properties. This study provides guidelines for the required profiles of potential dual-active antimalarial agents and facilitates the development of effective transmission-blocking compounds., Competing Interests: Competing interests D.L. is a senior director in drug discovery at the Medicines for Malaria Venture (M.M.V.). M.M.V. funded part of this study. The other authors declare no competing interest., (© 2024. The Author(s).)

Previously, we successfully introduced laeA gene into a fungal strain in order to significantly increase the production of a bioactive compound, allowing use to discover novel biological activity. To demonstrate the universal applicability of the laeA gene introduction strategy for taping the potential of fungal secondary metabolism, in this present study, we created a library of microorganisms which we had the laeA gene inserted, and from that library we aimed to isolate compounds which are produced at significantly greater quantities compared to the respective wild type strains. From this investigation, we were able to isolate sclerotinin A (1) from Pochonia sp. KTF-0504 strain. We revealed that 1 showed anti-malarial activity against Plasmodium falciparum parasite strains. On the other hands, 1 showed no anti-fungal activity against multidrug-sensitive budding yeast. Our study implies that the utilization of the laeA gene in fungi is a versatile method for the discovery of drug candidates., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Y.A., T.T., and K.I. are employees of Kikkoman Corporation. Kitasato University received research funding from Kikkoman Corporation., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To achieve malaria eradication, new preventative agents that act differently to front-line treatment drugs are needed. To identify potential chemoprevention starting points we screened a sub-set of the CSIRO Australia Compound Collection for compounds with slow-action in vitro activity against Plasmodium falciparum. This work identified N,N-dialkyl-5-alkylsulfonyl-1,3,4-oxadiazol-2-amines as a new antiplasmodial chemotype (e.g., 1 96 h IC 50 550 nM; 3 96 h IC 50 160 nM) with a different action to delayed-death slow-action drugs. A series of analogues were synthesized from thiotetrazoles and carbomoyl derivatives using Huisgen 1,3,4-oxadiazole synthesis followed by oxidation of the resultant thioethers to target sulfones. Structure activity relationship analysis of analogues identified compounds with potent and selective in vitro activity against drug-sensitive and multi-drug resistant Plasmodium parasites (e.g., 31 and 32 96 h IC 50 <40 nM; SI > 2500). Subsequent studies in mice with compound 1, which had the best microsomal stability of the compounds assessed (T 1/2 >255 min), demonstrated rapid clearance and poor oral in vivo efficacy in a P. berghei murine malaria model. These data indicate that while N,N-dialkyl-5-alkylsulfonyl-1,3,4-oxadiazol-2-amines are a novel class of slow-acting antiplasmodial agents, the further development of this chemotype for malaria chemoprophylaxis will require pharmacokinetic profile improvements., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Katherine Andrews, Tina Skinner-Adams, Oliver Hutt, John Ryan, Andrew Riches reports financial support was provided by National Health and Medical Research Council of Australia. Anjana Rai reports financial support was provided by Griffith University GUIPRS and GUPRS PhD scholarships. Katherine Andrews reports equipment, drugs, or supplies was provided by Australian Red Cross Lifeblood. Katherine Andrews reports writing assistance was provided by Monash Institute of Pharmaceutical Sciences Centre for Drug Candidate Optimisation. Katherine Andrews reports equipment, drugs, or supplies was provided by Compounds Australia, Griffith University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

This study aimed to analyze polymorphisms in Pfcrt, Pfmdr1, and Pfk13 genes' markers of resistance to Artemisinin-based combination therapy (ACT), in Plasmodium falciparum isolates from southern Brazzaville, 15 years after the adoption of ACT in the Republic of Congo. A total of 369 microscopy-confirmed malaria-infected individuals were enrolled from March to October 2021 in the community and in health facilities during a cross-sectional study. The K76T mutation in the Pfcrt gene, N86Y and Y184F mutations in the Pfmdr1 gene were investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) while the codons region (1005-1300) of the Pfmdr1gene, and Pfk13 gene were sequenced. The prevalences of K76T, N86Y, Y184F mutations were 26.0%, 6.8%, and 27.7%, respectively. However, no mutations were detected in codons 1034, 1042, and 1246 of the Pfmdr1 gene. None of the mutations previously associated with artemisinin-based resistance were detected in the Pfk13 gene. The results reveal a significant decrease in the prevalence of K76T, N86Y, Y184F mutations, in Plasmodium falciparum isolates following the change of therapeutic policy. As artemisinin resistance is emerging throughout Africa, continued surveillance for early detection of these mutations and relevant partner markers of drug resistance are recommended in the Republic of Congo., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase Pf CLK3 with the reversible inhibitor TCMDC-135051 ( 1 ), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

Malaria has been a deadly enemy of mankind throughout history, affecting over 200 million people annually, along with approximately half a million deaths. Resistance to current therapies is of great concern, and there is a dire need for novel and well-tolerated antimalarials that operate by clinically unexploited mechanisms. We have previously reported that both tambjamines and prodiginines are highly potent novel antiplasmodial agents, but they required rigor optimizations to enhance the oral efficacy, safety, and physicochemical properties. Here, we launched a comprehensive structure-activity relationship study for B-ring-functionalized tambjamines and prodiginines with 54 novel analogues systematically designed and synthesized. A number of compounds exhibited remarkable antiplasmodial activities against asexual erythrocytic Plasmodium parasites, with improved safety and metabolic profiles. Notably, several prodiginines cured erythrocytic Plasmodium yoelii infections after oral 25 mg/kg × 4 days in a murine model and provided partial protection against liver stage Plasmodium berghei sporozoite-induced infection in mice.

The potency of frontline antimalarial drug artemisinin (ART) derivatives is triggered by heme-induced cleavage of the endoperoxide bond to form reactive heme-ART alkoxy radicals and covalent heme-ART adducts, which are highly toxic to the parasite. ART-resistant (ART-R) parasites with mutations in the Plasmodium falciparum Kelch-containing protein Kelch13 (PfKekch13) exhibit impaired hemoglobin uptake, reduced yield of hemoglobin-derived heme, and thus decreased ART activation. However, any direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that the purified recombinant PfKelch13 wild-type (WT) protein displays measurable binding affinity for iron and heme, the main effectors for ART activation. The heme-binding property is also exhibited by the native PfKelch13 protein from parasite culture. The two ART-R recombinant PfKelch13 mutants (C580Y and R539T) display weaker heme binding affinities compared to the ART-sensitive WT and A578S mutant proteins, which further translates into reduced yield of heme-ART derivatives when ART is incubated with the heme molecules bound to the mutant PfKelch13 proteins. In conclusion, this study provides the first evidence for ART activation via the heme-binding propensity of PfKelch13. This mechanism may contribute to the modulation of ART-R levels in malaria parasites through a novel function of PfKelch13., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The development of parasite resistance to both artemisinin derivatives and their partner drugs jeopardizes the effectiveness of the artemisinin combination therapy. Thus, the discovery of new antimalarial drugs, with new mechanisms of action, is urgently needed. We recently disclosed that β-carboline 1a was orally efficacious in Plasmodium berghei -infected mice and that it showed low cross-resistance between susceptible Plasmodium falciparum and four different drug-resistant strains. In this report, we describe the synthesis and in vitro antimalarial evaluation of 91 new derivatives of 1a . The asexual blood stage growth inhibition data show a clear preference for a 3,4-dihalogenated, 3,5-dihalogenated, 3,4,5-trichloro-, or 4-trifluoromethyphenyl ring at the C1-position. The most potent compound, 3,4,5-trichlorophenyl-substituted 42a , is twice as potent as 1a . Six potent analogues were assessed for their drug-like properties, and four of these were subjected to in vitro barcoded cross-resistance profiling. Compounds 1a , 1m , 42a , and 42m showed no cross-resistance to 32 resistance mutations on the Dd2 genetic background and 10 resistance mutations on the 3D7 genetic background. These data suggest that compounds in this scaffold possess a novel mechanism of antimalarial action.

Background: The most widely used anti-malarial drug artemisinin (ART) is metabolized extensively, but the therapeutic capacity of its major metabolite remains unknown. Whether the major metabolite of ART (ART-M) contributes to its antiplasmodial potency was investigated in this study., Methods: The metabolite identification and enzyme phenotyping of ART were performed using human liver microsomes (HLMs). The stereostructure of the major metabolite ART-M was elucidated by spectroscopic and X-ray crystallographic analysis. The anti-malarial activity of ART-M against two reference Plasmodium strains (Pf3D7 and PfDd2) was evaluated. The pharmacokinetic profiles of ART and its metabolite ART-M were investigated in healthy Chinese subjects after a recommended two-day oral dose of ART plus piperaquine. Pharmacodynamic parameters based on minimum inhibitory concentration (MIC 50 ) and free plasma concentration were employed to evaluate the therapeutic potency of ART-M, including fAUC 0-t /MIC 50 , fC max /MIC 50 and T > MIC 50 ., Results: A major metabolite 10β-hydroxyartemisinin (ART-M) was found for ART in human, and CYP3A4/3A5 was the major enzymes responsible for ART 10β-hydroxylation. Compared with ART (MIC 50 , 10.1 nM against Pf3D7), weaker antiplasmodial activity was found for ART-M (MIC 50 , 61.4 nM against Pf3D7). However, a 3.5-fold higher maximal free plasma concentration was achieved for ART-M (fC max , 180.0 nM vs. 51.8 nM for ART). ART-M displayed comparable antiplasmodial potency to ART, in terms of fAUC 0-t /MIC 50 (12.5 h), fC max /MIC 50 (2.8) and T > MIC 50 (5 h)., Conclusions: The major metabolite 10β-hydroxyartemisinin contributes to the antiplasmodial efficacy of ART, which should be considered when evaluation of ART dosing regimens and/or clinical outcomes., (© 2024. The Author(s).)

Background: Artemisinin-based combination therapy (ACT) is currently recommended for treatment of uncomplicated malaria. However, the emergence and spread of partial artemisinin resistance threatens their effectiveness for malaria treatment in sub-Saharan Africa where the burden of malaria is highest. Early detection and reporting of validated molecular markers (pfk13 mutations) in Plasmodium falciparum is useful for tracking the emergence and spread of partial artemisinin resistance to inform containment efforts., Methods: Genomic surveillance was conducted at 50 surveillance sites across four regions of Uganda in Karamoja, Lango, Acholi and West Nile from June 2021 to August 2023. Symptomatic malaria suspected patients were recruited and screened for presence of parasites. In addition, dried blood spots (DBS) were collected for parasite genomic analysis with PCR and sequencing. Out of 563 available dried blood spots (DBS), a random subset of 240 P. falciparum mono-infections, confirmed by a multiplex PCR were selected and used for detecting the pfk13 mutations by Sanger sequencing using Big Dye Terminator method. Regional variations in the proportions of pfk13 mutations were assessed using the chi square or Fisher's exact tests while Kruskal-Wallis test was used to compare absolute parasite DNA levels between wild type and mutant parasites., Results: Overall, 238/240 samples (99.2%) contained sufficient DNA and were successfully sequenced. Three mutations were identified within the sequenced samples; pfk13 C469Y in 32/238 (13.5%) samples, pfk13 A675V in 14/238 (5.9%) and pfk13 S522C in (1/238 (0.42%) samples across the four surveyed regions. The prevalence of pfk13 C469Y mutation was significantly higher in Karamoja region (23.3%) compared to other regions, P = 0.007. The majority of parasite isolates circulating in West Nile are of wild type (98.3), P = 0.002. Relative parasite DNA quantity did not differ in samples carrying the wild type, C469Y and A675V alleles (Kruskal-Wallis test, P = 0.6373)., Conclusion: Detection of validated molecular markers of artemisinin partial resistance in multiple geographical locations in this setting provides additional evidence of emerging threat of artemisinin partial resistance in Uganda. In view of these findings, periodic genomic surveillance is recommended to detect and monitor levels of pfk13 mutations in other regions in parallel with TES to assess potential implication on delayed parasite clearance and associated treatment failure in this setting. Future studies should consider identification of potential drivers of artemisinin partial resistance in the different malaria transmission settings in Uganda., (© 2024. The Author(s).)

The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013 and 2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample from 2016 carrying K13 622I, no other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations were observed. However, in the more recent years (2016-2017) five novel K13-propeller-domain mutations, C532S, A578S, Q613E, D680N and G718S were identified at low prevalence. Moreover, 13% (CI, 9.6-17.2) of isolates had the AP2MU 160N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015 and 2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 NFD haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia., (© 2024. The Author(s).)

Dynamics of epigenetic modifications such as acetylation and deacetylation of histone proteins have been shown to be crucial for the life cycle development and survival of Plasmodium falciparum, the deadliest malaria parasite. In this study, we present a novel series of peptoid-based histone deacetylase (HDAC) inhibitors incorporating nitrogen-containing bicyclic heteroaryl residues as a new generation of antiplasmodial peptoid-based HDAC inhibitors. We synthesized the HDAC inhibitors by an efficient multicomponent protocol based on the Ugi four-component reaction. The subsequent screening of 16 compounds from our mini-library identified 6i as the most promising candidate, demonstrating potent activity against asexual blood-stage parasites (IC 50 Pf3D7 = 30 nM; IC 50 PfDd2 = 98 nM), low submicromolar activity against liver-stage parasites (IC 50 PbEEF = 0.25 μM), excellent microsomal stability (t 1/2  > 60 min), and low cytotoxicity to HEK293 cells (IC 50  = 136 μM)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Sulfadoxine-pyrimethamine (SP) is a key drug recommended by the World Health Organization for the chemoprevention of malaria. However, the strategy is affected by the parasite resistance to SP. This study evaluated Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, associated with SP resistance, from 508 P. falciparum isolates imported from West African countries to Henan Province, China, during 2012-2022. High mutant prevalence of the genes Pfdhfr (94.7%) and Pfdhps (96.8%) was observed. The mutants Pfdhfr N51I, C59R, S108N, and Pfdhps A437G were at high frequency in all countries analyzed. The overall prevalence of the mutant Pfdhps K540E was low (3.4%), but with a high frequency in Liberia (24.3%). The frequency of mutants Pfdhps I431V, A581G, and A613S was 11.7%, 9.8%, and 16.2%, respectively, all of which had the highest mutant prevalence in Nigeria. The mutant Pfdhps A581G and A613S were identified in the absence of K540E. The partially resistant haplotype (I 51 R 59 N 108 - G 437 ) was the most common (72.6%), and the fully resistant haplotype (I 51 R 59 N 108 - G 437 E 540 ) had a low prevalence of 3.4% and mainly occurred in Liberia. No super resistant haplotype was identified. The mutant Pfdhps I431V and the octuple mutant haplotype I 51 R 59 N 108 - V 431 A 436 G 437 G 581 S 613 deserve more attention. In areas of high SP resistance, the intervention still reduces low birthweight and maternal anaemia. SP should continue to be used in areas of high SP resistance until more effective alternatives for malaria chemoprevention are found. It is important to continuously monitor the molecular markers associated with SP resistance to better implement intermittent preventive treatment policies in pregnancy (IPTp) and infants (IPTi)., (© 2024. The Author(s).)

Background: Ganaplacide, also known as KAF156, is among the new antimalarial drug candidates that have successfully reached Phase III clinical trials, and is proposed in combination with lumefantrine. This combination could replace the current front-line artemisinin-based combination therapies (ACTs) in case of Plasmodium falciparum resistance to both artemisinins and partner drugs. Indeed, the African continent, where the malaria burden is the highest, is currently experiencing worrying multiple emergences and spread of artemisinin resistance, which urges for the exploration of the antiparasitic properties of KAF156 in this context., Objectives and Methods: The objectives of this work were firstly to evaluate the risk of cross-resistance between artemisinins and KAF156 alone, and in combination with lumefantrine, using a panel of artemisinin-resistant strains carrying different pfk13 mutations and markers of other antiplasmodial drug resistances; secondly to explore in vitro the relevance of combining KAF156 and lumefantrine with artemisinins, based on the model of triple ACTs., Results: Our results highlighted that KAF156 activity was not impaired by mutations in pfk13, pfcrt, pfmdr1, pfmdr2, pfdhps and pfdhfr genes or by pfmdr1 amplification. Moreover, we demonstrated that KAF156 alone and in combination with lumefantrine was active against artemisinin-resistant parasites, including when they are quiescent., Conclusions: All these in vitro results evidence that multi-drug resistant parasites currently in circulation in the field might not affect KAF156 efficacy, and are encouraging signs for KAF156 use in a triple ACT to preserve the use of artemisinins for as long as possible., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: Distinguishing Plasmodium falciparum recrudescence from new infections is crucial for the assessment of antimalarial drug efficacy against P falciparum. We aimed to compare the efficacy of different genotyping methods to assess their effect on drug efficacy estimates, particularly in patients from high-transmission settings with polyclonal infections., Methods: In this head-to-head comparison study, we compared five different genotyping methods currently used: fast capillary electrophoresis (F-CE) using msp1, msp2, and glurp; high-resolution capillary electrophoresis (H-CE) using msp1, msp2, and glurp; H-CE using microsatellites; targeted amplicon deep sequencing (TADS) using single nucleotide polymorphism (SNP)-rich markers; and high-resolution melting (HRM) analysis using msp1 and msp2. We assessed their sensitivity in detecting minority clones in polyclonal infections, their reproducibility, and the genetic diversity of the markers used. Our study used four well characterised P falciparum laboratory strains mixed in varying ratios, and 20 paired samples collected from an in-vivo clinical trial. The experiments were performed at the Swiss Tropical and Public Health Institute in Basel, Switzerland between May 5, 2020, and Aug 23, 2021., Findings: H-CE using msp1 and msp2 and TADS revealed the highest sensitivity in detecting minority clones (up to ratios of 1:100 for H-CE and 50:1:1:1 for TADS in the FCB1:HB3 and 3D7:K1:HB3:FCB1 laboratory strain mixtures, respectively), highest reproducibility (intra-assay: 99% and 91% for H-CE and TADS, respectively; inter-assay: 98% and 92% for H-CE and TADS, respectively), and highest genetic diversity in the used markers (up to 36 and 32 unique genotypes in 20 paired samples for H-CE using msp2 and TADS using cpmp, respectively). Microsatellites assessed by H-CE had a lower genetic diversity compared with msp1, msp2, and glurp assessed by H-CE and the SNP-rich markers assessed by TADS, with a maximum of 13 unique genotypes, and some genotypes having allelic frequencies larger than 30%. Markers used by TADS gave the most consistent results in distinguishing recrudescence from new infection across all methods (in 18 of 20 pairs of samples vs 15 of 20 pairs for H-CE)., Interpretation: WHO currently recommends replacing glurp with microsatellites. However, in this study, the replacement of glurp with microsatellites did not change the genotyping outcome, probably due to the lower genetic diversity of microsatellites. More studies with large sample sizes are required to identify the most suitable microsatellites that could replace glurp. Our study indicates that TADS should be considered the gold standard for genotyping to distinguish recrudescence from new infection, and that it should be used to validate other methods., Funding: Swiss Tropical and Public Health Institute., Competing Interests: Declaration of interests AS, CB, MC, and CN have provided genotyping services to Novartis. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The human malaria parasite Plasmodium falciparum causes the most severe form of malaria in endemic regions and is transmitted via mosquito bites. To better understand the biology of this deadly pathogen, a variety of P. falciparum reporter lines have been generated using transgenic approaches to express reporter proteins, such as fluorescent proteins and luciferases. This review discusses the advances in recently generated P. falciparum transgenic reporter lines, which will aid in the investigation of parasite physiology and the discovery of novel antimalarial drugs. Future prospects for the generation of new and superior human malaria parasite reporter lines are also discussed, and unresolved questions in malaria biology are highlighted to help boost support for the development and implementation of malaria treatments., Competing Interests: Declaration of interests Shinya Miyazaki declares grant/research funding from Shionogi & Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The genetics of Plasmodium as an intracellular, mostly haploid, sexually reproducing, eukaryotic organism with a complex life cycle, presents unprecedented challenges in studying drug resistance. This article summarizes current knowledge on the genetic basis of artemisinin resistance (AR) - a main component of current drug therapies for falciparum malaria. Although centered on nonsynonymous single-nucleotide polymorphisms (nsSNPs), we describe multifaceted resistance mechanisms as part of a complex, cumulative genetic trait that involves regulation of expression by a wide array of polymorphisms in noncoding regions. These genetic variations alter transcriptome profiles linked to Plasmodium's development and population dynamics, ultimately influencing the emergence and spread of the resistance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Twelve new sulphonamide (Cys-Gly) dipeptide carboxamide derivatives 17a-17l were designed, prepared and characterized through spectroscopic techniques and their pharmacological properties investigated. The molecular docking analyses revealed good interactions of the derivatives with the desired amino residues active pockets. In vitro antimicrobial, in vivo antimalarial, haematological and other related tests (liver and kidney) were also conducted. Compounds 17b exhibited good minimum inhibitory concentration (MIC) results (0.9-11) mg/mL for the studied organisms when compared with ciprofloxacin and fluconazole. Derivatives 17a -17l showed parasitaemia inhibition in the range (31.11-67.78) % on the fourth day after treating the animals with 40 mg/kg of the compounds. Derivative 17b also displayed the highest parasitaemia inhibition (67.78 %) comparable with the standard (Lumenfantrine) 75.27 %. The prepared derivatives showed promising pharmacological properties with regards to hematological, liver and kidney function tests., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: In 2021, nationwide malaria molecular surveillance revealed a high prevalence of a validated artemisinin resistance marker, the kelch13 (k13) Arg561His mutation, in the Kagera region of northwestern Tanzania. We aimed to investigate the efficacy of artemether-lumefantrine and artesunate-amodiaquine and to confirm the presence of artemisinin partial resistance (ART-R) in the Karagwe district of this region., Methods: This single-arm, therapeutic efficacy study was carried out at the Bukangara dispensary in the Karagwe district of the Kagera region in northwestern Tanzania. Eligible participants were aged between 6 months and 120 months, had confirmed Plasmodium falciparum asexual parasitaemia, and met other inclusion criteria according to WHO's standard protocol. Participants were enrolled, treated sequentially with either artemether-lumefantrine or artesunate-amodiaquine, and assessed clinically and parasitologically for 28 days as per WHO protocol. Parasitaemia was measured every 8 h until day 3, on day 7, and then during weekly follow-up visits until day 28. Mutations in the k13 gene and extended haplotypes with the mutations were analysed, and comparisons were made with previous samples collected in the same region of Kagera and in Rwanda and southeast Asia. The primary endpoint was PCR-corrected cure rate., Findings: Between April 29 and Sept 1, 2022, 343 patients were screened, of whom 176 were enrolled: 88 in each treatment group. The PCR-corrected cure rate was 98% (95% CI 91-100) in the artemether-lumefantrine group and 100% (96-100) in the artesunate-amodiaquine group. Persistent parasitaemia on day 3 occurred in 11 (13%) of 88 patients in the artemether-lumefantrine group and 17 (19%) of 88 patients in the artesunate-amodiaquine group. Arg561His mutations on day 0 and parasitaemia on day 3 were reported in eight (9%) of 87 patients in the artemether-lumefantrine group and ten (12%) of 86 patients in the artesunate-amodiaquine group. The median parasite clearance half-life in patients harbouring parasites with Arg561His mutation was 6·1 h in the artemether-lumefantrine group and 6·0 h in the artesunate-amodiaquine group. Parasites with the Arg561His mutation were not similar to those from southeast Asia and Rwanda but had similar haplotypes to parasites reported in the same Tanzanian region of Kagera in 2021., Interpretation: This study confirms the presence of ART-R in Tanzania, although artemether-lumefantrine and artesunate-amodiaquine showed high efficacy. A context-specific response strategy and vigilance to detect the reduced efficacy of current antimalarial treatments and ART-R in other parts of the country are urgently needed., Funding: The Bill & Melinda Gates Foundation and the US National Institutes of Health., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Two undescribed frenolicins H and I ( 1 and 2 ) along with six previously described frenolicin analogues [frenolicins A ( 3 ), B ( 4 ), UCF76-B ( 5 ), E - G ( 6 - 8 )] and two anthraquinones [3,8-dihydroxy-1-propylanthraquinone-2-carboxylic acid ( 9 ) and 3,8-dihydroxy-1-propylanthraquinone ( 10 )] were isolated from a longkong bark eating caterpillar-derived Streptomyces sp. TBRC17107. The chemical structures were determined by NMR spectroscopic information and HRESIMS data. Frenolicins H ( 1 ) and I ( 2 ) showed weak cytotoxicity against malignant and non-malignant cells. Frenolicins A ( 3 ) and B ( 4 ) showed antimalarial activity against Plasmodium falciparum (IC 50 17.4 and 1.37  μ M), antibacterial activity against Bacillus cereus and Staphylococcus aureus (MIC 50.0 and 0.20  μ g/mL). Only frenolicin B had anti-plant pathogenic fungal activity against Collectotrichum acutatum and Alternaria brassicicola with MIC values of MIC 1.56 and 6.25  μ g/mL, respectively. Frenolicins A and G possessed anti- Mycobacterium tuberculosis with equal MICs of 25.0  μ g/mL.

Background: The diagnosis and management of malaria in non-endemic countries presents a continuing challenge. Plasmodium falciparum, which is capable of rapidly inducing severe and life-threatening multiorgan disease, is the species most frequently diagnosed in Europe and North America., Objectives: To summarise the more relevant diagnostic findings and clinical features of malaria observed in non-endemic settings and to provide an update of the key management decision points using three illustrative clinical scenarios of uncomplicated and severe malaria., Sources: The discussion is based on a relevant literature search spanning the last 20 years. Recommendations are based on available clinical guidelines including those of the WHO, observational studies conducted in non-endemic settings, and, when available, extrapolation from randomised studies from malaria-endemic settings., Content: The following topics are covered: diagnosis, including the use of molecular biology; clinical characteristics; management with a specific focus on complicated (severe) and uncomplicated malaria; and areas of resistance to available antimalarial drugs., Implications: Malaria imported to non-endemic settings, especially P. falciparum malaria, is sometimes initially overlooked and the delayed diagnosis is responsible for every year of preventable deaths. This review aims to raise awareness of malaria outside endemic countries and to provide clinicians with a practical guide for efficient diagnosis and targeted therapy for the different species involved., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

S alacia debilis Walp., ( Celastraceae ) is used traditionally in West Africa for the treatment of malaria. However, no scientific reports validating these effects and its active constituents are on record. Therefore, this study is aimed at evaluating the antimalarial effects, of its ethanolic extract and isolated compounds against Plasmodium falciparum 3D7 and P. berghei ANKA strains. Using chromatographic, spectrometric and spectroscopic techniques three compounds were isolated and characterised. The extract of S. debilis was active against P. falciparum 3D7, in an in vitro assay with IC 50 of 12.0 ± 0.32 µg/ml. The three isolated compounds, namely 1,10-dihydroxy-6 H -benzo[ c ]chromen-6-one ( 1 ), 8- hydroxy-3,4-dimethoxydibenzo[ b , d ]furan-1-carboxylic acid ( 2 ) and benzyl-2-methoxybenzoate ( 3 ), also showed antimalarial activity against Plasmodium berghei ANKA strain in curative and suppressive in vivo assays. The ethanolic extract and isolated compounds of S. debilis possess antimalarial effects. The isolated compounds may be responsible, at least in part, for the observed activities of the extract.

On the basis of a streamlined route to the pyrroloiminoquinone (PIQ) core, we made 16 natural products spread across four classes of biosynthetically related alkaloid natural products, and multiple structural analogs, all in ≤8 steps longest linear sequence (LLS). The strategy features a Larock indole synthesis as the key operation in a five-step synthesis of a key methoxy-PIQ intermediate. Critically, this compound was readily diverged via selective methylation of either (or both) of the imine-like or pyrrole nitrogens, which then permitted further divergence by either O- demethylation to o- quinone natural products or displacement of the methoxy group with a range of amine nucleophiles. Based on a single, early report of their potential utility against the malaria parasite, we assayed these compounds against several strains of Plasmodium falciparum , as well as two species of the related protozoan parasite Babesia . In combination with evaluations of their human cytotoxicity, we identified several compounds with potent (low-nM IC 50 ) antimalarial and antibabesial activities that are much less toxic toward mammalian cells and are therefore promising lead compounds for antiprotozoal drug discovery.

Natural product ring distortion strategies have enabled rapid access to unique libraries of stereochemically complex compounds to explore new chemical space and increase our understanding of biological processes related to human disease. Herein is described the development of a ring-cleavage strategy using the indole alkaloids yohimbine, apovincamine, vinburnine, and reserpine that were reacted with a diversity of chloroformates paired with various alcohol/thiol nucleophiles to enable the rapid synthesis of 47 novel small molecules. Ring cleavage reactions of yohimbine and reserpine produced two diastereomeric products in moderate to excellent yields, whereas apovincamine and vinburnine produced a single diastereomeric product in significantly lower yields. Free energy calculations indicated that diastereoselectivity regarding select ring cleavage reactions from yohimbine and apovincamine is dictated by the geometry and three-dimensional structure of reactive cationic intermediates. These compounds were screened for antiplasmodial activity due to the need for novel antimalarial agents. Reserpine derivative 41 was found to exhibit interesting antiplasmodial activities against Plasmodium falciparum parasites (EC 50 = 0.50 μM against Dd2 cultures), while its diastereomer 40 was found to be three-fold less active (EC 50 = 1.78 μM). Overall, these studies demonstrate that the ring distortion of available indole alkaloids can lead to unique compound collections with re-engineered biological activities for exploring and potentially treating human disease.

Artemisinin-based combination therapy (ACT) is the mainstay of effective treatment of Plasmodium falciparum malaria. However, the long-term utility of ACTs is imperiled by widespread partial artemisinin resistance in Southeast Asia and its recent emergence in parts of East Africa. This underscores the need to identify chemotypes with new modes of action (MoAs) to circumvent resistance to ACTs. In this study, we characterized the asexual blood stage antiplasmodial activity and resistance mechanisms of LDT-623, a 4-aminoquinoline (4-AQ). We also detected LDT-623 activity against multiple stages (liver schizonts, stage IV-V gametocytes, and ookinetes) of Plasmodium's life cycle, a feature unlike other 4-AQs such as chloroquine (CQ) and piperaquine (PPQ). Using heme fractionation profiling and drug uptake studies in PfCRT-containing proteoliposomes, we observed inhibition of hemozoin formation and PfCRT-mediated transport, which constitute characteristic features of 4-AQs' MoA. We also found minimal cross-resistance to LDT-623 in a panel of mutant pfcrt or pfmdr1 lines, but not the PfCRT F145I mutant that is highly resistant to PPQ resistance yet is very unfit. No P. falciparum parasites were recovered in an in vitro resistance selection study, suggesting a high barrier for resistance to emerge. Finally, a competitive growth assay comprising >50 barcoded parasite lines with mutated resistance mediators or major drug targets found no evidence of cross-resistance. Our findings support further exploration of this promising 4-AQ., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferreira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The natural product 9-methoxystrobilurin G (9MG) from Favolaschia spp basidiomycetes is a potent and selective antimalarial. The mechanism of action of 9MG is unknown. We induced 9MG resistance in Plasmodium falciparum 3D7 and Dd2 strains and identified mutations associated with resistance by genome sequencing. All 9MG-resistant clones possessed missense mutations in the cytochrome b (CYTB) gene, a key component of mitochondrial complex III. The mutations map to the quinol oxidation site of CYTB, which is also the target of antimalarials such as atovaquone. In a complementary approach to identify protein targets of 9MG, a photoactivatable derivative of 9MG was synthesized and applied in chemoproteomic-based target profiling. Three components of mitochondrial complex III (QCR7, QCR9, and COX15) were specifically enriched consistent with 9MG targeting CYTB and complex III function in P. falciparum . Inhibition of complex III activity by 9MG was confirmed by ubiquinone cytochrome c reductase assay using P. falciparum extract. The findings from this study may be useful for developing novel antimalarials targeting CYTB.

Macrocycles are prominent among drugs for treatment of infectious disease, with many originating from natural products. Herein we report on the discovery of a series of macrocycles structurally related to the natural product hymenocardine. Members of this series were found to inhibit the growth of Plasmodium falciparum , the parasite responsible for most malaria cases, and of four kinetoplastid parasites. Notably, macrocycles more potent than miltefosine, the only oral drug used for the treatment of the neglected tropical disease visceral leishmaniasis, were identified in a phenotypic screen of Leishmania infantum . In vitro profiling highlighted that potent inhibitors had satisfactory cell permeability with a low efflux ratio, indicating their potential for oral administration, but low solubility and metabolic stability. Analysis of predicted crystal structures suggests that optimization should focus on the reduction of π-π crystal packing interactions to reduce the strong crystalline interactions and improve the solubility of the most potent lead.

Inhibition of the lactate transporter PfFNT is a valid novel mode of action against malaria parasites. Current pyridine-substituted pentafluoro-3-hydroxy-pent-2-en-1-ones act as substrate analogs with submicromolar EC 50 in vitro, and >99.7% activity in mice. The recently solved structure of a PfFNT-inhibitor complex visualized the binding mode. Here, we extended the inhibitor layout by series of amine- and anilide-linked pyridine p-substituents to generate additional interactions in the cytoplasmic vestibule. Virtual docking indicated hydrogen bonding to Tyr31 and Ser102. Fluorescence cross-correlation spectroscopy yielded respectively enhanced target affinity. Strikingly, the in vitro activity increased by 1 order of magnitude to 14.8 nM at negligible cytotoxicity. While p -amine substitutions were rapidly metabolized, the more stable p -acetanilide cleared 99.7% of parasites at 4 × 50 mg kg -1 in a mouse malaria model. Future stabilization of the p-substitution against metabolism may translate the gain in in vitro potency to the in vivo situation.

Seasonal Malaria Chemoprevention consisting of monthly administration of amodiaquine/sulfadoxine-pyrimethamine to children aged 3-59 months during the transmission season could promote SP-resistance. Mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes were assessed before and after SMC adoption in Burkina Faso. A total of 769 dried blood spots were selected from studies conducted in Nanoro, Burkina Faso, between 2010 and 2020. Of those, 299 were pre-SMC (2010-2012) and 470 were post-SMC-samples. Pfdhps and Pfdhfr genes were PCR-amplified and sequenced. A systematic review/meta-analysis of published studies conducted in Burkina Faso (2009-2023) was additionally performed. In Nanoro, the prevalence of Pfdhfr triple mutations (CIRNI) rose from 43.6% pre-SMC to 89.4% post-SMC (p < 0.0001). There was no mutation in Pfdhfr 164 and Pfdhps 540; Pfdhps A437G mutation increased from 63.9% (2010-2012) to 84.7% (2020) (p < 0.0001). The VAGKGS haplotype was 2.8% (2020). Pfdhfr/Pfdhps quintuple mutant IRN-436A437G rose from 18.6% (2010-2012) to 58.3% (2020) (p < 0.0001). Meta-analysis results from Burkina Faso showed an increase in mutations at Pfdhfr N51I, C59R, S108N, and Pfdhps A437G after SMC adoption. Post-SMC, the pyrimethamine-resistance marker prevalence increased, while the sulfadoxine-resistance marker prevalence remained stable. Detection of emerging PfdhpsVAGKGS haplotypes in 2020 underscores the importance of continuous SP-resistance monitoring., (© 2024. The Author(s).)

New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance; thus, new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20-residue span at the base of the Pf KRS ATP-binding pocket. The superior transfection efficiency of Plasmodium knowlesi was exploited to generate a high coverage parasite library expressing Pf KRS bearing all possible amino acid changes within this region of the enzyme. The selection of the library with Pf KRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modeling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed., Importance: An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS). Screening of this high-coverage library with characterized Pf KRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance., Competing Interests: The authors declare no conflict of interest.

Malaria is a deadly disease caused by Plasmodium parasites. While potent drugs are available in the market for malaria treatment, over the years, Plasmodium parasites have successfully developed resistance against many, if not all, front-line drugs. This poses a serious threat to global malaria eradication efforts, and the continued discovery of new drugs is necessary to tackle this debilitating disease. With recent unprecedented progress in machine learning techniques, single-cell transcriptomic in Plasmodium offers a powerful tool for identifying crucial proteins as a drug target and subsequent computational prediction of potential drugs. In this study, We have implemented a mutual-information-based feature reduction algorithm with a classification algorithm to select important proteins from transcriptomic datasets (sexual and asexual stages) for Plasmodium falciparum and then constructed the protein-protein interaction (PPI) networks of the proteins. The analysis of this PPI network revealed key proteins vital for the survival of Plasmodium falciparum. Based on the function and identification of a few strong binding sites on a couple of these key proteins, we computationally predicted a set of potential drug molecules using a deep learning-based technique. Lead drug molecules that satisfy ADMET and drug-likeliness properties are finally reported out of the generated drugs. The study offers a general computational pipeline to identify crucial proteins using scRNA-seq data sets and further development of potential new drugs., (© 2024. The Author(s).)

Currently, primaquine is the only malaria transmission-blocking drug recommended by the WHO. Recent efforts have highlighted the importance of discovering new agents that regulate malarial transmission, with particular interest in agents that can be administered in a single low dose, ideally with a discrete and Plasmodium -selective mechanism of action. Here, our team demonstrates an approach to identify malaria transmission-blocking agents through a combination of in vitro screening and in vivo analyses. Using a panel of natural products, our approach identified potent transmission blockers, as illustrated by the discovery of the transmission-blocking efficacy of brusatol. As a member of a large family of biologically active natural products, this discovery provides a critical next step toward developing methods to rapidly identify quassinoids and related agents with valuable pharmacological therapeutic properties.

The apicoplast is an essential organelle for the viability of apicomplexan parasites Plasmodium falciparum or Toxoplasma gondii , which has been proposed as a suitable drug target for the development of new antiplasmodial drug-candidates. Plasmodione, an antimalarial redox-active lead drug is active at low nM concentrations on several blood stages of Plasmodium such as early rings and gametocytes. Nevertheless, its precise biological targets remain unknown. Here, we described the synthesis and the evaluation of new heteroaromatic analogues of plasmodione, active on asexual blood P. falciparum stages and T. gondii tachyzoites. Using a bioimaging-based analysis, we followed the morphological alterations of T. gondii tachyzoites and revealed a specific loss of the apicoplast upon drug treatment. Lipidomic and fluxomic analyses determined that drug treatment severely impacts apicoplast-hosted FASII activity in T. gondii tachyzoites, further supporting that the apicoplast is a primary target of plasmodione analogues. To follow the drug localization, "clickable" analogues of plasmodione were designed as tools for fluorescence imaging through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Short-time incubation of two probes with P. falciparum trophozoites and T. gondii tachyzoites showed that the clicked products localize within, or in the vicinity of, the apicoplast of both Apicomplexa parasites. In P. falciparum , the fluorescence signal was also associated with the mitochondrion, suggesting that bioactivation and activity of plasmodione and related analogues are potentially associated with these two organelles in malaria parasites.

The methyl-d-erythritol phosphate (MEP) pathway has emerged as an interesting target in the fight against antimicrobial resistance. The pathway is essential in many human pathogens, including Plasmodium falciparum ( Pf ), but is absent in human cells. In the present study, we report on the discovery of a new chemical class targeting IspD, the third enzyme in the pathway. Exploration of the structure-activity relationship yielded inhibitors with potency in the low-nanomolar range. Moreover, we investigated the whole-cell activity, mode of inhibition, metabolic, and plasma stability of this compound class, and conducted in vivo pharmacokinetic profiling on selected compounds. Lastly, we disclosed a new mass spectrometry (MS)-based enzymatic assay for direct IspD activity determination, circumventing the need for auxiliary enzymes. In summary, we have identified a readily synthesizable compound class, demonstrating excellent activity and a promising profile, positioning it as a valuable tool compound for advancing research on IspD.

Dobinin K is a novel eudesmane sesquiterpenoids compound isolated from the root of Dobinea delavayi and displays potential antiplasmodial activity in vivo. Here, we evaluate the antiplasmodial activity of dobinin K in vitro and study its acting mechanism. The antiplasmodial activity of dobinin K in vitro was evaluated by concentration-, time-dependent, and stage-specific parasite inhibition assay. The potential target of dobinin K on Plasmodium falciparum was predicted by transcriptome analysis. Apoptosis of P. falciparum was detected by Giemsa, Hoechst 33258, and TUNEL staining assay. The reactive oxygen species (ROS) level, oxygen consumption, and mitochondrial membrane potential of P. falciparum were assessed by DCFH-DA, R01, and JC-1 fluorescent dye, respectively. The effect of dobinin K on the mitochondrial electron transport chain (ETC) was investigated by enzyme activity analysis and the binding abilities of dobinin K with different enzymes were learned by molecular docking. Dobinin K inhibited the growth of P. falciparum in a concentration-, time-dependent, and stage-specific manner. The predicted mechanism of dobinin K was related to the redox system of P. falciparum . Dobinin K increased intracellular ROS levels of P. falciparum and induced their apoptosis. After dobinin K treatment, P. falciparum mitochondria lost their function, which was presented as decreased oxygen consumption and depolarization of the membrane potential. Among five dehydrogenases in P. falciparum ETC, dobinin K displayed the best inhibitory power on NDH2 activity. Our findings indicate that the antiplasmodial effect of dobinin K in vitro is mediated by the enhancement of the ROS level in P. falciparum and the disruption of its mitochondrial function.

BEI Resources, a National Institute of Allergy and Infectious Diseases-funded program managed by the American Type Culture Collection, serves researchers worldwide through the provision of a centralized repository for the acquisition, production, characterization, preservation, storage, and distribution of standardized biological resources targeting National Institutes of Health priority pathogens including bacteria, viruses, pathogenic fungi, and parasitic protozoa. These reference materials are critical for the development of diagnostics, vaccines, and therapeutics and are available to qualified registered investigators and institutions worldwide. Bioresources within BEI include well-characterized malaria isolates as part of the Malaria Research and Reference Reagent Resource Center (MR4). These isolates are critical for screening antimalarial compounds, conducting drug resistance studies, and for resistance surveillance and management. In our efforts to enhance the characterization of MR4 P. falciparum isolates, we measured antimalarial susceptibility of >100 isolates against a panel of standard antimalarial compounds. Our results provide valuable information to assist current and prospective users of the BEI Resources repository in making data-driven requests of isolates to meet their research needs., Competing Interests: The authors declare no conflict of interest.

A revamped in vitro compound identification and activity profiling approach is required to meet the large unmet need for new anti-malarial drugs to combat parasite drug resistance. Although compound hit identification utilizing high-throughput screening of large compound libraries is well established, the ability to rapidly prioritize such large numbers for further development is limited. Determining the speed of action of anti-malarial drug candidates is a vital component of malaria drug discovery, which currently occurs predominantly in lead optimization and development. This is due in part to the capacity of current methods which have low throughput due to the complexity and labor intensity of the approaches. Here, we provide an adaptable screening paradigm utilizing automated high content imaging, including the development of an automated schizont maturation assay, which collectively can identify anti-malarial compounds, classify activity into fast and slow acting, and provide an indication of the parasite stage specificity, with high-throughput capability. By frontloading these critical biological parameters much earlier in the drug discovery pipeline, it has the potential to reduce lead compound attrition rates later in the development process. The capability of the approach in its alternative formats is demonstrated using three Medicines for Malaria Venture open access compound "boxes," namely Pathogen Box (malaria set-125 compounds), Global Health Priority Box [Malaria Box 2 (80 compounds) and zoonotic neglected diseases (80 compounds)], and the Pandemic Response Box (400 compounds). From a total of 685 compounds tested, 79 were identified as having fast ring-stage-specific activity comparable to that of artemisinin and therefore of high priority for further consideration and development., Competing Interests: The authors declare no conflict of interest.

UCT594 is a 2-aminopyrazine carboxylic acid Plasmodium phosphatidylinositol 4-kinase inhibitor with potent asexual blood-stage activity, the potential for interrupting transmission, as well as liver-stage activities. Herein, we investigated pharmacokinetic/pharmacodynamic (PK/PD) relationships relative to blood-stage activity toward predicting the human dose. Dose-fractionation studies were conducted in the Plasmodium falciparum NSG mouse model to determine the PK/PD indices of UCT594, using the in vivo minimum parasiticidal concentration as a threshold. UCT594 demonstrated concentration-dependent killing in the P. falciparum -infected NSG mouse model. Using this data and the preclinical pharmacokinetic data led to a low predicted human dose of <50 mg. This makes UCT594 an attractive potential antimalarial drug., Competing Interests: The authors declare no conflict of interest.

Protein arginine methyltransferases (PRMTs) play critical roles in Plasmodium falciparum , a protozoan causing the deadliest form of malaria, making them potential targets for novel antimalarial drugs. Here, we screened 11 novel PRMT inhibitors against P. falciparum asexual growth and found that onametostat, an inhibitor for type II PRMTs, exhibited strong antimalarial activity with a half-maximal inhibitory concentration (IC 50 ) value of 1.69 ± 0.04 µM. In vitro methyltransferase activities of purified PfPRMT5 were inhibited by onametostat, and a shift of IC 50 to onametostat was found in the PfPRTM5 disruptant parasite line, indicating that PfPRTM5 is the primary target of onametostat. Consistent with the function of PfPRMT5 in mediating symmetric dimethylation of histone H3R2 (H3R2me2s) and in regulating invasion-related genes, onametostat treatment led to the reduction of H3R2me2s level in P. falciparum and caused the defects on the parasite's invasion of red blood cells. This study provides a starting point for identifying specific PRMT inhibitors with the potential to serve as novel antimalarial drugs., Competing Interests: The authors declare no conflict of interest.

Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Organic compounds with antibacterial and antiparasitic properties are gaining significance for biomedical applications. This study focuses on the solvent-free synthesis (green synthesis) of 1,4-naphthoquinone or 2,3-dichloro-1,4-naphthoquinone with different phenylamines using silica gel as an acid solid support. The study also includes in silico PASS predictions and the discovery of antibacterial and antiparasitic properties of phenylaminonaphthoquinone derivatives 1 - 12 , which can be further applied in drug discovery and development. These activities were discussed in terms of molecular descriptors such as hydrophobicity, molar refractivity, and half-wave potentials. The in vitro antimicrobial potential of the synthesized compounds 1 - 12 was evaluated against a panel of six bacterial strains (three Gram-positive: Staphylococcus aureus , Proteus mirabilis , and Enterococcus faecalis ; and three Gram-negative bacteria: Escherichia coli , Salmonella typhimurium , and Klebsiella pneumoniae ). Six compounds ( 1 , 3 , 5 , 7 , 10, and 11 ) showed better activity toward S. aureus with MIC values between 3.2 and 5.7 μg/mL compared to cefazolin (MIC = 4.2 μg/mL) and cefotaxime (MIC = 8.9 μg/mL), two cephalosporin antibiotics. Regarding in vitro antiplasmodial activity, compounds 1 and 3 were the most active against the Plasmodium falciparum strain 3D7 (chloroquine-sensitive), displaying IC 50 values of 0.16 and 0.0049 μg/mL, respectively, compared to chloroquine (0.33 μg/mL). In strain FCR-3 (chloroquine-resistant), most of the compounds showed good activity, with compounds 3 (0.12 μg/mL) and 11 (0.55 μg/mL) being particularly noteworthy. Additionally, docking studies were used to better rationalize the action and prediction of the binding modes of these compounds. Finally, absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions were performed.

Malaria, a persistent and ancient adversary, continues to impact vast regions worldwide, afflicting millions and severely affecting human health and well-being. Recently, despite significant progress in combating this parasitic disease, malaria remains a major global health concern, especially in areas with limited resources and vulnerable populations. Consequently, identifying and developing effective agents to combat malaria and its associated dysfunctions is essential therefore the two new Schiff base ligands incorporated Co(II), Ni(II), Cu(II) and Zn(II) ions were synthesized and thoroughly characterized. The synthesized compounds were assessed for in vitro anti-malarial and antimicrobial efficacy, compounds (9, 10) demonstrated highest potential with IC 50 =1.08±0.09 to 1.18±0.04 μM against P. falciparum and MIC=0.0058 μmol/mL against C. albicans and E. coli, respectively. The complexes (5, 6) were effectively reduce mitigate oxidative stress with lowest IC 50 value of 2.69±0.12 to 2.87±0.09 μM. Moreover, the biological findings were reinforced by a molecular docking investigation involving the potential compounds (2, 7-10) against dihydroorotate dehydrogenase and sterol 14-alpha demethylase proteins which exposed complex's excellent biological response than their parent ligands. ADMET profiling was used to confirm the compounds' oral drug-like features. This research offers promising prospects for future multi-functional drug innovations targeting malaria, pathogenic infections, and oxidative stress., (© 2024 Wiley-VCH GmbH.)

Background: The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs., Methods: In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6 months and older, in 13 regions of mainland Tanzania from Jan 31 to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting P falciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda., Findings: 6855 (88·1%) of 7782 capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50 of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. A region of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80 of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75 of 2861 genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide., Interpretation: These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa., Funding: This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Malaria is a disease caused mostly by Plasmodium falciparum, affects millions of people each year. The kinases are validated targets for malaria infection. In this study, we investigate for real and hypothetical compounds that can inhibit cyclic guanosine monophosphate (CGMP)-dependent protein kinase using molecular docking via combined similarity analysis, molecular dynamics simulations, quantitative structure activity relationship (QSAR). Using Tanimoto similarity scores, ∼8.4 million compounds were screened. Compounds that have at least 70% similarity are used in further analysis. These compounds are assessed by means of docking, MMBPSA, MMGBSA and ANI&#95;LIE. Based on consensus of different free energy methods and docking we revealed two potential inhibitors that can be useful for treatment of malaria. Apart from screening of real compounds, we have also selected the 10 most plausible hypothetical compounds by performing QSAR. By QSAR proposed pharmacophores, we generated over 247 hypothetical compounds and among them 19 molecules with lower QSAR predicted IC50 values and high docking scores were selected for further analysis. We selected the top 10 inhibitor candidates and performed MD simulations for free energy calculations like the protocol applied for real compounds. According to the free energy calculations, we suggest 2 real (C 34 H 29 F 5 N 8 O 4 S and C 30 H 27 F 2 N 7 O 2 S 2 , PubChem IDs: 140564801 and 89035196, respectively) and 2 hypothetical (C 23 H 27 FN 6 O 2 S, MOL3 and C 23 H 25 FN 6 O 2 S, MOL4) compounds that can be effective inhibitors against the protein kinase of Plasmodium falciparum.Communicated by Ramaswamy H. Sarma.

A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Antimalarial drug resistance is a major obstacle in the ongoing quest against malaria. The disease affects half of the world's population. The majority of them are toddlers and pregnant women. Needed a potent compound to act on drug-resistant Pf at appropriate concentrations without endangering the host. Envisaged solving this issue through rational drug design by creating a novel hybrid drug possessing two pharmacophores that can act on two marvellous and independent aims within the cell. Synthesized a new series of substituted 4-phenyl-1,2,3,6-tetrahydropyridine (THP) 8-Aminoquinoline-based hybrid analogs which have been integrated with quinoline, chloroquine, pamaquine, and primaquine, which exhibited antimalarial activity against Pf. Out of thirteen 4-phenyl-1,2,3,6-THP appended 8-Aminoquinoline derivatives, the compounds 1j, 1e, 1b, and 1l have exhibited good antimalarial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (RKL-9) strain with the minimum inhibitory concentration. Compound 1b was the most effective and showed consistently good potency against the drug-resistant (RKL-9) strain, although all other arrays showed good antimalarial efficacy. Additional docking and molecular dynamics studies were carried out at several targeting sites to quantify the structural parameters necessary for the activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Three new steroids, turranin M, N and O ( 1 - 3 ), together with four known limonoids, nymania 1 ( 4 ), rubralin B ( 5 ), aphapolynin C ( 6 ) and Trichillia substance Tr B ( 7 ), were isolated from the leaves of Turraea obtusifolia . Their chemical structures were elucidated using NMR and MS. Rubralin B ( 5 ) displayed good activity against the asexual parasites from the drug sensitive Plasmodium falciparum NF54 strain with an IC 50 value of 3.47 µg/mL (4.57 µM), nymania 1 ( 4 ) showed a weak activity (IC 50 13.36 µg/mL (19.40 µM)) and the rest of compounds had IC 50 > 20 µg/mL.

Two new heptapeptides, [1-7-NαC]-crocaorbs A1 (1) and A2 (2), were isolated from the latex of Croton campanulatus. Their structures were determined using NMR spectroscopic techniques, ESI-HRMS data, Marfey's method, and further refined using molecular dynamics with simulated annealing (MD/SA). Molecular dynamics calculations of peptides 1 and 2 demonstrated greater stability in simulations using a biological solvent compared to those using DMSO. Compound 1, the most abundant peptide in latex, was assessed for NO production, antiplasmodial and cytotoxicity activities. The peptide significantly increased nitric oxide (NO) production at concentrations of 40, 20 or 10 μM (17.932 ± 1.1, 18.270 ± 0.9, 18.499 ± 0.7, respectively). Its antiplasmodial activity exhibited limited efficacy, with only 5% inhibition of Plasmodium falciparum 3D7 growth at a concentration of 50 μM. Also, it exhibited no cytotoxic effects in the J774A.1 murine macrophages cell line. This study represents the first report of a phytochemical investigation of the species C. campanulatus, which showed orbitides with distinctive sequences in contrast to other peptides described for the genus Croton and contributes to the study of structural diversity within this particular class of compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Malaria remains a global health problem, and the standard membrane feeding assay (SMFA) is a key functional assay for development of new interventions to stop malaria transmission from human to mosquito. For SMFA, media with ~ 10% of human serum has been used for infectious gametocyte cultures, however, there are multiple challenges to obtain a suitable human serum. Here we show a human-serum-free culture medium (HSF), which was a mixture of two stem cell culture media and AlbuMAX, supported infectious gametocyte growth. Moreover, the HSF-induced gametocytes elicited significantly higher numbers of oocysts compared to gametocytes cultured with conventional human serum medium (Conv). While some caution is required when comparing percent transmission reducing activity data generated from HSF-SMFA and Conv-SMFA, the HSF method can facilitate the establishment of gametocyte cultures or SMFA by bypassing the need for human serum. Thus, this study will support future development of P. falciparum transmission-blocking interventions., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

We report the discovery of MED6-189, an analog of the kalihinol family of isocyanoterpene natural products that is effective against drug-sensitive and drug-resistant Plasmodium falciparum strains, blocking both asexual replication and sexual differentiation. In vivo studies using a humanized mouse model of malaria confirm strong efficacy of the compound in animals with no apparent hemolytic activity or toxicity. Complementary chemical, molecular, and genomics analyses revealed that MED6-189 targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses revealed that a mutation in PfSec13 , which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. Its high potency, excellent therapeutic profile, and distinctive mode of action make MED6-189 an excellent addition to the antimalarial drug pipeline.