Transcriptional profiling of hepatocytes infected with the replicative form of the malaria parasite Plasmodium cynomolgi.

BACKGROUND: The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. The transcriptional profiling of P. cynomolgi liver stages was previously reported and revealed many important biological features of the parasite but left out the host response to malaria infection. METHODS: Previously published RNA sequencing data were used to quantify the expression of host genes in rhesus macaque hepatocytes infected with P. cynomolgi in comparison to either cells from uninfected samples or uninfected bystander cells. RESULTS: Although the dataset could not be used to resolve the transcriptional profile of hypnozoite-infected hepatocytes, it provided a snapshot of the host response to liver stage schizonts at 9-10 day post-infection and identified specific host pathways that are modulated during the exo-erythrocytic stage of P. cynomolgi. CONCLUSIONS: This study constitutes a valuable resource characterizing the hepatocyte response to P. cynomolgi infection and provides a framework to build on future research that aims at understanding hepatocyte-parasite interactions during relapsing malaria infection.

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite.

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.

Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Antirelapse Compounds.

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.

Identification of adjuvants for clinical trials performed with Plasmodium falciparum AMA1 in rabbits.

BACKGROUND: In this study, seven adjuvants were compared for use with Plasmodium falciparum DiCo-Apical Membrane Antigen 1 (Pf-DiCo-AMA1), with the aim to identify an ideal adjuvant which yields high antibody titres and potentially broadens the responses in clinical trials. The following adjuvant formulations were evaluated: SE, SE-GLA, Liposomes, Liposomes-GLA, CoVaccine HT™, ImSaVac-P and ImSaVac-P o/w. The study was performed in rabbits, which were immunized with FVO-AMA1 in combination with one of the seven adjuvants. Antibody levels (humoral responses) and functional activity of the antibodies induced against malaria vaccine candidate AMA1 were evaluated. Thus, in this study the ideal adjuvant is expected to induce high functional antibody levels, a long-lived response, and a broad cross-strain activity. RESULTS: AMA1 formulated in all adjuvants was immunogenic. However, the magnitude of the immune responses differed between the seven adjuvants. The highest IgG levels were observed for the CoVaccine HT™ group, this was statistically significant for all four AMA1 variants versus all other adjuvant groups. No differences were observed in the breadth of the humoral response, i.e., increased recognition of AMA1 variants. Also, Growth Inhibition Activity (GIA) for both Plasmodium falciparum strains (FCR3 - homologous to FVO AMA1 protein and NF54 - heterologous to FVO AMA1 protein) were significantly higher in the CoVaccine HT™ group as compared to the other adjuvant groups. CONCLUSIONS: In brief, all seven vaccine - adjuvant formulations were immunogenic. The magnitude of the immune responses differed between the seven adjuvants. No statistically significant differences were observed in the breadth of the humoral response, nor in longevity of the response. Nevertheless, AMA1 formulated in CoVaccine HT™ appeared as the best adjuvant for use in clinical trials.

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

From marginal to essential: the golden thread between nutrient sensing, medium composition and Plasmodium vivax maturation in in vitro culture.

Historically neglected, due to its biological peculiarities, the absence of a continuous long-term in vitro blood stage culture system and a propensity towards high morbidity rather than mortality, Plasmodium vivax was put back on the agenda during the last decade by the paradigm shift in the fight against malaria from malaria control to malaria eradication. While the incidence of the deadliest form of malaria, Plasmodium falciparum malaria, has declined since this paradigm shift took hold, the prospects of eradication are now threatened by the increase in the incidence of other human malaria parasite species. Plasmodium vivax is geographically the most widely distributed human malaria parasite, characterized by millions of clinical cases every year and responsible for a massive economic burden. The urgent need to tackle the unique biological challenges posed by this parasite led to renewed efforts aimed at establishing a continuous, long-term in vitro P. vivax blood stage culture. Based on recent discoveries on the role of nutrient sensing in Plasmodium's pathophysiology, this review article critically assesses the extensive body of literature concerning Plasmodium culture conditions with a specific focus on culture media used in attempts to culture different Plasmodium spp. Hereby, the effect of specific media components on the parasite's in vitro fitness and the maturation of the parasite's host cell, the reticulocyte, is analysed. Challenging the wide-held belief that it is sufficient to find the right parasite isolate and give it the right type of cells to invade for P. vivax to grow in vitro, this review contends that a healthy side-by-side maturation of both the parasite and its host cell, the reticulocyte, is necessary in the adaptation of P. vivax to in vitro growth and argues that culture conditions and the media in particular play an essential role in this maturation process.

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

Plasmodium knowlesi: a relevant, versatile experimental malaria model.

The primate malaria Plasmodium knowlesi has a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite-host interactions. The adaptation to long-term in vitro continuous blood stage culture in rhesus monkey, Macaca fascicularis and human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence that P. knowlesi is an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.

PI4 Kinase Is a Prophylactic but Not Radical Curative Target in Plasmodium vivax-Type Malaria Parasites.

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally. Both the KDU691 and LMV599 compounds were fully protective when administered prophylactically, and the more potent compound LMV599 achieved protection as a single oral dose of 25 mg/kg of body weight. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that was sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess these compounds' activity on established hypnozoites and predict radical cure in vivo.

Proteomic and genetic analyses demonstrate that Plasmodium berghei blood stages export a large and diverse repertoire of proteins.

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria.

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 µM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 µM, and PQ, 0.84 µM; for developing liver stages, KAI407, 0.64 µM, and PQ, 0.37 µM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

A Diversity Covering (DiCo) Plasmodium vivax apical membrane antigen-1 vaccine adjuvanted with RFASE/RSL10 yields high levels of growth-inhibitory antibodies.

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax-induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10.

Azithromycin disrupts apicoplast biogenesis in replicating and dormant liver stages of the relapsing malaria parasites Plasmodium vivax and Plasmodium cynomolgi.

The control and elimination of malaria caused by Plasmodium vivax is hampered by the threat of relapsed infection resulting from the activation of dormant hepatic hypnozoites. Currently, only the 8-aminoquinolines, primaquine and tafenoquine, have been approved for the elimination of hypnozoites, although their use is hampered by potential toxicity. Therefore, an alternative radical curative drug that safely eliminates hypnozoites is a pressing need. This study assessed the potential hypnozoiticidal activity of the antibiotic azithromycin, which is thought to exert antimalarial activity by inhibiting prokaryote-like ribosomal translation within the apicoplast, an indispensable organelle. The results show that azithromycin inhibited apicoplast development during liver-stage schizogony in P. vivax and Plasmodium cynomolgi, leading to impaired parasite maturation. More importantly, this study found that azithromycin is likely to impair the hypnozoite's apicoplast, resulting in the loss of this organelle. Subsequently, using a recently developed long-term hepatocyte culture system, this study found that this loss likely induces a delay in the hypnozoite activation rate, and that those parasites that do proceed to schizogony display liver-stage arrest prior to differentiating into hepatic merozoites, thus potentially preventing relapse. Overall, this work provides evidence for the potential use of azithromycin for the radical cure of relapsing malaria, and identifies apicoplast functions as potential drug targets in quiescent hypnozoites.

Diversity covering AMA1-MSP119 fusion proteins as malaria vaccines.

To overcome polymorphism in the malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1), fusion protein chimeras comprised of three diversity-covering (DiCo) PfAMA1 molecules (D1, D2, and D3) and two allelic variants of the C-terminal 19-kDa region of merozoite surface protein 1 (MSP119) (variants M1 and M2) were generated. A mixture of fusion proteins (D1M1/D2M2D3) and the D1M1D2M2D3 fusion were compared to a single-unit mixture (D1/D2/D3/M1) in an immunological study in groups of rabbits. Following immunization, titers of antibodies (Abs) against four naturally occurring PfAMA1 alleles were high for all groups, as were growth inhibition assay (GIA) levels against two antigenically distinct laboratory parasite strains. Fusion of AMA1 to MSP119 did not suppress levels of antibodies against the AMA1 component. In addition, the breadth of antibody responses was unaffected. Anti-AMA1 antibodies were largely responsible for parasite growth inhibition, as shown in reversal-of-inhibition experiments by adding competing AMA1 antigen. For all groups, titration of the MSP119 antigen into the GIA led to only a small decrease in parasite inhibition, although titers of antibodies against MSP119 were increased 15-fold for the groups immunized with fusion proteins. GIA with affinity-purified anti-MSP119 antibodies showed that the 50% inhibitory concentrations of the anti-MSP119 antibody preparations were in the same order of magnitude for all animals tested, leading to the conclusion that fusing MSP119 to PfAMA1 leads to a small but significant increase in functional antibody levels. This study shows that combination of multiple vaccine candidates in fusion proteins may lead to improved characteristics of the vaccine.

A novel live-dead staining methodology to study malaria parasite viability.

BACKGROUND: Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead. METHODS: Different commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive. RESULTS: Here, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing. CONCLUSIONS: The JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture.

Transfection Models to Investigate Plasmodium vivax-Type Dormant Liver Stage Parasites.

Plasmodium vivax causes the second highest number of malaria morbidity and mortality cases in humans. Several biological traits of this parasite species, including the formation of dormant stages (hypnozoites) that persist inside the liver for prolonged periods of time, present an obstacle for intervention measures and create a barrier for the elimination of malaria. Research into the biology of hypnozoites requires efficient systems for parasite transmission, liver stage cultivation and genetic modification. However, P. vivax research is hampered by the lack of an in vitro blood stage culture system, rendering it reliant on in vivo-derived, mainly patient, material for transmission and liver stage culture. This has also resulted in limited capability for genetic modification, creating a bottleneck in investigations into the mechanisms underlying the persistence of the parasite inside the liver. This bottleneck can be overcome through optimal use of the closely related and experimentally more amenable nonhuman primate (NHP) parasite, Plasmodium cynomolgi, as a model system. In this review, we discuss the genetic modification tools and liver stage cultivation platforms available for studying P. vivax persistent stages and highlight how their combined use may advance our understanding of hypnozoite biology.

Antimalarial Agents Targeting Plasmodium falciparum Carbonic Anhydrase: Towards Artesunate Hybrid Compounds with Dual Mechanism of Action.

Malaria continues to be a major public health challenge worldwide and, as part of the global effort toward malaria eradication, plasmodium carbonic anhydrases (CAs) have recently been proposed as potential targets for malaria treatment. In this study, a series of eight hybrid compounds combining the Artesunate core with a sulfonamide moiety were synthesized and evaluated for their inhibition potency against the widely expressed human (h) CAs I, II and the isoform from P. falciparum (PfCA). All derivatives demonstrated high inhibition potency against PfCA, achieving a KI value in the sub-nanomolar range (0.35 nM). Two Compounds showed a selectivity index of 4.1 and 3.1, respectively, against this protozoan isoform compared to hCA II. Three Derivatives showed no cytotoxic effects on human gingival fibroblasts at 50 µM with a high killing rate against both P. falciparum and P. knowlesi strains with IC50 in the sub-nanomolar range, providing a wide therapeutic window. Our findings suggest that these compounds may serve as promising leads for developing new antimalarial drugs and warrant further investigation, including activity against antimalarial-resistant strains, mode of action studies, and in vivo efficacy assessment in preclinical mouse models of malaria.