Aspartyl protease inhibition interferes with Plasmodium falciparum asexual blood-stage and early gametocyte development

Aspartyl protease inhibition interferes with Plasmodium falciparum asexual blood-stage and early gametocyte development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Aspartyl protease inhibition interferes with Plasmodium falciparum asexual blood-stage and early gametocyte development Gamolthip Niramolyanun, Chonnipa Praikongkatham, Rachaneeporn Jenwithisuk, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6591841/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Aug, 2025 Read the published version in Malaria Journal → Version 1 posted 8 You are reading this latest preprint version Abstract Background Plasmodium falciparum is the most influential species of malaria parasites, capable of causing severe illness and mortality, especially in pregnant women and children under the age of 5. Global distribution of disease impacted on billions of endemic people and travelers. Asexual stage and gametocyte cause harmful manifestations, impacting the patients and contributing to the spread of the disease in the community, respectively. Moreover, most recent therapeutic drugs did not affect the gametocyte. The discovery of a new drug with dual actions on both stages could elucidate a cost-effective way to combat malaria. Within a human host, the parasite possesses many activities for its survival, such as invasion, egress, hemoglobin degradation, and protein trafficking, many of which are related to aspartyl protease, revealing the potential for antimalarial drug targets. Methods Pepstatin A, the representative of the board-spectrum aspartyl protease inhibitor, was utilized to investigate the effects of aspartyl protease inhibition on parasite development. The experiments were separately performed in vitro for different developmental stages of parasites, including the asexual blood-stage, early gametocytes, late gametocytes, and gamete. To demonstrate the effects of pepstatin A, the number of intact parasites and their stage distribution were counted under the microscope and calculated as a percentage of inhibition compared to the control. Additionally, the morphology of pepstatin A-treated parasites was observed to identify cellular alterations in the parasites. Results Pepstatin A at 100 µM inhibited the asexual stage and early-stage gametocyte development by 47% and 73%, respectively. They exhibited morphological defects, including chromatin condensation, vacuolization and hemozoin clumping in both asexual blood-stage and early-stage gametocyte. However, it could not influence the late-stage gametocyte development and gamete formation. Conclusion The inhibition of aspartyl protease by pepstatin A moderately affected both asexual blood-stage and early-stage gametocyte development. Morphological changes on treated parasites implied the effect of pepstatin A on hemoglobin degradation process, suggesting its potential for reducing the severity of the disease and minimizing malaria transmission. However, further research and development are required to use aspartyl protease as a drug target, focusing on identifying and modifying the drug to be more sensitive and effective. Aspartyl protease Developmental inhibition Gametocyte Malaria Transmission Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Malaria is an infectious tropical and subtropical disease caused by the Plasmodium parasite. Plasmodium falciparum , the causative agent of human malaria, can manipulate and develop through several stages following red blood cell (RBC) invasion, including ring, trophozoite, schizont, and five stages of gametocyte [ 1 ]. Significantly, the intraerythrocytic stage was the most hazardous, causing a coma and death in approximately 500,000 people each year [ 2 ]. Moreover, the mature stage V gametocyte was a precursor stage that mediate malaria transmission [ 3 ]. Although there are various strategies to counteract malaria, such as developing new pharmaceuticals for treatment and prevention, inventing new innovations for malaria management, and educating the local community in endemic areas, there are no 100% effective malaria eradication strategies. Furthermore, the majority of antimalarial medicines had no effect on gametocytes. Malaria transmission to different communities continued for weeks after asexual stage parasites had been cleared [ 4 ]. As a result, the development of medications that target the key mechanisms in both the asexual stage parasite and the gametocyte may be the most cost-effective malaria eradication strategy. The important source of nutrients for the parasite growth in the intraerythrocytic stage was the amino acid derived from the hemoglobin degradation process [ 5 ]. Previous research showed that this mechanism was essential for both asexual and gametocyte development. In this process, various protease enzymes worked together in a cascade to degrade the large molecules of hemoglobin into small molecules of amino acids [ 6 – 9 ]. This function in the parasite cellular mechanism suggested that inhibiting enzymes involved in this process could impact on the development of the intraerythrocytic parasites. One type of protease involved in the hemoglobin breakdown process was aspartyl protease [ 5 , 10 ]. Aspartyl proteases in P. falciparum were made up of ten members, plasmepsin I to X, and they played a crucial role in both the asexual stage parasite and the gametocyte. These proteases contribute to the parasite's survival and growth within the erythrocyte. They were not only participated in hemoglobin breakdown but also contributed to other critical activities such as protein export, and they may be involved in parasite invasion and egress [ 11 – 16 ]. Thus, the aspartyl protease could be a powerful enzyme critical to the parasite's survival, indicating its potential as a useful target for blocking parasite development and transmission. Pepstatin A was a broad-spectrum aspartyl protease inhibitor that has been demonstrated to block hemoglobin degradation enzymes and to reduce the growth of the Babesia parasite [ 17 ], which belonged to the same Apicomplexa phylum as the Plasmodium parasite. Moreover, its effect on the asexual stage of the P. falciparum 3D7 strain had been demonstrated [ 18 , 19 ]. For this reason, it can be hypothesized that the use of pepstatin A could help elucidate the effects of aspartyl protease inhibition on the development of asexual blood-stage parasites, gametocytes, and gametes, especially in transmission stages that have not yet been investigated. In this study, pepstatin A was co-cultured with the intraerythrocytic P. falciparum AMB47 Thai isolate to examine aspartyl protease inhibitory effects on various developmental stages, including asexual blood stage, early, late gametocyte, and gamete. Notably, the morphology of the treated parasites was illustrated for describing the possible cellular process interfered by pepstatin A. Methods In vitro culture of Plasmodium falciparum P. falciparum AMB47, a gametocyte-productive strain from Thailand, was cultivated using the method described by Read with some modifications [ 20 ]. In brief, the parasite's asexual stage was constantly cultured at 5% hematocrit (HCT) under mixed gas conditions (90% N 2 , 5% O 2 and 5% CO 2 ) at 37 ºC. The complete culture medium for the asexual stage of P. falciparum consists of RPMI 1640 powdered medium with L-Glutamine (Gibco), 2 g/L sodium bicarbonate (Mallinckrodt), 25 mM HEPES (Sigma), 2 g/L dextrose (M&B), 50 mg/L hypoxanthine (Sigma), 10% heat-inactivated human serum type A positive and 10 mg/L gentamicin (Siam pharmaceutical). The parasite was synchronized with 5% sorbitol (PanReac AppliChem ITW Reagents) to convert the mixed stage culture to ring stage [ 21 ]. The gametocyte culture followed the method outlined by Bounkeua (2010) method with some changes [ 22 ]. It was maintained under the same conditions as the asexual stage culture, excluding gentamicin. Initially, the parasitemia of the asexual stage culture, ranging from 5–8%, was diluted to achieve a parasitemia of 0.4–0.6%. It was then cultured at 6% HCT until the parasitemia was up to more than 5%. At this point, the hematocrit culture percentage was reduced to 3.5% HCT (Day1). To prevent undesired gametogenesis, all gametocyte culture processes were carried out on a 37ºC hot plate. Mature stage V gametocytes were typically observed in the culture on Days 12–14. Drug and inhibitor preparation Pepstatin A (Sigma), an aspartyl protease inhibitor, was prepared at a concentration of 1 mM in 5% Dimethyl sulfoxide (DMSO). The positive control for the asexual stage inhibition experiment was 100 nM Artesunate (ATS), and the control for the gametocyte development experiment was 100 nM Chloroquine (CQ). They were stored at -20ºC before the experiment. The negative control used in this investigation was 0.5% DMSO. Development inhibition for asexual stage development In a 96-well plate, the synchronized late ring stage at 1% parasitemia was co-cultured with pepstatin A concentrations ranging from 0.1 to 100 µM, along with negative and positive controls, for 26–28 hr. After incubation, the treated parasites were harvested, and Giemsa-stained thin blood smears were prepared to investigate the defects of the parasite under light microscope. Early-stage gametocyte development inhibition (EGDI) and late-stage gametocyte development inhibition (LGDI) The experiments were carried out using the modified method of Adjalley (2011) [ 23 ]. Various concentrations of pepstatin A were treated in the 2% HCT of gametocyte culture daily for three consecutive days. The investigation of the early-stage gametocyte development inhibition (EGDI) began on Day 3 of gametocyte development when the parasites were in stage I and II gametocytes. Late-stage gametocyte development inhibition (LGDI) was initiated on Day 8 when the parasites were in stage III and IV gametocytes. Giemsa-stained thin blood smears were prepared for light microscopic examination after three days of treatment. Gamete formation inhibition The interval from day 12 to 14 of gametocyte culture is an adequate period to generate gamete. The gametocyte was pre-incubated with 10 µM or 100 µM of pepstatin A for 15 min at 37 ºC [ 24 ]. Afterward, they were spun down and immediately mixed with 21ºC ookinete medium in a 2:1 ratio for 15 min at room temperature. The ookinete medium contains incomplete medium, 20% non-heat inactivated serum type A positive, and 100 µM of xanthurenic acid to induce gamete formation. The number of exflagellation centers was counted using a hemocytometer, and Giemsa-stained thin blood smears were prepared to examine the number of macrogamete formations. Light microscopic observation Under a light microscope, the percentage of parasitemia and morphological changes were examined after treating the parasites with pepstatin A. The number of normal parasites in 10,000 total red blood cells (total RBCs) was counted and calculated as a percentage of inhibition relative to the untreated control. The formula for calculating the percentage of inhibition; The morphological investigation The morphological changes of the parasite were observed and investigated as shown in Table 1 and 2 . Shape, nucleus, hemozoin, and vacuolization were the key constituents studied. Table 1 The criteria for morphological characterization on asexual blood stage parasite Composition Normal Defect Ring Trophozoite Schizont Nucleus 1 or 2 nuclei ≤ 2 nuclei > 2 nuclei Chromatin condensation, pyknosis, karyorrhexis and karyolysis Hemozoin No hemozoin formation Brown pigment Dark brown pigment Clumped black hemozoin Vacuolization No vacuole formation Tiny size, ≤ 3 vacuole formation Enlarged or increased the number (with/without the content) Table 2 The criteria for morphological characterization on gametocyte Composition Normal Defect I II III IV V Shape Difficult to differentiate from trophozoite D-shape Elongated D-shape with blunt-ends Elongated shape with pointed ends Crescent-shape with blunt-ends Shrinkage or swelling Nucleus Elongate nucleus which located in the terminal site Male gametocyte has the nucleus larger than female Chromatin condensation, pyknosis, karyorrhexis and karyolysis Hemozoin Distribute throughout the cytoplasm of gametocyte Clumped hemozoin or accumulation in the same food vacuole Vacuolization Tiny size, difficult to observe in the gametocyte by light microscope Enlarged or increased the number (with/without the content) Statistical analysis The statistical analysis to compare the significant difference was performed by one-way ANOVA. The half-maximal inhibitory concentration (IC 50 ) was determined through Probit analysis from IBM® SPSS® statistics 24. Results The effect of aspartyl protease inhibition on asexual blood-stage. From the past until now, several aspartyl protease inhibitors have been synthesized and tested for their ability to inhibit this influential enzyme. Among these inhibitors, pepstatin A has shown the ability to inhibit all plasmepsins (Table 3 ). Therefore, pepstatin A was selected to investigate the inhibition of aspartyl proteases that could interfere with the development of the asexual blood-stage parasite. Table 3 Summary of plasmepsin inhibitor testing in Plasmodium spp. Inhibitor Target plasmepsin Experiment Reference Note Inhibitor Target plasmepsin Experiment Reference Note Azacyclic plasmepsin inhibitors Plasmepsin II In vitro enzyme activity [ 59 ] Fluorescence-based proteolysis assay KNI-764 Plasmepsin IV In vitro enzyme activity [ 60 ] Pm Crystallization Plasmepsin IV Crystallization Pm Plasmepsin IV In vitro enzyme activity Fluorescence-based proteolysis assay KNI-10006 Plasmepsin I Crystallization [ 61 ] Crystallization Plasmepsin I Molecular Docking [ 62 ] Azole-based inhibitor Plasmepsin II Molecular Docking [ 63 ] Plasmepsin II Molecular Docking [ 62 ] In vitro enzyme activity Plasmepsin IV Molecular Docking Plasmepsin IX Molecular Docking KNI-10395 HAP Crystallization [ 64 ] In vitro enzyme activity Pepstatin A All plasmepsins Molecular Docking [ 65 ] Plasmepsin X Molecular Docking Hemoglobin degradation enzyme In vitro enzyme activity [ 6 ] In vitro enzyme activity Plasmepsin II Crystallization [ 66 ] Canavanine Plasmepsin V In vitro enzyme activity [ 67 ] Fluorescence-based proteolysis assay Crystallization [ 68 ] Crystallization Crystallization [ 69 ] Molecular Docking HAP Crystallization [ 70 ] Hydroxyethylamine derivative Plasmepsin I In vitro enzyme activity [ 71 ] Plasmepsin IV Dimerization [ 69 ] Pf,Pv Plasmepsin II In vitro enzyme activity [ 72 ] Crystallization [ 73 ] Pv Molecular Docking Plasmepsin V In vitro enzyme activity [ 14 ] Partially inhibited Crystallization [ 74 ] In vitro enzyme activity [ 75 ] Plasmepsin IV In vitro enzyme activity [ 72 ] Plasmepsin IX MD simulations [ 76 ] Molecular Docking Plasmepsin X MD simulations In vitro enzyme activity [ 77 ] Pepstatin A analogue Plasmepsin II In vitro enzyme activity [ 78 ] Molecular Docking Plasmepsin II Crystallization [ 78 ] Plasmepsin V In vitro enzyme activity [ 79 ] PEXEL peptidomimetic inhibitors Plasmepsin V In vitro enzyme activity [ 80 ] Fluorescence-based proteolysis assay Plasmepsin IX Parasite egress inhibition [ 81 ] Pb Crystallization Parasite invasion inhibition Pb Molecular Docking In vitro enzyme activity Pb RS367 Plasmepsin II Crystallization [ 82 ] Plasmepsin X Parasite egress inhibition Pb RS370 Plasmepsin II Crystallization [ 82 ] Parasite invasion inhibition Pb HIV-1 inhibitors Plasmepsin II Crystallization In vitro enzyme activity [ 83 ] Plasmepsin X In vitro enzyme activity Pb Plasmepsin X In vitro enzyme activity The life cycle of P. falciparum required around 48 hr for its development, progressing from the ring to the trophozoite and finally the schizont stage. In this study, the late ring stage (10–16 hr) was co-cultured with various concentrations of pepstatin A. After 26–28 hr of incubation, the parasite showed 8.2 ± 6.4% inhibition in 0.5% DMSO treatment, 2.2 ± 8.6%, 3.3 ± 10.8%, 25.5 ± 5.6%, 46.7 ± 13.4% inhibition for pepstatin A treatments ranging from 0.1, 1, 10, to 100 µM, and 94.8 ± 4.0% inhibition for ATS treatment, in comparison to the untreated control (Fig. 1 A). The parasite in the negative group developed into a typical trophozoite and schizont, characterized by ≥ one nucleus with the brown pigment of hemozoin. The treatment with 100 µM of pepstatin A inhibited development by 46.7% at the IC 50 greater than 100 µM (Fig. 1 A). Although the IC 50 for pepstatin A was higher than expected, it exhibited a trend of development inhibition, as demonstrated by the reducing proportion and % parasitemia of developing parasites compared to controls (Fig. 1 B). Furthermore, the conversion rate of parasite from ring to trophozoite and schizont stage, indicative of normal parasite development, was revealed to be significantly reduced in 100 µM of pepstatin A when compared to no treated parasite (Fig. 1 C). Pepstatin A treatment caused chromatin condensation, vacuolization, and hemozoin clumping in asexual stage development. The morphological investigation was a simple yet advantageous study that helped to discover or predict the relationship between the parasite's defect after treatment and the underlying mechanism. In this study, we also investigated the morphological defects of the asexual blood-stage parasite under a light microscope after drugs treatment. The normal ring stage before treatment represented 1–2 nuclei without hemozoin formation (0 h). After 26–28 hr, the parasite in the no treated control and 0.5% DMSO treatment group developed into trophozoite and schizont, characterized by ≥ two nuclei with normal dark brown hemozoin pigment. Following 100 µM pepstatin A treatment, three primary morphological defects— chromatin condensation, vacuolization, and hemozoin clumping —were identified in the asexual blood-stage. In 100 nM ATS treatment, dead parasites were recognized by the dark blue color of the pyknotic nucleus. (Fig. 2 ). In general, the asexual blood-stage hemozoin pigment was brown to dark brown in color and accumulated in the same location. In this investigation, a high dose of pepstatin A treatment (100 µM) caused hemozoin clumping and turned the malaria pigment into black color (Fig. 2 ). The normal food vacuoles in parasites were usually found in a tiny size. However, this study demonstrated that pepstatin A treatment increased both size and the number of vacuole formations in the parasites (Fig. 2 ). Importantly, the sign of cell death also was observed in pepstatin A-treated parasites as show the chromatin condensation starting from the edge of nucleus or forming ring condensation (Fig. 2 ). Targeting aspartyl protease by pepstatin A affected early-stage gametocyte development but not late-stage gametocyte development. Aspartyl protease was involved not only in the asexual blood-stage but also in the gametocyte. The effect of pepstatin A treatment on gametocyte development was examined. P. falciparum undergoes five stages of gametocyte development, stage I through V, each with a unique metabolism. In this study, investigations on gametocyte development were divided into early-stage gametocyte (stage I to III) and late-stage gametocyte (stage IV and V). Pepstatin A was co-cultured in 10-fold dilutions ranging from 100 µM to 0.1 µM, as in the asexual stage experiment. After three days of treatment, the gametocytes in the negative control developed normally from stage I or II to stage II or III in the EGDI test and from stage III or IV to stage IV or V in the LGDI assay. Pepstatin A treatment highlighted the importance of aspartyl protease in gametocyte development, demonstrating that early-stage gametocyte development was inhibited by 100 µM pepstatin A (IC 50 = 53.9 µM). In comparison to the notreated control, the early-stage gametocyte showed 1.6 ± 13.8% inhibition in 0.5% DMSO treatment, 10.9 ± 10.2%, 15.5 ± 10%, 42.5 ± 5.0% and 72.6 ± 7.1% inhibition with pepstatin A treatment ranging from 0.1, 1, 10, to 100 µM, respectively. CQ treatment exhibited 89.09 ± 8.83% inhibition (Fig. 3 A). Moreover, the percentage of early-stage gametocyte inhibition was also related to the reduction of % parasitemia of stage II and III gametocyte (Fig. 3 B) and conversion rate of stage I&II to stage II&III gametocyte (Fig. 3 C). On the other hand, the pepstatin A treatment had no effect on late-stage gametocyte development (IC 50 > 100 µM). After 3 days of treatment, the late-stage gametocyte showed 0 ± 19.5% inhibition in DMSO treatment, 6.9 ± 3.6%, 14.7 ± 11.7%, 17.8 ± 10.3%, and 31.1 ± 13.5% inhibition after pepstatin A treatment ranging from 0.1, 1, 10, to 100 µM, respectively. The positive control, CQ, exhibited 63.1 ± 10.4% inhibition (Fig. 3 D). Although development inhibition increased in a dose-dependent manner (Fig. 3 D) and the conversion of gametocyte stage III &IV to stage IV and V showed the significant reduction compared to no treated control (Fig. 3 F), it had no effect on gametocyte development to stage V. (Fig. 3 E). Pepstatin A treatment induced morphological changes exclusively in the early stages of gametocyte development. Morphological defects in both early and late-stage gametocytes were examined under a light microscope. The normal morphology of a stage II gametocyte characterized by a D-shape with blunt-ends and representing the normal distribution of hemozoin, was observed before treatment (0 h). After three days of continuous treatment, the no treated control and 0.5% DMSO-treated parasites progressed to stage II and III gametocytes, exhibiting an elongated D-shape with blunt ends and a normal hemozoin distribution. The defective stage III gametocyte could be found in 100 nM CQ and 100 µM pepstatin A treatment group. The gametocyte from the 100 nM CQ treatment group represented the hemozoin clumping. Chromatin condensation, hemozoin clumping and vacuole formation were distinctly evident in the pepstatin A treatment (Fig. 4 A). In late-stage gametocytes, 0-hr stage III gametocytes displayed an elongated D-shape with blunt ends and normal hemozoin distribution. After three days of continuous treatment under all conditions, parasites developed into normal stage IV and V gametocytes. Stage IV gametocytes had an elongated shape with pointed ends and a normal distribution of hemozoin pigment, while mature stage V gametocytes exhibited a crescent shape with normal hemozoin distribution at the center (Fig. 4 B) Notably, abnormalities were exclusively observed in early-stage gametocytes following treatment with a high dose of pepstatin A (100 µM); no abnormalities were found in late-stage gametocytes. The hemozoin pigment distribution in gametocytes differs from that in asexual stage parasites, as it is distributed throughout the cytoplasm of gametocytes rather than collecting in a specific area. In this work, a high dose of pepstatin A treatment (100 µM) induced hemozoin aggregation in the same vacuole (Fig. 4 A). Additionally, similar to the asexual stage treatment, chromatin condensation indicated by dark purple color concentrated at the inner surface of the nuclear membrane and vacuole formation characterized by increase in both size and number were observed in early-stage gametocytes. The effect of aspartyl protease inhibition by pepstatin A during gamete formation The transmission of the mature gametocyte from the patient to another human requires the critical process of gamete formation inside a female Anopheles mosquito. This process involved in the transformation of male and female gametocytes into microgametes and macrogametes [ 25 ]. It occurred after the gametocytes were activated by a change in pH, a drop in temperature, or exposure to xanthurenic acid within the mosquito [ 26 , 27 ]. The process of microgamete formation was called exflagellation. The parasite replicates its genome to create an octoploid and develops eight flagella to find the macrogamete. The macrogamete was differentiated from the female gametocyte. The female gametocyte started to round up and emerged from the host red blood cell after being activated. To determine whether pepstatin A could inhibit gamete formation, mature gametocytes were pre-incubated with pepstatin A for 15 min. After the activation of gametocyte, the results showed that pepstatin A could not inhibit the process of gamete formation. There were no significant differences between the pepstatin A treatments and the no treated control (Fig. 5 A and 5 B). Although there was a significant difference in the number between male gametocytes and male gamete formation, there was no significant difference in the number of male gamete formation between no treated control and pepstatin A treatment. Moreover, the morphology of macrogametes was normal as represented by no identification of RBC membrane surrounding parasites (Fig. 3 C). The macrogamete normally exhibited a circular shape, dispersed hemozoin, a cytoplasm stained blue, and a single mass of chromatin similar to previously identified [ 28 ]. Discussion The malaria parasite has numerous mechanisms for survival in the human host. Each stage of parasite development has a unique mechanism that is important for that stage. For example, the invasion mechanism is for the parasite's invasive stages (merozoite, ookinete, and sporozoite), the egress mechanism is for schizont, mature gametocyte, and oocyst, and the hemoglobin degradation process is for parasite growth in asexual blood-stage and early-stage gametocyte. Although each stage of the parasite has its own mechanism, those mechanisms may share a key molecule. Aspartyl protease is a good example of a molecule involved in many important processes. It participated in the hemoglobin degradation, protein exportation and might be involved in invasion and egress of the parasites [ 11 – 15 , 29 ]. Thus, the aspartyl protease represented a group of critical enzymes important for parasite survival. Significantly, the previous study demonstrated that inhibiting two specific aspartyl enzymes inhibited the parasite at multiple stages [ 30 ]. This study explored the effects of pepstatin A, a broad-spectrum aspartyl protease inhibitor. Previous studies demonstrated the effect of pepstatin A in Plasmodium falciparum , confirming the impact of aspartyl protease inhibition on asexual blood-stage development [ 18 , 19 ]. In this study, in addition to influencing the asexual blood stage of the parasite, pepstatin A also induced morphological changes in early gametocyte development. However, no effect was observed on late gametocyte development and gamete formation. Previous research supported this finding by highlighting the dependency of the parasite's development in the asexual stage and early-stage gametocyte on the hemoglobin degradation process, which was not required for late gametocyte development [ 8 , 9 ]. Unfortunately, the previous study also indicated that, while pepstatin A poorly entered the food vacuole [ 31 , 32 ]. This observation could be related to the high concentration of IC 50 in this study and the ineffectiveness of pepstatin A in inhibiting aspartyl protease in late-stage gametocyte and gamete formation. This observation was supported by the use of another aspartyl protease inhibitor, 1,2-Epoxy-3-(p-nitrophenoxy) propane (EPNP), which demonstrated that treatment could reduce the number of both male and female gamete [ 33 ]. Although pepstatin A did not inhibit gametocyte maturation or gamete formation, the previous study showed that aspartyl protease inhibition can interfere with the sexual stage [ 33 ], as well as influenced on the development of both asexual blood-stage and early-stage gametocytes. Therefore, it implied that the inhibition of aspartyl protease by pepstatin A can minimize the severity of disease caused by asexual blood-stage development inhibition and reduce the possibility of transmission caused by gametocyte development being blocked from the early stage (Fig. 6 ). The first morphological defect visible in parasites treated with pepstatin A was vacuole formation. It was identified as cytoplasmic vacuolization in asexual blood-stage and early-stage gametocytes after treatment with pepstatin A. Vacuolization was usually associated with autophagy [ 34 – 36 ]. Generally, autophagy occurred to remove the damaged or aged organelles [ 37 ]. It was also related to cell death and became more prevalent as the cells lose nourishment [ 38 – 40 ]. Previous research demonstrated that vacuolization occurred in Plasmodium falciparum after antimalarial drug incubation [ 41 , 42 ]. Pepstatin A was also found to be capable of inhibiting autophagic breakdown [ 43 , 44 ]. Aspartyl protease participated in hemoglobin degradation, the parasite's primary strategy for producing amino acids for growth. Thus, the inhibition of this target enzyme by pepstatin A might disrupt the hemoglobin breakdown process, resulting in amino acid loss. Finally, the parasite died since it is unable to compensate for starvation through the autophagic breakdown process. The next defect that was found in pepstatin A- treated parasite is a defect in nucleus. Previous studies demonstrated the parasite treated with pepstatin A exhibited pyknotic nuclei. This evidence supports our finding of chromatin condensation. It was shown in the dark blue color of chromatin condensation on the inner surface of nuclear membrane, forming the pattern of ring condensation. It is the stage I of the process of cell apoptosis [ 45 ]. The malaria pigment hemozoin originated from the heme detoxification process [ 46 ], transforming toxic heme, a byproduct of hemoglobin breakdown, into the non-toxic crystallized form of β-hematin or hemozoin [ 47 – 49 ]. The erythrocytic stage of the parasite could consume up to 80% of host hemoglobin [ 50 ], indicating substantial heme synthesis. Fitch's findings suggested that a mere 0.1% of heme derived from erythrocyte hemoglobin could lyse the parasite in 10 min [ 51 ]. Free-form heme in a ferrous state caused membrane damage, block proteases, and led to parasite mortality [ 52 ]. Thus, the parasite needed the detoxification process to get rid of heme. Unfortunately, the parasite lacked heme oxygenase activity, which was the heme degradation enzyme found in the vertebrates [ 53 ]. However, the parasite could utilize another detoxification process called hemozoin synthesis [ 54 ]. In this study, asexual blood-stage and early gametocytes exhibited hemozoin clumping after pepstatin A treatment. This defect might relate to the hemozoin synthesis, as aspartyl protease contributes to 60–80% of the globin degradation process in the purified digestive vacuole [ 49 , 55 ]. Additionally, plasmepsin II, histo aspartic protease or plasmepsin III and plasmepsin IV played a role in hemozoin synthesis by forming the protein complex with falcipain 2 and Heme Detoxification Protein (HDP) [ 55 , 56 ]. Apart from the defect in hemozoin synthesis due to aspartyl protease inhibition, hemozoin clumping could be a result from a process known as hemozoin depolymerization, as demonstrated in 1997 studied by Pandey and colleagues. They showed that chloroquine could depolymerize purified hemozoin to heme, similarly representing the defect in hemozoin clumping [ 57 ]. Therefore, further study of the hemozoin clumping process was necessary, as the mechanism generating this defect remained unclear [ 54 , 55 , 58 ]. Conclusion Although pepstatin A did not completely inhibit both asexual blood stage and gametocyte, it moderately affected the growth of asexual blood-stage and early-stage gametocytes development. Moreover, this study provided insights into the morphological changes of P. falciparum after aspartyl protease inhibition by pepstatin A. It exhibited chromatin condensation, vacuolization and hemozoin clumping in both asexual blood-stage and early-stage gametocyte, which are related to defects in the hemoglobin degradation process. Given its ability for aspartyl protease inhibition, it becomes a valuable target for an antimalarial drug. However, further studies on drug development or combination therapy are needed to increase the efficiency of aspartyl protease inhibition. Abbreviations ATS: Artesunate CQ: Chloroquine DMSO: Dimethyl sulfoxide EGDI: Early-stage gametocyte development inhibition HCT: Hematocrit HDP: Heme Detoxification Protein IC 50 : The half-maximal inhibitory concentration LGDI: Late-stage gametocyte development inhibition RBC: Red blood cell RBCs: Red blood cells Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research work was supported by Mahidol University (Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF)), and the CIF and CNI Grant, Faculty of Science, Mahidol University. Authors' contribution s GN has designed the work, performed the experiments, interpreted data, drafted the manuscript, and revised the manuscript. CP is the colleague that also performed all experiments. RJ, WR, and JS have made contributions to the conception and advice on the experiments. VP and NK have major contributions to conceptualization, experimental design, data analysis, conclusion, and revised the manuscript. NK has approved the submitted version. All authors have contributed to the approval of the submitted version of manuscript. Acknowledgements We would like to thank all staff from Department of Pathobiology, Faculty of Science and Mahidol Vivax Research Unit (MVRU), Faculty of Tropical Medicine, Mahidol University for their kindly support at all the time to finish in this experiment. References Hawking F, Wilson ME, Gammage K. Evidence for cyclic development and short-lived maturity in the gametocytes of Plasmodium falciparum . Trans R Soc Trop Med Hyg. 1971;65(5):549–59. https://doi.org/10.1016/0035-9203(71)90036-8 . World Health Organization. World malaria report 2023. Geneva; 2023. Thomson JG, Robertson A. The structure and development of Plasmodium falciparum gametocytes in the internal organs and peripheral circulation. Trans R Soc Trop Med Hyg. 1935;29(1):31–40. https://doi.org/10.1016/S0035-9203(35)90015-3 . Schneider P, Bousema JT, Gouagna LC, Otieno S, Van De Vegte-Bolmer M, Omar SA, et al. Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6591841","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453981006,"identity":"77157b27-3d67-496c-a76e-6cdc9b41453e","order_by":0,"name":"Gamolthip Niramolyanun","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Gamolthip","middleName":"","lastName":"Niramolyanun","suffix":""},{"id":453981007,"identity":"35366641-4d6e-4d2b-978d-ffd8609802b3","order_by":1,"name":"Chonnipa Praikongkatham","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Chonnipa","middleName":"","lastName":"Praikongkatham","suffix":""},{"id":453981008,"identity":"65c7cce7-ed77-4527-91b4-815f7b64a45f","order_by":2,"name":"Rachaneeporn Jenwithisuk","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Rachaneeporn","middleName":"","lastName":"Jenwithisuk","suffix":""},{"id":453981009,"identity":"e36849ed-fe75-40fa-9815-3ce1a9280259","order_by":3,"name":"Wanlapa Roobsoong","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Wanlapa","middleName":"","lastName":"Roobsoong","suffix":""},{"id":453981010,"identity":"919287e3-fdcf-4a75-8035-31f9ae205f8d","order_by":4,"name":"Jetsumon Sattabongkot","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Jetsumon","middleName":"","lastName":"Sattabongkot","suffix":""},{"id":453981011,"identity":"46141bb7-548a-48ea-a635-d942e1014737","order_by":5,"name":"Viriya Pankao","email":"","orcid":"","institution":"Thammasat University","correspondingAuthor":false,"prefix":"","firstName":"Viriya","middleName":"","lastName":"Pankao","suffix":""},{"id":453981012,"identity":"2a0736d1-e9a5-43a8-9ac8-63de33ea43f4","order_by":6,"name":"Niwat Kangwanrangsan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDACZjCZwMDA3gBkGzAz8EHED+DXcgCkhecARAsbxAw8WhhgWiQSQNqJ0GLOzmP4+QNDmry55OvEzwUF1nJsDMwPPzD+uINTi2Uzj7HEAYYcw52zczdLzzBIN2ZjYDOWYEh4hlOLwWHeDUAtFYwbbudukOYxOJzYxsBgBnTYYXxaNv8AarHfcPPs5t8QLezfCGnZBnJY4oYbvNugtvAQsoX/m8UZg7TkDWdyt1nzgPzCzFMskZCGR8v5Y8k3KiqSbTccP7v5Ns8fazl+9vaNHz7Y4NYC1YjMAUVuAgENo2AUjIJRMArwAwCQv1Die9HVVgAAAABJRU5ErkJggg==","orcid":"","institution":"Mahidol University","correspondingAuthor":true,"prefix":"","firstName":"Niwat","middleName":"","lastName":"Kangwanrangsan","suffix":""}],"badges":[],"createdAt":"2025-05-05 06:53:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6591841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6591841/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12936-025-05518-z","type":"published","date":"2025-08-21T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82601797,"identity":"63b47dd8-50d7-45c4-913e-91ea145d9a1c","added_by":"auto","created_at":"2025-05-13 09:44:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":401276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePepstatin A treatment inhibits the development of asexual blood-stages.\u003c/strong\u003e The bar graph represents the percentage of development inhibition after treatment compared to the untreated control (A). The parasites were staged under a light microscope both before (0 h) and after treatment. The distribution of the asexual stage and %parasitemia in each condition is shown by the 100% stacked column (B).\u003cstrong\u003e \u003c/strong\u003eThe conversion rate graph of parasites from ring to trophozoite and schizont (C). The signification was indicated by an asterisk. (n = 6 biological replicates, \u003cem\u003eF\u003c/em\u003e \u003csub\u003e(6, 35) \u003c/sub\u003e= 58.26, *\u003cem\u003eP\u003c/em\u003e = 0.0494, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 by ordinary one-way ANOVA). ATS, artesunate; DMSO, dimethylsulfoxide.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6591841/v1/25596562a98e229805b9ccb2.png"},{"id":82601812,"identity":"2bc48fc5-0296-499d-b68d-5a8d9417ce91","added_by":"auto","created_at":"2025-05-13 09:44:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":769905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe morphological changes of the asexual blood-stage after treatment.\u003c/strong\u003e The Giemsa-stained thin smears were observed under a light microscope to investigate the parasite's defect after treatment. The parasites were photographed before (0 h) and after treatment (26-28 h), including no treated control, 0.5% DMSO, 100 µM pepstatin A, and 100 nM ATS. Arrow indicated abnormal vacuole formation, red arrowhead denoted hemozoin clumping, and black arrowhead specified chromatin condensation. A micron bar indicated 10 µm. ATS, artesunate; DMSO, dimethylsulfoxide.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6591841/v1/af3194b1a2b2b65c64d97fa4.png"},{"id":82601791,"identity":"ffccc921-b411-4d89-813f-db8eeeb2edd3","added_by":"auto","created_at":"2025-05-13 09:44:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":722143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe gametocyte development inhibition after drug treatment.\u003c/strong\u003e Early-stage gametocyte treatment (A-C). Late-stage gametocyte treatment (D-F). The bar graphs represented the early-stage gametocyte development inhibition (EGDI) relative to no treated control (A). For staging, gametocytes were examined under a light microscope both before (0 h) and after treatment. The distribution of the early-stage gametocyte and %gametocytemia in each condition is shown by the 100% stacked column (B).\u003cstrong\u003e \u003c/strong\u003eThe bar graph shows the conversion rate of gametocyte from stage I\u0026amp;II to stage II\u0026amp;III (C) (n = 6 biological replicates,\u003cem\u003e F\u003c/em\u003e \u003csub\u003e(6, 30) \u003c/sub\u003e= 47.22, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 by one-way ANOVA). The bar graph depicts late-stage gametocyte development inhibition (LGDI) relative to the no treated control (D). The distribution of gametocyte stages and %gametocytemia before treatment (0 h) and after treatment in the LGDI assay (E). The bar graph illustrates the conversion rate of gametocytes from stage III\u0026amp;IV to stage IV\u0026amp;V when compared to the no treated control (F) (n = 6 biological replicates, \u003cem\u003eF\u003c/em\u003e \u003csub\u003e(6, 35) \u003c/sub\u003e= 17.33, **\u003cem\u003eP\u003c/em\u003e =0.0017, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 by ordinary one-way ANOVA). CQ, chloroquine; DMSO, dimethylsulfoxide\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6591841/v1/de55e6e042b9b576c4eacc66.png"},{"id":82601793,"identity":"2534af0c-e608-4fb4-a41f-5819a0c70447","added_by":"auto","created_at":"2025-05-13 09:44:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":589891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe morphological defects investigated in pepstatin A-treated gametocytes. \u003c/strong\u003eA thin smear of Giemsa's stain was examined under a light microscope to investigate the parasite's defect after treatment. The parasite was photographed before (0 h) and after treatment, including no treated control, 0.5% DMSO, 100 µM pepstatin A, and 100 nM CQ. The morphology of gametocyte in the early-stage development inhibition (EGDI) experiment (A). The morphology of gametocyte in the late-stage development inhibition (LGDI) experiment (B). Hemozoin clumping is indicated by the red arrowhead, black arrowhead indicated chromatin condensation and vacuole formation was specified by arrow. The micron bar indicated 10 µm. CQ, chloroquine; DMSO, dimethylsulfoxide\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6591841/v1/dad23ab7d36ecf192b757e1a.png"},{"id":82605347,"identity":"87fc478b-dbc3-4009-83af-d2186d1434de","added_by":"auto","created_at":"2025-05-13 10:00:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":573913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGamete formation after treatment.\u003c/strong\u003e The number of male gametocytes and exflagellation events was represented per 10,000 total RBCs in each group from the exflagellation assay (A). The number of female gametocytes and macrogamete formations was represented per 10,000 total RBCs in each group from the Giemsa-stained blood smear (B). \u0026nbsp;Gametocyte morphology prior to activation and gamete formation in each condition (C). n = 3 biological replicates for gametocyte, and 6 biological replicates for gamete, \u003cem\u003eF\u003c/em\u003e \u003csub\u003e(3, 17) \u003c/sub\u003e= 692.6, \u0026nbsp;***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 by ordinary one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6591841/v1/f10794af9eb6e64964ed4088.png"},{"id":82601800,"identity":"472d381d-d590-44bd-8442-d8b668797acb","added_by":"auto","created_at":"2025-05-13 09:44:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":281467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of aspartyl protease inhibition by pepstatin A treatment.\u003c/strong\u003eWithin 48 hr, the asexual blood-stage developed from the ring stage to the trophozoite and schizont stages. Some parasite populations grew into gametocytes, which further formed five stages of development in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. The early-stage gametocyte was defined as the stage I through stage III gametocyte, while late-stage gametocytes were those that progressed from stage IV to mature stage V. Only mature stage V gametocytes could then egress from the host RBC and produced the gamete, which naturally occurred within the mosquito. This study depicted the dual effect of aspartyl protease inhibition by pepstatin A, mainly interfering with the asexual blood-stage and early gametocyte development. This suggested that aspartyl protease inhibition by pepstatin A could minimize the severity of disease caused by asexual stage development as well as reducing the chance of disease transmission to mosquitoes.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6591841/v1/13101ae473d9af3ef2a2e3da.png"},{"id":89847119,"identity":"e36717f7-7ab4-476a-92b4-b7c25d88a569","added_by":"auto","created_at":"2025-08-25 16:40:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4913782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6591841/v1/a4a24bd2-8ad5-4b98-ae72-6a479e77a9ad.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aspartyl protease inhibition interferes with Plasmodium falciparum asexual blood-stage and early gametocyte development","fulltext":[{"header":"Background","content":"\u003cp\u003eMalaria is an infectious tropical and subtropical disease caused by the \u003cem\u003ePlasmodium\u003c/em\u003e parasite. \u003cem\u003ePlasmodium falciparum\u003c/em\u003e, the causative agent of human malaria, can manipulate and develop through several stages following red blood cell (RBC) invasion, including ring, trophozoite, schizont, and five stages of gametocyte [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Significantly, the intraerythrocytic stage was the most hazardous, causing a coma and death in approximately 500,000 people each year [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, the mature stage V gametocyte was a precursor stage that mediate malaria transmission [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough there are various strategies to counteract malaria, such as developing new pharmaceuticals for treatment and prevention, inventing new innovations for malaria management, and educating the local community in endemic areas, there are no 100% effective malaria eradication strategies. Furthermore, the majority of antimalarial medicines had no effect on gametocytes. Malaria transmission to different communities continued for weeks after asexual stage parasites had been cleared [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As a result, the development of medications that target the key mechanisms in both the asexual stage parasite and the gametocyte may be the most cost-effective malaria eradication strategy.\u003c/p\u003e \u003cp\u003eThe important source of nutrients for the parasite growth in the intraerythrocytic stage was the amino acid derived from the hemoglobin degradation process [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Previous research showed that this mechanism was essential for both asexual and gametocyte development. In this process, various protease enzymes worked together in a cascade to degrade the large molecules of hemoglobin into small molecules of amino acids [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This function in the parasite cellular mechanism suggested that inhibiting enzymes involved in this process could impact on the development of the intraerythrocytic parasites.\u003c/p\u003e \u003cp\u003eOne type of protease involved in the hemoglobin breakdown process was aspartyl protease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Aspartyl proteases in \u003cem\u003eP. falciparum\u003c/em\u003e were made up of ten members, plasmepsin I to X, and they played a crucial role in both the asexual stage parasite and the gametocyte. These proteases contribute to the parasite's survival and growth within the erythrocyte. They were not only participated in hemoglobin breakdown but also contributed to other critical activities such as protein export, and they may be involved in parasite invasion and egress [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Thus, the aspartyl protease could be a powerful enzyme critical to the parasite's survival, indicating its potential as a useful target for blocking parasite development and transmission.\u003c/p\u003e \u003cp\u003ePepstatin A was a broad-spectrum aspartyl protease inhibitor that has been demonstrated to block hemoglobin degradation enzymes and to reduce the growth of the \u003cem\u003eBabesia\u003c/em\u003e parasite [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], which belonged to the same Apicomplexa phylum as the \u003cem\u003ePlasmodium\u003c/em\u003e parasite. Moreover, its effect on the asexual stage of the \u003cem\u003eP. falciparum\u003c/em\u003e 3D7 strain had been demonstrated [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For this reason, it can be hypothesized that the use of pepstatin A could help elucidate the effects of aspartyl protease inhibition on the development of asexual blood-stage parasites, gametocytes, and gametes, especially in transmission stages that have not yet been investigated. In this study, pepstatin A was co-cultured with the intraerythrocytic \u003cem\u003eP. falciparum\u003c/em\u003e AMB47 Thai isolate to examine aspartyl protease inhibitory effects on various developmental stages, including asexual blood stage, early, late gametocyte, and gamete. Notably, the morphology of the treated parasites was illustrated for describing the possible cellular process interfered by pepstatin A.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003eculture of\u003c/strong\u003e \u003cstrong\u003ePlasmodium falciparum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e AMB47, a gametocyte-productive strain from Thailand, was cultivated using the method described by Read with some modifications [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. In brief, the parasite\u0026apos;s asexual stage was constantly cultured at 5% hematocrit (HCT) under mixed gas conditions (90% N\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e) at 37 \u0026ordm;C. The complete culture medium for the asexual stage of \u003cem\u003eP. falciparum\u003c/em\u003e consists of RPMI 1640 powdered medium with L-Glutamine (Gibco), 2 g/L sodium bicarbonate (Mallinckrodt), 25 mM HEPES (Sigma), 2 g/L dextrose (M\u0026amp;B), 50 mg/L hypoxanthine (Sigma), 10% heat-inactivated human serum type A positive and 10 mg/L gentamicin (Siam pharmaceutical). The parasite was synchronized with 5% sorbitol (PanReac AppliChem ITW Reagents) to convert the mixed stage culture to ring stage [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe gametocyte culture followed the method outlined by Bounkeua (2010) method with some changes [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. It was maintained under the same conditions as the asexual stage culture, excluding gentamicin. Initially, the parasitemia of the asexual stage culture, ranging from 5\u0026ndash;8%, was diluted to achieve a parasitemia of 0.4\u0026ndash;0.6%. It was then cultured at 6% HCT until the parasitemia was up to more than 5%. At this point, the hematocrit culture percentage was reduced to 3.5% HCT (Day1). To prevent undesired gametogenesis, all gametocyte culture processes were carried out on a 37\u0026ordm;C hot plate. Mature stage V gametocytes were typically observed in the culture on Days 12\u0026ndash;14.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eDrug and inhibitor preparation\u003c/h2\u003e\n \u003cp\u003ePepstatin A (Sigma), an aspartyl protease inhibitor, was prepared at a concentration of 1 mM in 5% Dimethyl sulfoxide (DMSO). The positive control for the asexual stage inhibition experiment was 100 nM Artesunate (ATS), and the control for the gametocyte development experiment was 100 nM Chloroquine (CQ). They were stored at -20\u0026ordm;C before the experiment. The negative control used in this investigation was 0.5% DMSO.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDevelopment inhibition for asexual stage development\u003c/h3\u003e\n\u003cp\u003eIn a 96-well plate, the synchronized late ring stage at 1% parasitemia was co-cultured with pepstatin A concentrations ranging from 0.1 to 100 \u0026micro;M, along with negative and positive controls, for 26\u0026ndash;28 hr. After incubation, the treated parasites were harvested, and Giemsa-stained thin blood smears were prepared to investigate the defects of the parasite under light microscope.\u003c/p\u003e\n\u003ch3\u003eEarly-stage gametocyte development inhibition (EGDI) and late-stage gametocyte development inhibition (LGDI)\u003c/h3\u003e\n\u003cp\u003eThe experiments were carried out using the modified method of Adjalley (2011) [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Various concentrations of pepstatin A were treated in the 2% HCT of gametocyte culture daily for three consecutive days. The investigation of the early-stage gametocyte development inhibition (EGDI) began on Day 3 of gametocyte development when the parasites were in stage I and II gametocytes. Late-stage gametocyte development inhibition (LGDI) was initiated on Day 8 when the parasites were in stage III and IV gametocytes. Giemsa-stained thin blood smears were prepared for light microscopic examination after three days of treatment.\u003c/p\u003e\n\u003ch3\u003eGamete formation inhibition\u003c/h3\u003e\n\u003cp\u003eThe interval from day 12 to 14 of gametocyte culture is an adequate period to generate gamete. The gametocyte was pre-incubated with 10 \u0026micro;M or 100 \u0026micro;M of pepstatin A for 15 min at 37 \u0026ordm;C [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Afterward, they were spun down and immediately mixed with 21\u0026ordm;C ookinete medium in a 2:1 ratio for 15 min at room temperature. The ookinete medium contains incomplete medium, 20% non-heat inactivated serum type A positive, and 100 \u0026micro;M of xanthurenic acid to induce gamete formation. The number of exflagellation centers was counted using a hemocytometer, and Giemsa-stained thin blood smears were prepared to examine the number of macrogamete formations.\u003c/p\u003e\n\u003ch3\u003eLight microscopic observation\u003c/h3\u003e\n\u003cp\u003eUnder a light microscope, the percentage of parasitemia and morphological changes were examined after treating the parasites with pepstatin A. The number of normal parasites in 10,000 total red blood cells (total RBCs) was counted and calculated as a percentage of inhibition relative to the untreated control.\u003c/p\u003e\n\u003cp\u003eThe formula for calculating the percentage of inhibition;\u003c/p\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1747044451.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eThe morphological investigation\u003c/h2\u003e\n \u003cp\u003eThe morphological changes of the parasite were observed and investigated as shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Shape, nucleus, hemozoin, and vacuolization were the key constituents studied.\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe criteria for morphological characterization on asexual blood stage parasite\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eComposition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDefect\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRing\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTrophozoite\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSchizont\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 or 2 nuclei\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;2 nuclei\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;2 nuclei\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChromatin condensation, pyknosis, karyorrhexis and karyolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHemozoin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo hemozoin formation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrown pigment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDark brown pigment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClumped black hemozoin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVacuolization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo vacuole formation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTiny size, \u0026le; 3 vacuole formation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEnlarged or increased the number (with/without the content)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe criteria for morphological characterization on gametocyte\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eComposition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDefect\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIV\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eV\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDifficult to differentiate from\u003c/p\u003e\n \u003cp\u003etrophozoite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD-shape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eElongated D-shape with blunt-ends\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eElongated shape with pointed ends\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrescent-shape with blunt-ends\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShrinkage or swelling\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eElongate nucleus which located in the terminal site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eMale gametocyte has the nucleus larger than female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChromatin condensation, pyknosis, karyorrhexis and karyolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHemozoin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eDistribute throughout the cytoplasm of gametocyte\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClumped hemozoin or accumulation in the same food vacuole\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVacuolization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eTiny size, difficult to observe in the gametocyte by light microscope\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEnlarged or increased the number (with/without the content)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe statistical analysis to compare the significant difference was performed by one-way ANOVA. The half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) was determined through Probit analysis from IBM\u0026reg; SPSS\u0026reg; statistics 24.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe effect of aspartyl protease inhibition on asexual blood-stage.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFrom the past until now, several aspartyl protease inhibitors have been synthesized and tested for their ability to inhibit this influential enzyme. Among these inhibitors, pepstatin A has shown the ability to inhibit all plasmepsins (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, pepstatin A was selected to investigate the inhibition of aspartyl proteases that could interfere with the development of the asexual blood-stage parasite.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of plasmepsin inhibitor testing in \u003cem\u003ePlasmodium\u003c/em\u003e spp.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInhibitor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTarget plasmepsin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExperiment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNote\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInhibitor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTarget plasmepsin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eExperiment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNote\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eAzacyclic plasmepsin inhibitors\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFluorescence-based proteolysis assay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eKNI-764\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003ePm\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003ePm\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePlasmepsin IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFluorescence-based proteolysis assay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eKNI-10006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cb\u003eAzole-based inhibitor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePlasmepsin IX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eKNI-10395\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003ePepstatin A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAll plasmepsins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePlasmepsin X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHemoglobin degradation enzyme\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eCanavanine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePlasmepsin V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFluorescence-based proteolysis assay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"14\" rowspan=\"15\"\u003e \u003cp\u003e\u003cb\u003eHydroxyethylamine derivative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasmepsin I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eDimerization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003ePf,Pv\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003ePv\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePartially inhibited\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePlasmepsin IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin IX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMD simulations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMD simulations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003ePepstatin A\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eanalogue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasmepsin V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003ePEXEL peptidomimetic inhibitors\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFluorescence-based proteolysis assay\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePlasmepsin IX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eParasite egress inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eParasite invasion inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMolecular Docking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eRS367\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePlasmepsin X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eParasite egress inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eRS370\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eParasite invasion inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eHIV-1 inhibitors\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCrystallization\u003c/p\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasmepsin X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmepsin X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u0026nbsp;enzyme activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe life cycle of \u003cem\u003eP. falciparum\u003c/em\u003e required around 48 hr for its development, progressing from the ring to the trophozoite and finally the schizont stage. In this study, the late ring stage (10\u0026ndash;16 hr) was co-cultured with various concentrations of pepstatin A. After 26\u0026ndash;28 hr of incubation, the parasite showed 8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4% inhibition in 0.5% DMSO treatment, 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6%, 3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8%, 25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6%, 46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;13.4% inhibition for pepstatin A treatments ranging from 0.1, 1, 10, to 100 \u0026micro;M, and 94.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0% inhibition for ATS treatment, in comparison to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The parasite in the negative group developed into a typical trophozoite and schizont, characterized by \u0026ge;\u0026thinsp;one nucleus with the brown pigment of hemozoin.\u003c/p\u003e \u003cp\u003eThe treatment with 100 \u0026micro;M of pepstatin A inhibited development by 46.7% at the IC\u003csub\u003e50\u003c/sub\u003e greater than 100 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Although the IC\u003csub\u003e50\u003c/sub\u003e for pepstatin A was higher than expected, it exhibited a trend of development inhibition, as demonstrated by the reducing proportion and % parasitemia of developing parasites compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, the conversion rate of parasite from ring to trophozoite and schizont stage, indicative of normal parasite development, was revealed to be significantly reduced in 100 \u0026micro;M of pepstatin A when compared to no treated parasite (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePepstatin A treatment caused chromatin condensation, vacuolization, and hemozoin clumping in asexual stage development.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe morphological investigation was a simple yet advantageous study that helped to discover or predict the relationship between the parasite's defect after treatment and the underlying mechanism. In this study, we also investigated the morphological defects of the asexual blood-stage parasite under a light microscope after drugs treatment. The normal ring stage before treatment represented 1\u0026ndash;2 nuclei without hemozoin formation (0 h). After 26\u0026ndash;28 hr, the parasite in the no treated control and 0.5% DMSO treatment group developed into trophozoite and schizont, characterized by \u0026ge;\u0026thinsp;two nuclei with normal dark brown hemozoin pigment. Following 100 \u0026micro;M pepstatin A treatment, three primary morphological defects\u0026mdash; chromatin condensation, vacuolization, and hemozoin clumping \u0026mdash;were identified in the asexual blood-stage. In 100 nM ATS treatment, dead parasites were recognized by the dark blue color of the pyknotic nucleus. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn general, the asexual blood-stage hemozoin pigment was brown to dark brown in color and accumulated in the same location. In this investigation, a high dose of pepstatin A treatment (100 \u0026micro;M) caused hemozoin clumping and turned the malaria pigment into black color (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The normal food vacuoles in parasites were usually found in a tiny size. However, this study demonstrated that pepstatin A treatment increased both size and the number of vacuole formations in the parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Importantly, the sign of cell death also was observed in pepstatin A-treated parasites as show the chromatin condensation starting from the edge of nucleus or forming ring condensation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTargeting aspartyl protease by pepstatin A affected early-stage gametocyte development but not late-stage gametocyte development.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAspartyl protease was involved not only in the asexual blood-stage but also in the gametocyte. The effect of pepstatin A treatment on gametocyte development was examined. \u003cem\u003eP. falciparum\u003c/em\u003e undergoes five stages of gametocyte development, stage I through V, each with a unique metabolism. In this study, investigations on gametocyte development were divided into early-stage gametocyte (stage I to III) and late-stage gametocyte (stage IV and V).\u003c/p\u003e \u003cp\u003ePepstatin A was co-cultured in 10-fold dilutions ranging from 100 \u0026micro;M to 0.1 \u0026micro;M, as in the asexual stage experiment. After three days of treatment, the gametocytes in the negative control developed normally from stage I or II to stage II or III in the EGDI test and from stage III or IV to stage IV or V in the LGDI assay. Pepstatin A treatment highlighted the importance of aspartyl protease in gametocyte development, demonstrating that early-stage gametocyte development was inhibited by 100 \u0026micro;M pepstatin A (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;53.9 \u0026micro;M). In comparison to the notreated control, the early-stage gametocyte showed 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8% inhibition in 0.5% DMSO treatment, 10.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2%, 15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, 42.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0% and 72.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1% inhibition with pepstatin A treatment ranging from 0.1, 1, 10, to 100 \u0026micro;M, respectively. CQ treatment exhibited 89.09\u0026thinsp;\u0026plusmn;\u0026thinsp;8.83% inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, the percentage of early-stage gametocyte inhibition was also related to the reduction of % parasitemia of stage II and III gametocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and conversion rate of stage I\u0026amp;II to stage II\u0026amp;III gametocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eOn the other hand, the pepstatin A treatment had no effect on late-stage gametocyte development (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;100 \u0026micro;M). After 3 days of treatment, the late-stage gametocyte showed 0\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5% inhibition in DMSO treatment, 6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6%, 14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7%, 17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3%, and 31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;13.5% inhibition after pepstatin A treatment ranging from 0.1, 1, 10, to 100 \u0026micro;M, respectively. The positive control, CQ, exhibited 63.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4% inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Although development inhibition increased in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and the conversion of gametocyte stage III \u0026amp;IV to stage IV and V showed the significant reduction compared to no treated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), it had no effect on gametocyte development to stage V. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePepstatin A treatment induced morphological changes exclusively in the early stages of gametocyte development.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMorphological defects in both early and late-stage gametocytes were examined under a light microscope. The normal morphology of a stage II gametocyte characterized by a D-shape with blunt-ends and representing the normal distribution of hemozoin, was observed before treatment (0 h). After three days of continuous treatment, the no treated control and 0.5% DMSO-treated parasites progressed to stage II and III gametocytes, exhibiting an elongated D-shape with blunt ends and a normal hemozoin distribution. The defective stage III gametocyte could be found in 100 nM CQ and 100 \u0026micro;M pepstatin A treatment group. The gametocyte from the 100 nM CQ treatment group represented the hemozoin clumping. Chromatin condensation, hemozoin clumping and vacuole formation were distinctly evident in the pepstatin A treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn late-stage gametocytes, 0-hr stage III gametocytes displayed an elongated D-shape with blunt ends and normal hemozoin distribution. After three days of continuous treatment under all conditions, parasites developed into normal stage IV and V gametocytes. Stage IV gametocytes had an elongated shape with pointed ends and a normal distribution of hemozoin pigment, while mature stage V gametocytes exhibited a crescent shape with normal hemozoin distribution at the center (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) Notably, abnormalities were exclusively observed in early-stage gametocytes following treatment with a high dose of pepstatin A (100 \u0026micro;M); no abnormalities were found in late-stage gametocytes.\u003c/p\u003e \u003cp\u003eThe hemozoin pigment distribution in gametocytes differs from that in asexual stage parasites, as it is distributed throughout the cytoplasm of gametocytes rather than collecting in a specific area. In this work, a high dose of pepstatin A treatment (100 \u0026micro;M) induced hemozoin aggregation in the same vacuole (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, similar to the asexual stage treatment, chromatin condensation indicated by dark purple color concentrated at the inner surface of the nuclear membrane and vacuole formation characterized by increase in both size and number were observed in early-stage gametocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of aspartyl protease inhibition by pepstatin A during gamete formation\u003c/h2\u003e \u003cp\u003eThe transmission of the mature gametocyte from the patient to another human requires the critical process of gamete formation inside a female \u003cem\u003eAnopheles\u003c/em\u003e mosquito. This process involved in the transformation of male and female gametocytes into microgametes and macrogametes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It occurred after the gametocytes were activated by a change in pH, a drop in temperature, or exposure to xanthurenic acid within the mosquito [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The process of microgamete formation was called exflagellation. The parasite replicates its genome to create an octoploid and develops eight flagella to find the macrogamete. The macrogamete was differentiated from the female gametocyte. The female gametocyte started to round up and emerged from the host red blood cell after being activated.\u003c/p\u003e \u003cp\u003eTo determine whether pepstatin A could inhibit gamete formation, mature gametocytes were pre-incubated with pepstatin A for 15 min. After the activation of gametocyte, the results showed that pepstatin A could not inhibit the process of gamete formation. There were no significant differences between the pepstatin A treatments and the no treated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Although there was a significant difference in the number between male gametocytes and male gamete formation, there was no significant difference in the number of male gamete formation between no treated control and pepstatin A treatment. Moreover, the morphology of macrogametes was normal as represented by no identification of RBC membrane surrounding parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The macrogamete normally exhibited a circular shape, dispersed hemozoin, a cytoplasm stained blue, and a single mass of chromatin similar to previously identified [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe malaria parasite has numerous mechanisms for survival in the human host. Each stage of parasite development has a unique mechanism that is important for that stage. For example, the invasion mechanism is for the parasite's invasive stages (merozoite, ookinete, and sporozoite), the egress mechanism is for schizont, mature gametocyte, and oocyst, and the hemoglobin degradation process is for parasite growth in asexual blood-stage and early-stage gametocyte. Although each stage of the parasite has its own mechanism, those mechanisms may share a key molecule.\u003c/p\u003e \u003cp\u003eAspartyl protease is a good example of a molecule involved in many important processes. It participated in the hemoglobin degradation, protein exportation and might be involved in invasion and egress of the parasites [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, the aspartyl protease represented a group of critical enzymes important for parasite survival. Significantly, the previous study demonstrated that inhibiting two specific aspartyl enzymes inhibited the parasite at multiple stages [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This study explored the effects of pepstatin A, a broad-spectrum aspartyl protease inhibitor. Previous studies demonstrated the effect of pepstatin A in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e, confirming the impact of aspartyl protease inhibition on asexual blood-stage development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, in addition to influencing the asexual blood stage of the parasite, pepstatin A also induced morphological changes in early gametocyte development. However, no effect was observed on late gametocyte development and gamete formation. Previous research supported this finding by highlighting the dependency of the parasite's development in the asexual stage and early-stage gametocyte on the hemoglobin degradation process, which was not required for late gametocyte development [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Unfortunately, the previous study also indicated that, while pepstatin A poorly entered the food vacuole [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This observation could be related to the high concentration of IC\u003csub\u003e50\u003c/sub\u003e in this study and the ineffectiveness of pepstatin A in inhibiting aspartyl protease in late-stage gametocyte and gamete formation. This observation was supported by the use of another aspartyl protease inhibitor, 1,2-Epoxy-3-(p-nitrophenoxy) propane (EPNP), which demonstrated that treatment could reduce the number of both male and female gamete [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although pepstatin A did not inhibit gametocyte maturation or gamete formation, the previous study showed that aspartyl protease inhibition can interfere with the sexual stage [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], as well as influenced on the development of both asexual blood-stage and early-stage gametocytes. Therefore, it implied that the inhibition of aspartyl protease by pepstatin A can minimize the severity of disease caused by asexual blood-stage development inhibition and reduce the possibility of transmission caused by gametocyte development being blocked from the early stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe first morphological defect visible in parasites treated with pepstatin A was vacuole formation. It was identified as cytoplasmic vacuolization in asexual blood-stage and early-stage gametocytes after treatment with pepstatin A. Vacuolization was usually associated with autophagy [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Generally, autophagy occurred to remove the damaged or aged organelles [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It was also related to cell death and became more prevalent as the cells lose nourishment [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous research demonstrated that vacuolization occurred in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e after antimalarial drug incubation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Pepstatin A was also found to be capable of inhibiting autophagic breakdown [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Aspartyl protease participated in hemoglobin degradation, the parasite's primary strategy for producing amino acids for growth. Thus, the inhibition of this target enzyme by pepstatin A might disrupt the hemoglobin breakdown process, resulting in amino acid loss. Finally, the parasite died since it is unable to compensate for starvation through the autophagic breakdown process.\u003c/p\u003e \u003cp\u003eThe next defect that was found in pepstatin A- treated parasite is a defect in nucleus. Previous studies demonstrated the parasite treated with pepstatin A exhibited pyknotic nuclei. This evidence supports our finding of chromatin condensation. It was shown in the dark blue color of chromatin condensation on the inner surface of nuclear membrane, forming the pattern of ring condensation. It is the stage I of the process of cell apoptosis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe malaria pigment hemozoin originated from the heme detoxification process [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], transforming toxic heme, a byproduct of hemoglobin breakdown, into the non-toxic crystallized form of β-hematin or hemozoin [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The erythrocytic stage of the parasite could consume up to 80% of host hemoglobin [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], indicating substantial heme synthesis. Fitch's findings suggested that a mere 0.1% of heme derived from erythrocyte hemoglobin could lyse the parasite in 10 min [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Free-form heme in a ferrous state caused membrane damage, block proteases, and led to parasite mortality [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Thus, the parasite needed the detoxification process to get rid of heme. Unfortunately, the parasite lacked heme oxygenase activity, which was the heme degradation enzyme found in the vertebrates [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, the parasite could utilize another detoxification process called hemozoin synthesis [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this study, asexual blood-stage and early gametocytes exhibited hemozoin clumping after pepstatin A treatment. This defect might relate to the hemozoin synthesis, as aspartyl protease contributes to 60\u0026ndash;80% of the globin degradation process in the purified digestive vacuole [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, plasmepsin II, histo aspartic protease or plasmepsin III and plasmepsin IV played a role in hemozoin synthesis by forming the protein complex with falcipain 2 and Heme Detoxification Protein (HDP) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Apart from the defect in hemozoin synthesis due to aspartyl protease inhibition, hemozoin clumping could be a result from a process known as hemozoin depolymerization, as demonstrated in 1997 studied by Pandey and colleagues. They showed that chloroquine could depolymerize purified hemozoin to heme, similarly representing the defect in hemozoin clumping [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, further study of the hemozoin clumping process was necessary, as the mechanism generating this defect remained unclear [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAlthough pepstatin A did not completely inhibit both asexual blood stage and gametocyte, it moderately affected the growth of asexual blood-stage and early-stage gametocytes development. Moreover, this study provided insights into the morphological changes of \u003cem\u003eP. falciparum\u003c/em\u003e after aspartyl protease inhibition by pepstatin A. It exhibited chromatin condensation, vacuolization and hemozoin clumping in both asexual blood-stage and early-stage gametocyte, which are related to defects in the hemoglobin degradation process. Given its ability for aspartyl protease inhibition, it becomes a valuable target for an antimalarial drug. However, further studies on drug development or combination therapy are needed to increase the efficiency of aspartyl protease inhibition.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eATS:\u003c/strong\u003e Artesunate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCQ:\u003c/strong\u003e Chloroquine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDMSO:\u003c/strong\u003e Dimethyl sulfoxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEGDI:\u0026nbsp;\u003c/strong\u003eEarly-stage gametocyte development inhibition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHCT:\u0026nbsp;\u003c/strong\u003eHematocrit\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHDP:\u003c/strong\u003e Heme Detoxification Protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIC\u003csub\u003e50\u003c/sub\u003e:\u003c/strong\u003e The half-maximal inhibitory concentration\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLGDI:\u0026nbsp;\u003c/strong\u003eLate-stage gametocyte development inhibition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRBC:\u0026nbsp;\u003c/strong\u003eRed blood cell\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRBCs:\u0026nbsp;\u003c/strong\u003eRed blood cells\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was supported by Mahidol University (Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF)), and the CIF and CNI Grant, Faculty of Science, Mahidol University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors' contribution\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGN has designed the work, performed the experiments, interpreted data, drafted the manuscript, and revised the manuscript. CP is the colleague that also performed all experiments. RJ, WR, and JS have made contributions to the conception and advice on the experiments. VP\u0026nbsp;and NK have major contributions to conceptualization, experimental design, data analysis, conclusion, and revised the manuscript. NK has approved the submitted version. All authors have contributed to the approval of the submitted version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all staff from Department of Pathobiology, Faculty of Science and Mahidol Vivax Research Unit (MVRU), Faculty of Tropical Medicine, Mahidol University for their kindly support at all the time to finish in this experiment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHawking F, Wilson ME, Gammage K. 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Curr Res Struct Biology. 2024;7:100128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.crstbi.2024.100128\u003c/span\u003e\u003cspan address=\"10.1016/j.crstbi.2024.100128\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aspartyl protease, Developmental inhibition, Gametocyte, Malaria, Transmission","lastPublishedDoi":"10.21203/rs.3.rs-6591841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6591841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is the most influential species of malaria parasites, capable of causing severe illness and mortality, especially in pregnant women and children under the age of 5. Global distribution of disease impacted on billions of endemic people and travelers. Asexual stage and gametocyte cause harmful manifestations, impacting the patients and contributing to the spread of the disease in the community, respectively. Moreover, most recent therapeutic drugs did not affect the gametocyte. The discovery of a new drug with dual actions on both stages could elucidate a cost-effective way to combat malaria. Within a human host, the parasite possesses many activities for its survival, such as invasion, egress, hemoglobin degradation, and protein trafficking, many of which are related to aspartyl protease, revealing the potential for antimalarial drug targets.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePepstatin A, the representative of the board-spectrum aspartyl protease inhibitor, was utilized to investigate the effects of aspartyl protease inhibition on parasite development. The experiments were separately performed \u003cem\u003ein vitro\u003c/em\u003e for different developmental stages of parasites, including the asexual blood-stage, early gametocytes, late gametocytes, and gamete. To demonstrate the effects of pepstatin A, the number of intact parasites and their stage distribution were counted under the microscope and calculated as a percentage of inhibition compared to the control. Additionally, the morphology of pepstatin A-treated parasites was observed to identify cellular alterations in the parasites.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePepstatin A at 100 \u0026micro;M inhibited the asexual stage and early-stage gametocyte development by 47% and 73%, respectively. They exhibited morphological defects, including chromatin condensation, vacuolization and hemozoin clumping in both asexual blood-stage and early-stage gametocyte. However, it could not influence the late-stage gametocyte development and gamete formation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe inhibition of aspartyl protease by pepstatin A moderately affected both asexual blood-stage and early-stage gametocyte development. Morphological changes on treated parasites implied the effect of pepstatin A on hemoglobin degradation process, suggesting its potential for reducing the severity of the disease and minimizing malaria transmission. However, further research and development are required to use aspartyl protease as a drug target, focusing on identifying and modifying the drug to be more sensitive and effective.\u003c/p\u003e","manuscriptTitle":"Aspartyl protease inhibition interferes with Plasmodium falciparum asexual blood-stage and early gametocyte development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 09:44:37","doi":"10.21203/rs.3.rs-6591841/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-27T18:24:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T17:50:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315167463472792641519736258059655465444","date":"2025-06-27T15:10:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178370610406765067074681331254492599963","date":"2025-05-15T13:03:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-06T18:34:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-05T06:59:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-05T06:54:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2025-05-05T06:46:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48fa8297-925f-4929-8787-657dfecc3861","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:31:00+00:00","versionOfRecord":{"articleIdentity":"rs-6591841","link":"https://doi.org/10.1186/s12936-025-05518-z","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2025-08-21 15:56:58","publishedOnDateReadable":"August 21st, 2025"},"versionCreatedAt":"2025-05-13 09:44:37","video":"","vorDoi":"10.1186/s12936-025-05518-z","vorDoiUrl":"https://doi.org/10.1186/s12936-025-05518-z","workflowStages":[]},"version":"v1","identity":"rs-6591841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6591841","identity":"rs-6591841","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}