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 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 | [52] | Fluorescence-based proteolysis assay | Hydroxyethylamine derivative (Cont.) | Plasmepsin X | In vitro enzyme activity | [53] | Pb |
Crystallization | | KNI-764 | Plasmepsin IV | In vitro enzyme activity | [54] | Pm |
Plasmepsin IV | In vitro enzyme activity | Fluorescence-based proteolysis assay | Plasmepsin IV | Crystallization | Pm |
Crystallization | | KNI-10006 | Plasmepsin I | Crystallization | [55] | |
Azole-based inhibitor | Plasmepsin II | Molecular Docking | [56] | | Molecular Docking | [57] | |
In vitro enzyme activity | | Plasmepsin II | Molecular Docking | |
Plasmepsin IX | Molecular Docking | | Plasmepsin IV | Molecular Docking | |
In vitro enzyme activity | | KNI-10395 | HAP | Crystallization | [58] | |
Plasmepsin X | Molecular Docking | | Pepstatin A | All plasmepsins | Molecular Docking | [59] | |
In vitro enzyme activity | | Hemoglobin degradation enzyme | In vitro enzyme activity | [6] | |
Canavanine | Plasmepsin V | In vitro enzyme activity | [60] | Fluorescence-based proteolysis assay | Plasmepsin II | Crystallization | [61] | |
Crystallization | | Crystallization | [62] | |
Molecular Docking | | Crystallization | [63] | |
Hydroxyethylamine derivative | Plasmepsin I | In vitro enzyme activity | [64] | | HAP | Crystallization | [65] | |
Plasmepsin II | In vitro enzyme activity | [66] | | Plasmepsin IV | Dimerization | [63] | Pf,Pv |
Molecular Docking | | Crystallization | [67] | Pv |
Crystallization | [68] | | Plasmepsin V | In vitro enzyme activity | [14] | Partially inhibited |
Plasmepsin IV | In vitro enzyme activity | [66] | | In vitro enzyme activity | [69] | |
Molecular Docking | | Plasmepsin IX | MD simulations | [70] | |
In vitro enzyme activity | [71] | | Plasmepsin X | MD simulations | |
Molecular Docking | | Pepstatin A analogue | Plasmepsin II | In vitro enzyme activity | [72] | |
Plasmepsin V | In vitro enzyme activity | [73] | | Plasmepsin II | Crystallization | |
Plasmepsin IX | Parasite egress inhibition | [53] | Pb | PEXEL peptidomimetic inhibitors | Plasmepsin V | In vitro enzyme activity | [74] | Fluorescence-based proteolysis assay |
Parasite invasion inhibition | Pb | Crystallization | |
In vitro enzyme activity | Pb | Molecular Docking | |
Plasmepsin X | Parasite egress inhibition | Pb | RS367 | Plasmepsin II | Crystallization | [75] | |
Parasite invasion inhibition | Pb | RS370 | Plasmepsin II | Crystallization | [75] | |
The life cycle of P. falciparum requires around 48 hours for its development, progressing from the ring to the trophozoite and finally the schizont stage. In this study, the late ring stage (10–16 hours) was co-cultured with various concentrations of pepstatin A. After 26–28 hours 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. 1A). 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% and had an IC50 greater than 100 µM (Fig. 1A). Although the IC50 for pepstatin A was higher than expected, it exhibited a trend of development inhibition, as demonstrated by the reduced proportion and % parasitemia of developing parasites compared to controls (Fig. 1B). 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. 1C).
Pepstatin A treatment causes the hemozoin clumping and vacuolization in asexual stage development.
The morphological investigation is a simple yet advantageous study that helps 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 hours, 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, two primary morphological defects—hemozoin clumping and vacuolization—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 is brown to dark brown in color and accumulate in the same location. In this investigation, a high dose of pepstatin A treatment (100 µM) caused hemozoin clumping to black color (Fig. 2). The small food vacuole in parasite usually can be found, but it will present in a tiny size. This study found that pepstatin A treatment increased both size and the number of vacuole formation (Fig. 2).
Targeting aspartyl protease by pepstatin A affected early-stage gametocyte development but not late-stage gametocyte development.
Aspartyl protease is involved not only in the asexual blood-stage but also in the gametocyte stage. 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, gametocyte development was divided into two stages: early-stage gametocyte and late-stage gametocyte. Stage I to III represent early-stage gametocytes, while stages IV and V represent late-stage gametocytes.
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 (IC50 = 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. 3A). Moreover, the percentage of early-stage gametocyte inhibition was also related to the reduction of % parasitemia of stage II and III gametocyte (Fig. 3B) and conversion rate of stage I&II to stage II&III gametocyte (Fig. 3C).
On the other hand, the pepstatin A treatment had no effect on late-stage gametocyte development (IC50 > 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. 3D). Although development inhibition increased in a dose-dependent manner (Fig. 3D) and the conversion of gametocyte stage III &IV to stage IV and V showed the significant reduction compared to no treated control (Fig. 3F), it had no effect on gametocyte development to stage V. (Fig. 3E).
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 is 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. Hemozoin clumping and vacuole formation were evident in the pepstatin A treatment (Fig. 4A).
In late-stage gametocytes, 0-hour 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. 4B)
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 (Fig. 4).
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, high dose of pepstatin A treatment (100 µM) induced hemozoin aggregation in the same vacuole (Fig. 4A). Additionally, similar to the asexual stage treatment, vacuole formation was observed in early-stage gametocytes characterized by increase in both size and number (Fig. 4).
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 involves the transformation of male and female gametocytes into microgametes (male) and macrogametes (female) [22]. It occurs after the gametocytes have been activated by a change in pH, a drop in temperature, or exposure to xanthurenic acid within the mosquito [23, 24]. The process of microgamete formation is called exflagellation. The parasite replicates its genome to create an octoploid and develops eight flagella to find the macrogamete. The macrogamete is differentiated from the female gametocyte. The female gametocyte starts to round up and emerge from the host red blood cell after being activated.
To determine whether pepstatin A can inhibit gamete formation, mature gametocytes were pre-incubated with pepstatin A for 15 minutes. After the activation of gametocyte, the results showed that pepstatin A could not inhibit the process of gamete formation. There are no statistically significant differences between the pepstatin A treatment and the no treated control (Fig. 5A and 5B). 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 macrogamete was normal (Fig. 3C). The absence of the RBC membrane represents the success of gametocyte egress from the host red blood cell. The macrogamete exhibited a circular shape, dispersed hemozoin, a cytoplasm stained blue, and a single mass of chromatin [25].