The potential role of CI-679 against artemisinin-resistant Plasmodium falciparum

doi:10.21203/rs.3.rs-968572/v1

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The potential role of CI-679 against artemisinin-resistant Plasmodium falciparum

https://doi.org/10.21203/rs.3.rs-968572/v1

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Background

The emergence of artemisinin-resistant Plasmodium falciparum (P. falciparum) in the southeast of Asia hampers the control of malaria. It is urgent to develop novel antimalarial drug with mechanism different from that of artemisinin.

Methods

The efficacy of Nitroquine (CI-679), which is thought to antifolate, against blood stage parasites in the Plasmodium yoelii (P. yoelii) -infected BALB/C mice model was firstly identified. Then, in vitro assay was performed to investigate its killing effect on 3D7 P. falciparum strain. Finally, the potential effect of CI-679 was also observed on artemisinin-resistant P. falciparum, which is constructed by introducing the C580Y mutation in K13 of the 3D7 using the CRISPR-cas9 technology.

Results

CI-679 could significantly suppress the growth of rodent malaria parasite P. yoelii BY265 at a dose-dependent manner, and even potently kill the parasites in mice when administrated at the peak of parasitemia. Furthermore, CI-679 could also efficiently inhibit the growth of P. falciparum in vitro, with more sensitivity against late phase of parasite. Strikingly, CI-679 can even kill the artemisinin-resistant P. falciparum, which is constructed by the mutation of C580Y at K13 gene, as sensitive as the 3D7 parent parasites.

Conclusion

CI-679 could be explored as an alternative antimalaria drug against artemisinin-resistant P. falciparum, as well as a potential partner of artemisinin-based combination therapies against malaria parasite in the future.

Parasitology

CI-679

Nitroquine

artemisinin-resistant

Plasmodium falciparum

malaria

Malaria is the most important parasitic disease of human beings. It is transmitted in 87 endemic countries, affected about 3 billion people and caused 229 million cases and 409 thousand deaths in 2019, the majority being children under 5 years old living in Sub-Saharan Africa [1]. This situation is expected to worsen as the ongoing SARS-COV-2 pandemic affects malaria treatment and prevention measures [2]. Five species of the genus Plasmodium cause all malarial infections in human beings. P. vivax is the most widespread species, whereas P. falciparum is responsible for the most deaths [1]. Pregnant women and nonimmune naïve hosts are particularly susceptible to severe malaria, which is associated with complications such as severe anemia and/or sequestration of parasitized red blood cells (RBCs) into key organs, including brain [3]. The deployment of first-line artemisinin-based combination therapies (ACTs) has made a substantial contribution to promoting control and elimination strategies [4]. In the absence of an effective licensed malaria vaccine, the strategies are critically reliant on the continued clinical efficacy of ACTs [3].

However, the initial observation of artemisinin clinical failure in Southeast Asia along the Thai-Myanmar and Thai-Cambodia borders was reported in 2008 [5, 6]. Great efforts have been made to reveal the artemisinin-resistant mechanism of P. falciparum, and kelch 13 (Pfk13) was identified as the artemisinin resistance (AR) molecular marker [7]. Pfk13 resides on chromosome 13 of the P. falciparum genome, mapping to the β-propeller domain of the encoded kelch-like protein, PF3D7_1343700 [7]. The propeller domain of the kelch superfamily encodes proteins harbor multiple protein-protein interaction sites and mediates diverse cellular functions, including ubiquitin-regulated protein degradation and oxidative stress responses [8] and the Pfk13 is associated with slow parasite clearance rates [9–11]. Over 20 mutations in Pfk13 have been identified after treatment with artemisinin derivatives across 15 locations in Southeast Asia [12], among which, at least 11 kelch13 mutations has been validated as molecular markers for artemisinin resistance according to WHO criteria [13, 14] and the most prevalent Pfk13 mutation is C580Y [7, 15–17]. The emergency and spread of resistance to artemisinin has posed a major hindrance to the strategies towards malaria control and elimination [17–20] and potentially derailing current control efforts. Thus, there is an urgent need for new antimalarial drugs with different mechanism, especially those that act against currently artemisinin resistant strains.

In the early 1960s, a collaborative program of synthesis of folic acid analogs in which the pteridine ring was replaced by a quinazoline moiety was launched by the Departments of Chemistry of Parke Davis and Company, Ann Arbor, Michigan and Hounslow, Middlesex, England [21]. There interests soon focused on 2,4-diamino-6-substituted derivatives for their promising properties to inhibit the activities of dihydrofolate reductases and thymidylate synthetases of mammal and bacteria [22, 23]. Representative 2,4-diamino-6-substituted quinazolines were further determined for antiparasitic activities and PAM-1392 [2,4-diamino-6-(3,4-dichlorobenzylamino)-quinazoline] [22] and CI-679 (2,4-diamino-6-[(3,4-dichlorobenzyl)-nitrosoamino]-quinazoline) [24] attracted special attention. CI-679 was approximately 40 times as effective as PAM-1392 in suppressing infections with the parent sensitive strain of Plasmodium berghei and approximately 10 times as effective as PAM-1392 in suppressing and curing infections with blood schizonts of Plasmodium cynomolgi [25, 26]. Then, CI-679, along with artemisinin of the same time, was selected as an important part of Project 523, a national project set up by the Chinese government in 1967 [27, 28]. However, the research and development of CI-679 were retarded by the success in artemisinin. As the developing and spread resistance to artemisinin, and the mechanism of killing parasite by CI-679 is divergent from that of artemisinin, there has been a resurgence of interest in CI-679.

Here, we describe the development of CI-679 for activity against P. yoelii in murine model and artemisinin-sensitive P. falciparum 3D7 and artemisinin-resistant P. falciparum C580Y in vitro.

Study design

The program objectives were to characterize the efficacy of CI-679. Efficacy was evaluated against the P. yoelii in murine model in vivo and artemisinin-sensitive P. falciparum 3D7 and artemisinin-resistant P. falciparum C580Y in vitro. Studies in vivo were designed to evaluate efficacy and how fast the compound kills parasites. Studies in vitro were designed to evaluate efficacy across the full range of blood stage of P. falciparum 3D7 and against P. falciparum C580Y.

Parasite and mice

BALB/C female mice at 6 weeks of age, were purchased from Beijing Huafukang Bioscience Company Limited. P. yoelii strain BY-265 is maintained in our laboratories. It has been used for several decades to test susceptibility to a wide range of antimalarial drugs. For infection, BALB/C female mice were injected with 1 × 10⁶ rodent malaria parasite P. yoelii strain BY-265 via intravenous injection (i.v.) [29]. Animal experiments were approved by the institutional animal care and use committees for each of the experimental sites. All studies were conducted according to the appropriate legislation and institutional policies on the care and use of animals.

Parasite culture and the establishment of P. falciparum C580Y

3D7 P. falciparum were cultured as previously described with 5% O₂ and 5% CO₂ at 37 ℃ [30]. Parasites were synchronized with 5% sorbitol solution for ring stage or purified on a 40/70% percoll discontinuous gradient for schizont stage.

The resistant strain P. falciparum C580Y was constructed through mutation of C580Y in K13 of 3D7 using Crispr-Cas9 technology [31]. The K13_C580Y mutation (with shield mutations) and the Sir2B shield mutations were introduced into the Cas9i system by plasmid pL6cs-K13-Sir2B. For transfection, fresh human type O erythrocytes were electroporated in cytomix (120 mM KCl, 10 mM KH₂PO₄, 25 mM HEPES, 2 mM EGTA, 0.15 mM CaCl₂, 5 mM MgCl₂, pH 7.6) with 100 µg of each plasmid under standard electroporation parameters [32] and enriched late schizonts were mixed with the electroporated erythrocytes immediately. Positive selection drugs were applied 72 h post-transfection, and media and drugs were refreshed every day for the first 7 days. The final concentrations of drugs were 2.5 µg/ml for BSD (Sigma), 2.5 nM for WR99210 (Sigma), and 1.5 µM for DSM1 [33], respectively. The desired transgenic 3D7 strain K13::Sir2B-mut^Cas9i was obtained in about 20 days. The correct introduction of K13_C580Y mutation and shield mutations was identified as reported [31].

In vivo efficacy of CI-679 in the P. yoelii-infected BALB/C mice

BALB/C mices were infected with P. yoelii strain BY-265 as described above. To observe the dose-dependent efficacy of CI-679, the mice were treated with or without 2.5, 5.0 and 10.0 mg/kg/day of CI-679 (dissolve in DMSO for storage and dissolve in PBS when used) via intragastric injection (i.g.) respectively for 3-day-treatment. To investigate the treatment effect of CI-679, the mice were treated with 2.5 mg/kg/day of CI-679 via intragastric injection on 5-6 days post infection (dpi) and 3-5 dpi for its role on peak and rising stage of parasitemia, respectively, for 3-day-treatment.

The determination of parasitemia and mice survival rate

Blood smears were made daily from the tail venous blood of mice and stained by Giemsa. Parasitemia was counted from at least 5,000 RBCs by microscopy. Blood smears and mortality data were taken on 1 dpi and thereafter till clear of parasitemia of blank control, at which time all treated mice surviving that were blood film negative were considered cured.

In vitro blood-stage parasites inhibition assay

A total of 0-3 h ring-stage survival assay (RSA^{0–3 h}) assay was performed to evaluate the activity of CI-679 against the P. falciparum sensitive 3D7 and artemisinin resistance C580Y as previously described [34]. In brief, parasites were synchronized several times to acquire the accurate 0-3 h post-invasion rings. The RSA^{0–3 h} was done immediately with 0-3 h post-invasion rings, the trophozoite-stage survival assay (TSA^{24–27 h}) with 24-27 h post-invasion rings, and the schizont-stage survival assay (SSA^{36–39 h}) with 36-39 h post-invasion trophozoites. In each assay, the accurately synchronized parasites were exposed to 0/640 ng/ml discontinuous gradient (0, 0.01, 0.04, 0.16, 0.64, 2.5, 10, 40, 160, 640 ng/ml) CI-679 or 0.1% DMSO (the solvent) for 6 h, washed with incomplete medium to remove the drug, and cultured for another 66 h, 42 h and 30 h, respectively, after drug removal. Blood films were made to assess the survival rates of the strains microscopically. Each assay was repeated for 3 times independently, with each group setting three technical repetitions. Each technical repetition was counted for 40,000 erythrocytes.

For EC50 determinations, data were fitted to the log(inhibitor) versus response − variable slope (four-parameter) model in GraphPad Prism 9.0.

Statistical analysis

Graphical representation and statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Inc.). Unless otherwise stated, the results are shown as means ± s.e.m. from three independent experiments in duplicate. The two-way ANOVA was used to compare parasitemia between CI-679 treatments and the control group mice. The two-tailed Student’s t - test was used to test the differences of proliferation in vitro. The log-rank (Matel–Cox) test was used to compare the survival distributions of two groups. For parasite EC50 determination, data were fitted to the specified model as described in Materials and Methods using nonlinear least squares analysis. A P value threshold of 5% was used to indicate statistical significance. Biological replicates (n) indicated in figure legends refers to the number of mice or number of independent cultures. Sample sizes were chosen on the basis of historical data, no statistical methods were used to predetermine sample size.

CI-679 suppresses the growth of P. yoelii in a dose-dependent manner

Firstly, we had an insight into the efficacy of CI-679 against rodent malaria. We undertook a detailed analysis of the intragastric dose-dependent profile in the P. yoelii BALB/C mouse 3-day dosing model. Mice in all groups were inoculated intravenously with 1×10⁶ parasitized red blood cells (pRBCs). Parasitized mice in experimental groups treated with CI-679 by intragastric injection (i.g.) at 2.5, 5.0, 10.0 mg per kg of body weight 2 hours after inoculation for 3 consecutive days thereafter. The control group was given equal volume of PBS. All mice survived the challenge were able to control parasitemia without the need for antimalarial drug treatment (Fig. 1a). Parasitemia was evaluated from 1 day post infection (dpi) until the control mice were cleared of parasitemia. We found that as low as a dose of 2.5 mg per kg was fully protective (Fig. 1a) and all the mice were survived (Fig. 1b) in experimental groups.

CI-679 efficiently inhibit the growth of P. yoelii even administrated at the peak of parasitemia

The previous experiment suggested parasites infected mice treated with CI-679 by intragastric injection at 2.5 mg per kg of body weight for 3 consecutive days could be cured and the first parasitemia peak in control group was reached between 5 to 7 dpi. We choose this window to investigate the role of CI-679 even administrated at the peak of parasitemia. We found that all mice treated with CI-679 were clear of parasitemia soon after administration (7 dpi) (Fig. 2a) and all survived till the mice in control were free from parasitemia (Fig. 2b). We further investigated the role of CI-679 as the parasitemia rising up (3-5 dpi). As expected, all mice treated with CI-679 were clear of parasitemia after administration (6 dpi) (Fig. 2c) and survived (Fig. 2d).

CI-679 efficiently inhibit the growth of sensitive P. falciparum strain in vitro

To further identify the efficacy of CI-679 against different blood stages of P. falciparum, we evaluated the activity of CI-679 against the ring, trophozoite and schizont stages using highly synchronized P. falciparum parasites drug-sensitive strain 3D7. Parasites were synchronized to ring, trophozoite and schizont stage in a classical manner [35]. Parasites were exposed to CI-679 at 0/640 ng/ml discontinuous gradient for a 6-h treatment at ring (0-6 hours post invasion, hpi), trophozoite (24-30 hpi) and schizont (36-42 hpi) stages (Fig. 3a). Further measurements of parasitemia at 72 hpi indicated that the treatment at the trophozoite and schizont stages were more active to parasite killing than that at ring stage (Fig. 3b). This suggested that the mechanism of CI-679 may be associated with the development of trophozoites and schizonts in red blood cells.

CI-679 efficiently kills artemisinin-resistant P. falciparum strain

Having demonstrated the excellent inhibitory activities of CI-679 on P. falciparum drug sensitive 3D7, we wanted to determine the effect of CI-679 on artemisinin-resistant parasites. As the mutation of C580Y in K13 gene confer the resistance of P. falciparum to artemisinin, and C580Y mutation has been confirmed to be correlated to artemisinin resistance characterized by high RSA^{0–3 h} survival rates[7], we introduced the K13_C580Y mutation into the Cas9i system by plasmid pL6cs-K13-Sir2B (Fig. 4a). The level of artemisinin resistance was assessed as previously reported [31]. Both P. falciparum 3D7 and C580Y parasites were exposed to CI-679 at 0/640 ng/ml discontinuous gradient. Using the standard 72 h drug assay, CI-679 exhibited similar activities against both P. falciparum 3D7 and C580Y parasites (Fig. 4b). Taken together, our results suggested CI-679 had a potent inhibitory activity against artemisinin-resistant malaria parasites.

The P. falciparum parasites have independently developed partial resistance to artemisinin, the core compound of the best available antimalarial drugs, in several foci in the Greater Mekong subregion (GMS), Africa, Oceania and Latin America [1, 36, 37]. As a consequence, the increasing prevalence of artemisinin-resistance and the consequent emergence of resistance to the partner drugs in ACTs may well reverse the substantial recent gains in malaria control [15]. New antimalarial drugs are urgently needed.

Recent data indicate that the average cost and time for a new drug from idea to market are about 3 billion dollars and 14 years, respectively [38, 39]. Although the malaria drug development pipeline is in a healthier state for many years at present, there is only one 8-aminoquinoline, tafenoquine, newly approved by FDA against liver stages of malaria [40, 41]. An alternative strategy is to evaluate the antimalarial activity of molecules that are already licensed for new medical indications, an approach known as drug repurposing that has been successfully applied in oncology [42]. Since the drugs are already in routine clinical use for other purposes early phase clinical trials can be bypassed, saving time and money. Thus, this strategy offers a promising alternative to traditional drug development pipelines, and can help fast and less expensive solutions to realize malaria elimination and eradication.

Patients with P. falciparum hyperparasitemia are at increased risk for treatment failure, severe malaria and death [43]. Parasitemia level can serve as a predictor for severe malaria [44]. Herein, we show that CI-679 could suppress the growth of rodent malaria parasite P. yoelii BY265 at a dose-dependent manner, and even rapidly kill the parasite in mice when administrated at the peak of parasitemia. Furthermore, CI-679 has a wide therapeutic window, both effective at the rising and peak stage of parasitemia, makes it can be widely available as a partner drug in a fixed-combination. The property could make severe malaria and death much less likely.

Continued research and development to make new drugs available to replace current drugs as resistance emerges is one of the several high priority research areas identified to be addressed urgently regardless of whether the world mobilizes for a renewed effort to eliminate malaria [45]. As the increasing threat of the emergency and spread of resistance to artemisinin, finding the optimal combination of interventions to maximize impact and mitigate the risk of resistance is a critical challenge in malaria elimination [46]. For eradication to be effective, the concept of a drug combination Single Encounter Radical Cure and Prophylaxis (SERCaP) was developed [45], signify clinical candidates will necessarily require combination with mechanistically distinct drug partner(s) to alleviate the potential for drug resistance. CI-679 can kill the artemisinin-resistant P. falciparum, which is constructed by the mutation of C580Y at K13 gene. Therefore, CI-679 could be explored as an alternative antimalarial agent against artemisinin-resistant P. falciparum and a potential combination partner of ACTs against malaria parasite.

The ring stages can develop into schizonts through the trophozoites and the schizonts burst and release daughter merozoites by the end of blood stage, each of which can invade blood cells and repeat the cycle [3]. In the blood stage, Plasmodium parasites replicate rapidly and may replicate exponentially to > 10¹² parasites per patient. This rapid growth requires sustained pools of nucleotides for the synthesis of DNA and RNA [47]. Folate is essential for the survival and growth of the malaria parasite and DNA synthesis [48] and the folate metabolic pathway constitutes an antimalarial target. CI-679 could efficiently inhibit the growth of P. falciparum in vitro, with more sensitivity against late phase of blood stage. This result was as expected, for CI-679 was a product of the program of the synthesis of folic acid analogs. The development of parasites in late phase of blood stage might be impeded by the insufficient DNA synthesis ascribed to the administration of CI-679.

Although CI-679 is potent against P. yoelii and P. falciparum, even artemisinin-resistant C580Y, further pharmacokinetic properties, tolerability and safety need to be characterized to support its preclinical development and to enable the compound to advance to human clinical trials. In addition, as the rising concern of resistance to antifolates, possible drug resistance mechanisms based on in vitro selection should also be included.

In summary, CI-679 could be explored as an alternative antimalarial drug against artemisinin-resistant P. falciparum, as well as a potential combination partner of ACTs against Plasmodium parasite. As the money- and time-consuming progress in the antimalarial drugs research and development, repurposing the agents already known might be an efficient strategy to combat the P. falciparum drug resistance issues.

Acknowledgements

Not applicable.

Funding

This work was supported by the State Key Program of the National Natural Science Foundation of China (No. 81830067), the National Natural Science Foundation of China (No. 81772226) and the Natural Science Foundation of Chongqing, China (No. cstc2018jcyjAX0443).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

WYX, QFZ, JZ and NT conceived and designed the experiments. NT, YMZ, YD, YF and HXL performed the experiments. NT and YMZ analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Authors' details

¹Department of Pathogenic Biology, Army Medical University (The Third Military Medical University), Chongqing 400038, P.R. China. ²Research Center for Translational Medicine, Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China.

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The potential role of CI-679 against artemisinin-resistant Plasmodium falciparum

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Study design

Parasite and mice

Parasite culture and the establishment of P. falciparum C580Y

In vivo efficacy of CI-679 in the P. yoelii-infected BALB/C mice

The determination of parasitemia and mice survival rate

In vitro blood-stage parasites inhibition assay

Statistical analysis

Results

Discussion

Conclusions

Declarations

References

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