Insights in Chloroquine Action: Perspectives and Implications in Malaria and COVID-19

Abstract

Malaria is a threat to human mankind and kills about half a million people every year. On the other hand, COVID-19 resulted in several hundred thousand deaths since December 2019 and remains without an efficient and safe treatment. The antimalarials chloroquine (CQ) and its analog, hydroxychloroquine (HCQ), have been tested for COVID-19 treatment, and several conflicting evidence has been obtained. Therefore, the aim of this review was to summarize the evidence regarding action mechanisms of these compounds against Plasmodium and SARS-CoV-2 infection, together with cytometry applications. CQ and HCQ act on the renin angiotensin system, with possible implications on the cardiorespiratory system. In this context, flow and image cytometry emerge as powerful technologies to investigate the mechanism of therapeutic candidates, as well as for the identification of the immune response and prognostics of disease severity. Data from the large randomized trials support the conclusion that CQ and HCQ do not provide any clinical improvements in disease severity and progression of SARS-CoV-2 patients, as well as they do not present any solid evidence of increased serious side effects. These drugs are safe and effective antimalarials agents, but in SARS-CoV-2 patients, they need further studies in the context of clinical trials. © 2020 International Society for Advancement of Cytometry.

Keywords: Plasmodium; SARS-CoV-2; autophagy; clinical trials; renin angiotensin system; side effect; viral invasion.

Figure 1

Suggested modes of action of chloroquine against Plasmodium falciparum parasites. Chloroquine can (1) prevent the formation of hemozoin by masking functional groups of hematin and the growing hemozoin crystal resulting in accumulation in the food vacuole (FV) as CQ²⁺ and (2) inhibit the activity of PfEXP1 which is involved in reduced glutathione (GSH)‐mediated detoxification of heme. CQ resistance in P. falciparum parasites is believed to be inferred by mutations in PfCRT and PfMRP1 transporters that promote efflux of CQ out of the FV and the parasite, respectively. RBC, red blood cell; PVM, parasitophorous vacuole membrane; PM, parasite membrane; FV, food vacuole; CQ, chloroquine; CQ²⁺, protonated chloroquine as present in the FV; Hb, hemoglobin; EXP1, Plasmodium falciparum exported protein 1 (a glutathione‐S‐transferase); CRT, Plasmodium falciparum CQ resistance transporter; MRP1, Plasmodium falciparum multidrug resistance‐associated protein 1. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Interference of chloroquine and hydroxychloroquine in the renin‐angiotensin system (RAS). Angiotensinogen, produced in the liver, is cleaved by the renin protease produced in the kidney. Cleavage of Ang I by ACE produces the active octapeptide Ang II that acts via the AT1R, inducing vasoconstriction, production of aldosterone, increased inflammation, oxidative stress, fibrosis, and vascular permeability. Ang II levels are regulated by ACE2 that cleaves Ang II and produces Ang 1–7, a heptapeptide that acts via the Mas receptor, inducing vasodilatation and cardioprotective effects, while decreasing oxidative stress, inflammation and arrhythmias. Expression of ACE2, the SARS‐CoV‐2 cell receptor, is decreased by the endocytosis process that allows viral entry. CQ and HCQ inhibit viral entry by impairing terminal glycosylation of ACE2, which may reduce enzyme activity, elevating Ang II concentration and favoring the pressor axis. CQ, chloroquine; HCQ, hydroxychloroquine; AGT, angiotensinogen; Ang I, angiotensin I; Ang II, angiotensin II; ACE, angiotensin‐converting enzyme; ACE2, angiotensin‐converting enzyme 2; AT1R, Ang II receptor type 1; MasR, Mas receptor. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Cytometry applications for studying molecular interactions of coronavirus infection and pathology. (A) Antibody or drug interference of spike binding to ACE2 receptor can be evaluated by flow cytometry. The spike ectodomain is tagged to the human Fc domain (spike‐Fc), while ACE2 receptor expression was visualized by using an GFP‐coding vector. To study, whether drugs or monoclonal antibodies (mAbs) bind to the spike protein, these molecules can be preincubated with spike‐Fc to form complexes. After that, this mix can be incubated with cells expressing ACE2‐GFP and goat anti‐human IgG antibodies conjugated with fluorophore. If the tested antibody or drug interferes with spike/ACE2 interaction, only single‐positive cells will be observed in cytometry analysis. The influence of CQ and HCQ on spike/ACE2 interaction can also be evaluated by similar methods. (B) The influence of SARS‐CoV‐2 and possible treatments on lysosomal pH and autophagy can be investigated by cytometry analysis. CQ and HCQ, for example, may increase the endosomal and lysosomal pH, as evidenced by pH‐sensitive dyes. Moreover, CQ, HCQ and other potential anti‐SARS‐CoV‐2 drugs (corticosteroids, emtricitabine/tenofovir, interferon α‐2b, lopinavir/ritonavir and ruxolitinib) decrease autophagy by several mechanisms, including the inhibition of autophagosome fusion with lysosomes. This inhibition triggers autophagosome accumulation evidenced by high LC3‐II levels (detected as high MFI in flow cytometry and high number of LC3‐II⁺ points in image cytometry). Receptor binding domain, RBD; Ab, antibody; mAb, mouse antibody; MFI, median fluorescence intensity; CQ, chloroquine; HCQ, hydroxychloroquine; ACE2, angiotensin‐converting enzyme 2; LC3‐II, microtubule‐associated protein 1 light chain 3‐II. [Color figure can be viewed at wileyonlinelibrary.com]