key: cord-1004722-rhkr6t3w authors: Pillat, Micheli Mainardi; Krüger, Arne; Guimarães, Lara Mendes Ferreira; Lameu, Claudiana; de Souza, Edmarcia Elisa; Wrenger, Carsten; Ulrich, Henning title: Insights in chloroquine action: perspectives and implications in Malaria and COVID‐19 date: 2020-07-19 journal: Cytometry A DOI: 10.1002/cyto.a.24190 sha: 9be31456dfe3fcf620d1f16b33f7817a39164944 doc_id: 1004722 cord_uid: rhkr6t3w 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 analogue, 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 beneficial in disease severity and progression in SARS‐CoV‐2 patients, as well as, 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 a clinical trials. This article is protected by copyright. All rights reserved. Showing effects unrelated to heme-detoxification underpins the pleiotropic character of the drugs. The emergence of chloroquine-resistant strains urged the synthesis of CQ derivatives to overcome the health threat posed by malaria (5) . While quinine (QN) was originally identified as a natural compound, it was soon replaced by the cost-effective and safe 4aminoquinolines Chloroquine and Hydroxychloroquine (HCQ). QN is still used, though, as it is effective against CQ-resistant Plasmodium strains (2) . Amodiaquine (ADQ), also a 4aminoquinoline and QN derivative, was synthesized in the early 1940s and is still effective against CQ-resistant strains. It is used as a partner drug in the front-line malaria treatment artemisinin combination therapies (ACTs) (2, 4) . Among the CQ derivatives, primaquine is the only 8-aminoquinoline and was synthesized in the 1950s. Apart from the others, it potentially attacks the liver stages of P. vivax and P. ovale (5) . Mefloquine was screened after the emergence of CQ-resistant Plasmodium strains as antimalarial drug of choice by the Walter Reed Army Institute of Research (WRAIR) in the 1980s. It belongs to the class of amino alcohols and is, like ADQ, still in use, as a partner drug for ACTs. Further, mefloquine is still effective against CQ-resistant strains in which an increased expression of PfEXP1 upon mefloquine treatment (14, 5, 2) . Lumefantrine and Halofantrine also belong to the amino alcohol class of CQ analogues. Lumefantrine is used in combination with artemether in ACTs while Halofantrine is only used in rare cases, due to its high toxicity (16, 5) . Piperaquine was first synthesized in the 1980s as another bis-4-aminoquinoline analogue of CQ, linking together two quinoline molecules by their piperazine rings. This was thought to increase the positive charge and generate a bulky molecule that gets entrapped in the DV more efficiently (5) . (Plasmodium falciparum multidrug resistance-associated protein) and PfCRT (Plasmodium falciparum Chloroquine Resistance Transporters), respectively (21) . PfCRT was identified in 2000 to be one of the main driving forces of CQ resistance (20) . Its underlying gene pfcrt is highly polymorphic, with up to 20 codon variations known leading to altered amino acid sequences (22) . The most important mutation conferring CQ resistance is K76T, despite the inability to confer resistance alone. According to the charged drug leak hypothesis, though, the K76T mutation introduces with threonine and uncharged amino acid, removing a positive charge (carried by lysine), which allows the double positively charged CQ to exit down its concentration gradient (23) . Apart from its role in resistance mechanism, field studies corroborate the significance if K76T Accepted Article linked to further genes (26) . To complicate the molecular interplay even more, clinical studies revealed that mutations in PfCRT also influence the expression of up to 45 unrelated genes, whose role in the overall resistance could not be determined yet (27) . This might be the parasite's response to cover the fitness loss accompanied by PfCRT mutations (20) . 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 any efficient and safe treatment. The antimalarials CQ and its analogue HCQ have been tested for COVID-19 treatment. The first evidence that they might present effects anti-SARS-CoV-2 came from an in vitro assay (6) . Since then, several mechanistic studies and clinical trials have been performed around the world. In mechanistic studies in mammalian cells infected with different virus, CQ has presented several effects, including prevention of autophagy (28) , neutralization of acidic compartments such as lysosomes and endosomes, diminished endocytosis (by reducing PICALM expression) (29) , and acting as zinc ionophore facilitating extracellular zinc influx, which inhibits RNA polymerase (30) . Another mechanism may involve inhibition of virion assembly in endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-like structures (29) . In fact, in the past, CQ was tested against several viruses, including the coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle Eastern respiratory syndrome (MERS), and demonstrated important antiviral effects in vitro (31, 32, 33) . However, until today, no therapeutic effects have been observed in humans (31) . angiotensin-converting enzyme (ACE) converting Ang I into angiotensin II (Ang II), whose actions are mediated by Ang II receptor type 1 (AT1R) and Ang II receptor type 2 (AT2R); (III) angiotensin-converting enzyme 2 (ACE2), which hydrolyzes Ang II into Angiotensin-(1-7) (Ang 1-7) that exerts its biological function through the Mas receptor (Mas R) (58, 60) . Then, the activity of ACE elevates Ang II concentration, whilst ACE2 catalyzes the cleavage of Ang II into Ang 1-7, characterizing the pressor axis (ACE/Ang II/AT1R) and the depressor axis (ACE2/Ang 1-7/Mas R) (61), respectively. The alterations of activity and/or expression of one of these components cause an imbalance of RAS, hence, inducing cardiorespiratory problems. This article is protected by copyright. All rights reserved. ACE2, one of the main components of RAS, is also the invasion receptor for SARS-CoV-2, which bound to this enzyme enters the cell mainly through endocytosis, promoting loss of ACE2 function (62, 63) . Low ACE2 activity increases the ACE1/Ang II/AT1R pressor axis at the expense of the depressor ACE2/Ang1-7/Mas R axis, rising the concentration of Ang II and reducing the Ang 1-7 concentration. Ang II binding to the AT1R stimulates blood pressure increase, vascular permeability, inflammatory cells into tissues and cytokine production (64) . Furthermore, activation of NAD(P)H oxidase, stimulated by AT1R activation, produces reactive oxygen species, mitochondrial dysfunction, and cellular injury (65, 66 This article is protected by copyright. All rights reserved. However, up to now there is no favorable scientific evidence to support the use of any dose of CQ and HCQ in patients with COVID-19 (57; 77). In contrast, there are studies that suggest potential harm of patients infected with SARS-CoV-2 by the HCQ treatment, which may be associated with a significant occurrence of ventricular arrhythmias (78) and increased risk of QT prolongation (78, 79). ACE2 is expressed in similar quantities at the cell surface when treated with CQ, however, the impaired glycosylation might reduce ACE2 activity (80) . Although enzyme activity is not relevant for virus infection, it is extremely important for Ang II conversion into Ang 1-7 that is a physiological antiarrhythmic agent (72) . COVID-19 patients present downregulation of ACE2 in plasmatic membrane by SARS-CoV-2 interaction. We hypothesized that loss of ACE2 functions corroborates to CQ-impaired ACE2 glycosylation, leading to arrhythmia, increase of oxidative stress, vascular permeability and fibrosis, as well as proinflammatory cytokine productions ( Figure 2 ). All these consequences may be attributed to the imbalance of RAS, due to the increase of pressor axis, aggravating ARDS and elevating death risk in COVID-19 patients. CQ and HCQ pharmacokinetics shows large distribution together with slow elimination from the body, enabling toxic effects of this drug (34) . HCQ has one hydroxyl group more than CQ and is associated with a lower incidence of adverse effects with chronic use (81 Cytometry applications focus at the investigation of virus-cell surface interactions as well as determination of viral load to study the efficiencies of drug and vaccine candidates. Effects of CQ and derivates on the endosomal pH possibly can also be evaluated using imaging with pH-sensitive fluorescence dyes coupled to transferrin. Endocytosis of the complex formed by transferrin and its receptor and subsequent endocytic trafficking (101) will take the dye into the endosomes, enabling pH measurements in this organelle. In this context, endosome pH measurements, following conjugation of both rhodamine and fluorescein can be performed, by using flow or imaging cytometry, to determine the ratio of This article is protected by copyright. All rights reserved. Accepted Article pH-sensitive fluorescein over pH-insensitive rhodamine fluorescence emissions (102) . Another strategy of using nanoparticles for delivery of pH-sensitive fluorophores into endosomes was described by Benjaminsen and co-workers (103) . 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