Hydroxychloroquine: mechanism of action inhibiting SARS-CoV2 entry

Abstract

Background: SARS-coronavirus 2 (SARS-CoV-2) is currently causing a worldwide pandemic. Potential drugs identified for the treatment of SARS-CoV-2 infection include chloroquine (CQ), its derivative hydroxychloroquine (HCQ), and the anesthetic propofol. Their mechanism of action in SARS-CoV-2 infection is poorly understood. Recently, anesthetics, both general and local, were shown to disrupt ordered lipid domains. These same lipid domains recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to an endocytic entry point and their disruption by cholesterol depletion decreases ACE2 recruitment and viral entry. Methods: Viral entry was determined using a SARS-CoV-2 pseudovirus (SARS2-PV) and a luciferase reporter gene expressed by the virus after treatment of the cells with 50 micromolar propofol, tetracaine, HCQ, and erythromycin. HCQ disruption of monosialotetrahexosylganglioside1 (GM1) lipid rafts, phosphatidylinositol 4,5-bisphosphate (PIP2) domains, and ACE2 receptor at nanoscale distances was monitored by direct stochastic reconstruction microscopy (dSTORM). Cells were fixed, permeabilized, and then labeled with either fluorescent cholera toxin B (CTxB) or antibody and then fixed again prior to imaging. Cluster analysis of dSTORM images was used to determine size and number and cross pair correlation was used to determine trafficking of endogenously expressed ACE2 in and out of lipid domains. Results: Propofol, tetracaine, and HCQ inhibit SARS2-PV viral entry. HCQ directly perturbs both GM1 lipid rafts and PIP2 domains. GM1 rafts increased in size and number similar to anesthetic disruption of lipid rafts; PIP2 domains decreased in size and number. HCQ blocked both GM1 and PIP2 domains ability to attract and cluster ACE2. Conclusions: We conclude HCQ is an anesthetic-like compound that disrupts GM1 lipid rafts similar to propofol and other local or general anesthetics. Furthermore, we conclude disruption of GM1 raft function, and not the concentration of GM1 raft molecules, governs the antiviral properties of HCQ. HCQ disruption of the membrane appears to also disrupt the production of host defense peptide, hence an antimicrobial such as erythromycin could be an important combined treatment. Nonetheless erythromycin has anti-SARS-CoV-2 activity and may combine with HCQ to reduce infection.

The interplay between emerging human coronavirus infections and autophagy

“…As a new emerging hCoV, it is largely unknown how SARS-CoV-2 is targeted by or manipulates autophagy. Accumulating evidence suggests that SARS-CoV-2 infection may perturb the autophagic process ( localization [70,71]. It is doubtful that their putative effects on autophagy inhibition are necessarily causal for their anti-SARS-CoV-2 activity in specific cells.…”

Pathogenesis‐directed therapy of 2019 novel coronavirus disease

“…In particular, the spike (S) protein cannot be cleaved by host cell proteases such as cathepsins because these proteases are inhibited due to the increased pH within the lysosome 297 . In addition, chloroquine and/or hydroxychloroquine inhibit sialic acid synthesis by inhibiting quinine reductase‐2 298 . Chloroquine and/or hydroxychloroquine reduce glycosylation of ACE2 and thus interfere with binding of the spike protein of SARS‐CoV‐2 288,299 .…”

“…297 Moreover, in the lung, chloroquine and/or hydroxychloroquine are able to diffuse across cell membranes to acidic lysosomal vesicles in the cytoplasm and become trapped in these lysosomes when pronated. 298 The chloroquine and/or hydroxychloroquine thus increases the acidic environment within the lysosome such that the cell cannot perform certain functions such as endocytosis, exosome release, or phagolysosomal fusion. 297,298 In particular, the spike (S) protein cannot be cleaved by host cell proteases such as cathepsins because these proteases are inhibited due to the increased pH within the lysosome.…”

“…298 The chloroquine and/or hydroxychloroquine thus increases the acidic environment within the lysosome such that the cell cannot perform certain functions such as endocytosis, exosome release, or phagolysosomal fusion. 297,298 In particular, the spike (S) protein cannot be cleaved by host cell proteases such as cathepsins because these proteases are inhibited due to the increased pH within the lysosome. 297 In addition, chloroquine and/or hydroxychloroquine inhibit sialic acid synthesis by inhibiting quinine reductase-2.…”

“…297 In addition, chloroquine and/or hydroxychloroquine inhibit sialic acid synthesis by inhibiting quinine reductase-2. 298 Chloroquine and/or hydroxychloroquine reduce glycosylation of ACE2 and thus interfere with binding of the spike protein of SARS-CoV-2. 288,299 Finally, hydroxychloroquine appears to disrupt lipid rafts and thus interferes with ACE2 localization.…”