key: cord-0700715-ad5b7h34
authors: Yuen, Chun‐Kit; Wong, Wan‐Man; Mak, Long‐Fung; Wang, Xiaohui; Chu, Hin; Yuen, Kwok‐Yung; Kok, Kin‐Hang
title: Suppression of SARS‐CoV‐2 infection in ex‐vivo human lung tissues by targeting class III phosphoinositide 3‐kinase
date: 2020-10-30
journal: J Med Virol
DOI: 10.1002/jmv.26583
sha: 5eb4905ebdc596a7830df332b01055267eaea011
doc_id: 700715
cord_uid: ad5b7h34

The novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) emerged at the end of 2019 and caused the coronavirus disease 19 (COVID‐19) pandemic due to its high transmissibility and early immunosuppression. Previous studies on other betacoronaviruses suggested that betacoronavirus infection is associated with the host autophagy pathway. However, it is unclear whether any components of autophagy or virophagy can be therapeutic targets for COVID‐19 treatment. In this report, we examined the antiviral effect of four well‐characterized small molecule inhibitors that target the key cellular factors involved in key steps of the autophagy pathway. They include small molecules targeting the ULK1/Atg1 complex involved in the induction stage of autophagy (ULK1 inhibitor SBI0206965), the ATG14/Beclin1/VPS34 complex involved in the nucleation step of autophagy (class III PI3‐kinase inhibitor VPS34‐IN1), and a widely‐used autophagy inhibitor that persistently inhibits class I and temporary inhibits class III PI3‐kinase (3‐MA) and a clinically approved autophagy inhibitor that suppresses autophagy by inhibiting lysosomal acidification and prevents the formation of autophagolysosome (HCQ). Surprisingly, not all the tested autophagy inhibitors suppressed SARS‐CoV‐2 infection. We showed that inhibition of class III PI3‐kinase involved in the initiation step of both canonical and noncanonical autophagy potently suppressed SARS‐CoV‐2 at a nano‐molar level. In addition, this specific kinase inhibitor VPS34‐IN1, and its bioavailable analogue VVPS34‐IN1, potently inhibited SARS‐CoV‐2 infection in ex vivo human lung tissues. Taken together, class III PI3‐kinase may be a possible target for COVID‐19 therapeutic development.

belongs to the subgenus Merbecovirus. 1, 2 It is alarming that SARS-CoV-2 that causes the coronavirus disease pandemic is already the third highly pathogenic human coronavirus since the first appearance of SARS-CoV in 2003. More understanding of the virology of coronaviruses is urgently needed for combating the COVID-19 pandemic and the possible upcoming coronavirus diseases.

Autophagy is an essential cellular pathway important for the clearance and recycling of intracellular materials. The activation of autophagy is induced by metabolic stress including nutrient deprivation and hypoxia, which causes inhibition of the autophagy negative regulator mammalian target of rapamycin (mTOR). 3 LC3-II and membrane elongation of the phagophore. The phagophore eventually seals to form the autophagosome, which then fuses with the lysosome for breaking down the enclosed content. It is noteworthy that autophagy, apart from the recycling of useful materials, is also an important mechanism for the elimination of intracellular pathogens including viruses, a process termed virophagy. 5, 6 Although autophagy is involved in the clearance of invading pathogens, some viruses are able to evade, and even benefit from autophagy. [5] [6] [7] [8] [9] It has been speculated that coronavirus replication is associated with autophagy, although the exact interaction is still poorly understood. Coronavirus infection causes the formation of numerous membranous structures including double-membrane vesicles (DMVs), which is coincidentally a hallmark of autophagy. [10] [11] [12] Evidence, although still debatable, showed that coronavirus nsp6 can mediate the formation of DMVs. [13] [14] [15] [16] Moreover, SARS-CoV nsp6 has been reported to partially co-localize with LC3 in DMVs. 17 In view of the implied relationship between autophagy and coronavirus, previous studies have attempted to elucidate the possibility of inhibiting coronavirus infection by manipulating the autophagy pathway. 18 Nonetheless, contrasting results were observed across studies using various gain-of-function and loss-of-function approaches. 18, 19 On one hand, this might be due to discrepancies between the systems used in these studies, it is also possible that coronaviruses may take advantage of only particular component(s) of the autophagy pathway instead of the entire autophagy machinery. It is therefore worthwhile to dissect the molecular interactions between coronavirus and autophagy.

In view of the association between autophagy and coronavirus infection, we speculated whether suppression of coronavirus replication could be attained by interrupting the autophagy pathway. 

Cells seeded on chamber slides were fixed with 4% paraformaldehyde, NP-40 permeabilized, and blocked with 5% normal donkey serum (Jackson ImmunoResearch). Viral NP proteins were stained using our in-house mouse anti-NP antibody, and then Alexa Flour-488 conjugated donkey anti-mouse immunoglobulin G (Abcam). The immunofluorescence signals were detected using a Carl Zeiss LSM 880 confocal microscope (Zeiss).

Cytotoxicity was assayed by an LDH-Glo Cytotoxicity Assay kit (Promega). Culture supernatant diluted 1:100 with lactate dehydrogenase (LDH) storage buffer was mixed and incubated with an equal volume of LDH detection reagent. After 60 min of incubation at room temperature, luciferase activity was measured using a multi-well plate reader (Beckman Coulter).

Human lung tissues were obtained with written consent from patients undergoing surgical operations at the Queen Mary Hospital, Hong Kong. This study has been approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW13-364). The culture method has been described previously. 20 Briefly, freshly obtained tissues were dissected into small cubes and maintained in a basal medium of 

Autophagy is a multi-step mechanism for clearing and recycling of cellular materials ( Figure 1A) . Although it has been implicated that autophagy plays an intimate role with coronavirus infection, the interplay between autophagy and SARS-CoV-2 infection is poorly understood. 18, 19 To dissect the involvement of autophagy during in SARS-CoV-2 replication ( Figure 1C ). 3-MA, which mainly inhibits class I PI3-kinase, caused minimal inhibition ( Figure 1D ). HCQ, consistent with previous reports, 21 infection. 20 We, therefore, sought to examine the inhibitory effect of Vps34-IN1 in noncancerous lung tissues freshly isolated from human patients ( Figure 3A,B) . After dissection, the lung tissues were Here, we further showed that VVps34-IN1 was also able to impede SARS-CoV-2 infection in our ex-vivo human lung tissue culture model ( Figure 3D) . Taken together, we demonstrated in this study that the class III PI3-kinase Vps34 might be a host target hijacked by SARS- Vps34-IN1 is a potent inhibitor of class III PI3-K Vps34 with high specificity. It was first discovered by Alessi's group at the University of Dundee and has been clearly demonstrated no significant nonspecific inhibition on 340 protein kinases and 25 lipid kinases including all class I and class II PI3Ks. 25 It is noted that the Vps34-IN1

has been well-characterized not only in in vitro assays 25 but also in a subsequent in vivo animal study. 26 This potent and specific Vps34-IN1 inhibitor has also been applied for specific inhibition of Vps34 in numerous studies. [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] Therefore, the potential off-target effect of class III PI3-K inhibitor, Vps34-IN1, used in this study will be minimal.

In addition, another group studying the chemistry optimization of the Vps34-IN1 ( Figure 3E) found that its derivative VVps34-IN1 (also known as compound 19; Figure 3F ) maintains the high selectivity and in vitro potency. 24 The compound 19 was further characterized in C57BL/6 and nude mice, demonstrating its good bioavailability and in vivo inhibition of autophagy. However, no direct comparison of Vps34-IN1 and VVps34-IN1 was included in that report. 24 As we found that SARS-CoV-2 inhibition by Vps34-IN1 was slightly better than that of VVps34-IN1 in ex vivo human lung tissues ( Figure 3C,D) , it will be of great interest to determine the pharmacokinetic profile of Vps34-IN1 in animal models such as hACE2-transgenic mice and hamsters.

Next, the inhibition of SARS-CoV-2 infection was further con- 

Coronaviridae Study Group of the International Committee on Taxonomy of V. The species severe acute respiratory syndromerelated coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Novel human coronavirus (SARS-CoV-2): a lesson from animal coronaviruses

Autophagy as a regulated pathway of cellular degradation

Endoplasmic reticulum stress triggers autophagy

Macroautophagy-friend or foe of viral replication?

Autophagy and viruses: adversaries or allies?

Autophagy and autophagy-related proteins in the immune system

Viruses and the autophagy machinery

The autophagic machinery in viral exocytosis

Autophagy during viral infection-a double-edged sword

Autophagy and viral infection

Manipulation of autophagy by (+) RNA viruses

SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro

SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum

Detection of nonstructural protein 6 in murine coronavirus-infected cells and analysis of the transmembrane topology by using bioinformatics and molecular approaches

Expression and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral RNA replication

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate

Canonical and noncanonical autophagy as potential targets for COVID-19

Targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in COVID-19

Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19

Autophagy as an emerging target for COVID-19: lessons from an old friend, chloroquine

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro

Potent, selective, and orally bioavailable inhibitors of VPS34 provide chemical tools to modulate autophagy in vivo

Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase

Vps34 PI 3-kinase inactivation enhances insulin sensitivity through reprogramming of mitochondrial metabolism

Defining the subcellular distribution and metabolic channeling of phosphatidylinositol

PIK3C3 regulates the expansion of liver CSCs and PIK3C3 inhibition counteracts liver cancer stem cell activity induced by PI3K inhibitor

The identification and characterisation of autophagy inhibitors from the published kinase inhibitor sets

Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes

The ER-localized transmembrane protein TMEM39A/SUSR2 regulates autophagy by controlling the trafficking of the PtdIns(4)P phosphatase SAC1

Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling

Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion

Feedback activation of SGK3 and AKT contributes to rapamycin resistance by reactivating mTORC1/4EBP1 axis via TSC2 in breast cancer

Her2-targeted therapy induces autophagy in esophageal adenocarcinoma cells

Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature

Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R

WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome

Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles

Suppression of SARS-CoV-2 infection in ex-vivo human lung tissues by targeting class III phosphoinositide 3-kinase