Short linear motif candidates in the cell entry system used by

SARS-CoV-2 and their potential therapeutic implications

Bálint Mészáros1 (ORCID: 0000-0003-0919-4449)

Hugo Sámano-Sánchez1 (ORCID: 0000-0003-4744-4787)

Jesús Alvarado-Valverde1,2 (ORCID: 0000-0003-0752-1151)

Jelena Čalyševa1,2 (ORCID: 0000-0003-1047-4157)

Elizabeth Martínez-Pérez1,3 (ORCID: 0000-0003-4411-976X)

Renato Alves1 (ORCID: 0000-0002-7212-0234)

Manjeet Kumar1 (ORCID: 0000-0002-3004-2151) Email: manjeet.kumar@embl.de

Friedrich Rippmann4 (ORCID: 0000-0002-4604-9251)

Lucía B. Chemes5 (ORCID: 0000-0003-0192-9906) Email: lchemes@iib.unsam.edu.ar

Toby J. Gibson1 (ORCID: 0000-0003-0657-5166) Email: toby.gibson@embl.de

1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany 2Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences 3Laboratorio de bioinformática estructural, Fundación Instituto Leloir, C1405BWE, Buenos Aires, Argentina 4Computational Chemistry & Biology, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany 5Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín. Av. 25 de Mayo y Francia S/N,

CP1650 Buenos Aires, Argentina

Keywords: COVID-19 / Coronavirus / Receptor-Mediated Endocytosis / Autophagy

/ SLiM / ELM Server / Host-Pathogen Interaction / ACE2 / Integrins / Integrin αvβ3 /

Integrin α5β1 / Host Targeted Therapy / Tyrosine Kinase

1

Abstract

The primary cell surface receptor for SARS-CoV-2 is the angiotensin-converting

enzyme 2 (ACE2). Recently it has been noticed that the viral Spike protein has an RGD

motif, suggesting that cell surface integrins may be co-receptors. We examined the

sequences of ACE2 and integrins with the Eukaryotic Linear Motif resource, ELM, and

were presented with candidate short linear motifs (SLiMs) in their short, unstructured,

cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy,

cytoskeleton and cell signalling. These SLiM candidates are highly conserved in

vertebrates. They suggest potential interactions with the AP2 µ2 subunit as well as I-BAR,

LC3, PDZ, PTB and SH2 domains found in signalling and regulatory proteins present in

epithelial lung cells. Several motifs overlap in the tail sequences, suggesting that they

may act as molecular switches, often involving tyrosine phosphorylation status. Candidate

LIR motifs are present in the tails of ACE2 and integrin β3, suggesting that these proteins

can directly recruit autophagy components. We also noticed that the extracellular part of

ACE2 has a conserved MIDAS structural motif, which are commonly used by β integrins

for ligand binding, potentially supporting the proposal that integrins and ACE2 share

common ligands. The findings presented here identify several molecular links and

testable hypotheses that might help uncover the mechanisms of SARS-CoV-2

attachment, entry and replication, and strengthen the possibility that it might be possible

to develop host-directed therapies to dampen the efficiency of viral entry and hamper

disease progression. The strong sequence conservation means that these putative SLiMs

are good candidates: Nevertheless, SLiMs must always be validated by experimentation

before they can be stated to be functional.

Introduction

The COVID-19 pandemic is caused by severe acute respiratory syndrome

coronavirus 2 (SARS-CoV-2), an enveloped, single-stranded RNA virus. It had infected

more than two and a half million people and caused circa 170,000 deaths globally by mid-

April 2020. SARS-CoV-2 belongs to the Coronaviridae family, whose members are

common human pathogens responsible for the common cold, as well as for some

emerging severe respiratory diseases. Among them are the SARS-CoV and the Middle

East respiratory syndrome coronavirus (MERS-CoV), the former of which caused over

8,000 cases in 2003 with a fatality rate of ~10%, and the latter caused about 2,500

infections in 2012 with a fatality rate of 37% (de Wit et al., 2016). Another coronavirus,

infectious bronchitis virus (IBV), infects birds and has been used as a model in

coronavirus research (Sisk et al., 2018). SARS-CoV-2, like SARS-CoV (Li et al., 2003),

uses the angiotensin converting enzyme 2 (ACE2) as a receptor to attach to the host cells

(Hoffmann et al., 2020; Wrapp et al., 2020; P. Zhou et al., 2020). ACE2 is a single pass

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type I membrane protein with a short cytosolic C-terminal region for which the

functionality, however, is mostly unknown.

Earlier results clearly show that the SARS-CoV-2 receptor binding domain (RBD) of

the Spike protein interacts with ACE2 for cellular entry. However, type II alveolar cells

(AT2) – the main targets of SARS-CoV-2 in the lung (Zou et al., 2020) – express a

relatively low amount of ACE2, which points to the existence of co-receptors being

targeted by the virus in parallel. One such candidate are integrins that bind a large variety

of ligands harbouring an RGD sequence motif, as recent analysis of the RBD identified a

possibly functional RGD motif (Sigrist et al., 2020).

Integrins are major cell attachment receptors, which are known to be targeted by a

range of viruses - including HIV, herpes simplex virus-2, Epstein-Barr virus (EBV) and the

foot and mouth virus - for cell entry and activation of linked intracellular pathways (Hussein

et al., 2015; Stewart and Nemerow, 2007; Triantafilou et al., 2001). Integrins are special

types of receptors, as they propagate signals in both directions; extracellular ligands can

induce cytoplasmic pathway activation, but intracellular binding on the cytosolic tails can

influence the structure of the ectodomains and hence ligand-binding affinity. The

complexity of integrin signalling stems in the dimeric structure of integrins, as they are

composed of two subunits, α and β. For the RGD-binding integrins, the ligand binding

surface lies at the interface of the two integrin subunits with both subunits making contacts

with the ligand. These RGD motifs are recognized by at least 8 out of the 24 human

integrins, and the flanking residues next to the core RGD motif are known to play a huge

role in selectivity (Kapp et al., 2017). Several viral proteins contain RGD (or RGD-like)

short linear motifs (SLiMs) for integrin modulation; and in addition, some viruses can not

only use integrins on the host cell surface, but HIV and SIV can also incorporate integrins

into their own membranes for mediating interactions with the host (Guzzo et al., 2017).

Therefore, integrins can potentially be targeted at both the extracellular and the

intracellular side to combat pathogenic hijacking.

Viruses, as obligate intracellular entities, need to interfere with major cellular

processes like vesicular trafficking, cell cycle, cellular transport, protein degradation or

signal transduction to satisfy their replication, enzymatic, metabolic and transport needs

(Davey et al., 2011). To achieve this, a large number of host processes are hijacked using

SLiMs often located in intrinsically disordered regions to establish protein-protein

interactions with host proteins or undergo post-translational modifications, like tyrosine

phosphorylation. For example, cellular signalling relies heavily on the use of SLiMs (Van

Roey et al., 2013, 2012). The low affinity and cooperativity of SLiM-based molecular

processes allow reversible and transient interactions that can work as switches between

distinct functional states and are regulated both in time and space (Gibson, 2009; Scott

and Pawson, 2009). Conditional switching of SLiMs, for example through

phosphorylation, can induce the exchange of binding partners for a protein, thus

mediating molecular decision-making in response to signals reporting on the cell state

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(Van Roey et al., 2012). The central resource for SLiMs is the Eukaryotic Linear Motif

database (ELM; http://elm.eu.org/), also serving as an exploratory server for over 280

manually curated SLiM classes with experimental evidence, each defined by a POSIX

regular expression (Kumar et al., 2020).

As explained above, a major strategy of viruses is to abuse the host system by using

mimics of eukaryotic SLiMs to compete with extracellular or intracellular binding partners

or to sequester host proteins (Davey et al., 2011). This dependence of viruses and many

other pathogens on SLiM-mediated functions suggests that there is an opportunity to drug

the cell systems where these interactions are being hijacked (Sámano-Sánchez and

Gibson, 2020). For example, tyrosine kinase inhibitors, often used in anticancer therapy,

have shown promising coronavirus replication inhibition in infectious cell culture systems

(Coleman et al., 2016; Dong et al., 2020; Shin et al., 2018; Sisk et al., 2018). In the

remainder of the introduction, we will describe some of the major pathways hijacked by

viruses to accomplish cell attachment, entry and replication, which are suggested by our

results to be relevant to SARS-CoV-2 infection.

Receptor Mediated Endocytosis (RME) is a cellular import process triggered by cell

surface receptor proteins, including any cargoes attached to them, in which a large

vesicular structure is assembled entirely through cooperative low affinity interactions of

SLiMs and phospholipid head groups with their globular protein domain partners. The

vesicles are strong and stable, yet flexible and dynamically assembled and disassembled.

The external triggering of surface receptors (many of which have the YxxPhi or NPxY

tyrosine sorting motifs) is transmitted across the plasma membrane, inducing local

enzymatic modification of lipid head-groups from PI4P to (PI(4,5)P2) by the PIPK1 kinase.

The local enrichment of (PI(4,5)P2) enables binding of domains such as ENTH in Epsins

which can begin to curve the membrane and assemble clathrin cages using their Clathrin

Box motif and also attract additional adapter proteins via yet more SLiMs. In turn,

additional sets of SLiM-bearing proteins stimulate the actin filament formation and

attachment, necessary to fold and pull the invagination into the cytosol. Later, Dynamin

binds directly to (PI(4,5)P2) on the membrane to complete the scission process. Once in

the cytosol, the clathrin-coated vesicles are soon dismantled and the contents are

included into the early endosomes. (For recent reviews of the process, see (Haucke and

Kozlov, 2018; Kaksonen and Roux, 2018; Senju and Lappalainen, 2019). Many viruses

enter the cell via endocytosis, using many different cell surface receptors (Grove and

Marsh, 2011). Viruses such as HIV and Hepatitis C virus depend on the recognition of

more than one receptor for entry, but in many cases the stoichiometry of receptor

engagement is unknown. Coronaviruses can enter cells through different routes that

include receptor mediated endocytosis or fusion at the membrane (de Haan and Rottier,

2006). In the case of SARS-CoV, the main entry route is endocytic, and depends on

endosome acidification (Huang et al., 2006; Simmons et al., 2004). However, protease-

mediated activation of the Spike protein relieves the pH-dependence of viral entry,

4

indicating that acidification is not a requirement per se, but acts by inducing the

endosomal Spike protein cleavage required for viral fusion (Matsuyama et al., 2005;

Simmons et al., 2005). Spike protein cleavage can be done by the transmembrane

protease serine 2 (TMPRSS2) at the cell surface, or by Cathepsin L within endosomes

(de Wilde et al., 2018). The same entry route and proteases are utilized by SARS-CoV-

2, and the main entry route also seems to be endocytic (Hoffmann et al., 2020; Ou et al.,

2020).

Autophagy is an evolutionarily conserved process in eukaryotes with multiple cellular

roles that include the regulation of cellular homeostasis through the catabolism of cell

components, immune development and the host cell response to infection through

pathogen phagocytosis (Deretic and Levine, 2009). Viruses have evolved mechanisms

to block the host cell antiviral response, and can further hijack autophagy components to

promote their survival and replication. This can be done through viral mimicry of host

proteins coordinating autophagy or through the direct inhibition of the host autophagy

machinery (Kudchodkar and Levine, 2009). Coronaviruses (CoVs) exploit the autophagy

machinery through different mechanisms (Cong et al., 2017; Maier and Britton, 2012).

For example, MERS-CoV targets the BECN1 autophagy regulator for degradation,

blocking the fusion of autophagosomes and lysosomes and protecting the virus from

degradation (Gassen et al., 2019). Coronaviruses repurpose cellular membranes to

create double membrane vesicles (DMVs) onto which the replication-transcription

complex (RTC) is assembled, a process that involves recruitment of multiple autophagy

components (Cong et al., 2017; Prentice et al., 2004; V’kovski et al., 2019).

Betacoronavirus mouse hepatitis virus (MHV) RTCs assemble by recruiting LC3-I, a non-

lipidated form of the LC3 autophagy protein (Cong et al., 2017; Reggiori et al., 2010), and

SARS-CoV RTCs also colocalize with LC3 (Prentice et al., 2004). Proximity-based mass

spectrometry on the MHV replication complex further revealed that the RTC environment

repurposes components from the host autophagy, vesicular trafficking and translation

machineries (V’kovski et al., 2019)

In the present work, we identify a set of conserved SLiM candidates in the ACE2 and

integrin proteins, which are likely to act in the cell entry system of SARS-CoV-2. These

motifs can provide molecular links to understand how the virus recognizes target

membranes, enters into cells, and how it repurposes intracellular membrane components

to drive its replication. These molecular links might provide novel clues towards drugging

SARS-CoV-2 infections. We first focus on the extracellular SLiMs, before moving across

the membrane to examine the cytosolic potential of the receptor tails.

5

Extracellular receptor interplay and viral hijacking in the

ACE2/integrin system

Integrins are candidates for acting as co-receptors for SARS-CoV-2 entry

The ability of SARS-CoV-2 RBD to bind integrins via the RGD motif (see Table 1)

has not yet been assessed directly. However, there are several features that make the

Spike-integrin interaction plausible, including sequence- and structure-level information,

expression profiles, the presence of accessory motifs and protein-protein interactions.

Evolution of the RGD motif in Spike receptor binding domain

Aligning close homologues of the RBD from the Coronaviridae family (Figure 1A)

shows that the motif candidate is located in a locally less conserved region, hinting at the

rapid evolvability of the site. Several coronavirus RBDs contain KGD at this site, which is

known to be a lower affinity integrin binding site, first identified in snake venom

disintegrins, such as barbourin (Minoux et al., 2000). A KGD motif residing in the EBV

gH/gL protein has also been shown to be essential for entry into epithelial and B cells

(Chen et al., 2012). Figure 1B shows the tree derived from the Spike protein sequence

alignment, highlighting that the SARS-CoV-2 RGD motif might have evolved from an

earlier KGD motif, and might present a distinct step of adaptation if the motif is indeed an

integrin attachment site.

6

Figure 1: The RGD motif of the SARS-CoV-2 spike protein. A) Multiple sequence alignment of

a part of the SARS-CoV-2 Spike RBD region using 9 homologous sequences from closely related

viruses and showing the conservation of the RGD motif. Red box marks the conservation range

of the [RK]GD motif. Viruses' names, UniProt IDs and sequence numberings are listed on the left

side of the alignment. The location of the region shown in the alignment is indicated in a

representative diagram of the Spike protein. B) Guide neighbour joining tree of the multiple

sequence alignment with the sequences containing the potential low- and high-affinity integrin

binding motifs (KGD and RGD) shown in orange and red boxes, respectively. C) Structure of the

SARS-CoV-2 RBD as seen in the ACE2-bound form (PDB ID:6m17). The RGD motif is shown in

red sticks. Regions in direct contact with ACE2 are shown in blue. Residues with missing atomic

coordinates (indicating flexibility) in the unbound trimeric Spike protein structures (PDB IDs: 6vsb,

6vxx and 6vyb) are shown in transparency. Alignment and tree prepared in Jalview (Waterhouse

et al., 2009) with Clustal colours. Structure was visualized using UCSF Chimera (Pettersen et al.,

2004).

The RBD-integrin interaction is structurally feasible

At the time of reporting the RGD motif, no SARS-CoV-2 Spike structures were

available, so the authors used structural homology modelling to determine that the RGD

motif is surface accessible (Sigrist et al., 2020). Since then, several RBD structures have

been determined, both in unbound (Walls et al., 2020; Wrapp et al., 2020) and ACE2

complexed forms using electron microscopy (Yan et al., 2020) and X-ray diffraction (Lan

et al., 2020), allowing for the direct structural assessment of the possibility of binding to

integrins. Figure 1C shows the RGD motif together with the residues involved in direct

binding of ACE2. The RGD motif is located in the vicinity of the ACE2 binding site,

however, based on uncomplexed structures of the RBD, the residues that surround the

RGD site are flexible. This indicates that while ACE2 binding blocks the RGD motif,

without ACE2 the RGD is surface accessible and interaction with integrins are not

sterically blocked.

As Spike exists as a trimer on the virion surface, different copies of the RBD can in

theory interact with ACE2 and integrins at the same time. There is no solved complex

structure of the ACE2-integrin complex. However, further structural consideration may

indicate whether the Spike-ACE2 and the Spike-integrin interactions can coexist. The

ectodomains of both ACE2 and integrins in the open conformation are roughly the same

size measured from the membrane, being in the 150-200 Å range (based on available

structures PDB:6m17 (Yan et al., 2020) and PDB:3ije (Xiong et al., 2009)). This means

that the RGD-binding site of integrins and the RBD binding regions of ACE2 are relatively

close in space. In addition, in the more open ‘up’ conformation of the RBDs, the distance

between pairs of RBDs is about 100 Å (based on the structure PDB:6vsb reported in

(Wrapp et al., 2020)), which is probably larger than the distance between the ACE2

binding region and the integrin ligand binding site, estimated from the individual integrin

and ACE2 structures.

7

α5β1 and αvβ3 integrins are potential targets for SARS-CoV-2 RBD

The RGD motif is recognized by several integrins, and specificity is determined

mostly by the flanking residues around the core motif. As evidenced by crystallized

integrin dimer:ligand complexes, the residue preceding RGD is in contact with the α

subunit, while the residue after the core motif interacts with the β subunit. The immediate

context of the SARS-CoV-2 RBD motif is 402-IRGDE-406, which can give an indication

about possible integrin targets. IRGD can be found in several native integrin-binding

partners, including FREM1 (Kiyozumi et al., 2012), MFAP4 (Pilecki et al., 2015) and

IGFBP1/2 (Cavaillé et al., 2006; Wang et al., 2006). These extracellular matrix proteins

target integrins with αv, α5 and α8 subunits. RGDE is present in the native human integrin

ligands TGF-β1, osteolectin, collagen α-1(VI) chain, PSBG-9 and polydom, and in vitro

and in vivo binding studies on the specificity profiles of these proteins (Cescon et al.,

2015; Rattila et al., 2019; Sato-Nishiuchi et al., 2012; Shanley et al., 2013; Shen et al.,

2019; Tumbarello et al., 2012) highlighted a post-RGD Glu to be efficient in binding to β1,

β2 and β3 integrin subunits. Correlating these preferences with possible α and β integrin

subunit pairings points to the most likely candidate target integrins for SARS-CoV-2 being

αvβ1, αvβ3, α5β1 and α8β1.

Motif-domain interactions are typically under heavy spatio-temporal regulation.

Hence the SARS-CoV-2 RBD-integrin binding can only occur if the possible target

integrins are expressed on AT2 cells. Both α5β1 (Pilewski et al., 1997) and αvβ3

(Caccavari et al., 2010; Nakamura et al., 2002; Singh et al., 2000) have been observed

in lung epithelial cells and have been shown to be implicated in disease emergence and

progression, including emphysema, non-small cell lung cancer and mechanical injury of

the lungs (Teoh et al., 2015), marking these two integrins as prime suspects for targets

of the RBD.

Native ACE2/integrin interplay

It has been shown that in heart tissues, ACE2 is able to bind the β1 subunit of

integrins in an RGD-independent manner, enhancing cell adhesion and regulating integrin

signalling via the focal adhesion kinase (FAK) (Clarke et al., 2012). The RGD

independence of the interaction means that while ACE2 and integrins are in complex, the

RGD binding site of the integrin is unoccupied, further supporting a potential

integrin:ACE2:Spike ternary interaction.

Apart from the known interplay between ACE2 and integrins, there are additional

features that indicate an even tighter crosstalk between the two receptors. RGD-mediated

binding to integrins is metal mediated (via divalent cations like Mg2+ or Mn2+), and all

integrins have a so-called ‘metal ion-dependent adhesion site’ (MIDAS) motif (DxSxS)

(Lee et al., 1995). The integrin MIDAS structural motif is located near the ligand binding

site on the β subunit and is essential for binding, as sidechains belonging to the motif and

an acidic residue from the ligand coordinate the metal ion together (Zhang and Chen,

8

2012). ACE2 also has a similar DxSxS motif (see Table 1), that might facilitate interactions

with ligands that are recognized by integrins, possibly creating an overlap between the

ligand binding profiles and regulation of the two receptors. In the known structures where

Spike is bound to ACE2 the RGD motif is not in contact with the ACE2 MIDAS (Yan et

al., 2020). However, the MIDAS motif is highly conserved across species (see Figure 2),

and surface exposed. Consequently it may still be involved in mediating an interaction

with an RGD-like motif, potentially serving as a parallel mechanism for binding the Spike

protein.

Figure 2: Alignment of ACE2 illustrating conservation of the MIDAS motif. Multiple sequence

alignment of a part of the ACE2 extracellular region using 21 homologous sequences from

different vertebrates (mammals, birds and reptiles) and showing the conservation of the Dx[ST]xS

motif (main residues displayed above). Red boxes mark the conservation range of the MIDAS

motif in all sequences. Organism names, UniProt IDs and sequence numberings are listed on the

left side of the alignment. The location of the region shown in the alignment is indicated in a

representative diagram of the ACE2 protein. Figure prepared with Jalview using Clustal colours.

Protease usage by SARS-CoV-2

ACE2 and several integrin subunits require proteolytic cleavage for biological activity

(see Table 1). Integrin subunits α3, α5, α6 and αv are cleaved by furin or furin-like

proprotein convertases (PCs) during maturation (Lehmann et al., 1996; Lissitzky et al.,

2000). Nearly all PCs contain an RGD motif, and while its role in integrin binding is not

clear, the motif has been shown to be required for proper functioning for several PCs (Lou

et al., 2007; Lusson et al., 1997; Rovère et al., 1999). The SARS-CoV-2 Spike protein

contains a furin-like cleavage site that is absent from closely related Spike proteins,

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immediately following the RBD (Coutard et al., 2020). This cleavage results in increased

virulence, possibly by allowing greater movement of the RBD potentially aiding in

exploring a larger space around the RBD-binding region of ACE2.

ACE2 is cleaved by several proteases, including TMPRSS2 (Heurich et al., 2014).

ACE2 binds to TMPRSS2, forming a receptor-protease complex (Shulla et al., 2011).

TMPRSS2 is also known to cleave the Spike protein of both SARS-CoV and MERS-CoV

(Iwata-Yoshikawa et al., 2019), augmenting their entry into the host cell (Heurich et al.,

2014). Furthermore, similar results have been found for SARS-CoV-2, where TMPRSS2

was found to be fundamental for cell entry (Hoffmann et al., 2020). This dependence is

most probably two-fold: on one hand TMPRSS2 is needed for ACE2 activation, on the

other hand, SARS-CoV-2 Spike protein also contains a TMPRSS2 cleavage site (Meng

et al., 2020).

SLiM candidates in the ACE2 receptor intrinsically disordered

tail The ACE2 sequence (UniProt:ACE2_HUMAN) was entered in the ELM server

(Kumar et al., 2020) and returned several relevant candidate SLiMs in the short cytosolic

C-terminal tail. Because SLiMs are so short, it is difficult to obtain significant results in

sequence searches. Contextual information, including cell compartment localisation and

functional relevance, is important in deciding whether a motif candidate is worth testing

experimentally (Gibson et al., 2015). Furthermore, in intrinsically unstructured protein

sequence, amino acid conservation is indicative of functional interactions. Therefore, an

alignment was prepared of vertebrate ACE2 proteins. All of the detected motif matches

(shown in Table 1 together with potential binding partner domains defined using Pfam (El-

Gebali et al., 2019) and InterPro (Mitchell et al., 2019)) were conserved in mammals, most

were conserved with birds and mammals and some were conserved with extant reptiles

(Figure 3). These groups diverged from one another >300 million years ago (Kemp, 2005)

indicating a strong conservation of all candidate motifs. In addition, the functional contexts

of these motifs are biologically coherent, involving signalling by tyrosine kinases,

endocytosis, autophagy and actin filament induction (Table 1). In the following

subsections we briefly summarise each of the conserved motifs and their possible role in

the viral entry mechanism.

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Figure 3: Alignment of ACE2 illustrating conserved motifs in the cytosolic C-terminal tail

following the transmembrane helix. Multiple sequence alignment of ACE2 transmembrane and

C-terminus regions using 21 homologous sequences from different vertebrates (mammals, birds

and reptiles) and showing their motif conservation. The names (bold) and key residues of the

motifs are displayed above the alignment (ɸ stands for a bulky hydrophobic residue), including a

conserved tyrosine (bold) and excluded positions (red and crossed). Red boxes mark the

conservation range of the PDZ-binding motif (PBM) (all sequences) and NPY motif (in mammals

and birds). Organism names, UniProt IDs and sequence numberings are listed on the left side of

the alignment. The location of the region shown in the alignment is indicated in a representative

diagram of the ACE2 protein. Figure prepared with Jalview using Clustal colours.

The YxxPhi endocytic sorting signal

The YxxPhi motif binds the μ2 subunit (UniProt:AP2M1_HUMAN) of the endocytosis

AP-2 adaptors by β-augmentation (Owen and Evans, 1998). It is found in numerous cell

surface receptors which have intrinsically disordered C-terminal tails (Bonifacino and

Traub, 2003). A small selection is listed in the database entry ELM:TRG_ENDOCYTIC_2,

and while the motif has not been validated in ACE2, it is highly conserved (Figure 3).

When the Tyr is phosphorylated, this motif becomes an SH2 binding site, while in the apo

form it binds the μ2 adapter. Therefore, this motif can operate as a molecular switch. The

residue following the Tyr makes a β-strand interaction and therefore cannot be a proline

(PDB:1bxx). The Phi position requires a bulky hydrophobic residue. The motif pattern can

be represented by the regular expression Y[^P].[LMVIF] and this motif is conserved in

ACE2 from all mammals except monotremes. Thus the mammalian ACE2, which

internalises the coronavirus, has a SLiM candidate for internalisation appropriately

located within its cytosolic tail.

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The NCK SH2 binding motif

The region encompassing the YxxPhi motif overlaps with a Src homology 2 (SH2)

domain binding motif (Figure 3) that is created upon phosphorylation of Tyr 781. SH2

domain binding motifs are characterized by an invariant phosphotyrosine (pY) that is

created following tyrosine kinase activation, and allows binding to more than 100 types of

SH2 domains present in human proteins (Tinti et al., 2013). The pY residue is

accompanied by additional binding determinants that frequently involve hydrophobic

residues at the pY+3 position, but can also involve other combinations, such as Asn at

pY+2 in Grb2-specific SH2 motifs, or hydrophobic residues at pY+4 in STAP-1 SH2 motifs

(Huang et al., 2008; Kaneko et al., 2010). Most motifs are also characterized by the

exclusion of residues at certain positions following the pY, and in general, SH2-binding

motifs show a high degree of cross-specificity (Huang et al., 2008; Liu et al., 2010).

Cell culture infection assays with different Coronaviruses, including SARS-CoV, have

shown susceptibility to tyrosine kinase inhibitors, indicating the involvement of host

tyrosine phosphorylation (Coleman et al., 2016; Dong et al., 2020; Shin et al., 2018; Sisk

et al., 2018). The sequence found in ACE2 (781-YASID-785) best matches the binding

specificity for the SH2 domain present in NCK1/2 proteins, which belong to the Class IA

SH2 domains (Kaneko et al., 2010). Proteins known to contain this motif are listed in entry

ELM:LIG_SH2_NCK1_1. NCK proteins are adaptor proteins that modulate actin

cytoskeleton dynamics (Buday et al., 2002). For example, NCK is recruited to the cell

membrane by the Nephrin protein, following tyrosine signalling that creates several NCK

SH2 binding motifs in Nephrin (Blasutig et al., 2008). Once recruited to the membrane,

NCK activates Wiskott-Aldrich syndrome (WASP) family proteins through the use of a

helical binding motif (ELM:LIG_GBD_CHELIX_1) that relieves WASP autoinhibition and

allows the recruitment of the actin regulatory protein complex Arp2/3, which leads to the

initiation of actin polymerization (Okrut et al., 2015). The NCK binding motif is exploited

by two known human pathogens, namely enteropathogenic Escherichia coli and vaccinia

virus (Frese et al., 2006). The residues present at pY+1, pY+2 and pY+4 rule out that the

ACE2 YASID motif can be a strong Grb2, CRK or STAP1 SH2 domain binder, and binding

to STAT1/2/3 SH2 domains is also unlikely due to the lack of adequate specificity

determinants. Other SH2 domains (e.g. Src-related) could be recruited by ACE2, and

experimental validation will be required to test these hypotheses.

The NPY IBAR binding motif

Tyrosine 781 in ACE2 also overlaps a phosphorylation-independent NPY motif

(ELM:LIG_IBAR_NPY_1). This motif was initially described in the bacterial secreted

protein translocated intimin receptor (Tir) from pathogenic strains of E. coli like

enterohaemorrhagic E. coli (EHEC). The NPY tripeptide recognizes and binds with a 60

μM affinity to inverse Bin-Amphiphysin-Rvs (I-BAR) domains in adaptor proteins like

insulin receptor substrate protein of 53 kDa (IRSp53) and its homolog insulin receptor

12

tyrosine kinase substrate (IRTKS) (Campellone et al., 2006; de Groot et al., 2011). I-BAR

domains bind to the plasma membrane to favour weak membrane protrusions, and the

preference of I-BAR domains for negative membrane curvatures enables a positive

feedback loop that can result in the formation of lamellipodia, filopodia and other types of

membrane protrusions (Chen et al., 2015; Prévost et al., 2015; Zhao et al., 2011). IRSp53

and IRTKS are modular proteins that contain SH3 domains which in turn recognize PxxP

SLiMs in actin filament regulators like Mena, Eps8 and mDia1 (Ahmed et al., 2010)

resulting in the formation of membrane protrusions through actin filament formation

(Campellone et al., 2006; Chen et al., 2015; Prévost et al., 2015; Zhao et al., 2011).

Moreover, IRSp53 has an additional Cdc42-binding motif that can result in a direct WASP

activation (Ahmed et al., 2010). During EHEC infection, the bacteria uses the NPY motif

in the transmembrane protein Tir to recruit IRSp53 (Campellone et al., 2006). IRSp53

acts as a scaffold to localize the injected bacterial protein EspFU to the bacterial

attachment site, cytosolic side, through the binding of a PxxP motif in EspFU to the IRSp53

SH3 domain. Through the use of the same helical SLiM present in NCK

(ELM:LIG_GBD_CHELIX_1), EspFU acts as a potent WASP activator, inducing the actin

polymerization that contributes to the pedestal formation characteristic of EHEC infections

(Cheng et al., 2008; Sallee et al., 2008). The NPY SLiM, although not yet experimentally

validated in any human protein, is potentially functional in proteins like SHANK2 or the

microtubule-binding CLIP-associating protein 1 (CLASP1), based on protein conservation

and functional association (de Groot et al., 2011). The putative NPY motif in ACE2 is

conserved in all analysed mammalian and bird homologs (Figure 3), suggesting a direct

interaction with host I-BAR-containing proteins such as IRSp53 or IRTKS, which are

expressed in lung tissues (Uhlén et al., 2015).

The I-BAR domain-binding motif in the cytosolic region of ACE2 could be relevant for

SARS-CoV-2 infection in the following scenario. During viral cell entry, the NPY motif

could recruit I-BAR-containing proteins such as IRSp53 or IRTKS, resulting in membrane

protrusion formation that could be exploited for viral entry or in cell to cell transmission. It

is known that the hijack of the filopodia formation network is beneficial for the entry and

spreading of many enveloped viruses (reviewed in Chang et al., 2016), but whether this

process is active during coronavirus infection is still unclear. A second route might

cooperate with the NPY motif in the recruitment of actin cytoskeleton components. A

direct interaction between the SARS-CoV Spike protein cytosolic side C-terminal domain

and the Ezrin FERM domain can occur during the opening of the viral fusion pore and

has been proposed to restrain viral infection (Millet et al., 2012). Ezrin is a protein involved

in cell morphology and apical membrane remodelling that acts as a membrane-

cytoskeleton linker. Ezrin recruits F-actin through its C-terminal domain, and can also bind

to IRSp53 located at negatively curved membranes (Saleh et al., 2009; Tsai et al., 2018),

suggesting that while the NPY motif acts at earlier stages of viral attachment, the Spike

13

protein/Ezrin interaction might work during or after viral fusion, to promote the recruitment

of actin regulatory components to viral fusion sites.

The apoPTB domain-binding motif

Certain members of the PTB domain family were discovered to bind to

phosphorylated NPxY motifs, hence the designation Phospho-Tyrosine Binding domain

(Zhou et al., 1995). The NPxY motifs in cytosolic tails of receptors, including integrins, are

regarded as endocytosis sorting signals (Bonifacino and Traub, 2003). It was later

discovered that PTB domains in the internalisation adapter protein Dab1 could also bind

non-phosphorylated Nxx[FY] motifs (apoPTB motif) and that this might be the case for

the majority of PTBs (Uhlik et al., 2005). Representative receptors with apoPTB motifs

are in the database entry ELM:LIG_PTB_Apo_2. In ACE2, the core NxxF motif is

conserved in all land vertebrates (Figure 3). For Dab1 apoPTB motifs, there is a

hydrophobic requirement two residues before the Asn. In ACE2, this is predominantly a

charged residue: Therefore, if this strikingly conserved NxxF is an apoPTB motif, it should

bind a protein other than Dab1 and proteins with related specificities. The apoPTB motif

binds as a short β-strand (β-augmentation) followed by a β-turn. Proline is rejected at one

strand-forming position and therefore the regular expression for this motif would be

[^P].N..F for land vertebrates or [EQ].N..F for mammals. As with the phosphorylated

versions, the apo-motifs are tightly connected to endocytosis (Uhlik et al., 2005).

The LC3-interacting region (LIR) autophagy motif

Autophagy, the recycling of cellular material, is vital for cellular homeostasis. Many

pathogens must control the autophagy response to establish productive infection (Deretic

and Levine, 2009). It has been shown that coronaviruses, including human CoVs, subvert

autophagy components to promote viral replication at DMVs associated to the RTC

(Cottam et al., 2014; Fung and Liu, 2019; Gassen et al., 2019; Reggiori et al., 2010). The

LIR motif is required for the interaction of a target protein with Atg8 or its homologues

LC3 and GABARAP to facilitate autophagy of the target via the autophagosome

(Birgisdottir et al., 2013). The LIR motif has been catalogued in the ELM resource entry

ELM:LIG_LIR_Gen_1 which detected a candidate motif in the human ACE2 cytosolic tail

sequence (Figure 3). After the LIR motif was annotated in ELM, a more recently solved

LC3-LIR structure (PDB:5cx3) showed that the interacting peptide is longer, with one or

two more hydrophobic interactions (Olsvik et al., 2015). LIR enters a hydrophobic groove

bordered by positively charged residues. A core [WFY]xx[ILMV] enters the deepest part

of the groove. On either side of the core, the interacting residues can be flexibly spaced.

The core must be preceded by a negatively-charged residue (which might be enabled by

phosphorylation). Further, the motif core is followed by a flexibly spaced hydrophobic

residue. There is often a negatively-charged residue preceding this hydrophobic position:

it can make favourable interactions with counter charges but is not an absolute

14

requirement, so is not included in the revised motif pattern. Based on the structure

(PDB:5cx3) and some SPOT arrays (Alemu et al., 2012; Olsvik et al., 2015; Rasmussen

et al., 2019), the regular expression [EDST].{0,2}[WFY][^RKP][^PG][ILMV].{0,4}[LIVFM]

matches the motifs annotated in ELM. The revised motif is conserved in the mammalian

ACE2 cytosolic tail, but not in birds or reptiles. The ACE2 LIR motif candidate can

potentially enable the incoming coronavirus to attract autophagy elements such as LC3

to the structures where the virus replicates and assembles. In line with this, a non-

lipidated form of the LC3 protein has been shown to be associated with the RTCs of MHV

and SARS-CoV (Cong et al., 2017; Prentice et al., 2004; Reggiori et al., 2010). This brings

up the interesting possibility that ACE2 remains associated with the membranous

structures where SARS-CoV-2 replicates at later infection stages, assisting in the

repurposing of autophagy components required for viral replication.

The TxF$ PDZ-binding motif

Amongst other motif-binding modules, PDZ domains come in great abundance in

human and other multicellular animals (Ernst et al., 2009). PDZ domains take part in a

variety of biological processes including cellular signalling and activity at the synapse

(Manjunath et al., 2018). These domains bind SLiMs by β-strand augmentation which are

called PDZ-binding motifs or PBMs, most commonly known to be found in the C-terminus

of fully or partially disordered proteins. These interactions are widely studied and their link

to various diseases and infections has been previously established (Christensen et al.,

2019). A PBM candidate is also found in the very C-terminus of the cytosolic tail of

vertebrate ACE2 proteins (Figure 3). Motifs following a pattern [ST].[ACVILF]$ are a

common PDZ-binding motif variant, described in the ELM resource entry

ELM:LIG_PDZ_Class_1. There are multiple functional examples of this motif. However,

in the ACE2 protein the matching sequence is not characterized. ACE2 has a disordered

tail facing the cytosol, where a number of different PDZ domains could be its potential

binders (Manjunath et al., 2018).

Two PDZs in two different adapter proteins – Na(+)/H(+) exchange regulatory

cofactor NHERF3 and SH3 and multiple ankyrin repeat domains protein 1 SHANK1 –

have been previously identified to be able to bind TxF$ sequences (“$” stands for the C-

terminal end) (Ernst et al., 2014), which makes them both candidates for an interaction

with the ACE2 C-terminus. NHERF3 is co-localised with ACE2 in intestinal tissue, and its

PDZ domains were previously validated to interact with PBMs in transmembrane proteins

on the cytosolic side of the membrane (Gisler et al., 2003), so it is possible they come in

proximity with the ACE2 tail containing the TxF$ motif, and possibly bind it as a part of

ion exchange regulation of small molecule transport activities. NHERF3 is known for its

involvement in sodium ion-dependent transporter activity (Srivastava et al., 2019), and

ACE2 was also shown to interact with a sodium-dependent transporter (Yan et al., 2020),

which could be one of the leads towards unravelling the possible interaction between

15

NHERF3 and ACE2. NHERF3 (gene name PDZK1) and ACE2 share a network of

experimentally validated protein-protein interactions localized at the cell membrane

(Figure 4). These proteins share localisation and function characteristics, and some of

them could turn out to be the missing puzzle pieces to connect ACE2 and NHERF3 as

possible interactors.

All in all, whether NHERF3 is the PDZ-containing protein interacting with ACE2 or not

is an open question, but there should be very little doubt that ACE2 exhibits PDZ-binding

activity in the cell, since the motif is highly conserved, very specific and already known to

appear in membrane-associated proteins with C-terminal tails facing the cytosol.

Figure 4: Experimentally validated interaction network between NHERF3 (PDZK1) and

ACE2. C-terminal PBMs are displayed in white boxes. Common functionalities of the proteins

marked by different colours. Thickness of nodes is proportional to the confidence behind the

experimental evidence. The network was built using the STRING resource (https://string-db.org)

(Szklarczyk et al., 2019).

The phosphorylated Tyr781 in the ACE2 tail

Tyrosine 781 is a part of the motif patterns for four of the motifs listed above but must

be phosphorylated to act as an SH2-binding motif. Therefore, we searched the ACE2

literature for reports of phosphorylation but were unable to find any with strong site

identification. Examination of the human ACE2 entry in the database PhosphoSitePlus

(Hornbeck et al., 2019) revealed that high-throughput (HTP) phosphoproteomic studies,

but no low-throughput (LTP) studies, identify pTyr781. As shown in Figure 5, thirteen HTP

measurements identified phosphorylation at Tyr781 and this residue is the only ACE2

phosphosite that is highly reproducible across multiple HTP datasets. For example,

pTyr781 was one of 318 unique phosphopeptides belonging to 215 proteins analysed

16

from an erlotinib-treated breast cancer cell line model (Tzouros et al., 2013). Therefore,

this site indeed fulfils the phosphorylation requirement to be an SH2-binding motif.

Figure 5: The summary for the ACE2 C-terminal tail provided by PhosphoSitePlus. No low-

throughput (LTP) studies have been recorded in the database for ACE2. Thirteen high-throughput

(HTP) studies have identified pTyr781. Phosphosites reported in the extracellular part of ACE2

have only been reported once each and therefore are likely to be misidentified peptides.

A potential four way molecular switch at Tyr781 in the ACE2 tail

As described above, four sequence motifs overlap in the region surrounding

Tyr781: the YxxPhi endocytic sorting signal (ELM:TRG_ENDOCYTIC_2), an NCK SH2

binding motif (ELM:LIG_SH2_NCK_1), an NPY I-BAR binding motif

(ELM:LIG_IBAR_NPY_1) and the LIR autophagy motif (ELM:LIG_LIR_Gen_1). While the

YxxPhi, NPY and LIR motifs require a non-phosphorylated state of Tyr781, the NCK motif

requires Tyr781 phosphorylation, creating the opportunity for a four way molecular

phospho-switch acting in this region of ACE2 that directs different steps of the SARS-

CoV-2 infection cycle. The state of this switch can be controlled by tyrosine kinase activity

involving the Src/Abl and other tyrosine kinases known to be upregulated during

endosomal processes (Reinecke and Caplan, 2014) and viral infection (Davey et al.,

2011) including in coronaviruses (Coleman et al., 2016; Dong et al., 2020; Shin et al.,

2018; Sisk et al., 2018). Similar two-way switches have been described before, as with

the CTLA-4 receptors, where Src-family tyrosine kinases dictate the binding preferences

of overlapping YxxPhi and SH2 binding motifs. In the non-phosphorylated state

endocytosis is favoured, whereas T cell activation brings about Tyr phosphorylation,

shutting down endosomal recycling and initiating signalling through the recruitment of

17

SH2-domain containing proteins (Bradshaw et al., 1997; Miyatake et al., 1998; Ohno et

al., 1996; Owen and Evans, 1998; Shiratori et al., 1997).

During early stages of viral infection, and following viral attachment to the host cell

membrane, the YxxPhi motif could activate the early events of receptor-mediated

endocytosis by binding the µ subunit of the AP2 complex, which in turn recruits clathrin

and other endocytic components to the viral attachment sites. Following the formation of

the clathrin coat and initial invagination, actin polymerization is required to assist in the

internalization of the endocytic vesicles. In addition, some viruses can ‘surf’ along

filopodia by myosin-mediated actin cytoskeleton movements that transport the viral

particles to the entry sites at the cell body, ultimately increasing their entry rate (reviewed

in Chang et al., 2016). The actin hijack related to endocytic uptake and the formation of

membrane protrusions could be coordinated in one or several stages by the NCK SH2

and NPY motifs respectively, by recruiting WASP and I-BAR proteins to the viral

attachment site. This might be enacted by a Tyr781 phosphorylation switch that is

regulated in time with early attachment characterized by non-phosphorylated Tyr781 that

allows the YxxPhi and NPY motifs to be active, and a later phase where Tyr781 becomes

phosphorylated, inactivating the YxxPhi and NPY motifs and bringing the NCK SH2 motif

into play. An alternative scenario might be enabled by the multimeric nature of the Spike

protein and by attachment of several viral particles to a membrane domain, leading to

adjacent ACE2 tails on the intracellular side that expose both phosphorylated and non-

phosphorylated motifs, allowing these three signalling steps to take place simultaneously.

The presence of several parallel routes for the recruitment of cytoskeleton components

involving the NPY and NCK motifs could provide robustness needed to ensure the actin-

reorganization required for the uptake of virus-containing vesicles into the cytosol.

Following endocytosis and fusion, viral components are released into the cell and viral

replication takes place. During this phase, the last component of the switch could come

into play, when the ACE2 protein that remains bound to Spike protein-coated membranes

could promote the hijack of autophagy components necessary to assemble the viral

replication factories. At this time, non-phosphorylated ACE2 tails would recruit LC3 to

replication structures through their LIR motifs.

Known and candidate motifs in the integrin β tails

Integrin β tails are short cytosolic C-terminal intrinsically disordered regions, similar

to the analysed region of ACE2. The two most probable integrin β subunit candidates at

play in SARS-CoV-2 viral entry are β3 and β1. The C-terminal tail of both subunits share

a high degree of sequence similarity, and similarly to ACE2, contain several known and

candidate SLiMs (see Table 1 and Figure 6) that propagate signals in the cytoplasm and

regulate integrin activity not just through intracellular pathways, but also changing the

18

structural state of the ectodomains determining ligand binding capacity (Anthis and

Campbell, 2011).

The membrane proximal tyrosine kinase interacting region

Integrin β tails contain a highly charged patch in their membrane proximal region.

This region is indispensable for the interaction between integrins and tyrosine kinases,

including the Src-family kinase Fyn (Reddy et al., 2008) and the focal adhesion kinase

(FAK), most probably via the direct interaction with paxillin (Liu et al., 2002). Through

these interactions, integrins regulate cytoskeletal remodelling (Lv et al., 2016) and the

promotion of cell survival (Tang et al., 2007), as well as regulation of focal adhesion

assembly and cell protrusion formation (Hu et al., 2014). In turn, FAK regulates integrin

recycling and endosomal trafficking (Mana et al., 2020; Nader et al., 2016).

Currently, there is no consensus sequence motif describing these interactions,

although a definition of HDR[KR]E has been proposed (Legate and Fässler, 2009), fitting

integrins β1, β3, β5 and β6. This motif is under heavy regulation by several mechanisms.

First, the interaction with tyrosine kinases seem to involve additional residues N-terminal

of the charged motif core – most notably the conserved lysine preceding the hydrophobic

patch (Reddy et al., 1998) – that are only accessible in the active state of the integrin

dimer, as these regions are buried in the membrane otherwise (Stefansson et al., 2004).

Second, the D residue of the motif forms a salt bridge with the cytosolic tail of the α subunit

of the integrin in the inactive conformation of the receptor. Thus, this motif region is

dependent on integrin activation regulated by ligand binding and intracellular interactions

mediated by the C-terminally located NPxY motifs.

The NPxY motifs

Both the β1 and β3 tails contain two regions that match the apoPTB motif defined in

the ELM resource (ELM:LIG_PTB_Apo_2). Furthermore, these regions are known to

have Tyr phosphorylation, matching the phosphorylated motif definition as well

(ELM:LIG_PTB_Phospho_1). These regions are known to be able to form β-turns, and

are recognition sites for phosphotyrosine-binding domains. NPxY motifs (or NxxY in the

case of the second motif of the β3 tail) are the major sorting signals mediating interactions

with FERM domains for regulating endosomal trafficking (Ghai et al., 2013). In integrin β

tails these motifs recruit adaptor proteins and clathrin, serving as sorting signals (Ohno

et al., 1995), and the NPxY motifs in the β1 tail have a direct connection to viral entry for

reovirus (Maginnis et al., 2008).

Interactions mediated by the NPxY motif switches

The first NPxY motif binds talin-1, serving as a connection between the plasma

membrane and the major cytoskeletal structures (Horwitz et al., 1986). Considering the

expression profiles of talins, the most likely interaction partner of lung-expressed integrins

19

is talin-1. Talin-1 contains a FERM domain, similarly to Ezrin, which establishes a direct

interaction with the SARS-CoV Spike protein upon viral fusion (Millet et al., 2012).

However, the interaction between the RBD and integrins offers the virus an earlier point

of interference with the cytoskeletal system, being able to modulate it cooperatively with

the ACE2 actin-regulatory elements (NPY and NCK SH2 motifs) before and during

cellular entry. The talin/integrin interaction however presents a feedback loop: the binding

of talin on the cytoplasmic side induces a structural rearrangement on the ectodomains

of integrins, enabling a higher affinity interaction with RGD motif containing ligands

(Tadokoro et al., 2003).

The first NPxY motif is also a binding site for Dok1, a negative regulator of integrin

activation. Dok1 is in direct competition with talin for binding integrins (Tadokoro et al.,

2003). The competition is fundamentally influenced by phosphorylation on Tyr773 of the

NPxY motif. The non-phosphorylated motif has a higher affinity towards talin, whereas

phosphorylation prefers Dok1 (Oxley et al., 2008); thus Tyr773 acts as a phospho-switch

that regulates integrin activation.

The first NPxY motif also presents a site for a largely phosphorylation-independent

interaction with the Integrin Cytoplasmic domain-Associated Protein-1 (ICAP-1). ICAP-1

is a fundamental regulator of the assembly of focal adhesions (FA) and ICAP-1

knockdown reduced FA assembly (Alvarez et al., 2008), possibly working in conjunction

with the membrane-proximal charged motif. ICAP-1 seems to be specific for β1, and

hence the therapeutic considerations for targeting this pathway requires the verification

of the type of integrins expressed on AT2 cells (and other related cell types).

The second NPxY motif is a binding site for kindlin (Ma et al., 2008). This interaction

requires the integrin tail to be non-phosphorylated and phosphorylation on Tyr785 (for β3)

can switch off the interaction with kindlin-2 (Bledzka et al., 2010). Kindlin binding (together

with talin binding) is a crucial step in integrin activation, and hence regulates the

availability of integrins for extracellular ligands (Herz et al., 2006), and was also

suggested to play a role in TGF-β1 signalling (Kloeker et al., 2004).

Building a complex switching mechanism from two phospho-switches

The two NPxY(-like) motifs in the integrin β tails not only constitute two separate

phospho-switches, but also act in synergy to give rise to more complex regulation. Filamin

and the PTB domain region of Shc1 each bind to both NPxY motifs (Deshmukh et al.,

2010; Liu et al., 2015). Shc is an adaptor protein playing a key role in MAPK and Ras

signalling pathways, and its interaction with integrin β3 requires both phosphorylations on

Tyr773 and Tyr785 (Audero et al., 2004; Deshmukh et al., 2010). In contrast, binding of

the Ig domain of filamin-A requires both tyrosines to be in a non-phosphorylated state.

The filamin-A interaction can be considered as a main shutdown switch in integrin

signalling, as this interaction induces the closed conformation of the integrin ectodomains,

decreasing the chance of ligand binding (Liu et al., 2015). In addition, binding partners

20

utilizing both NPxY motifs may also serve as stronger modulators of endosomal

trafficking, switching on enhanced signals.

Potential LIR autophagy motif in integrin β3

The connection between autophagy and cell adhesion has already been described,

showing that both reduced FAK signalling (Sandilands et al., 2011) and detachment from

the extracellular matrix via integrins (Vlahakis and Debnath, 2017) enhances autophagy.

Atg deficient cells have enhanced migration properties, and at the molecular level there

seems to be a direct connection between Atg proteins and integrins as well: autophagy

stimulation increases the co-localization of β1 integrin-containing vesicles with LC3-

stained autophagic vacuoles, while autophagy inhibition decreases the degradation of

internalized β1 integrins (Tuloup-Minguez et al., 2013). In Drosophila cells it has been

shown that the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) plays a

connecting role between integrin recycling and the efficiency of phagocytic and

autophagic clearance (Nagel et al., 2017). However, molecular details about how this

connection is brought about are unclear.

Sequence analysis of integrin β3 tails show a potential Atg-interacting LIR motif,

similarly to the ACE2 tail. Low throughput phosphorylation assays have determined that

both Tyr773 and Tyr785 (required for the functional PTB NPxY motif) are in fact

phosphorylated in vivo. However, such assays have also determined additional

phosphorylation sites in the β3 tail, Thr777, Ser778, Thr779 and Thr784. These

phosphorylations aren't connected to the NPxY motif switches in any known way.

However, the three phosphorylations between residues 777-779 and the following

sequence region matches the LIR autophagy motif also found in the ACE2 tail. While the

current motif definition does not exactly fit the β1 tail, there is also low throughput

phosphorylation assay data (Wennerberg et al., 1998) for the existence of these

phosphorylations in the corresponding residues, hinting at the possibility of the presence

of a slightly modified motif. For both β1 and β3 tails, the phosphorylation provides the

negative charge required N-terminal of the FxxIxY LIR motif hydrophobic core.

Phosphopeptides spanning the candidate region should reveal whether the LIR motif-like

region is a functional Atg-binding site in integrin β1, and also whether the multiple

phosphorylations act as a rheostat, modulating the affinity of the interaction. The motif

found in integrin β3 is also present in integrin β2, and the motif candidate identified in

integrin β1 is also present in integrin β6.

21

Figure 6: Alignment of human integrins illustrating conserved motifs in the cytosolic C-

terminal tail. A) Multiple sequence alignment of human integrin C-terminus regions, not including

the two divergent β tails (β4 and β8). The alignment shows their motif conservation of the NPxY

and LIR motifs (key residues displayed above). Red boxes mark the conservation range of the

PTB motif in all sequences and the location of the LIR motif in integrin β3. Protein names, UniProt

IDs and sequence numberings are listed on the left side of the alignment. B-C) Summary of the

PTMs on the integrin β1 and β3 C-terminal tails. Details of the experimental evidence for the PTB

tyrosine phosphorylations are highlighted: pY783 & pY795 for β1 and pY773 & pY785 for β3.

Graphs obtained from PhosphoSitePlus (09/04/2020).

22

Potential synergy between the ACE2 and integrin intracellular

motifs

Bringing together the candidate SLiMs identified in the integrin β and ACE2 tails

potentially strengthens the functional links between them, and provides an emergent

picture of SLiM-driven cooperative switches driving viral attachment, entry and replication

(Figure 7). Following attachment of Spike to the receptors, the two NPxY motifs in the

integrin β subunit could act cooperatively with the apoPTB and YxxPhi motifs in ACE2 as

sorting signals that mediate the internalization of viral particles into endosomes. The

presence of several endocytic motifs in close proximity would strengthen the interaction

with the endocytosis apparatus creating a high-avidity environment for recruitment of

RME components (Bonifacino and Traub, 2003). During this time, the phosphorylated

integrin NPxY motifs would also reinforce viral attachment through inside out signalling,

stabilizing the integrin ectodomain in the open, high affinity ligand-binding conformation.

As discussed previously, RME also involves the recruitment of adaptor molecules that

activate rearrangements of the actin cytoskeleton required for the internalization of the

endocytic vesicle. At this stage, the NPY and NCK SH2 motifs in ACE2 would recruit

several molecules that mediate actin polymerization signalling, prominently I-BAR

containing proteins IRSp53 and IRTKS, and the WASP-Arp2/3 complex. While most of

this actin signalling would serve to allow viral entry, additional actin recruitment processes

could occur following viral fusion, such as that initiated by the interaction between the

Spike protein and Ezrin. Finally, at later stages of infection, both integrins and ACE2 might

remain attached to virus-associated DMVs and other replication-competent membranes

where the RTC assembles. At this stage, ACE2 and integrins might cooperatively mediate

the recruitment of autophagy components such as LC3, through the LIR motifs located in

the cytosolic tails of both molecules.

23

Figure 7: Model of the proposed interplay between motifs in the interface between SARS-

CoV-2 and human AT2 cell to achieve receptor mediated endocytosis. Receptors of the

SARS-CoV-2 (pink) and human AT2 cell (light blue), motifs involved in viral recognition and entry

are shown in boxes. Elements shown in one of the monomers of a homotrimer (Spike) or

homodimer (ACE2) are also present in the other proteins forming that complex. Lines below motif

boxes represent each of the overlapping motifs in that specific region. Arrows indicate the related

cellular process and the protein known to interact with their respective motif is indicated in

24

parenthesis. Phosphorylation sites are shown as red circles with the respective sequence position

indicated. For the integrin β tail, the PTB/apoPTB phospho switch is depicted as two separate

versions of the same motif region, and the subscripts represent the motif order in the sequence.

The colour code is as follows: cleavage sites (brown), for motifs: apoPTB/PTB (orange), endocytic

sorting signal (purple), I-BAR binding (red), LIR (blue), MIDAS (yellow), NCK (green), PBM

(magenta), RGD (light red). SLiMs mediating interactions are marked with coloured aquares,

protease cleavage sites are marked with hexagons, structural motifs are marked with ovals. Motifs

marked with † are experimentally validated.

Short linear motifs and their potential therapeutic implications

The analysis of candidate SLiMs in ACE2 and integrins suggests that SARS-CoV-

2 hijacks both receptors, co-opting their SLiMs to drive viral attachment, entry and

replication. This creates an opportunity for drugging these interactions, or the processes

they control through Host Directed Therapies (HDTs), to prevent viral entry. A list of

potentially useful drugs is presented in Table 2, together with ChEMBL accessions

(Mendez et al., 2019).

The sequence RGD is used by a large number of viruses for cell attachment, via

integrins (Hussein et al., 2015). RGD mimics have been developed as inhibitors of

integrin-extracellular matrix protein interaction for a variety of diseases. A cyclic RGD

peptide (c-RGDfV, cilengitide) has been developed clinically for glioblastoma treatment

and other cancers. It proved safe, but did not enhance the survival benefit (Stupp et al.,

2014). SARS-CoV-2 has a unique RGD sequence in the ACE2 binding region of its Spike

protein. It has been speculated that integrins may have a potential role for infectivity

(Sigrist et al., 2020). Therefore, integrin inhibitors like RGD mimetics might be able to

block the RGD binding site(s) on target cells and block the attachment of the virus.

Another application that has been suggested is bacterial sepsis (sepsis is also a dreaded

complication in COVID-19 patients), and experimental evidence in animals is available

(Garciarena et al., 2017). Cilengitide is relatively specific for integrin αvβ3 and also active

on αvβ5, αvβ6. The antibody abituzumab (aka DI 17E6) is a pan-av antibody, i.e. also

active against other αv integrins, and may consequently be better suited for blocking virus

entry. It has been clinically tested in several cancer indications (Élez et al., 2015; Hussain

et al., 2016). Cilengitide and abituzumab are made available for in vitro testing in the

context of the SARS-CoV-2 pandemic by the company who developed it initially (contact:

compound_donation@merckgroup.com).

As discussed above, tyrosine kinase mediated phosphorylation plays an important

role in virus entry and maturation, and several tyrosine kinase inhibitors have been

developed in the clinic and show effects on viral infection. For example, saracatinib, a Src

and Abl inhibitor which has completed several clinical trials, including some cancers,

inhibited replication of different coronaviruses including MERS-CoV, SARS-CoV and

HCoV-229E in cell culture infection experiments (Shin et al., 2018). After internalization

25

and endosomal trafficking, imatinib, an Abl inhibitor, prevented fusion of SARS-CoV and

MERS-CoV virions at the endosomal membrane in infected cell culture experiments

(Coleman et al., 2016). Using the avian model virus, IBV, imatinib and two other Abl

inhibitors, GNF2 and GNF5, prevented the fusion of the Spike protein to the membrane

of the target cell as well as cell-cell fusion and syncytia formation (Sisk et al., 2018). More

recently, tyrphostin A9, a platelet-derived growth factor receptor (PDGFR) tyrosine kinase

inhibitor came out from a high-throughput screening using cytopathic effect as readout, it

also showed in vitro inhibitory capacity to transmissible gastroenteritis virus (TGEV), an

alpha coronavirus that infects pigs (Dong et al., 2020). The authors also showed that

tyrphostin A9 has a broad spectrum, being active against three other tested

coronaviruses: MHV in L929 cells, porcine epidemic diarrhea virus in Vero cells and feline

infectious peritonitis virus in CCL-94 cells. The mode of action was found to be through

p38 MAPK, at the post-adsorption stage. As FAK has been implicated in viral entry for

other viruses including influenza A (Elbahesh et al., 2014), experimental drugs targeting

FAK, including some in clinical trials (de Jonge et al., 2019), can be considered for

studying the potential Spike-induced integrin signalling. Currently, 39 tyrosine kinase

inhibitors are approved by the FDA: eleven target non-receptor protein-tyrosine kinases

and 28 inhibit receptor protein-tyrosine kinases (Roskoski, 2020). Consequently, tyrosine

kinase inhibitors may be good candidates to test for their effect on SARS-CoV-2.

A number of protease inhibitors are currently discussed for SARS-CoV-2 treatment.

Serine protease inhibitor camostat mesylate is active against TMPRSS2 and blocks cell

entry (Hoffmann et al., 2020). Only the Spike protein of SARS-CoV-2 contains a furin

cleavage sequence (PRRARS|V). Consequently, furin convertase inhibitors are

considered as antiviral agents (Shiryaev et al., 2007).

Many viruses enter the cell via endocytosis, and a number of candidate SLiMs

relevant for SARS-CoV-2 infection are related to endocytosis (see above).

Chlorpromazine, an antipsychotic (via dopamine D2 antagonism) dating from the 1950s,

is also a potent endocytosis inhibitor (which may explain some of its marked side effects,

which can include low white blood cell levels). It promotes the assembly of adaptor

proteins and clathrin on endosomal membranes thus depleting them from the plasma

membrane, leading to a block in clathrin-mediated endocytosis (Wang et al., 1993). The

potential use of endocytosis inhibitors such as Amiodarone (Stadler et al., 2008) and

chlorpromazine in coronavirus infection is further discussed here (Yang and Shen, 2020).

The situation with targeting autophagy seems unclear. Autophagy activators might

help the cell to consume incoming virus - or speed up the establishment of the viral

replication complexes and accelerate disease. Autophagy inhibitors might work in later

stages of infection to dampen viral production, but this will depend on whether autophagy

is active at the time or if the constituent components have been captured and effectively

shut down. Several inhibitors/activators have been reported which can target autophagy

and multiple auxiliary signals feeding into the process of autophagy (Table 2). One such

26

axis is via the mTORC1 complex. Active mTORC1 keeps the autophagy process inhibited

by phosphorylating the ULK complex which is a key regulator in autophagy. Inhibition of

mTORC1 activates autophagy. Multiple FDA approved mTOR inhibitors are known and

include Rapamycin and Everolimus. Rapamycin has been shown to be effective in cell

culture for countering MERS-CoV infection and might assist in tackling SARS-CoV-2

infection (Kindrachuk et al., 2015) although the stage of infection might be crucial for the

desired outcome. Simvastatin is another drug which is known to upregulate autophagy

via the mTOR pathway (Wei et al., 2013). Simvastatin has also been reported to alleviate

airway inflammation in a mouse asthma model (Gu et al., 2017). Another autophagy

modulator is Niclosamide which regulates autophagy by targeting the autophagy regulator

Beclin1 via SKP2 E3-ligase in MERS-CoV infection. In this case, reduced Beclin1 levels

lead to blocking fusion of autophagosomes and lysosomes and hence the virus protects

itself in the host (Gassen et al., 2019). Overall, inhibiting SKP2 by Niclosamide relieves

Beclin1, allowing autophagosome-lysosome fusion and resumption of autophagy to

reduce the MERS-CoV production. In addition, niclosamide valinomycin has been shown

to target SARS-CoV in cell cultures as well (Wu et al., 2004).

Discussion

A recent proteomic study expressed 26 tagged SARS-CoV-2 proteins individually to

create a viral-human protein-protein interaction map (Gordon et al., 2020). As a result, 69

compounds, some being FDA approved, are candidates for drug repurposing. The Spike

protein interacted with the GOLGA7-ZDHHC5 acyl-transferase complex, possibly for

palmitoylation on Spike's cytosolic tail. Spike did not pull down ACE2 or any cell surface

receptor protein, but these experiments are not an assay for viral entry, and therefore

many protein interactions related to the viral entry mechanism are likely missing from

these data. Therefore our observations of SLiM candidates in the viral attachment, entry

and replication system reflect additional areas of the cell where drug repurposing for host-

directed therapy might be explored. Although tyrosine kinase inhibitors have frequently

been shown to dampen pathogen invasion and disease progression in cell culture, there

has been little effort to move these findings into the clinic (Sámano-Sánchez and Gibson,

2020). Because of their widespread use in cancer, the safety profiles of tyrosine kinase

inhibitors are well known and we wonder whether this might be a neglected opportunity.

Drugging the cell to cure the pathogen using HDTs is unlikely to fully remove a virus.

This would also be undesirable, because the immune system must mount a defence in

order to prevent viral reinfection. Rather, dampening viral load during viral invasion or

replication should be the target, to give the host defences time to respond. It is well known

that drugs like Tamiflu that slows influenza exit, and therefore entry into uninfected cells,

can only have a strong effect when taken prophylactically or early in infection (Bassetti et

al., 2019). Depending on the importance of integrins in SARS-CoV-2 lung cell entry,

27

reducing viral entry is a possible role for cilengitide or other molecules that hamper

integrin or ACE2 binding. An endocytosis inhibitor might play a similar role and is

independent of receptor type. However, for any such inhibitor that passes the blood-brain

barrier, effects on mood and other brain operations are an inevitable side effect: Even so,

the endocytosis inhibitor chlorpromazine is a widely used drug with a well-known safety

profile (Solmi et al., 2017).

Due to the presence of the cell attachment motif RGD in SARS-CoV-2, integrin

inhibitors seem worthwhile to explore further. Cilengitide, a relatively selective integrin

αvβ3 and αvβ6 inhibitor (a cyclic peptide that proved safe in patients, but failed to show

a survival benefit in glioblastoma (Stupp et al., 2014)) might be useful in two phases: it

could block virus attachment to target cells, and it has also been proposed as a potential

treatment in sepsis (Garciarena et al., 2017). Sepsis was the most frequently observed

complication among COVID-19 patients in Wuhan (F. Zhou et al., 2020). Another potential

application of integrin inhibitors, especially integrin αvβ6, would be lung fibrosis - patients

on respirators tend to develop lung fibrosis, and show increased αvβ6 levels (Horan et

al., 2008). The antibody abituzumab (aka DI 17E6) is a pan-αv antibody with high potency

on αvβ6, i.e. also active against several αv integrins, and may consequently be better

suited for blocking virus entry, or may be suitable for lung fibrosis or sepsis protection. It

has been clinically tested in several cancer indications (Élez et al., 2015; Hussain et al.,

2016), and proved safe, but did not achieve a survival benefit in cancer.

Whether the enzymatic function of ACE2 has a role in SARS-CoV-2 infection is

unknown. However, it would be readily testable with available ACE2 inhibitors, like

Captopril, on the market since the 1970s (Enalapril would not be suited for in vitro testing,

as it needs to be activated in the liver to become the active ingredient). It might be

productive to test for synergy between molecules that block viral binding to ACE2 and

molecules that block binding to integrins.

Summary/Conclusion

We have presented evidence at the sequence level for SLiMs in ACE2 and β integrins

with the potential to function in viral attachment, entry and replication for SARS-CoV-2.

We identified several candidate molecular links and testable hypotheses that might help

uncover the (still poorly understood) mechanisms of SARS-CoV-2 entry and replication.

Because these motifs belong to host proteins acting as viral receptors, they are not

revealed by virus-centred proteomic assays. Most of these putative motifs lack direct

experimental evidence. That they may well be functional, however, is indicated by

sequence conservation, in some cases for hundreds of millions of years. In addition, these

motifs are in appropriate cellular contexts to interact with their respective partner proteins.

Experimental validation will yield insights into receptor-mediated endocytosis for SARS-

CoV-2 virus and, in addition, for the role of ACE2 in the normal cell, where it surely has

28

much more functionality than being an angiotensin converting enzyme. Overall, the

collection of candidate motifs in this system suggests that a range of host directed

therapies might be explored including RGD inhibition, tyrosine kinase inhibition,

endocytosis inhibition and autophagy inhibition and/or activation.

Acknowledgements

BM has received funding from the European Union’s Horizon 2020 research and

innovation programme under the Marie Skłodowska-Curie grant agreement No. 842490

(MIMIC). JČ is supported by the European Union's Horizon 2020 research and innovation

programme under the Marie Skłodowska-Curie grant agreement No. 675341 (PDZnet).

EM-P is a PhD student of CONICET, Argentina. RA is supported by BMBF-funded

Heidelberg Center for Human Bioinformatics (HD-HuB) within the German Network for

Bioinformatics Infrastructure (de.NBI #031A537B) and ELIXIR Germany. LBC is a

National Research Council Investigator (CONICET, Argentina). The work was supported

by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2017-1924) grant to

LBC. This paper is part of a project that has received funding from the European Union’s

Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie

grant agreement No. 778247 (IDPfun) to LBC and TJG. IDPfun also funded EM-P’s

placement at EMBL.

Region

Protein (UniProt

accession)M

otifELM

class1

Main

residues2

Regular expression

StartEnd

Sequence3

Binding dom

ain4

Interaction partner 5Interaction

type

RG

DLIG

_RG

DR

GD

RG

D403

405R

GD

Pfam:PF00362 and

Pfam:PF01839

RG

D binding integrins, m

ost probably host:virus

-R

RxR

-682

687R

RAR

*SV

-KxxKR

-811

817KPSKR

*SF

Multibasic

cleavage sites-

xKR-

888892

TKR*D

LPfam

:PF00082Furin-like PC

shost

MID

AS6

-D

xSxSD

.[TS].S145

149D

LSYS-

The acidic part of RG

D-like ligands

host

Furin (P09958)R

GD

LIG_R

GD

RG

DR

GD

498500

RG

DPfam

:PF00362 and Pfam

:PF01839Possibly R

GD

-binding integrin dimers

host

MID

AS6

-D

xSxSD

.[TS].S543

547D

ISNS

-U

nknown partner w

ith acidig residue via m

etal ion coordinationhost

Multibasic

cleavage site-

R-

697716

RTEVEKAIR

MSR

SRIN

DAFR

InterPro:IPR001254

TMPR

SS2host

I-BAR binding

LIG_IBAR

_NPY_1

NPY

NPY

779781

NPY

InterPro:IPR027681

I-BAR dom

ain containing proteins like IR

Sp53 or IRTKS

Endocytic sorting signal

TRG

_END

OC

YTIC_2YP

Y[^P].[LMVIF]

781784

YASIPfam

:PF00928AP-2 Adapter Protein com

plex subunit

NC

K SH2

bindingLIG

_SH2_N

CK_1

(Y)[DESTN

A][^GW

FY][VPAI][DEN

QS

TAGYFP]

781785

YASIDPfam

:PF00017SH2 D

omain of the N

CK adapter protein

LIR autophagy

LIG_LIR

_Gen_1

[EDST].{0,2}[W

FY][^RKP][^PG

][ILMV]

.{0,4}[LIVFM]

778786

ENPYASIDI

Pfam:PF02991

Related proteins LC

3, Atg8, GABAR

AP. There m

ay be some variation in LIR

motif

specificity

apoPTBLIG

_PTB_Apo_2N

xx[FY](.[^P].N

P.[FY])|(.[ILVMFY].N

..[FY].)789

796G

ENN

PGFQ

Pfam:PF08416

PTB-containing protein with a preference for N

xxF core motifs

PBMLIG

_PDZ_C

lass_1TxF$

[ST].[ACVILF]$

800805

DVQ

TSFPfam

:PF00595PDZ-containing proteins w

ith TxF$ preferences such as N

HER

F3 and SHAN

K1

767774

TANN

PLYKPfam

:PF00373Pfam

:PF00630

talins (high affinity)D

ok1 (low affinity)

filamin-A (binding to both apoPTB m

otifs sim

ultaneously)

779786

TFTNITYR

Pfam:PF00373

Pfam:PF00630

kindlinfilam

in-A (binding to both apoPTB motifs

simultaneously)

767773

TANN

PLYPfam

:PF08416Pfam

:PF00640Pfam

:PF02174

talins (low affinity)

Dok1 (high affinity)

Shc (binding to both PTB motifs

simultaneously)

779785

TFTNITY

Pfam:PF00640

Shc (binding to both apoPTB motifs

simultaneously)

LIR autophagy

LIG_LIR

_Gen_1

[EDST].{0,2}[W

FY][^RKP][^PG

][ILMV]

.{0,4}[LIVFM]

777783

TSTFTNIPfam

:PF02991Atg8 protein fam

ilyhost

777784

TGEN

PIYKPfam

:PF00373Pfam

:PF10480Pfam

:PF00630

talins (high affinity)D

ok1 (low affinity)

ICAP-1

filamin-A (binding to both apoPTB m

otifs sim

ultaneously)

789796

TVVNPKYE

Pfam:PF00373

Pfam:PF00630

kindlinfilam

in-A (binding to both apoPTB motifs

simultaneously)

777783

TGEN

PIYPfam

:PF10480Pfam

:PF00640Pfam

:PF02174

talins (low affinity)

Dok1 (high affinity)

ICAP-1

Shc (binding to both PTB motifs

simultaneously)

789795

TVVNPKY

Pfam:PF00640

Shc (binding to both PTB motifs

simultaneously)

verified motifs

1 Motif identifier as in the Eukaryotic Linear M

otif Resource

4 Defined using Pfam

(El-Gebali et al., 2019) or InterPro (M

itchell et al., 2019), where applicable

motif candidates

2 x - any residue, P5 PC

: proprotein convertases3 '*' m

arks cleavage points for protease-recognition motifs

6 Not a SLiM

but a structural motif

Table 1. Known and predicted short linear motifs (SLiMs) in the SARS-CoV-2/host interaction

29

SARS-C

oV-2 Spike protein (P0D

TC2)

Multibasic

cleavage sitesPfam

:PF00082 or InterPro:IPR

001254Furin-like PC

s / TMPR

SS2host:virus

ACE2 (Q9BYF1)

host

host

host

PTBLIG

_PTB_Phospho_1N

xx(Y)

(.[^P].NP.[FY])|(.[ILVM

FY].N..[FY].)

(.[^P].NP.[FY])|(.[ILVM

FY].N..[FY].)

LIG_PTB_Apo_2

LIG_PTB_Phospho_1

Nxx(Y)

(.[^P].NP.(Y))|(.[ILVM

FY].N..(Y))

Extra-cellular

Intra-cellular

apoPTBN

xx[FY]

(.[^P].NP.(Y))|(.[ILVM

FY].N..(Y))

apoPTB

PTB

LIG_PTB_Apo_2

Nxx[FY]

Table 2: Drugs acting on different processes involved in viral entry and infection.

Name of Drug Mode of action Clinical status

Other details ChEMBL ID1

Inhibitors of viral attachment

Camostat Inhibits TMPRSS2 Approved (Japan)

Shown to be relevant in pancreatic fibrosis

590799

Abituzumab Integrin inhibition (αvβ6, pan-αv)

Phase 2 in oncology

May also be relevant in sepsis, fibrosis

2109621

Cilengitide Integrin inhibition (αvβ3, αvβ5, αvβ6)

Phase 3 in oncology

May also be relevant in sepsis, fibrosis

429876

Endocytosis inhibitors

Amiodarone Inhibits late endosomes

FDA Approved Cell culture based evidence for SARS-CoV2

633

Chlorpromazine Blocks Clathrin-mediated endocytosis

FDA Approved Anti-psychotic drug routinely used as endocytosis inhibitor in cell culture

823

Imatinib Abl inhibitor FDA Approved First line treatment for chronic myeloid leukaemia

941

Teicoplanin Inhibits Cathepsin L (late endosomes and lysosome)

FDA Approved Glycopeptide antibiotic 2367892

BI-853520 FAK inhibitor Phase 1 FAK has been implicated in the Influenza A virus cell entry and replication3

3544961

Saracatinib Src and Abl inhibitor Phase 2 in oncology

Currently also considered for Alzheimer's disease

217092

Tyrphostin A9 PDGF receptor kinase inhibitor (plus other activities)

Preclinical Inhibits actin ring formation 78150

Autophagy modulators

Azithromycin Not known FDA Approved Approved for multiple bacterial infections

529

Chloroquine Hydoxy-Chloroquine

Lysosomal inhibition FDA Approved Antimalarial agent 76 1690

Metformin

NDUF modulation; mTOR pathway modulation (plus other activities?)

FDA Approved Approved for type 2 diabetes 1431

Rapamycin Everolimus

mTORC1 inhibition FDA Approved To prevent transplant rejection

1908360 413

Simvastatin Autophagy upregulation via mTOR

FDA Approved Treatment for dyslipidemia and atherosclerosis prevention

1064

Niclosamide Valinomycin (VAL)

Inhibits SKP2 Niclosamide - FDA Approved; VAL - Preclinical

Niclosamide - moderate effect; VAL targets SARS-CoV in cell culture4 - known to inhibit SKP2

1448

NVP-BEZ235/Dactolisib

Autophagy induction Phase 2 PI3k/Akt/mTOR 1879463

1Drug details are accessible by clicking ChEMBL ids (Mendez et al., 2019); 2(Stadler et al., 2008); 3(Elbahesh et al., 2014); 4(Wu et al., 2004)

30

31

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