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Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

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Science Signaling  12 Jan 2021:
Vol. 14, Issue 665, eabd0334
DOI: 10.1126/scisignal.abd0334
  • Fig. 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 homologous sequences from betacoronaviruses of various evolutionary distances and showing the location of potential integrin-binding motifs in black. Virus names together with the host organisms, UniProt accessions (*or GenBank accession in the case of RatG13), and sequence region numberings are shown 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, together with the location of the RGD motif and the region responsible for ACE2 binding. (B) Neighbor-joining tree of the multiple sequence alignment, with this particular set of sequences containing the potential high affinity, low affinity, and reverse integrin-binding motifs (RGD, KGD, and NGR) shown in red, orange, and green boxes, respectively. Only the sequence regions shown in (A) were used in the calculation of the tree. (C) Structure of the SARS-CoV-2 RBD as seen in the ACE2-bound form (PDB: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:6vsb, 6vxx, and 6vyb) are shown in transparency. Alignment and tree were prepared in Jalview (226) with Clustal colors. Structure was visualized using UCSF Chimera (228).

  • Fig. 2 Alignment of ACE2 illustrating conservation of the MIDAS motif.

    Multiple sequence alignment of a part of the ACE2 extracellular domain using 25 homologous sequences from different vertebrate lineages (mammals, birds, reptiles, and fish) and showing the conservation of the Dx[ST]xS motif as well as an NxT glycosylation site (main residues displayed above). A red box marks the conservation range of the MIDAS motif in all sequences but the hagfish. Organism names, UniProt IDs (UniParc for hagfish), 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 was prepared with Jalview using Clustal colors. TM, transmembrane; C-ter, C-terminal.

  • Fig. 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-terminal regions using 25 homologous sequences from different vertebrate lineages (mammals, birds, reptiles, and fish) 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, birds, and some fish). Organism names, UniProt IDs (UniParc for hagfish), 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 was prepared with Jalview using Clustal colors.

  • Fig. 4 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 phosphorylation on Tyr781. Phosphosites reported in the extracellular part of ACE2 have only been reported once each and therefore are likely to be misidentified peptides.

  • Fig. 5 Alignment of human integrins illustrating conserved motifs in the cytosolic C-terminal tail.

    (A) Multiple sequence alignment of human integrin C-terminal regions, not including the two most divergent β tails (β4 and β8). The alignment shows 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) Summary of the PTMs on the C-terminal tail of integrin β3. Details of the experimental evidence for the PTB tyrosine phosphorylations are highlighted: pTyr773 (pY773) and pTyr785 (pY785). Graph was obtained from PhosphoSitePlus.

  • Fig. 6 Model of the proposed interplay between motifs in the interface between SARS-CoV-2 and a human host cell to achieve RME.

    Receptors of the SARS-CoV-2 (gray) and a human host cell (light blue) motifs involved in viral recognition and entry are shown in colored 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 parenthesis. Phosphorylation sites are shown as inverted triangles, with the respective sequence position indicated. For the β-integrin 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. SLiMs mediating interactions are represented with boxes of different colors, protease cleavage sites with hexagons (PCs, furin-like proprotein convertases; T, TMPRSS2), phosphorylation sites with inverted triangles, and structural motifs with ovals. The color code is as follows: cleavage sites, yellow hexagon; apoPTB/PTB motif, orange; endocytic sorting signal motif, purple; I-BAR–binding motif, dark red; LIR motif, blue; MIDAS motif, gray; SH2 motif, green; PBM motif, magenta; RGD motif, bright red; and CendR motif, brown. † indicates that these motifs had been previously experimentally validated.

  • Table 1 Known and predicted SLiMs in SARS-CoV-2 host-entry interactions.

    Previously identified motifs are marked with (✓). Regular expressions follow POSIX definitions (23). The symbols ‘x’ and ‘.’ mark any residues in the definition of main residues and regular expressions.

    RegionProtein
    (UniProt
    accession)
    MotifELM class*Main
    residues
    Regular
    expression
    StartEndSequenceBinding
    domain
    Interaction
    partner§
    Interaction
    type
    ExtracellularSARS-CoV-2
    spike
    protein
    (P0DTC2)
    RGDLIG_RGDRGDRGD403405RGDPF00362 and
    PF01839
    RGD-binding
    integrins, most
    probably α5β1
    and αvβ3
    Host:virus
    Multibasic
    cleavage
    sites (✓)
    RRxR682687RRAR|SVPF00082 or
    IPR001254
    Furin-like PCs/
    TMPRSS2
    Host:virus
    KxxKR811817KPSKR|SF
    CendR (✓)LIG_NRP_
    CendR_1
    RxxR[RK].{0,2}[R]$682685RRARPF00754Neuropilin-1Host:virus
    Integrin αv
    (similar for
    other α
    chains)
    (P06756)
    Multibasic
    cleavage
    sites (✓)
    xKR888892TKR|DLPF00082Furin-like PCsHost
    Integrin β3
    (similar for
    other β
    chains)
    (P05106)
    MIDAS (✓)DxSxSD.[TS].S145149DLSYSThe acidic part
    of RGD-like
    ligands
    Host
    Furin
    (P09958)
    RGDLIG_RGDRGDRGD498500RGDPF00362 and
    PF01839
    Possibly
    RGD-binding
    integrin dimers
    Host
    MIDASDxSxSD.[TS].S543547DISNS-Unknown
    partner with
    acidic residue
    via metal ion
    coordination
    Host
    Multibasic
    cleavage
    site (✓)
    R697716RTEVEKAIRM
    SRSRINDAFR
    IPR001254TMPRSS2Host
    IntracellularACE2
    (Q9BYF1)
    I-BAR
    binding
    LIG_IBAR_
    NPY_1
    NPYNPY779781NPYIPR027681I-BAR domain–
    containing proteins
    like IRSp53 or IRTKS
    Host
    Endocytic
    sorting
    signal
    TRG_
    ENDOCYTIC_2
    YPxΦY[^P].[LMVIF]781784YASIPF00928Adapter protein
    complex μ2 subunit
    SH2
    binding
    YxxΦD((Y)[DE][^KRHG]
    [DESTAPILVMFYW]
    [^KR])|((Y)
    [NQSTAILVMFY]
    [^KRHG][ILV][^KR])
    781785YASIDPF00017SH2 domain of SFKs
    LIR
    autophagy
    LIG_LIR_
    Gen_1
    ExxYxxΦxΦ[EDST].{0,2}[WFY]
    [^RKP][^PG]
    [ILMV].{0,4}[LIVFM]
    778786ENPYASIDIPF02991Related proteins LC3,
    Atg8, GABARAP. There
    may be some variation
    in LIR motif specificity
    apoPTBLIG_PTB_
    Apo_2
    Nxx[FY](.[^P].NP.[FY])|(.
    [ILVMFY].N..[FY].)
    789796GENNPGFQPF08416PTB-containing protein
    with a preference for
    NxxF core motifs
    PBMLIG_PDZ_
    Class_1
    TxF$[ST].[ACVILF]$800805DVQTSFPF00595PDZ-containing proteins
    with TxF$ preferences
    such as NHERF3 and
    SHANK1
    Integrin β3
    (P05106)
    apoPTB (✓)LIG_PTB_
    Apo_2
    Nxx[FY](.[^P].NP.[FY])|(.
    [ILVMFY].N..[FY].)
    767774TANNPLYKPF00373
    PF00630
    Talins (high affinity)
    Dok1 (low affinity)
    Filamin-A (binding to
    both apoPTB motifs
    simultaneously)
    Host
    779786TFTNITYRPF00373
    PF00630
    Kindlin
    Filamin-A (binding to
    both apoPTB motifs
    simultaneously)
    PTB (✓)LIG_PTB_
    Phospho_1
    Nxx(Y)(.[^P].NP.(Y))|(.
    [ILVMFY].N..(Y))
    767773TANNPLYPF08416
    PF00640
    PF02174
    Talins (low affinity)
    Dok1 (high affinity)
    Shc (binding to both PTB
    motifs simultaneously)
    779785TFTNITYPF00640Shc (binding to both
    apoPTB motifs
    simultaneously)
    LIR
    autophagy
    LIG_LIR_
    Gen_1
    ExxYxxΦxΦ[EDST].{0,2}[WFY]
    [^RKP][^PG]
    [ILMV].{0,4}[LIVFM]
    777783TSTFTNIPF02991Atg8 protein familyHost
    Integrin β1
    (P05556)
    ApoPTB
    (✓)
    LIG_PTB_
    Apo_2
    Nxx[FY](.[^P].NP.[FY])|
    (.[ILVMFY].N..[FY].)
    777784TGENPIYKPF00373,
    PF10480
    PF00630
    Talins (high affinity)
    Dok1 (low affinity)
    ICAP-1
    Filamin-A (binding to
    both apoPTB motifs
    simultaneously)
    Host
    789796TVVNPKYEPF00373
    PF00630
    Kindlin
    Filamin-A (binding to
    both apoPTB motifs
    simultaneously)
    PTB (✓)LIG_PTB_
    Phospho_1
    Nxx(Y)(.[^P].NP.(Y))|(.
    [ILVMFY].N..(Y))
    777783TGENPIYPF10480
    PF00640
    PF02174
    Talins (low affinity)
    Dok1 (high affinity)
    ICAP-1
    Shc (binding to both PTB
    motifs simultaneously)
    789795TVVNPKYPF00640Shc (binding to both PTB
    motifs simultaneously)

    *Motif identifier as in the ELM resource.

    †“|” denotes cleavage points for protease-recognition motifs.

    ‡Defined through use of Pfam (103) or InterPro (104), where applicable.

    §PC, proprotein convertases.

    ║Not a SLiM but a structural motif.

    • Table 2 Drugs acting on various processes involved in viral entry and infection.

      Name of drugMode of actionClinical statusCOVID19
      ClinicalTrials.gov IDs*
      Other detailsChEMBL ID
      Inhibitors of viral attachment
      Camostat mesylateTMPRSS2 inhibitionApproved (Japan)NCT04355052,
      NCT04374019,
      NCT04353284,
      NCT04321096,
      NCT04470544
      Shown to be relevant in
      pancreatic fibrosis
      590799
      Nafamostat mesylateTMPRSS2 inhibitionApproved (Japan)NCT04473053Also inhibits human
      tryptase. Has strong
      anticoagulant effect
      3989553
      Decanoyl-RVKR-CMKFurin inhibitionPreclinicalHas been shown to
      inhibit CoV-2 spike
      cleavage at S1/S2 site
      by furin.§ Because the
      NRP1 receptor binds
      after cleavage, furin
      inhibition is potentially
      more relevant
      3126388
      AbituzumabIntegrin inhibition
      vβ6, pan-αv)
      Phase 2 in oncologyMay also be relevant in
      fibrosis
      2109621
      CilengitideIntegrin inhibition
      vβ3, αvβ5, αvβ6)
      Phase 3 in oncologyMay also be relevant in
      fibrosis, sepsis
      429876
      SuraminEntry/early replication
      inhibitor, mode of
      action unknown
      FDA approvedDrug of choice for
      treating African
      trypanosomiasis. Not
      without side effects
      413376
      Endocytosis inhibitors
      AmiodaroneInhibits late endosomesFDA approvedNCT04351763Cell culture–based
      evidence for SARS-CoV
      inhibition
      633
      ChlorpromazineInhibits dynamin,
      thereby blocking
      clathrin-mediated
      endocytosis
      FDA approvedNCT04354805,
      NCT04366739
      Antipsychotic drug
      routinely used as
      endocytosis inhibitor in
      cell culture
      823
      ApilimodInhibits PIKfyve, a
      regulator of endosomal
      trafficking
      Phase 2NCT04446377Blocks SARS-CoV-2
      infection. Good safety
      profile from (failed)
      clinical trials for
      immune disease
      application. Clinical
      trials are registered
      4297643
      ImatinibAbl inhibitorFDA approvedNCT04422678,
      NCT04394416,
      NCT04346147,
      NCT04357613
      First-line treatment for
      chronic myeloid
      leukemia
      941
      TeicoplaninInhibits cathepsin L
      (late endosomes and
      lysosome)
      FDA approvedGlycopeptide antibiotic2367892
      BaricitinibInhibits AAK1 and GAK
      endocytic kinases
      FDA approvedNCT04321993,
      NCT04340232,
      NCT04401579,
      NCT04362943,
      NCT04421027,
      NCT04390464,
      NCT04358614,
      NCT04346147,
      NCT04373044,
      NCT04393051,
      NCT04320277,
      NCT04399798
      Also inhibits JAK kinase2105759
      BI-853520FAK inhibitorPhase 1FAK has been
      implicated in the
      Influenza A virus cell
      entry and replication
      3544961
      SaracatinibSrc and Abl inhibitorPhase 2 in oncologyNow also considered
      for Alzheimer’s disease
      217092
      Tyrphostin A9PDGF receptor kinase
      inhibitor (plus other
      activities)
      PreclinicalInhibits actin ring
      formation
      78150
      Autophagy modulators
      MetforminNDUF modulation;
      mTOR pathway
      modulation (plus other
      activities?)
      FDA approvedNCT04510194Approved for type 2
      diabetes
      1431
      Rapamycin
      Everolimus
      mTORC1 inhibitionFDA approvedNCT04482712Used for preventing
      transplant rejection
      1908360
      413
      SimvastatinAutophagy up-
      regulation via mTOR
      FDA approvedNCT04348695,
      NCT02735707
      Treatment for
      dyslipidemia and
      atherosclerosis
      prevention
      1064
      Niclosamide
      Valinomycin (VAL)
      Inhibits SKP2Niclosamide—FDA
      approved;
      VAL—Preclinical
      NCT04372082,
      NCT04436458,
      NCT04399356
      Niclosamide—
      moderate effect; VAL
      targets MERS-CoV in
      cell culture,# known to
      inhibit SKP2
      1448
      NVP-BEZ235/dactolisibAutophagy inductionPhase 2PI3k/Akt/mTOR1879463
      Spautin-1Autophagy down-
      regulation by inhibition
      of USP10 and USP13
      PreclinicalInhibits dengue virus
      replication in tissue
      culture model.**
      Reported to have low
      toxicity in mice††
      2391504
      FluoxetineAffects endolysosomal
      acidification and
      cholesterol
      accumulation in the
      endosomes
      FDA approvedNCT04377308Used in treatment of
      major depressive
      disorder and obsessive
      compulsive disorder.
      Inhibits SARS-CoV-2
      replication in Calu-3
      and Vero E6 cells‡‡
      41

      *Clinical trial information can be checked by clicking the IDs (181).

      †Drug details are accessible by clicking ChEMBL IDs (180).

      ‡Reference (193).

      §Reference (90).

      ║Reference (197).

      ¶Reference (186).

      #Reference (207).

      **Reference (205).

      ††Reference (204).

      ‡‡Reference (206).

      Supplementary Materials

      • stke.sciencemag.org/cgi/content/full/14/665/eabd0334/DC1

        Text S1. Extended discussion on the potential of NRPs in SARS-CoV-2 cell entry.

        Fig. S1. The structural feasibility of simultaneous integrin and ACE2 binding by SARS-CoV-2 spike protein trimers.

        Fig. S2. Structural indication of a functional spike protein RBD:integrin αvβ6 interaction.

        Fig. S3. Alignment of human ACE2 transmembrane helix and C-terminal intracellular tail with homologous sequences of representative vertebrate collectrins from UniProt reference proteomes.

        Fig. S4. Alignment of homologous sequences of integrin β3 transmembrane helices and intracellular tails.

        Fig. S5. Alignment of homologous sequences of integrin β1 transmembrane helices and intracellular tails.

        Fig. S6. Alignment of homologous sequences of integrin β6 transmembrane helices and intracellular tails.

        Fig. S7. PTMs in β-integrin tails.

        Fig. S8. Transmembrane and intracellular regions of NRP1 and NRP2.

        Table S1. SH2 domain specificity for the candidate ACE2 tail SH2 motif.

        References (229235)

      • This PDF file includes:

        • Text S1. Extended discussion on the potential of NRPs in SARS-CoV-2 cell entry.
        • Fig. S1. The structural feasibility of simultaneous integrin and ACE2 binding by SARS-CoV-2 spike protein trimers.
        • Fig. S2. Structural indication of a functional spike protein RBD:integrin αvβ6 interaction.
        • Fig. S3. Alignment of human ACE2 transmembrane helix and C-terminal intracellular tail with homologous sequences of representative vertebrate collectrins from UniProt reference proteomes.
        • Fig. S4. Alignment of homologous sequences of integrin β3 transmembrane helices and intracellular tails.
        • Fig. S5. Alignment of homologous sequences of integrin β1 transmembrane helices and intracellular tails.
        • Fig. S6. Alignment of homologous sequences of integrin β6 transmembrane helices and intracellular tails.
        • Fig. S7. PTMs in β-integrin tails.
        • Fig. S8. Transmembrane and intracellular regions of NRP1 and NRP2.
        • Table S1. SH2 domain specificity for the candidate ACE2 tail SH2 motif.
        • References (229235)

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