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. 2012 Jun;94(3):288-96.
doi: 10.1016/j.antiviral.2011.12.012. Epub 2012 Jan 17.

A hexapeptide of the receptor-binding domain of SARS corona virus spike protein blocks viral entry into host cells via the human receptor ACE2

Anna-Winona Struck  1 , Marco Axmann, Susanne Pfefferle, Christian Drosten, Bernd Meyer
Affiliations

A hexapeptide of the receptor-binding domain of SARS corona virus spike protein blocks viral entry into host cells via the human receptor ACE2

Anna-Winona Struck et al. Antiviral Res. 2012 Jun.

Abstract

In vitro infection of Vero E6 cells by SARS coronavirus (SARS-CoV) is blocked by hexapeptide Tyr-Lys-Tyr-Arg-Tyr-Leu. The peptide also inhibits proliferation of coronavirus NL63. On human cells both viruses utilize angiotensin-converting enzyme 2 (ACE2) as entry receptor. Blocking the viral entry is specific as alpha virus Sindbis shows no reduction in infectivity. Peptide (438)YKYRYL(443) is part of the receptor-binding domain (RBD) of the spike protein of SARS-CoV. Peptide libraries were screened by surface plasmon resonance (SPR) to identify RBD binding epitopes. (438)YKYRYL(443) carries the dominant binding epitope and binds to ACE2 with K(D)=46 μM. The binding mode was further characterized by saturation transfer difference (STD) NMR spectroscopy and molecular dynamic simulations. Based on this information the peptide can be used as lead structure to design potential entry inhibitors against SARS-CoV and related viruses.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
SPR Screening. (A) SPR Response Units [RU] at equilibrium as a function of the concentration of RBD-11, RBD-14 and RBD-15 using immobilized ACE2 as receptor. A one site binding model was applied to fit the data and to calculate the dissociation constants. The peptides RBD-11, RBD-14 and RBD-15 bind to ACE2 with dissociation constants KD = 85 ± 14 μM for RBD-11, KD = 450 ± 36 μM for RBD-14 and KD = 672 ± 168 μM for RBD-15. It can be clearly seen that RBD-11 is the tightest binder, whereas RBD-14 and RBD-15 show only slightly lower binding constants. (B) SPR response units [RU] at equilibrium as a function of the concentration of RBD-11b (438YKYRYL443) using 100 fmol immobilized ACE2 as receptor. RBD-11b binds with KD = 46 ± 14 μM and shows a twofold tighter binding affinity than the corresponding dodecapeptide RBD-11. (C) Structure of the Hexapeptide RBD-11b.
Fig. 2
Fig. 2
SPR sensorgram data for the screening of selected RBD peptides. SPR sensorgrams using immobilized ACE2 as receptor. It can be clearly seen that RBD-11b shows the highest SPR response signal, whereas RBD-14b shows only weak interaction to ACE2. For RBD-15c is no interaction observable.
Fig. 3
Fig. 3
STD NMR spectroscopy of RBD-11b and ACE2. (A) Saturation transfer difference NMR spectra of RBD-11b binding to ACE2. (a) 1H STD NMR spectrum of an ACE2 solution (c = 0.83 μM) together with RBD-11b (c = 59 μM, 72 fold excess over ACE2) at 700 MHz (4 K scans) in d-TBS shows significant STD effects for the aromatic tyrosine residues between 6.7 and 7.1 ppm. Protons Hδ,δ´ of R441 (3.00 ppm) and L443 (0.72, 0.80 ppm) clearly receive saturation due to their proximity to the receptor molecule. STD NMR signals between 2.7 and 2.9 ppm show saturation of β-protons of all three tyrosine residues and of Lys Hε,ε´. (b) Normal 1H NMR spectrum of a 3.0 mM solution of the hexapeptide RBD-11b at 700 MHz. In all spectra water was suppressed by an excitation sculpting pulse sequence and spectra were acquired in d-TBS at 295 K in a 100 μL (Hwang and Shaka, 1995). (B) Determination of binding affinity from STD NMR titration data. The titration curve of the aromatic protons Hδ,δ´ of Tyr442 is shown. The KD value was determined from the STD NMR titration by using the one site binding model. The resulting KD value is 60 ± 25 μM. (C) STD NMR epitope mapping of RBD-11b. The spots indicate the range of relative STD% for the protons that were saturated according to their proximity to the human receptor protein ACE2. A strong binding is detected for the aromatic protons of the three tyrosines. The highest degree of saturation of 4.1 STD% (absolute) obtained for Hε,ε´ of Tyr440 was set to 100% relative STD.
Fig. 4
Fig. 4
Inhibition of virus replication in cells by different concentrations of RBD-11b. (A) Inhibition of SARS corona virus replication in VeroE6 cells. The bar plot indicates the relative level of RNA copies per mL as a function of the RBD-11b concentration (quantified by real-time PCR). The peptide shows a concentration dependent decrease of virus RNA. (B) Inhibition of NL63 virus in two cell lines. The results for CaCo2 cells are shown on the left side and the results for LLC-MK2 cells are shown on the right side of the figure. (C) Inhibition of alpha virus Sindbis replication in VeroE6 cells. No significant inhibitory effect is observed.
Fig. 5
Fig. 5
Synthetic alanine scan of the lead compound RBD-11b. (A) Sequence of alanine scan peptides. (B) The dissociation constants KD of the alanine scan. The KD values are obtained from thermodynamic data analysis of SPR studies. KD of the lead compound RBD-11b is show in dark gray, peptides with specific interaction in gray and peptides with no interaction to ACE2 are shown in light gray.
Fig. 6
Fig. 6
Molecular modeling studies of ACE2 and RBD-11b. Stereo view of the conformation of the peptide RBD-11b in the peptide receptor complex after 3 ns molecular dynamic simulation. The peptide conformation and the receptor surface are shown in atom color.
Fig. 7
Fig. 7
Schematic representation of the interaction between ACE2 and RBD-11b. The figure was calculated with the LIGPLOT program for the end frame of the MD simulation (Wallace et al., 1995).
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References

    1. Babcock G.J., Esshaki D.J., Thomas W.D.J., Ambrosino D.M. Amino acids 270–510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J. Virol. 2004;78:4552–4560. - PMC - PubMed
    1. Dimitrov D.S. The secret life of ACE2 as a receptor for the SARS virus. Cell. 2003;115:652–653. - PMC - PubMed
    1. Drosten C., Chiu L.L., Panning M., Leong H.N., Preiser W., Tam J.S., Gunther S., Kramme S., Emmerich P., Ng W.L., Schmitz H., Koay E.S. Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus. J. Clin. Microbiol. 2004;42:2043–2047. - PMC - PubMed
    1. Drosten C., Gunther S., Preiser W., van der Werf S., Brodt H.R., Becker S., Rabenau H., Panning M., Kolesnikova L., Fouchier R.A., Berger A., Burguiere A.M., Cinatl J., Eickmann M., Escriou N., Grywna K., Kramme S., Manuguerra J.C., Muller S., Rickerts V., Sturmer M., Vieth S., Klenk H.D., Osterhaus A.D., Schmitz H., Doerr H.W. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 2003;348:1967–1976. - PubMed
    1. Du L., He Y., Zhou Y., Liu S., Zheng B.J., Jiang S. The spike protein of SARS-CoV – a target for vaccine and therapeutic development. Nat. Rev. Microbiol. 2009;7:226–236. - PMC - PubMed

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