2006 Understanding Biology Using Peptides __ Design and Study of Novel Peptide Inhibitors against the SARS-Coronavirus S

335 Design and Study of Novel Peptide Inhibitors against the SARS- Coronavirus Spike Protein Zhe Yan, Brian Tripet and Robert S. Hodges Department of Biochemistry and Molecular Genetics. University of Colorado at Denver and Health Sciences Center, Aurora, CO, 80045, USA Introduction Severe Acute Respiratory Syndrome (SARS) is an acute respiratory illness caused by infection with a novel coronavirus (SARS-CoV). Infection by SARS coronaviruses requires fusion of the viral and cellular membranes, which is mediated by the viral envelope Spike (S) glycoprotein and receptors on the target cell. The S protein contains two hydrophobic repeat regions, denoted HRN and HRC, which oligomerize the S glycoprotein into a trimer in the native state, and when activated collapse into a six-helix bundle structure driving fusion of the host and viral membranes. We and others have previously reported that the HR regions of SARS- CoV S protein can associate to form a very stable helical six-stranded structure and residues 902–950 in HRN and 1151–1185 in HRC were identified to be crucial for their interaction [1-4]. Due to the severity and mortality (10%) witnessed in the fall of 2003 during the spread of the SARS-CoV pandemic, and the current lack of effective agents for the antiviral therapy of SARS-CoV infection, it has become imperative to learn as much as possible about this virus and the ability to prevent future infection. As successfully used in fusion inhibitor design for HIV [5], peptides derived from HRC can bind to the transiently exposed HRN coiled-coil trimer and block the formation of the six-helical bundle (Fig. 1), which ultimately leads to a loss of membrane-fusion activity. In this study, HRN (902-950) peptide of SARS-CoV S protein was chosen as the target for testing the interaction of HRC analogs. The 36-residue HRC peptide (1150-1185) was chosen as the region to design a series of HRC analogs, in order to increase their stability and binding affinity with HRN. These substitutions /modifications involved: (1) increasing helical propensity (HRC2 and HRC4); (2) increasing hydrophobicity in the hydrophobic core (HRC1 and HRC3); and (3) introducing a lactam bridge (HRC5). The sequences of these peptides are shown in Figure 2. Results and Discussion To examine the helical structure changes of the HRC analogs, we analyzed each peptide by circular dichroism spectroscopy (CD). Under physiological conditions, these analogs are well folded, soluble, have higher ?-helical content and are more stable than native HRC as expected (in Fig. 3A and Table 1). Understanding Biology Using Peptides Sylvie E. Blondelle (Editor) American Peptide Society, 2005 Fig. 1. Schematic model illustrating the action of SARS-CoV fusion inhibitors that target HRN. 336 Fig. 3. CD analysis of HRC analogs and interaction with HRN. To test the stability of HRN, HRC analogs, and complexes of HRN with HRC analogs, CD spectra at different temperatures were measured to determine the temperature denaturation profiles as illustrated in Figure 3B, and their Tm values are shown in Table 1. These results show that HRC and analogs can strongly bind to HRN. HRN–HRC5 complex was not completely unfolded, even at 95ºC. HRC2 has similar interaction with HRN as HRC. The complexes of HRN-HRC1 and HRN-HRC3 are less stable than HRN- HRC. HRN-HRC4 is the least stable complex. To verify complex formation between HRN and HRC analogs, we analyzed their interaction by 1150 1185 a d a d a d a d a d HRC Ac-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL-amide HRC1 Ac-DISGINASVVNIQKEIDRLNEVIKNLNESLIDLQEL-amide HRC2 Ac-DISGINASVVNIQKEIARLNEVAKALNESLIDLQEL-amide HRC3 Ac-DISGINASVVNIQKEIARLNEVIKALNESLIDLQEL-amide HRC4 Ac-DIAAINASVANIQKEIARLNEVAKALNESLAALQAL-amide HRC5 Ac-DISGINASVVNIQKEIERLNKVAKNLNESLIDLQEL-amide Fig. 2. Peptide sequences. Fig. 4. Analysis of HRN and HRC complexes by 15% tricine native PAGE. Concentration of individual peptides was 100 ?M. The samples were incubated at room temperature for 20 minutes before being analyzed by native PAGE. The peptide lanes are shown as: 1. HRC1; 2. HRN+HRC1; 3. HRC2; 4. HRN+HRC2; 5. HRC3; 6. HRN+HRC3; 7. HRC4; 8. HRN+HRC4; 9. HRC5; 10. HRN+HRC5; 11. HRC; 12. HRN+HRC; 13. HRN. 337 native PAGE. The results are shown in Figure 4 and Table 1. HRC or HRC analogs alone showed a band in the lower part of the gel. With the exception of HRC4, the mixture of HRC analogs and HRN showed two bands: the lower one had the same position as isolated HRC analog, and the upper band is higher order oligomeric complex formed by HRN and HRC analog. HRN alone showed no band because it carries a net positive charge under the native electrophoresis conditions, and consequently does not enter the gel. Conclusions 1. The substitutions/modifications have increased the HRC ?-helical structure and stability. As shown in Table 1, the results from CD and native PAGE suggested HRC2 and HRC5 have increased binding affinity with HRN. 2. The two analogs, HRC2 and HRC5, will now be tested as peptide fusion inhibitors in an antiviral activity assay. Acknowledgments This work was supported by NIH grants to R.S.H. (PO1AI059576) and the John Stewart Chair in Peptide Chemistry. We thank Dziuleta Cepeniene for help with peptide synthesis. References 1. Tripet B., et al. J. Biol. Chem. 279, 20836-20849 (2004). 2. Xu Y., et al. J. Biol. Chem. 279, 49414-49419 (2004). 3. Bosch, B.J., et al. Proc. Natl. Acad. Sci. USA 101, 8455-8460 (2004). 4. Chan, D.C. and Kim, P.S. Cell 93, 681-684 (1998). 5. Ingallinella, P., et al. Proc. Natl. Acad. Sci. USA 101, 8709-8714 (2004). Table 1. Summary of HRC analogs and interaction with HRN [?]222 a % ? helix b Complex with HRN Peptide Name Benign 50%TFE Benign 50%TFE Tm c (oC) Tm (oC) ?Tm (oC) Complex formation tested by Native PAGE HRC -21370 -29720 61 85 37 85 0 + HRC 1 -32060 -31510 92 90 57 74 -11 + HRC 2 -27060 -29740 77 85 48 86 +1 + HRC 3 -30900 -31450 88 90 66 75 -10 + HRC 4 -28560 -30990 82 89 74 59 -26 - HRC 5 -27770 -32320 80 92 41 88 +3 + a. [?]222 is the mean residue molar ellipticity (deg cm2 dmol-1) measured at 222 nm in a 100 mM PBS, pH 7.0, in the absence (benign) or presence of 50% TFE (v/v). Concentration of peptides was 70 ?M. b. % ? helix was calculated from [?]222 based on an ellipticity value for 100% ?-helical content derived from the equation, XHn = XH ? (1-k / n), where XH ? is –37,400, the wavelength dependent constant, k, is 2.5, and n is the number of residues in the helix. c. Tm is the temperature at which there is a 50% decrease in fraction folded compared to the fully folded coiled-coil as determined by CD at 5 oC.