A Fluorogenic Peptide Containing the Processing Site of Human SARS Corona Virus S‐Protein: Kinetic Evaluation and NMR Structure Elucidation

Abstract

Human severe acute respiratory syndrome coronavirus (hSARS-CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host-virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS-CoV spike protein (Abz-(755)Glu-Gln-Asp-Arg-Asn-Thr-Arg-Glu-Val-Phe-Ala-Gln(766)-Tyx-NH(2)) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC-inhibitor, alpha1-PDX, in a dose-dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. (1)H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C-terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R(761) E has been proposed.

Comparative analysis of photocaged RGDS peptides for cell patterning

“…To our best understanding, the synthetic scheme as proposed by us in this study for caged Gly has not been reported and in that respect it is novel. In the second stage of synthesis, these caged amino acids were incorporated at the proper location in an RGDS peptide chain via automated Fmoc‐solid phase chemistry 15. This incorporation often presents challenges in the generation of caged peptides.…”

“…Caged and native RGDS peptides were prepared by automated solid‐phase chemistry using the Intavis AG Bioanalytical Multipep Instrument (Germany) with normal HATU/DIEA mediated Fmoc chemistry as described earlier 15. Products were analyzed by SELDI‐TOF MS which showed a peak at m/z 565 for R[−]GDS and m/z 561 for RG[D]S which correlated well with the expected molecular weights.…”

Dynamic cell patterning of photoresponsive hyaluronic acid hydrogels

“…Caged RGDS and RGES peptides (denoted as R[‐]GDS and R[‐]GES, respectively) were synthesized via a liquid phase route as described previously . RGDS and RGES peptides were prepared by automated solid phase chemistry using an Intavis AG Bioanalytical Multipep Instrument with normal 2‐(1H‐7‐Azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyl uranium hexafluorophosphate methanaminium (HATU)/N,N‐diisopropylethylamine (DIEA) mediated Fmoc chemistry as described earlier …”

Hydrogel cell patterning incorporating photocaged RGDS peptides

“…Figure 3 outlines the synthesis of the caged RGDS peptide (1). The preparation of native RGDS peptide (steps not shown) was accomplished by normal HATU/DIPEA mediated Fmoc based solid phase chemistry as described earlier (Basak et al 2007). For the caged peptide (1), a 2-nitrobenzyl group was linked to the backbone amide nitrogen between the Arg and Gly residues.…”