An Efficient Method for the Synthesis of Peptide Aldehyde Libraries Employed in the Discovery of Reversible SARS Coronavirus Main Protease (SARS‐CoV M^pro) Inhibitors

Abstract

A method for the parallel solid-phase synthesis of peptide aldehydes has been developed. Protected amino acid aldehydes obtained by the racemization-free oxidation of amino alcohols with Dess-Martin periodinane were immobilized on threonyl resins as oxazolidines. Following Boc protection of the ring nitrogen to yield the N-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin. A peptide aldehyde library was designed for targeting the SARS coronavirus main protease, SARS-CoV M(pro)(also known as 3CL(pro)), on the basis of three different reported binding modes and supported by virtual screening. A set of 25 peptide aldehydes was prepared by this method and investigated in inhibition assays against SARS-CoV M(pro). Several potent inhibitors were found with IC(50) values in the low micromolar range. An IC(50) of 7.5 muM was found for AcNSTSQ-H and AcESTLQ-H. Interestingly, the most potent inhibitors seem to bind to SARS-CoV M(pro) in a noncanonical binding mode.

Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease

“…Interestingly, it turned out that some peptide aldehydes with an amino-acid sequence deviating from the consensus sequence embracing the cleavage sites of polyprotein substrates were surprisingly efficient as inhibitors. In particular, peptide aldehydes with P2 = Asp or Ser inhibited the main protease with a relatively low IC 50 (Al-Gharabli et al, 2006), although it is generally agreed that the S2 specificity subsite of the enzyme has a strong preference for large hydrophobic side-chains such as Leu, Phe, or Met (Fan et al, 2004(Fan et al, , 2005Lai et al, 2006). We therefore assumed that the hydrophilic P2 side-chain of these inhibitors would be oriented towards the solvent, rather than occupy the S2 pocket, and that the hydrophobic P3 residue would be binding to that pocket (Al-Gharabli et al, 2006;Schmidt et al, 2008).…”

“…In complex with the crystalline target enzyme, they provide a wealth of information on the specificity-defining subsites of the substrate-binding site. For this purpose, we have previously described peptide aldehydes as inhibitors of the SARS-coronavirus main protease (SARS-CoV M pro ) (Al-Gharabli et al, 2006). A major advantage of aldehydes over other peptide electrophiles is their reversible binding to the 0166-3542/$ -see front matter Ó 2011 Elsevier B.V. All rights reserved.…”

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

“…DLS provides fewer hits than biophysical assays, as only fragments with a fitting spatial orientation in the ligation product are detected as actives. Active fragment combinations were identified and optimized iteratively, with respect to affinity or, if second-sitespecific fragments are scrutinized with sets of proteins, with respect to specificity 20 . Other than in biophysical assays, nanomolar protein concentrations are sufficient and large collections of nucleophilic fragments can be tested in small volumes of liquids on microtitre plates.…”

Recent Advances in Targeting Viral Proteases for the Discovery of Novel Antivirals

“…In order to avoid the reactive chloromethyl ketone warhead, this reactive functional group was replaced by an aldehyde during the design of a library comprising peptidic inhibitors addressing the S5-S1 specificity pockets [112]. Such aldehyde-type inhibitors are less reactive and bind to the nucleophilic active-site cysteine in a reversible way.…”