attacking nucleophile and stabilization of a tetrahedral intermediate-transition state in 3C proteases closely resembles that of trypsin-like serine proteases, suggesting that the viral 3C proteases are related mechanistically to serine proteases rather than to the papain-like cysteine proteases. Picornaviral 3C proteases process a limited number of cleavage sites in the virally encoded polyprotein. Most cleavages occur between Gln-Gly peptide bonds with distinct differences in the efficiency of cleavage at various junction sites. Recombinant rhinovirus 3C protease has an absolute requirement for Gln-Gly cleavage junctions in peptide substrates ranging from 7 to 11 aa in length (12).
Picornaviral 3C proteases represent a unique class of enzymes that integrate characteristics of both serine and cysteine proteases with an unusual specificity for Gln-Gly cleavage junctions. The absence of known cellular homologues contributes to interest in 3C protease as a potentially important target for antiviral drug design. However, the vast serotypic diversity among rhinoviruses raises the question of whether or not a single agent can effectively target 3C proteases from the 100 or so rhinovirus serotypes capable of infecting humans. Primary sequence data are available for 3C proteases from 10 different rhinovirus serotypes, including the type 2 and type 14 enzymes that have less than 50% amino acid identity.
To address these diversity concerns before initiating a concerted drug-discovery effort, we undertook a program to obtain structural information on peptide-based inhibitors bound to 3C proteases from multiple rhinovirus serotypes. We wanted to identify the geometric and electronic factors that modulate protein/substrate (inhibitor) recognition, the extent to which specific residues that form the substrate (inhibitor) binding site of 3C protease are conserved across rhinovirus serotypes, and whether or not these binding-site residues are arranged similarly in 3C proteases from different virus serotypes.
Peptide Aldehydes Bound to Serotype 2 Rhinovirus 3C Protease. Peptide aldehydes have been used extensively as inhibitors of serine and cysteine proteases, although they typically have not proven effective as drug candidates because of their poor pharmacological properties. They bind as reversible adducts in which the nucleophilic cysteine or serine makes a covalent bond with the carbonyl carbon of the aldehyde, forming a stable tetrahedral species. Short peptidic aldehydes having sequences similar to canonical 3C protease cleavage sites have been reported as inhibitors of both rhinovirus and hepatitis A viral proteases (13–15). The combination of glutamine at P1 with aldehyde functionality causes cyclization on the aldehyde. (16). To circumvent this problem, replacements for the γ-carboxamide were sought that prevent internal cyclization but retain high affinity for the 3C protease S1 specificity pocket (15). Compound I (Fig. 1) is an N-terminal protected tripeptide aldehyde in which the -CH2C(O)NH2 of Gln is replaced with an N-acetyl isostere. Compound I is a 6-nM inhibitor of type 14 human rhinovirus 3C protease. Whereas the original x-ray structural studies of rhinovirus 3C protease were performed by using the serotype 14 enzyme (9), subsequent analysis of inhibitor binding was carried out mainly with type 2 3C protease, both because of the relative ease in obtaining cocrystals and their generally superior diffraction properties. Fig. 2 shows the 2.2-Å x-ray structure of compound I complexed with serotype 2 rhinovirus 3C protease (15).
The peptide aldehyde I binds to rhinovirus 3C protease in a partially extended conformation with inhibitor backbone atoms aligned for antiparallel β-sheet-type hydrogen bonding with an exposed β-strand (βE2) of the protein comprising
residues 162–165. The inhibitor’s P1 side chain lies in a shallow pocket bounded by βE2, by residues 142–144, and by His-161, the last of which donates a hydrogen bond to the N-acetyl oxygen. This oxygen accepts a second hydrogen bond from the side-chain hydroxyl of Thr-142. The inhibitor’s acetyl methyl group is close to the backbone carbonyl of Thr-142 (3.3 Å), suggesting that substrates or inhibitors having a similarly positioned P1 glutamine-like side chain could form a third hydrogen bond to enhance specific recognition of a γ-carboxamide group.
The P1 backbone amide makes a weak (3.2-Å) hydrogen bond with the carbonyl oxygen of Val-162. The deep S2 pocket easily accommodates the inhibitor’s bulky P2 Phe side chain, which is bounded on one side by the side chain of His-40 and on the other side by residues 127–130. Two ordered water molecules reside at the back of the S2 pocket. The side-chain hydroxyl and backbone NH of Ser-128 form hydrogen bonds with the inhibitor’s P2 NH and the carbonyl oxygen of the terminal benzyloxycarbonyl (CBZ) group, respectively. Two main-chain hydrogen bonds tether the inhibitor’s P3 Leu to backbone atoms of Gly-164, whereas the isobutyl side chain is mostly solvent-exposed. The benzyl portion of the CBZ group packs into a shallow hydrophobic pocket that probably accommodates a substrate’s P4 side chain. The side chain of Asn-165 is positioned directly above the benzene of CBZ, with its carboxamide NH pointing into the face of the aromatic ring, suggesting that some additional binding energy probably derives from this favorable amino-aromatic interaction (17).
The affinity of peptide aldehyde inhibitors for trypsin-like serine proteases has been attributed to their ability to form, with the active-site serine, hemiacetals that resemble the transition state in amide hydrolysis, with the oxyanion stabilized in a structurally conserved oxyanion hole. Considering the structural homology between 3C protease and trypsin-like serine proteases, we anticipated that the tetrahedral hemithioacetal oxygen of compound I when bound to 3C would be positioned similarly within the oxyanion hole. Indeed, we showed previously that 2,3-dioxindole inhibitors (see Fig. 1, compound II) form stable tetrahedral adducts with 3C pro-