Small-molecule inhibitors of the pseudaminic acid biosynthetic pathway: targeting motility as a key bacterial virulence factor.

Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 µM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 µM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 µM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.

Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases.

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.

Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease.

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS-PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k(cat)/K(M) of 602,000 M(-1)s(-1), PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme.

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.

Design of noncovalent inhibitors of human cathepsin L. From the 96-residue proregion to optimized tripeptides.

A novel series of noncovalent inhibitors of cathepsin L have been designed to mimic the mode of autoinhibition of procathepsin L. Just like the propeptide, these peptide-based inhibitors have a reverse-binding mode relative to a substrate and span both the S' and S subsites of the enzyme active site. In contrast to previous studies in which even moderate truncation of the full-length propeptide led to rapid reduction in potency, these blocked tripeptide-sized inhibitors maintain nanomolar potency. Moreover, these short peptides show higher selectivity (up to 310-fold) for inhibiting cathepsin L over K versus only 2-fold selectivity of the 96-residue propeptide of cathepsin L. A 1.9 A X-ray crystallographic structure of the complex of cathepsin L with one of the inhibitors confirms the designed reverse-binding mode of the inhibitor as well as its noncovalent nature. Enzymatic analysis also shows the inhibitors to be resistant to hydrolysis at elevated concentrations of the enzyme. The mode of inhibition of these molecules provides a general strategy for inhibiting other cathepsins as well as other proteases.