Elcira C. Villarreal

Small peptidic aldehyde inhibitors of human rhinovirus 3C protease

Abstract Small peptide aldehydes were designed to mimic the preferred substrate requirements for the human rhinovirus 3C protease. Di- and tripeptide aldehydes containing a methionine sulfone as a P 1 surrogate for glutamine show low micromolar enzyme inhibitory and antiviral tissue culture activity. LY338387, obtained in a short and efficient synthesis, appears to validate the protease as a therapeutic target.

[29] Purification and kinetic characterization of human cytomegalovirus assemblin

Publisher Summary This chapter describes expression and preparation of pure human cytomegalovirus (HCMV) assemblin with the help of a chelating peptide (CP) purification handle. Chelating peptide-immobilized metal ion affinity chromatography (CP-IMAC) uses an engineered metal-binding site or CP at either the N terminus or C terminus of a recombinant protein for a one-step affinity purification using IMAC. The CP sequences are easily incorporated into the protein with recombinant DNA cloning techniques. Cells are lysed with lysozyme and sonicated. Inclusion bodies are collected by centrifugation, lyophilized, and 10 mg/ml of solids dissolved in 0.5 M Tris, 7 M urea, pH 8.2. All purification steps are carried out in buffers containing 7 M urea, which maintains the enzyme in an unfolded inactive conformation. Blocking the cysteine residues as S-sulfonates facilitates the purification by solubilizing more protein from the inclusion bodies and preventing the formation of intermolecular disulfide bonds and aggregates. The activity of HCMV CP-assemblin is measured by following the hydrolysis of FITC, fluorescein isothiocyanate, a labeled peptide mimic of the maturational site of the assembly protein precursor.

Inhibition of human rhinovirus 3C protease by homophthalimides

Abstract Homophthalimides 2a and 3a were found to be inhibitors of Rhinovirus 3C protease through a blind screening effort. SAR studies resulted in compound 3g, which exhibited improved enzyme inhibition, in addition to whole cell antiviral activity. Molecular modeling studies suggest a preferred enzyme/inhibitor interaction, and LC/MS experiments confirmed tight/covalent binding of 3g to the enzyme.

Identification and Characterization of Human Rhinovirus-14 3C Protease Deamidation Isoform

A purified recombinant human rhinovirus-14 3C protease preparation contained only ∼50% active enzyme as titrated using specifically designed irreversible 3C protease inhibitors. Analysis of the purified 3C protein by isoelectric focusing showed differently charged 3C isoforms that had isoelectric points (pI) of 8.3 (55%) and 9.0 (45%), with the latter one being consistent with the predicted pI of the human rhinovirus-14 3C protein. Further analysis indicated that the pI 8.3 protein was the deamidated form of 3C, and it displayed ∼10-fold reduced cleavage activity relative to the original 3C protease sample. Peptide mapping followed by sequence analysis revealed that a single asparagine, Asn-164, was deamidated to aspartic acid in the pI 8.3 isoform. Converting Asn-164 to Asp by site-directed mutagenesis resulted in a mutated 3C protease with extremely low activity, as seen with the pI 8.3 isoform, indicating a role of Asn-164 in substrate recognition and binding. In addition, the deamidated 3C protease was found to be present in vivo, and its abundance was related to the viral replication cycle. Moreover, mutant virus carrying Asp-164 showed reduced viability in infected cells. Taken together, our data suggest that 3C protein deamidation plays a role in the regulation of its enzymatic activity.