Anti-poliovirus activity of protease inhibitor AG-7404, and assessment of in vitro activity in combination with antiviral capsid inhibitor compounds.

The National Research Council has recommended that at least one, preferably two, polio antiviral drugs be developed as a supplement to the tools currently available for control of polio outbreaks post-eradication. The primary application of such drugs is expected to be the resolution of chronic poliovirus excretion in persons with primary immunodeficiency disorders. We have assessed the in vitro activity of AG-7404 (also known as "compound 1"), an inhibitor of picornaviral 3C protease, against a large panel of programmatically important poliovirus strains and its activity in combination with two poliovirus capsid inhibitors, V-073 and BTA798. AG-7404 was active against all viruses in this panel, with EC50 values ranging from 0.080 to 0.674 µM. Similarly, BTA798 was active against all viruses in this panel, with EC50 values ranging from 0.003 to 0.591µM. By comparison, values for V-073 were 0.003-0.126 µM. BTA798 was active against V-073-resistant variants with an alanine to valine change in VP3 at position 24. However, BTA798 was inactive against the V-073-resistant strains with amino acid substitutions at VP1 amino acids 194 (equivalent to 192 in type 3) and 236. As expected from its different mechanism of action, AG-7404 was fully active against all V-073-resistant variants, with EC50 values ranging from 0.218 to 0.819 µM, compared to values of 0.202-0.407 µM for the V-073-susceptible parental strains. In vitro drug combination experiments demonstrated synergy between AG-7404 and either V-073 or BTA798, whereas the combination of the two capsid inhibitors acted additively.

StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase.

BACKGROUND: Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory--still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could help overcome these difficulties by facilitating the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. RESULTS: Here we present StralSV (structure-alignment sequence variability), a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus, and we demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique, or that share structural similarity with proteins that would be considered distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local structural alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. CONCLUSIONS: StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position. StralSV is provided as a web service at http://proteinmodel.org/AS2TS/STRALSV/.