Discovery of small molecule inhibitors of ubiquitin-like poxvirus proteinase I7L using homology modeling and covalent docking approaches

Abstract

Essential for viral replication and highly conserved among poxviridae, the vaccinia virus I7L ubiquitin-like proteinase (ULP) is an attractive target for development of smallpox antiviral drugs. At the same time, the I7L proteinase exemplifies several interesting challenges from the rational drug design perspective. In the absence of a published I7L X-ray structure, we have built a detailed 3D model of the I7L ligand binding site (S2-S2' pocket) based on exceptionally high structural conservation of this site in proteases of the ULP family. The accuracy and limitations of this model were assessed through comparative analysis of available X-ray structures of ULPs, as well as energy based conformational modeling. The 3D model of the I7L ligand binding site was used to perform covalent docking and VLS of a comprehensive library of about 230,000 available ketone and aldehyde compounds. Out of 456 predicted ligands, 97 inhibitors of I7L proteinase activity were confirmed in biochemical assays ( approximately 20% overall hit rate). These experimental results both validate our I7L ligand binding model and provide initial leads for rational optimization of poxvirus I7L proteinase inhibitors. Thus, fragments predicted to bind in the prime portion of the active site can be combined with fragments on non-prime side to yield compounds with improved activity and specificity.

Fig. 1

Covalent docking setup in ICM: (a) each ketone compound is converted, (b, c) yielding two possible thio-acyl stereoisomers. (d) Three thio-acyl atoms of the ligand intermediate are tethered to the corresponding three atoms of the template, derived form the X-ray structure of Ulp1-substrate complex (1euv)

Fig. 2

Structural model of I7L and its active site, based on the Ulp1 cysteine protease X-ray structure (PDB code 1euv). (a) Sequence-structure alignment between I7L and Ulp1, with highly conserved ULP residues “W-HW-Q-C” marked by ▼. (b) Superimposition of “W-HW-Q-C” motif side chains in four different ULPs structures (green: 1euv, magenta: 1xt9, cyan: 1th0, yellow: 1avp). Backbone contact residues of Ulp1 shown in thin wire representation with grey carbons. (c) Close up of I7L model active site. Five conserved residues are shown as sticks colored by atom type with green labels. Two residues of I7L active site (G260 and E325), not conserved in Ulp1 are shown as sticks with blue labels, the corresponding Ulp1 residues shown as red sticks. (d) Predicted overall structure of the I7L proteinase; ribbon is colored according residue identity with Ulp1 (green is fully conserved, yellow—similar residue). The Ulp1 substrate is shown as grey ribbon with C-terminal Gly-Gly residues highlighted in red

Fig. 3

Examples of predicted binding modes for the three most active I7L inhibitors. Conserved residues of I7L binding site are shown as green sticks. Positions of the thio-acyl template atoms are shown as magenta balls. Inhibitors are shown as sticks with yellow carbons. (a) ST-0194802 in non-prime pockets (S3, S2, S1) of the active site, (b) ST-0194680 in prime pockets (S1′, S2′), and (c) ST-0194797 in both prime and non-prime pockets (S2–S1–S1′)

Fig. 4

Inhibition curves for the three most active inhibitors of I7L proteolytic activity (compounds ST-680, ST-797, ST-802) and one of the “marginal” inhibitors (ST-884)

Fig. 5

Chemical structures of new I7L inhibitors and their ID numbers as shown in Table 3