DISCOVERY OF ALLOSTERIC INHIBITORS OF

WEST NILE VIRUS TWO-COMPONENT PROCESSING PROTEASE

 The corresponding paper reprint PDF is here.

West Nile Virus

West Nile virus (WNV) is a virus of the family Flaviviridae, which also includes Dengue, Hepatitis C, tick-borne encephalitis and some other viruses. It is found in both tropical and temperate regions. It mainly infects birds, but is known to infect humans, dogs, cats, bats. The main route of human infection is through the bite of an infected mosquito.

Drug Target

After the WNV virus infects host cell, its genomic DNA gets transcribed into a mRNA molecule, which is then translated into a single polyprotein molecule. To be useful for virus replication, this polyprotein molecule needs to be cut into separate functional proteins. This cutting function is accomplished by a viral enzyme, a viral processing protease. In the case of WNV, the protease belongs to a class of serine proteases (contains serine amino acid residue in its active site).

The vast majority of viruses require this  type of enzyme for new viruses production and further infection. An inhibition of this enzyme halts virus life cycle, and proved to be a successful approach to fight many dangerous viruses (e.g. HIV).

The common strategy to designing inhibitors against proteases is to inhibit directly the active site of the enzyme. While generally successful, this approach fails when applied to serine proteases. The failures are caused by poorly defined structural features, which can be exploited for designing specific high affinity inhibitors. For instance, it is very difficult to develop a specific serine protease inhibitor because serine proteases are highly homologous to each other within the active site. A non-specific serine protease inhibitor will be inhibiting other serine proteases, including those required for function of normal host cells (e.g. furin).

Targeting Strategy

Instead of following conventional inefficient drug discovery route, we have concentrated on trying to exploit particular WNV protease regulation mechanism. Specifically, our targeting strategy was to interfere with positioning of protease peptide cofactor. WNV protease is a two-component enzyme. It contains a larger catalytic subunit, and a small peptide-size cofactor (figure below).

The peptide cofactor can assume two distinct conformations: one reconstitutes the active form of WNV protease, while another is the inactive form. One can imagine that interfering with the peptide cofactor positioning which reconstitutes the active state of the enzyme should lead to its efficient inhibition. This interference can be introduced by a small molecule.

Designing small molecule against protein-protein interactions sites is considered to be the bottleneck of contemporary drug discovery. The constraints are thermodynamic in nature: a small molecule cannot compensate a free energy loss when a significant in area protein-protein interface is disrupted by its binding. However, when another protein-protein interface of similar or bigger area is created in the system as a result, the binding of a small molecule ligand becomes thermodynamically feasible.

The WNV two-component protease offers exactly this opportunity. The second “inactive” conformation of peptide provides necessary new protein-protein interface. We have successfully shown that exploiting these specific conformational transitions within the WNV viral protease leads to discovery of multiple high affinity specific small molecule inhibitors. These inhibitors are allosteric, i.e. interact with a site distinct from the active site. Although our hits are far from drug leads, the proof of principle is here.

Targeting more dangerous viruses

An important consequence of this work is that the same strategy can be applied to targeting significantly more dangerous viruses from the same family such as Dengue and Hepatitis C. This is the work in progress.

Anton Cheltsov, Ph.D.

anton@q-mol.com