The HCV NS5B polymerase, when complexed with certain inhibitors, adopts a conformation in which the finger loop region defined by amino acid residues 18 to 35 is displaced to expose a binding pocket defined generally by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 496, 500 and 503. This newly exposed binding pocket defines a novel target in the search of further chemical entities which are capable of binding to HCV NS5B and modulating, or preferably inhibiting, the polymerase activity of HCV NS5B.

Description of the Invention

SUMMARY OF THE INVENTION

It has now been found that the HCV polymerase, when complexed with certain inhibitors, adopts a conformation in which the finger loop region defined by amino acid residues 18 to 35 is displaced to expose a binding pocket defined generally by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503. This is in contrast to NS5B crystal structures disclosed in the prior art in which the finger loop defined by amino acid residues 18 to 35 conceals this binding pocket in its native state. This newly "exposed" binding pocket defines a novel target in the search of further chemical entities which are capable of binding to HCV NS5B and modulating, or preferably inhibiting, the polymerase activity of HCV NS5B.

Thus, in one aspect, the present invention provides an isolated and purified polypeptide comprising an HCV NS5B inhibitor binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket is exposed by displacement of a finger loop chain defined by at least amino acid residues 18 to 35 and said binding pocket retains its native functional configuration.

In a second aspect of the present invention, there is provided an isolated and purified HCV NS5B polypeptide analog comprising an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration and wherein said binding pocket is exposable.

In a further aspect, there is provided an isolated and purified HCV NS5B polypeptide consisting of an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424,425, 428, 429, 492, 493, 494, 495, 500 and 503 of native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration.

In another aspect, there is provided an HCV NS5B polypeptide variant comprising at least one amino acid mutation within a finger loop defined by amino acid residues 18 to 35 of an HCV NS5B, wherein said mutation provokes displacement of said finger loop to expose a binding pocket essentially defined by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of native HCV NS5B, or defined by a functionally equivalent analog thereof, and wherein said binding pocket retains its native functional configuration.

In another aspect of the present invention, there is provided an HCV NS5B polypeptide, or a functionally equivalent analog thereof, characterized by displacement of amino acid residues 18 to 35.

In another aspect of the present invention, there is provided an HCV NS5B polypeptide, or a functionally equivalent analog thereof, in which at least amino acid residues 18 to 35 have been deleted.

In another aspect of the invention, there is provided an HCV NS5B crystal structure comprising a binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of a native HCV NS5B, or defined by the structural coordinates of a functionally equivalent analog thereof, wherein a native finger loop chain defined by the structural coordinates of at least amino acids 18 to 35 is displaced to expose said binding pocket.

In another aspect of the present invention, there is provided a complex comprising an HCV NS5B polypeptide, polypeptide variant or polypeptide analog as defined above and a compound, wherein the compound associates with a binding pocket within the NS5B polypeptide, polypeptide variant or polypeptide analog, said binding pocket being defined by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of native HCV NS5B, or defined by a functionally equivalent analog thereof.

In further aspects of the present invention, a method of producing a crystallized HCV NS5B complex is provided, comprising an NS5B polypeptide, polypeptide variant or polypeptide analog as defined above and a compound binding to said polypeptide, polypeptide variant or polypeptide analog, wherein said compound is associated with an NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of a native HCV NS5B, or defined by a functionally equivalent analog thereof. This method comprises the steps of: a) incubating a purified HCV NS5B polypeptide in a crystallization buffer to obtain a crystallized NS5B polypeptide; b) solubilizing the compound; and c) soaking the crystallized NS5B polypeptide with the solubilized compound in a soaking buffer for a suitable soaking period to generate the HCV NS5B complex.

In an alternative method of preparing a crystallized HCV NS5B complex as defined above, the compound is added to a crystallization buffer containing crystallized HCV NS5B.

Another alternative method of preparing a crystallized HCV NS5B complex as defined above comprises the steps of: a) combining purified HCV NS5B with the compound in solubilized form to form an NS5B complex; and b) crystallizing the complex in a crystallization buffer.

In another aspect of the present invention, there is provided X-ray crystal structure coordinates of a complex comprising an HCV NS5B polypeptide, polypeptide variant or polypeptide analog as defined above and a compound, wherein the compound associates with a binding pocket within the NS5B polypeptide, polypeptide variant or polypeptide analog, said binding pocket being defined by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of native HCV NS5B, or defined by a functionally equivalent analog thereof.

In another aspect of the present invention, there is provided a computer-readable storage medium having stored thereon a model of the crystal structure of a complex comprising an HCV NS5B polypeptide, polypeptide variant or polypeptide analog as defined above and a compound, wherein said compound associates with an NS5B binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of a native HCV NS5B, or defined by structural coordinates of a functionally equivalent analog thereof.

In a further aspect of the present invention, there is provided a method of identifying a compound that may bind to HCV NS5B, comprising the steps of: a) applying a 3-dimensional molecular modeling algorithm to the structural coordinates of an HCV NS5B binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of native HCV NS5B, or defined by the structural coordinates of a functionally equivalent analog thereof, to determine the spatial coordinates of the binding pocket of HCV NS5B; and b) electronically screening stored spatial coordinates of the compound against the spatial coordinates of the HCV NS5B binding pocket to determine whether the compound may bind within the HCV NS5B binding pocket.

In another aspect of the present invention, there is provided a virtual screening method to identify potential HCV inhibitors comprising the steps of: a) constructing a computer model of an HCV NS5B binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of a native HCV NS5B, or defined by the structural coordinates of a functionally equivalent analog thereof; b) employing computational means to perform a fitting program operation between computer models of the compound to be evaluated and the NS5B binding pocket to provide an energy-minimized configuration of the compound in the binding pocket; and c) evaluating the results of the filting operation to quantify the association between the compound and the binding pocket, wherein a compound that associates with the binding pocket to yield a low energy, stable complex is a potential NS5B inhibitor.

In yet another aspect of the present invention, a method of screening candidate HCV NS5B inhibitor compounds is provided comprising the steps of: a) incubating an HCV NS5B polypeptide, polypeptide variant or polypeptide analog as defined above with a candidate inhibitor compound under conditions suitable for binding; and b) determining whether or not the candidate inhibitor compound binds to the polypeptide, wherein a compound that binds to the polypeptide is a potential HCV NS5B inhibitor.

In another aspect of the invention, a method of designing a compound which binds to an NS5B polypeptide, polypeptide variant or polypeptide analog as defined above is provided which comprises the step of: assessing the complementarity, i.e. the "fit", between the compound and a binding pocket in the NS5B polypeptide defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of a native HCV NS5B, or defined by a functionally equivalent analog thereof.

In another aspect of the invention, a method of producing a drug which inhibits RNA replication activity of HCV NS5B is provided which comprises identifying or designing a compound which fits into an NS5B binding pocket as defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket is exposed by displacement of a finger loop chain defined by at least amino acid residues 18 to 35.

DETAILED DESCRIPTION OF THE INVENTION

Preferred Embodiments

HCV NS5B Polypeptide

In a first aspect, the present invention provides an isolated and purified polypeptide comprising a functional HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one or more of: amino acid residues 37 and 496) of a native HCV NS5B, wherein said binding pocket is exposed by displacement of a finger loop chain defined by at least amino acid residues 18 to 35 and wherein said binding pocket retains its native functional configuration.

Within this aspect of this invention, there is also provided an isolated and purified HCV NS5B polypeptide consisting of an exposed HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one or more of: amino acid residues 37 and 496 ) of a native HCV NS5B, wherein said binding pocket retains its native functional configuration.

In this aspect of the invention, it is important that the present polypeptide incorporates a binding pocket that assumes its native configuration, i.e. the natural configuration that it assumes in the native HCV NS5B polymerase, in order for it to properly mimic the binding pocket and be a useful HCV NS5B inhibitor screening tool.

Further, in order for the polypeptide to function effectively as an HCV NS5B inhibitor screening tool, it is also important that the inhibitor binding pocket be exposed, or at least exposable, in order to allow inhibitor access to the binding pocket. In HCV NS5B, the inventors have determined that the binding pocket defined herein is exposed when at least amino acid residues 18 to 35 of an NS5B finger loop chain are displaced. Accordingly, in this aspect, a polypeptide is provided in which the binding pocket is in an exposed configuration.

The binding pocket of the present invention is defined herein by reference to amino acids by their position in the HCV NS5B protein due to expected sequence similarity from one HCV genotype or strain to another. The displaced or displaceable finger loop region is also defined by reference to amino acid positions, i.e. amino acids 18 to 35. It will be readily apparent to those of skill in the art that the numbering of amino acids in all HCV NS5B may be slightly different from that of the HCV NS5B exemplified herein due to amino acid insertions or deletions. The amino acid numbering presented herein is based on native HCV 1b NS5B polymerase sequence as shown SEQ ID NO: 1. However, corresponding amino acids in other HCV NS5B can be identified by visual inspection of the amino acid sequences or by using commercially available homology software programs such as Vector NTI (provided by InfoMax Inc.). In this regard, in order to identify the NS5B protein, it is notable that the first four amino acids of the HCV NS5B sequence are typically, -SMSY-(SEQ ID NO: 2), which are conserved from one variant to the next.

In one embodiment of the present invention, the binding pocket is defined by amino acid sequence as follows -- see Original Patent.

As will be appreciated by one of skill in the art, a functionally equivalent analog of the polypeptide is also within the scope of this aspect of the invention. One or more of the amino acid residues of the present polypeptide, either within the binding pocket or outside of the binding pocket domain, may be substituted with a functionally equivalent amino acid, generally a conservative amino acid replacement as set out in the `Table of Amino Acid Similarity` (see Original Patent), a synthetic amino acid analog thereof or a naturally occurring amino acid substitution as found in other HCV genotypes while still retaining the binding pocket in functional form or configuration. Amino acid deletions and/or insertions may also be made to the polypeptide. Such amino acid substitutions, insertions or deletions may render a polypeptide that is more practical for use in a screening assay, or a polypeptide that is more readily prepared. Cursory examples of naturally occurring amino acids substitutions within the binding pocket among different HCV genotypes include, but are in no way limited to, HCV genotype 1b NS5B has a T499V substitution; HCV genotype 1a NS5B has M36L, I424V and T499A substitutions; HCV genotype 3a NS5B has M36L, I424V, L425M, and V494C substitutions; HCV genotype 2b NS5B has M36K, L392I, A393S, I424V, L425I and V494A substitutions; HCV genotype 2a, 2k, 6b have a V494A substitution; HCV genotype 3b has a V494l substitution; HCV genotype 6a has a P495L substitution; and HCV genotype 4a has an A396V substitution.

It is notable that a binding pocket is generally defined by atoms of any amino acid of a polypeptide, e.g. NS5B, that are within 5 .ANG. of any atom of an inhibitor when the inhibitor is complexed with the polypeptide. Various computational analyses may be used to determine whether a polypeptide comprising a binding pocket as defined herein is sufficiently similar to the HCV NS5B binding pocket described above so as to be functional. Such analyses may be carried out in well known software applications, such as the Molecular Similarity applications of QUANTA [Molecular Simulations Inc, San Diego, Calif.], Sybyl [Tripos Associates, St. Louis, Mo.], InsightII [Accelrys], and MOE [Chemical Computing Group Inc., Montreal, Quebec, Canada].

There are a number of embodiments that stem from this aspect of the invention. For example, in addition to the amino acids set out above to define the binding pocket of the present invention, the binding pocket may additionally include one or more of amino acid residues 36, 426, 498 or 499 of the HCV NS5B. Preferably, each of these positions is occupied as follows: M36, M426, R498 and T/V499.

In another embodiment of this aspect, the polypeptide may contain the amino acid cluster of residues 36 and 37 of HCV NS5B in their native configuration. It is preferred that positions 36 and 37 are occupied by the following amino acids: M36, and V37, or has the amino acid sequence M-V.

In another embodiment, the polypeptide may contain the amino acid cluster of residues 392 to 399 of HCV NS5B in their native configuration. It is preferred that positions 392 to 399 are occupied by the following amino acids: L392, A393, R394, A395, A396, W397, E398 and T399, or has the amino acid sequence L-A-R-A-A-W-E-T (SEQ ID NO: 4).

In another embodiment, the polypeptide may contain the amino acid cluster of residues 424 to 429 of the HCV NS5B in their native configuration. It is preferred that positions 424 to 429 are occupied by the following amino acids: I424, L425, M426, T427, H428 and F429, or has the amino acid sequence, I-L-M-T-H-F (SEQ ID NO: 5).

In another embodiment, the polypeptide may contain the amino acid cluster of residues 492 to 503 of the HCV NS5B in their native NS5B configuration. In this regard, it is preferred that positions 492 to 503 are occupied by the following amino acids: L492, G493, V494, P495, P496, L497, R498, T499, W500, R501, H502 and R503, or has the amino acid sequence, L-G-V-P-P-L-R-T-W-R-H-R (SEQ ID NO: 6).

HCV NS5B Polypeptide Analog

In a second aspect, the present invention provides an isolated and purified HCV NS5B polypeptide analog comprising an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration and wherein said binding pocket is exposable.

An NS5B polypeptide analog as defined above which retains the functional configuration of the native binding pocket disclosed herein advantageously provides a mimic of the native NS5B polypeptide which can be designed to be superior to the native NS5B polypeptide for use in inhibitor screening assays. For example, the polypeptide analog can be designed such that it is more readily made and used, or is more stable. It can also be designed in order to provide a binding pocket that is more readily accessible.

The NS5B polypeptide may be altered to provide an NS5B polypeptide analog by amino acid substitution, deletion or insertion as described in more detail in the definitions set out above, and in particular, the definition of the term "analog". In this regard, it may be particularly desirable to modify the finger loop chain defined by amino acid residues 18 to 35 to provide a finger loop chain that is more readily displaced to expose the binding pocket of the present invention, or to provide a finger loop chain that is displaced or deleted to expose the binding pocket.

As set out above, the present HCV NS5B binding pocket is exposed when at least amino acid residues 18 to 35 of an NS5B finger loop chain are displaced. It has also been determined that certain compounds are capable of displacing the finger loop in order to access the binding pocket. Accordingly, a polypeptide analog is provided in which the binding pocket is exposable, i.e. concealed in its native NS5B configuration by a segment of the NS5B protein, such as the finger loop defined by amino acids 18 to 35, which can be displaced to expose the binding pocket, for example, by a compound with a propensity for the binding pocket.

There are a number of embodiments that stem from this aspect of the invention. For example, in addition to the amino acids set out above to define the binding pocket of the present invention, the binding pocket may additionally include one or more of amino acids residues 36, 426, 498 or 499 of the HCV NS5B. Preferably, the binding pocket includes all of these amino acid residues. Also preferably, each of these positions is occupied as follows: M36, M426, R498 and TN499.

In another embodiment of this aspect, the binding pocket of the polypeptide may contain amino acid residues 36 and 37 of HCV NS5B in their native configuration. It is preferred that positions 36 and 37 are occupied by the following amino acids: M36, and V37, or has the amino acid sequence M-V.

In another embodiment of this aspect, the binding pocket of the polypeptide may contain amino acid residues 392 to 399 of HCV NS5B in their native configuration. It is preferred that positions 392 to 399 are occupied by the following amino acids: L392, A393, R394, A395, A396, W397, E398 and T399, or has the amino acid sequence -- see Original Patent.

In another embodiment, the binding pocket of the polypeptide may contain the amino acid residues 424 to 429 of the HCV NS5B in their native configuration. It is preferred that positions 424 to 429 are occupied by the following amino acids: I424, L425, M426, T427, H428 and F429, or has the amino acid sequence, I-L-M-T-H-F (SEQ ID NO: 5).

In another embodiment, the binding pocket of the polypeptide may contain the amino acid residues 492 to 503 of the HCV NS5B in their native NS5B configuration. In this regard, it is preferred that positions 492 to 503 are occupied by the following amino acids: L492, G493, V494, P495, P496, L497, R498, T499, W500, R501, H502 and R503, or has the amino acid sequence -- see Original Patent.

In further embodiments, the preferred sequence of the binding pocket residues is in accordance with the sequence set out in SEQ ID NO: 1.

As set out above, the binding pocket and displaceable/displaced finger loop region are defined herein by reference to amino acid positions based on the HCV genotype 1b NS5B sequence shown in SEQ ID NO: 1 due to the high level of sequence homology that exists between HCV genotypes. However, one of skill in the art will appreciate that a slight variation in the position of one or more of the amino acid residues of the binding pocket or finger loop, for example a shift in position of each of the amino acids in the pocket by 1 or 2 (which may occur due to the insertion or deletion of one or more N-terminal amino acids) or a shift in a single amino acid elsewhere in the NS5B protein (e.g. a region which has no impact on binding pocket configuration), may still yield a binding pocket in accordance with the present invention and a finger loop that functions to expose the binding pocket. Accordingly, such position discrepancies are within the scope of the present invention.

HCV NS5B Polypeptide Variants

In another aspect, there is provided an HCV NS5B polypeptide variant comprising at least one amino acid mutation within a finger loop defined by amino acid residues 18 to 35, wherein said mutation provokes a displacement of said finger loop to expose a binding pocket essentially defined by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration.

An NS5B variant in accordance with this aspect of the invention may include any mutation which causes the finger loop defined by amino acids 18 to 35 to become displaced. For example, mutation of an amino acid which forms an association with one or more residues within the binding pocket as defined herein, may prevent such an association and thereby trigger the displacement or "opening" of the finger loop to expose the binding pocket.

In one embodiment of the present invention, at least one of the amino acid residues at positions 30 and 31 of HCV NS5B is mutated to provoke displacement of the finger loop. In a preferred embodiment, amino acid residue 30 is mutated to an amino acid residue other than leucine. More preferably, amino acid residue 30 is selected from: P, F, W, M, G, S, T, C, Y, N, Q, D, E, K, R and H.

Embodiments with respect to the binding pocket, and the additional amino acid residues that may comprise the binding pocket, as well as the specific sequences of the pocket residues, are set out above with respect to the NS5B polypeptides and analogs.

In another aspect of the present invention, there is provided an HCV NS5B polypeptide, or functionally equivalent analog thereof, characterized by displacement of amino acid residues 18 to 35.

In another aspect of the present invention, there is provided an HCV NS5B polypeptide, or functionally equivalent analog thereof, in which at least amino acid residues 18 to 35 have been deleted.

As previously set out, displacement or deletion of amino acid residues 18 to 35 exposes a novel binding pocket as defined herein which has significance in the development of HCV therapeutics.

HCV NS5B Crystal Structure

In a further aspect of the invention, there is provided an HCV NS5B crystal structure comprising a binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by the structural coordinates of a functionally equivalent analog thereof, wherein a native finger loop chain defined by the structural coordinates of at least amino acids 18 to 35 is displaced to expose said binding pocket. Preferably, the structural coordinates are as set forth in any one of FIG. 4, 5 or 6 (see Original Patent).

A structure in which an inhibitor binding pocket is exposed is a valuable tool for the design and development of candidate NS5B inhibitors because it provides a means to more clearly understand the configuration and overall nature of the binding pocket, knowledge that is crucial to guide the development of therapeutic NS5B inhibitors.

In a preferred embodiment, an HCV NS5B crystal structure is provided in which the binding pocket is additionally defined by amino acid residues 36, 426, 498 and 499. In a further preferred embodiment, an HCV NS5B crystal structure is provided in which the binding pocket is defined by clusters of amino acid residues 36-37, 392-399, 424-429 and 492-503.

HCV NS5B Complex

In another aspect of the present invention, there is provided a complex comprising an HCV NS5B polypeptide and a compound, wherein the compound associates with a binding pocket within the NS5B polypeptide which is defined by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of native HCV NS5B, or defined by a functionally equivalent analog thereof.

The NS5B inhibitor-binding pocket of the present invention is exposed on displacement of the finger loop region defined by amino acid residues 18 to 35. The binding pocket itself is defined by at least the amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of the HCV NS5B. However, it can further be defined by the addition of one or more of amino acid residues 36, 426, 498 and 499, or even further defined by the following clusters of amino acid residues: 36-37, 392-399, 424-429 and 492-503.

In accordance with this aspect of the invention, thus, the HCV NS5B polypeptide may be a native NS5B polypeptide, or it may be an HCV NS5B polypeptide, variant or analog selected from the group consisting of: i) an isolated and purified polypeptide comprising a functional HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket is exposed by displacement of a finger loop chain defined by at least amino acid residues 18 to 35 and wherein said binding pocket retains its native functional configuration; ii) an isolated and purified HCV NS5B polypeptide consisting of an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration; iii) an isolated and purified HCV NS5B polypeptide analog comprising an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration and wherein said binding pocket is exposable; iv) an HCV NS5B polypeptide variant comprising at least one amino acid mutation within a finger loop defined by amino acid residues 18 to 35, wherein said mutation provokes a displacement of said finger loop to expose a binding pocket essentially defined by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration; v) an HCV NS5B polypeptide, or a functionally equivalent analog thereof, characterized by displacement of amino acid residues 18 to 35; and vi) an HCV NS5B polypeptide, or a functionally equivalent analog thereof, in which at least amino acid residues 18 to 35 have been deleted.

In a preferred embodiment of this aspect, the complex comprises an HCV NS5B polypeptide, analog or variant as defined above, associated with a compound selected from the compound families described in the following patent documents: WO 01/047883, WO 02/004425, WO 03/000254, WO 03/007945, WO 03/010140, WO 03/010141 and WO 03/026587. In an alternative preferred embodiment of this aspect, the complex comprises an HCV NS5B polypeptide, analog or variant as defined above, associated with a compound selected from the compound families described in the U.S. co-pending applications Ser. Nos. 10/755,256, 10/755,544 and 60/546,213, herein incorporated by reference. Such compounds have not previously been shown to associate with the presently claimed binding pocket of NS5B to form an NS5B complex in accordance with the present invention.

In more preferred embodiments, the HCV NS5B complex comprises NS5B polypeptide, analog or variant associated with one of compounds A, B or C as set out below -- see Original Patent.

FIGS. 4, 5 and 6 show the structure coordinates of the HCV NS5B of SEQ ID No: 1 complexed with the compounds A, B and C, respectively. The manner of obtaining these structure coordinates, interpretation of the coordinates and their utility in understanding the protein structure and specifically the binding pocket as described herein, will be understood by those of skill in the art. Reference may also be made to standard texts such as Crystal Structure Analysis, Jenny Pickworth Glusker and Kenneth N. Trueblood, 2nd Ed. Oxford University Press, 1985, New York; and Principles of Protein Structure, G. E. Schulz and R. H. Schirmer, Springer-Verlag, 1985, New York which provide further guidance in this regard.

Moreover, as is appreciated by one of skill in the art, a set of structure coordinates for an enzyme-complex, as set out in FIGS. 4, 5 and 6, is a relative set of points that define a three-dimensional shape. It is possible, thus, that an entirely different set of coordinates could define a similar or identical shape, i.e. a functionally equivalent analog of the native NS5B binding pocket, and thus, be within the scope of the present invention. Moreover, slight variations in the individual coordinates will have little effect on overall shape. In terms of binding pockets, these variations would not be expected to significantly alter the nature of compounds that could associate with those pockets.

It is also noteworthy that modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within an acceptable standard of error such as a rmsd<1.0 .ANG. for the alpha carbons that comprise the binding pocket, as compared to the original coordinates, the resulting three-dimensional shape is considered to be the same. Thus, for example, a compound that bound to the active site binding pocket of NS5B described herein would also be expected to bind to another binding pocket whose structure coordinates defined a shape that fell within the acceptable error.

Method of Crystallizing

In another aspect of the present invention, there is provided a method for producing a crystallized HCV NS5B complex comprising an HCV NS5B polypeptide and a compound, wherein said compound forms an association within an NS5B binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of the HCV NS5B, or defined by a functionally equivalent analog thereof.

A preferred crystallization method comprises the steps of: a) incubating a purified HCV NS5B polypeptide in a crystallization buffer to obtain a crystallized NS5B polypeptide; b) solubilizing the compound; and c) soaking the crystallized polypeptide with the solubilized compound in a soaking buffer for a suitable soaking period to yield a crystallized NS5B complex.

In an alternative method of preparing a crystallized HCV NS5B complex as defined above, the compound is added directly to a crystallization buffer containing crystallized HCV NS5B.

In another alternative method, a crystallized HCV NS5B complex as defined above is prepared by co-crystallizing the NS5B protein with the compound. This method comprises the steps of: a) combining purified HCV NS5B polypeptide with the compound in solubilized form to form an NS5B complex; and b) crystallizing the complex in a crystallization buffer.

In a preferred embodiment of this aspect, the NS5B is complexed with a compound selected from the compound families identified above. More preferably, the compound is selected from one of Compounds A, B or C, as set out above, or a related compound thereof. As set out above, the crystallization method may involve direct addition of the compound to a crystallization buffer containing crystallized HCV NS5B or combining purified HCV NS5B polypeptide with the compound in solubilized form. When the solubilized form of the compound is used, the compound is preferably solubilized in 100% DMSO at a concentration of about 25 mM.

NS5B is admixed with the purification buffer at a concentration of about 5 mg/mL to about 15 mg/mL. Preferably, the NS5B is used at a concentration of about 7 to about 10 mg/mL in the purification buffer. The purification buffer, the nature of which is generally known in the art and which may include a salt and/or glycerol to stabilize the HCV NS5B, is used at a pH of between about 6 and about 9. The preferred pH of the purification buffer is about 7.5. A buffer such as, but not limited to, Tris-HCl, HEPES or bis-Tris can be used at a concentration of between about 0 mM and about 50 mM. Preferably, the buffer is Tris-HCl at a concentration of about 20 mM.

In order to stabilize the HCV NS5B, a salt such as NaCl, (NH.sub.4).sub.2SO.sub.4, or KCl can be added to the buffer at a concentration of about 200 mM to about 800 mM. Preferably, the salt is NaCl at a concentration of about 300 mM.

To further stabilize the HCV NS5B, glycerol can be added at a concentration of between about 0% and about 30%. Preferably, glycerol is at a concentration of about 10%.

More preferably, the purification buffer is at about pH 7.5 and contains Tris-HCl at a concentration of about 20 mM, glycerol at a concentration of about 10%, DTT at a concentration of about 5 mM, and NaCl at a concentration of about 300 mM.

The NS5B polypeptide can be crystallized using any one of various techniques known in the art, including for example, batch crystallization under oil, hanging drop vapor diffusion and sifting drop vapor diffusion techniques. The hanging drop vapor diffusion technique, as described in McPherson et al. (Preparation and Analysis of Protein Crystals, Krieger Pub. 1989) is the preferred method of crystallization for the purposes of the present invention. Briefly, this method of crystallization involves placing a droplet containing purified NS5B in a crystallization buffer over a reservoir solution. Vapor diffusion from the droplet increases protein concentration thereby promoting crystallization.

The crystallization buffer used may be selected from any one of a number of buffers known by those of skill in the art to be suitable for this purpose, including, but not limited to, MES, sodium phosphate, potassium phosphate, sodium acetate or sodium succinate at a concentration of about 50 mM to about 0.2 M. Preferably, the crystallization buffer is MES at a concentration of about 0.1 M.

The pH of the crystallization buffer is typically between about 4.5 and about 6.5, and preferably, the crystallization buffer is used at a pH of about 5.4.

The crystallization buffer may additionally contain at least one precipitating agent which facilitates crystallization of the NS5B. Examples of appropriate precipitating agents include, but are not limited to, PEG, PEG5K mme (monomethyl ether polyethylene glycol 5000), ammonium sulfate, MPD, isopropanol, ethanol, or tertiary butanol. The precipitating agent is generally used at a concentration of about 30% to about 40%. In a preferred embodiment, the precipitating agent is PEG5K mme at a concentration of about 21% and ammonium sulfate at a concentration of about 0.4 mM.

The crystallization of NS5B is conducted under standard conditions of crystallization. For example, the crystallization is carried out at a temperature of between about 0.degree. C. and about 22.degree. C. The preferred temperature under which to conduct the crystallization is between about 4.degree. C. and about 11.degree. C.

In the preferred crystallization method, solubilized compound is soaked into crystallized NS5B polypeptide in the presence of a soaking buffer. The soaking buffer can comprise any one of a number of standard buffers including, but not limited to, MES, Tris, sodium phosphate, sodium acetate and sodium succinate at a concentration of between about 50 mM to about 0.2 M. Preferably, the soaking buffer is used at a concentration of about 0.1 M. The pH of the soaking buffer is typically between about 5 and about 8, and preferably, the soaking buffer is used at a pH of about 7.0.

The protein content of the soaking buffer is supplemented to a concentration of up to about 10 mg/mL by addition of any suitable protein including, but not limited to, lysozyme, BSA or even additional NS5B.

The soaking buffer may contain additional agents which function as NS5B stabilizers. One or more salts such as NaCl, (NH.sub.4).sub.2SO.sub.4 or KCl can be added to the buffer at a concentration of about 100 mM to about 500 mM. Preferably, the salt is added at a concentration of between about 150 mM and about 300 nM. More preferably, NaCl and (NH.sub.4).sub.2SO.sub.4 are both added at concentration of about 210 mM and about 280 mM, respectively.

To further stabilize the HCV NS5B, glycerol can be added to the soaking buffer at a concentration of between about 10 and about 20%. Preferably, glycerol is added at a concentration of about 14%.

The soaking buffer may additionally contain at least one precipitating agent which facilitates crystallization. Examples of appropriate precipitating agents include, but are not limited to, PEG, PEG5K mme (monomethyl ether polyethylene glycol 5000), ammonium sulfate, MPD, isopropanol, ethanol, or tertiary butanol. The precipitating agent is generally used at a concentration of about 10% to about 18%. In a preferred embodiment, the precipitating agent is PEG5K mme at a concentration of about 14% and ammonium sulfate at a concentration of about 0.4 mM.

Crystallized NS5B is soaked with solubilized compound in a soaking buffer for a suitable soaking period of about 1 to about 12 hours, preferably about 3 to about 8 hours and most preferably for about 5 to about 6 hours. Soaking occurs at a temperature of between about 5 and about 15.degree. C., and preferably at a temperature of about 11.degree. C.

In one alternative crystallization method, the compound is added to crystallization buffer containing crystallized NS5B. In this method, the compound is simply sprinkled onto the buffer and allowed to solubilize and crystallize following a suitable period of incubation.

In another alternative crystallization method, NS5B and compound are co-crystallized in a crystallization buffer as described above for NS5B alone. In this method, the NS5B and solubilized compound are combined in a crystallization buffer and allowed to crystallize under crystallization conditions such as those described above.

In accordance with the present invention, it is important that the crystallized NS5B complex be amenable to X-ray crystallography. Using X-ray crystallography analysis, the crystals of the NS5B complex obtained belong to space group P2(1)2(1)2(1) with unit cell dimension of a=105.1, b=106.6 and c=133.5 and contain two molecules per asymmetric unit. Diffraction data were measured using a MicroMax007 home source x-ray generator equipped with a R-axis IV++ image plate area detector (Rigaku, Japan). Preferably, data to a resolution of 2.8 .ANG. were collected on a single crystal of the complex.

X-ray Coordinates

According to yet another aspect, there is provided X-ray crystal structure coordinates of an NS5B complex as defined above. Even more preferably, the set of structure coordinates for the NS5B complex are defined according to one of FIG. 4, FIG. 5 and FIG. 6.

The three-dimensional structure of an NS5B complex of this invention is defined by a set of structure coordinates as set forth in any one of FIG. 4, FIG. 5 and FIG. 6 (see Original Patent). The term "structure coordinates" refers to Cartesian coordinates derived from mathematical operations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scaltering centers) of the complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of the binding pocket known as the structure coordinates.

Those of skill in the art will understand that a set of structure coordinates for a protein or protein-inhibitor complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape.

The variations in coordinates may be generated by mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in FIG. 4, 5 or 6 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization or matrix operations to sets of the structure coordinates or any combination of the above.

Various computational analyses are necessary to determine whether a molecule or molecular complex or a portion thereof is sufficiently similar to all or parts of the HCV NS5B protein or the NS5B complex described herein to be considered equivalent. Such analyses may be carried out using current software applications, such as the Molecular Similarity applications of QUANTA [Molecular Simulations Inc, San Diego, Calif.], Sybyl [Tripos Associates, St. Louis, Mo.], InsightII [Accelrys], and MOE [Chemical Computing Group Inc., Montreal, Quebec, Canada].

The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalence in these structures; 3) perform a fitting (superposition) operation; and 4) analyze the results.

Computer-readable Storage Medium

Still, in another aspect of the present invention, there is provided a computer-readable storage medium having stored thereon a model of the crystal structure of an HCV NS5B complex comprising an HCV NS5B polypeptide and a compound, wherein said compound associates with an NS5B binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B as set forth in FIGS. 4, 5 and 6, or defined by structural coordinates of a functionally equivalent analog thereof.

Computer readable data storage media are well known to those skilled in the art and include, for example, hard-disk, CD-ROM, diskette ("floppy disks") and DVD.

In accordance with this aspect of the present invention, the structure coordinates of an NS5B complex, and portions thereof, can be stored in a machine-readable storage medium. Such data can be used for a variety of purposes, such as drug discovery and X-ray crystallographic analysis of the protein crystal.

In a preferred embodiment of this aspect, the HCV NS5B complex comprises NS5B complexed with a compound of a compound family identified above. More preferably, the NS5B complex comprises NS5B complexed with one of Compounds A, B or C.

As set out previously, the binding pocket may be additionally defined by the addition of one or more of the amino acid residues selected from 36, 426, 498 and 499, or even further defined by the amino acid clusters, 36-37, 392-399, 424-429 and 492-503.

The coordinate data of an NS5B complex, such as that set out in FIGS. 4, 5 and 6, when used in conjunction with a computer programmed with software to translate those coordinates into a 3-dimensional structure, may be used for a variety of purposes, especially for purposes relating to drug discovery. Software for generating such three-dimensional graphical representations are known and commercially available. Examples include Quanta and WebLite Viewer. The ready use of the coordinate data requires that it be stored in a computer-readable format. Thus, in accordance with the present invention, data capable of being displayed as a three dimensional structure is stored in a computer-readable storage medium which is capable of displaying a graphical three-dimensional representation of an HCV NS5B complex or of an HCV NS5B binding pocket as defined herein when used by a machine programmed with instructions for using said data.

The HCV NS5B X-ray coordinate data is useful for screening compounds for potential NS5B inhibitory activity. For example, the polypeptide NS5B binding pocket structure encoded by the data may be computationally evaluated for its ability to associate or bind with a given compound. Compounds determined to "fit" into the binding pocket defined herein via some type of association or bonding may also impede the biological activity of the HCV NS5B polymerase and, thus, represent a potential drug candidate. In addition, the data may be displayed in a graphical three-dimensional representation on a computer screen which allows visual examination of the HCV NS5B binding pocket as well as the association of compounds within the binding pocket in an NS5B complex.

Virtual Methods of Identifying Compounds that Associate/Bind HCV NS5B

In further aspects, the present invention provides virtual methods to evaluate the potential of a compound to complex with HCV NS5B. These methods represent a first screen in the search for compounds that can associate or bind to the binding pocket of the present invention, and ultimately in the search for a compound that has therapeutic effects due to an association/binding with the binding pocket that results in HCV inhibition.

Thus, in a further aspect of the present invention, a virtual screening method is provided to identify potential HCV inhibitors comprising the steps of: a) constructing a computer model of an HCV NS5B binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof; b) employing computational means to perform a filting program operation between computer models of the compound to be evaluated and the NS5B binding pocket to provide an energy-minimized configuration of the compound in the binding pocket; and c) evaluating the results of the fitting operation to quantify the association between the compound and the binding pocket, wherein a compound that associates with the binding pocket to yield a low energy, stable complex is a potential NS5B inhibitor.

In addition, the present invention provides a method of identifying compounds that can bind to HCV NS5B, comprising the steps of: a) applying a 3-dimensional molecular modeling algorithm to the structural coordinates of an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428,429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B to determine the spatial coordinates of the binding pocket of HCV NS5B; and b) electronically screening stored spatial coordinates of candidate compounds against the spatial coordinates of the HCV NS5B binding pocket to identify compounds that can bind within the HCV NS5B binding pocket.

In preferred embodiments of this aspect of the invention, the binding pocket may be defined additionally by one or more of amino acid residues 36, 426, 498 and 499, or by one or more of the amino acid clusters defined by amino acid residues 36-37, 392-399, 424-429 and 492-503.

In another preferred embodiment of the invention, the structural coordinates of the binding pocket are those set out in any one of FIGS. 4, 5 and 6, or functionally equivalent structural coordinates as would be appreciated by one of skill in the art.

According to this aspect of the invention, any given compound may be computationally evaluated for its ability to associate with the HCV NS5B binding pocket defined herein, and thus, its potential as an NS5B inhibitor determined. As alluded to above, a computer model of a polypeptide consisting of an HCV NS5B binding pocket as defined herein is constructed using well-known software such as QUANTA [Molecular Simulations Inc, San Diego, Calif.], Sybyl [Tripos Associates, St. Louis, Mo.], InsightII [Accelrys], MOE [Chemical Computing Group Inc., Montreal, Quebec, Canada]. Selected compounds to be evaluated may then be positioned in a variety of orientations, or docked, within the binding pocket. Docking may be accomplished using software such as GRID, DOCK, AUTODOCK, FlexX, and GOLD. When a compound is docked within the binding pocket to form a "virtual" representation of an NS5B complex, computational means may be further employed to generate quantitative and qualitative maps of the complex, including for example, pharmacophore maps, surface property maps (which map Conolly, Gaussian and van der Waals surfaces) and maps of Probabilistic Receptor Potentials using software such as QUANTA, Sybyl, InsightII, and MOE.

The efficiency with which a selected compound binds to the present HCV NS5B binding pocket may be tested and optimized by computational evaluation. The quality of the fit of a given compound within the NS5B binding pocket may be evaluated, for example, by shape, size and electrostatic complementarity as determined qualitatively by visual inspection or as determined quantitatively by the use of scoring functions such as LUDI, PLP, PMF, SCORE, GOLD and FlexX. These methods of qualitative and quantitative evaluation may be employed individually or in combination, for example, as in a consensus scoring manner.

Alternatively, binding efficiency can be determined based on the interaction energy of a complex formed by the binding or association of a compound with the HCV NS5B. For example, a compound determined to form a "low energy, stable complex" with NS5B, in the manner described herein, warrants further analysis as a potential NS5B inhibitor. The term "low energy, stable complex" as used herein is defined as an NS5B complex in which the van der Waals interaction energy value, i.e. the van der Waals energy of interaction between the compound and NS5B, is less than about 8000 kcal/mol. Van der Waals interaction energy value can be determined using the software MOE, and is based on the MMFF94 force field. Accordingly, a compound determined to form a complex having a van der Waals interaction energy value of less than about 8000 kcal/mol is a potential NS5B inhibitor. Preferably, a low energy, stable complex in accordance with the present invention will have a van der Waals interaction energy value of less than about 6000 kcal/mol, and more preferably, a value of less than about 4000 kcal/mol.

Method of Using the NS5B Polypeptide Variants/Analogs of the Invention

Once a series of compounds has been screened using virtual methods such as those described above, compounds determined to be potential HCV inhibitors can be further evaluated to determine the actual propensity of each to interact with the binding pocket of the present invention and to inhibit HCV.

Thus, in still another aspect of the invention, a method of screening candidate HCV NS5B inhibitor compounds is provided comprising the steps of: a) incubating a candidate inhibitor compound under conditions suitable for binding with a polypeptide selected from the group consisting of: i) an isolated and purified polypeptide comprising a functional HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket is exposed by displacement of a finger loop chain defined by at least amino acid residues 18 to 35 and wherein said binding pocket retains its native functional configuration; ii) an isolated and purified HCV NS5B polypeptide consisting of an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration; iii) an isolated and purified HCV NS5B polypeptide analog comprising an HCV NS5B binding pocket defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration and wherein said binding pocket is exposable; iv) an HCV NS5B polypeptide variant comprising at least one amino acid mutation within a finger loop defined by amino acid residues 18 to 35, wherein said mutation provokes a displacement of said finger loop to expose a binding pocket essentially defined by amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket retains its native functional configuration; v) an HCV NS5B polypeptide, or a functionally equivalent analog thereof, characterized by displacement of amino acid residues 18 to 35; and vi) an HCV NS5B polypeptide, or a functionally equivalent analog thereof, in which at least amino acid residues 18 to 35 have been deleted; and b) determining whether or not the candidate inhibitor compound binds to the polypeptide, wherein a compound that binds to the polypeptide is a potential HCV NS5B inhibitor.

Binding of the candidate compound within the defined HCV NS5B binding pocket can be determined using methods well-established in the art. For example, binding assays may be used in which the candidate compound is exposed to a polypeptide containing the NS5B binding pocket of the invention under conditions suitable for association or binding to occur. Binding is then assessed, for example using NMR or other known detection techniques.

Method of Designing an HCV NS5B Inhibitor

In further aspects of the present invention, there is provided methods of designing compounds which associate with the NS5B binding pocket defined herein. The present invention, thus, provides the opportunity to use molecular design techniques to identify, select or design potential inhibitors of HCV NS5B based on the structure of a novel binding pocket in NS5B. Such a predictive model is valuable in light of the high costs associated with the preparation and testing of many diverse compounds that may or may not bind to the HCV NS5B protein.

According to this invention, a potential NS5B inhibitor may be evaluated for its ability to bind an NS5B binding pocket as defined herein prior to its actual synthesis and testing. If a proposed compound is predicted to have insufficient interaction or association with the binding pocket, preparation and testing of the compound is obviated. However, if the computer modeling indicates a strong interaction, the compound may then be obtained and physically tested for its ability to bind. Testing to confirm binding may be performed using conventional assays within the purview of one of skill in the art.

In this regard, a method of designing a compound which binds to an NS5B polypeptide as defined above is provided which comprises the step of: assessing the complementarity, i.e. the "fit", between the compound and a binding pocket in the NS5B polypeptide defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof.

Similarly, a method of producing a drug which inhibits RNA replication activity of HCV NS5B is also provided which comprises identifying or designing a compound which fits into an NS5B binding pocket as defined by at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503 (and optionally one of: amino acid residues 37 and 496) of a native HCV NS5B, or defined by a functionally equivalent analog thereof, wherein said binding pocket is exposed by displacement of a finger loop chain defined by at least amino acid residues 18 to 35.

For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities, including inhibitory compounds, that are capable of filting into and/or binding with the novel NS5B binding pocket defined herein.

One particularly useful drug design technique enabled by this invention is iterative drug design. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein/compound complexes.

Those of skill in the art will realize that association of natural ligands or substrates with the binding pocket of their corresponding receptors or enzymes is the basis of many biological mechanisms of action. Similarly, many drugs exert their biological effects through association with the binding cavities of receptors and enzymes. Such associations may occur with all or any part of the binding pocket. An understanding of such associations will help lead to the design of drugs having more favorable associations with their target receptor or enzyme, and thus, improved biological effects. Therefore, this information is valuable in designing potential ligands or inhibitors of receptors or enzymes, such as inhibitors of HCV NS5B-like polypeptides, and more importantly, HCV NS5B.

In iterative drug design, crystals of a series of protein/compound complexes are obtained and then the three-dimensional structure of each complex is solved. Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three-dimensional structure of the complex, and comparing the associations between the new protein/compound complex and previously solved protein/compound complexes. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.
 

Claim 1 of 15 Claims

1. A method of identifying a compound that may bind to HCV NS5B, comprising the steps of: a) obtaining the structural coordinates of one of FIGS. 4 through 6; b) applying a 3-dimensional molecular modeling algorithm to the structural coordinates of an HCV NS5B binding pocket defined by the structural coordinates of at least amino acid residues 392, 393, 395, 396, 399, 424, 425, 428, 429, 492, 493, 494, 495, 500 and 503, and optionally one of: amino acid residues 37 and 496, of native HCV NS5B as shown in said Figure to determine the spatial coordinates of the binding pocket of HCV NS5B; and c) electronically screening stored spatial coordinates of the compound against the spatial coordinates of the HCV NS5B binding pocket to determine if the compound binds within the HCV NS5B binding pocket, wherein a compound identified by the electronic screening as a compound that binds to NS5B is identified as a compound that may bind to HCV NS5B.
 

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