SsDNA High fluorescence

FIG. 4. FRET-based helicase unwinding assay. The measured fluorescent signal depends on the interaction between the fluorescent tag and the quencher. Spatial separation of the two DNA strands through ATP driven unwinding increases the signal. An inhibitor would block the unwinding activity of the helicase.

NS5 polymerase

Dengue NS5 protein is the largest viral protein in the flavivirus genome and contains two enzymatic activities: an S-adenosyl methionine transferase (SAM) and an RNA-dependent RNA polymerase (RdRp) domain. The NS5 polymerase is the most conserved protein across dengue serotypes, with more than 75% sequence identity and it is essential for viral replication in all flaviviridae. Mutations within the GDD motif of this protein result in non-viable virus.

Polymerases have been the most useful target class for antiviral drug discovery. Numerous compounds targeting this class of enzymes, such as famciclovir (herpes simplex virus), tenofovir (HIV) or adefovir (hepatitis B) are in clinical use. While the above drugs work against different subtypes of polymerase enzymes, the first compounds inhibiting hepatitis C (HCV) RdRp have entered clinical trials or are in preclinical development. It is currently unclear whether these compounds will also inhibit the replication of flaviviruses, but scientists at Merck (Olsen et al 2004) have reported that compound A (a polymerase inhibitor of the nucleoside class) has antiviral activity on a number of members of flaviviridae (Table 4). We have examined 7-deaza-2'-'C-methyl adenosine (A) and one of its analogues (B) in our in-house cellular assay (Q.Y. Wang, personal communication) and confirmed their

TABLE 4 Cellular activity of Ac against Flaviviridae (mM)b

BVDVa

West Nile

Dengue 2

Yellow fever

EC50

0.3

4.5

15

15

CC50

>50

250

>320

>320

a Bovine diarrhoea virus. b Data from Olsen et al (2004). c 7-Deaza-2'-C-methyl-adenosine.

a Bovine diarrhoea virus. b Data from Olsen et al (2004). c 7-Deaza-2'-C-methyl-adenosine.

TABLE 5 Cellular activity of A and B against dengue 2c (m M)

ECS0

cc50

a 7-Deaza-2'-C-methyl-adenosine. b 2 '-C-methyl-adenosine. c TSV01.

d Values are averages of at least two independent determinations. e See description of assay in text.

activity on the dengue 2 virus (Table 5). The NITD cell-based assay is performed in a 96-well plate format, with BHK21 (BHK = baby hamster kidney) cells that are infected by dengue 2 virus in the presence of inhibitors. After 2 days of incubation, virus load is measured by quantifying the amount of viral envelope protein produced using ELISA.

Even though all of the compounds discussed above were derived from nucleo-sides, we have decided as a first priority to focus on non-nucleoside inhibitors for flavivirus RdRp. The reason for this strategy originates in the target product profile. Because all nucleosides are prodrugs which are converted in cells to the active triphosphate, the space for chemical innovation is restricted. Furthermore all work is done in cellular systems, which makes the tasks of optimizing parameters that dictate PK and tolerability slow and difficult. Because of the absence of structural information, we will follow a two-pronged strategy to find a lead. Taking advantage of recent developments in mass spectrometry, we have identified compounds that bind to the dengue NS5 protein. This is a very convenient and quick way to find potential chemical starting points, since there is no need for a sophisticated assay. A drawback of this technique is that not all hits will have the desired effect on the function of the protein. Nevertheless we have already identified very promising inhibitors of dengue 2 NS5 polymerase using this technology. These hits are currently going through a standard hit-to-lead process.

For the HTS campaign we have developed a Scintillation Proximity Assay (SPA) (Fig. 5) (Ferrari et al 1999), which will be used for the screening of the Novartis compound and natural product collections during the second half of 2005. In order to follow our platform strategy for this target, cellular assays to test the resulting compounds on other flaviruses are currently being set up in the NITD BSL-3 laboratory.

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