Validation Testing

It is essential to establish that the hits from the rapid mechanistic plate assay block DNA synthesis directed by Pol-8 and PF-8 specifically, e.g., by inhibiting the catalytic activity of Pol-8 or the interactions among Pol-8/PF-8, Pol-8/DNA, or PF-8/DNA. However, many compounds may inhibit DNA synthesis nonspecifically, e.g., by intercalating DNA. To confirm the specificity of the inhibition, and further, to distinguish whether the inhibition affects the catalytic Pol-8 vs the processive Pol-8/PF-8 interaction, requires greater detailed analyses. One informative test is the in vitro M13 DNA synthesis gel assay (21), which reveals the sizes of the DNA products resulting from processive DNA synthesis on agarose gels. The details of the M13 gel assay performed with herpesvirus polymerases and their cognate processivity factors are presented here, followed by an illustration of the assay using candidate inhibitors from the rapid mechanistic plate assay.

1. Preparation of the M13 primer-template.

a. Anneal the M13 Universal sequencing primer to M13mp18 (+) single-stranded DNA (Pharmacia) in the presence of 100 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.6.

b. Remove excess primer by filtration through a Centricon-100 spin filter (Amicon).

2. Performing the M13 DNA synthesis assay.

a. Using a 1.5-mL centrifuge tube for each reaction, add 2 |L of each protein (Pol-8 and/or PF-8), X |L of compound and (21 - X) |L of H2O to 25 |L of 2X buffer to make a 50 ||L final reaction volume that contains 100 mM (NH4)2SO4, 20 mM Tris-HCl, pH 7.5, 3 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 4% glycerol, 40 |g/mL bovine serum albumin, 60 ||M each of dATP, dGTP, and dTTP, 10 |M [a-32P]dCTP (3000 Ci/mmol; NEN), and 50 fmol of M13 primed template.

b. Incubate the reaction at 37°C for 60 min, and then stop it by adding 50 |L stop solution containing 1% SDS, 10 mM EDTA, 10 mM Tris-HCl, pH 8, and 200 |g/mL proteinase K followed by incubation at 37°C for 60 min.

3. Analyzing labeled M13-derived DNA products by gel electrophoresis.

a. Extract the DNA products by phenol/chloroform followed by ethanol precipitation in the presence of 1 M ammonium acetate.

b. Resuspend the precipitated DNA in 20 | L gel loading buffer (50 mM NaOH, 2.5 mM EDTA, 25% glycerol, and 0.025% bromocresol green) and then fractionate on a 1.3% alkaline agarose gel.

c. Dry the gel and subject it to autoradiography or analysis by a Phosphorlmager (Molecular Dynamics).

3.13.1. Example of Validation Testing

1. Twenty-eight putative inhibitors (hit compounds) were obtained by performing a rapid mechanistic plate assay screen of 2000 compounds from the NCI repository.

2. The specificity of these putative inhibitors, which were chosen on the basis of DIG-dUTP incorporation, were validated by the M13 gel assay, which allows newly synthesized DNA strands to be visualized as labeled products on a alkaline agarose gel. A subset of these compounds tested in the M13 assay is represented in Fig. 2.

3. Several of these compounds (represented by compound 130813) completely inhibited DNA synthesis directed by Pol-8/PF-8 in the M13 gel assay, whereas other compounds (represented by compound 86372) were partially effective (see upper panel in Fig. 2). Notably, one compound (i.e., compound 147744) that proved to be inhibitory in the rapid mechanistic plate assay failed to inhibit Pol-8/PF-8 in the M13 gel assay (see upper panel in Fig. 2).

Fig. 2. Use of the M13 gel assay to validate putative DNA synthesis inhibitors identified by the rapid mechanistic plate assay. M13 single-stranded DNA with an annealed oligonucleotide primer was incubated with the premix solution containing in vitro translated Pol-8 and PF-8 and [32P]dCTP for radiolabeling. The synthesized DNA products were fractionated on a 1.3% alkaline agarose gel and then analyzed by autoradiography. The control reaction (buffer, no compound) generated DNA strands corresponding to the full-length (FL) template (7249 nt) as well as characteristic shorter products (SP) that were several hundred nts in length. Numbers above each lane designate a particular test compound from the NCI repository. The effect of each compound on DNA synthesis by the Kaposi's sarcoma-associated virus (KSHV) proteins Pol-8 and PF-8 (upper panel) is directly compared with DNA synthesis by the herpes simplex virus (HSV)-1 proteins UL30 and UL42 (lower panel).

Fig. 2. Use of the M13 gel assay to validate putative DNA synthesis inhibitors identified by the rapid mechanistic plate assay. M13 single-stranded DNA with an annealed oligonucleotide primer was incubated with the premix solution containing in vitro translated Pol-8 and PF-8 and [32P]dCTP for radiolabeling. The synthesized DNA products were fractionated on a 1.3% alkaline agarose gel and then analyzed by autoradiography. The control reaction (buffer, no compound) generated DNA strands corresponding to the full-length (FL) template (7249 nt) as well as characteristic shorter products (SP) that were several hundred nts in length. Numbers above each lane designate a particular test compound from the NCI repository. The effect of each compound on DNA synthesis by the Kaposi's sarcoma-associated virus (KSHV) proteins Pol-8 and PF-8 (upper panel) is directly compared with DNA synthesis by the herpes simplex virus (HSV)-1 proteins UL30 and UL42 (lower panel).

4. To establish their validity and specificity further, this same panel of compounds was tested in the M13 gel assay for inhibition of DNA synthesis directed by the HSV-1 DNA polymerase (UL30) and processivity factor (UL42). The effect of most of the compounds on DNA synthesis by HSV-1 is similar to that observed with KSHV, with the major exception being compound 95609, which caused complete inhibition HSV-1 but only partially inhibited KSHV (see Fig. 2).

3.13.2. Interpretation of Validation Testing

In summary, the M13 gel assay serves to rigorously substantiate the compounds classified as inhibitory by the rapid mechanistic plate assay. Because the advantage of the rapid mechanistic plate assay is to examine thousands of compounds conveniently and relatively inexpensively, it is not surprising to find occasionally that a putative inhibitory compound actually proves not to be inhibitory using the more "visual" M13 gel assay (e.g., compound 147744; see Fig. 2) Importantly, unlike the mechanistic plate assay that employs a template of 100 nt, the M13 gel assay demands robust processivity by employing a full-length (FL) template of 7249 nt. It is noted that a large proportion of short products (SP) of several hundred nts are typically generated in this assay (see Fig. 2). The major question is whether inhibitors observed by both the rapid mechanistic plate assay and M13 gel assays are specific to the proteins in question. This demands testing the same compounds on other DNA polymerase/proces-sivity complexes, e.g., HSV-1. Inhibitors that completely block more than one polymerase/processivity complexes may be acting nonspecifically, e.g., by intercalating DNA or by inhibiting the conserved catalytic site present in many polymerases. Of interest are inhibitors that completely inhibit one poly-merase/processivity complex, but not another. For example, the complete inhibition of DNA synthesis directed by the HSV-1 proteins with compound 95609, but partial inhibition of the KSHV proteins, suggests that this inhibitor has greater specificity for HSV-1 than KSHV. Further requisite testing for specificity involves determining whether this inhibitor blocks DNA synthesis directed by other herpesvirus and other cellular DNA polymerases and processivity factors. Since the molecular structures of the NCI inhibitors have been characterized, new chemical compounds may be designed to obtain even greater specificity.

4. Notes

1. The ABTS substrate kit contains all the reagents necessary to prepare a working solution of 2,2'-azino-to (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). ABTS will produce a water-soluble, green-colored product upon reaction with horseradish peroxidase. The substrate is light-sensitive and must be kept in the dark both as a stock solution and as a working solution.

2. The total volume of a DNA synthesis reaction per well is 50 ||L. This includes 25.2 ||L of premix solution and 24.8 |L of the test compound with water to 50 |L final volume.

3. For Anti-DIG-POD stock and working solutions and for ABTS POD substrates, refer to the instruction manual of the DIG Detection ELISA (ABTS) kit (Roche).

4. Use separate premix solutions as negative controls in which Pol-8, PF-8, or both are absent and replaced with water.

5. The PF-8 and Pol-8 proteins can be either in vitro translated or purified from E. coli, as in the case of PF-8 (7) or from baculovirus, as in the case of Pol-8 (20).

6. To ensure a clean background, it is important to remove completely all the P/T binding solution.

7. The reaction may need to be mixed to distribute the test compound.

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