Biochemical screening assays to identify HIV-1 integrase inhibitors.

Human immunodeficiency virus type 1 (HIV-1) integrase is, in addition to reverse transcriptase and protease, an important enzymatic target for antiretroviral drug development. Integrase plays a critical role in the HIV-1 life cycle coordinating the integration of the reverse-transcribed viral DNA into the host genome. This integration step is the net result of two consecutive integrase-related processes. First, integrase removes a dinucleotide from the 3' viral DNA ends in a process called 3'-processing. Next, in a process called strand transfer, the viral DNA is integrated into the host genomic DNA. Early on, biochemical assays have played a critical role in understanding the function of HIV-1 integrase and the discovery of small-molecule inhibitors. In this chapter we describe two biochemical assays to identify inhibitors of the 3'-processing and strand transfer process of HIV-1 integrase.

A duplex real-time RT-PCR assay for profiling inhibitors of four dengue serotypes.

We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, ß-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells ß-actin gene is 100%.

Binding kinetics, uptake and intracellular accumulation of F105, an anti-gp120 human IgG1kappa monoclonal antibody, in HIV-1 infected cells.

The use of targeting moieties is a new and exciting field of scientific research for facilitating the specific delivery of therapeutic agents in HIV-infected patients. The interaction of a potential targeting moiety with its ligand is a crucial factor in the evaluation of a targeted approach for chemotherapeutic intervention. Therefore, we have further characterized the interaction between a potential targeting agent, the monoclonal human antibody F105, and its ligand gp120, a glycoprotein expressed on the surface of HIV-1 infected cells. We demonstrate the specificity of binding and entry of F105 to infected cells. F105 was rapidly taken up into the cell and accumulated in the Golgi apparatus. Kinetic analysis of the F105-gp120 interaction revealed an equilibrium dissociation constant (K(D)) of 0.62 nM, compared with the gp120-CD4 interaction where the K(D) was determined at 35 nM. Consequently, F105 displayed a higher gp120 affinity. This was due to a slower dissociation as compared with the natural ligand. These data further underline the potential of monoclonal antibodies as targeting agents, and offer new insights into the possibility of F105 as a targeting moiety for the delivery of antiretroviral drugs to HIV-1 infected cells.

Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology.

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.