Paul K. Tomich

Development of Activity Assays for High-Volume Evaluation of Human Immunodeficiency Virus (HIV) Protease Inhibitors in Rat Serum: Results with Ditekiren

We showed previously that a commercially available synthetic tetradecapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, produces authentic angiotensin I (Ang I) upon incubation with the HIV-1 protease (S. K. Sharma et al., Anal. Biochem. 198:363, 1991). Therefore, we developed an Ang-I based activity assay for HIV protease inhibitors based on the technology developed earlier (M. J. Ruwart et al., Pharm. Res. 7:407, 1990; S. K. Sharma et al., Anal. Biochem. 186:24, 1990) for tracking renin inhibitors in rat sera. Ditekiren was either extracted from sera with ethyl acetate or assayed after the interfering substances in sera were precipitated with acetonitrile. Purified recombinant HIV-1 protease was added to extracted rat serum and the enzymatic reaction was initiated in the presence of the tetradecapeptide substrate. The inhibition of Ang I production was measured by a commercially available RIA kit. The cleanup methodology also enabled a commercially available Proteinase Scintillation Proximity Assay (SPA, Amersham) to quantify ditekiren in rat serum through the addition of recombinant HIV-1 protease and cleavage of substrate from SPA beads. Results were confirmed by HPLC or by the renin assay for ditekiren, which inhibits both aspartyl proteases. These technologies should prove useful for assessing serum levels of HIV protease inhibitors in rat.

The Utility of Streptomycetes as Hosts for Gene Cloning

As in other biological areas, modern scientific techniques such as recombinant DNA methodology have helped to harvest the fruits of knowledge for those species belonging to the genus Streptomyces. This genus has long provided many useful products such as antibacterial and antineoplasia antibiotics (e.g., 1–4), immunomodulators (5) and a variety of enzymes such as proteases (6,7), restriction enzymes (8,9), amylases (10), xylanases (11), and agarases (12). In addition, Streptomyces ssp. deserve study in their own right because they have an interesting life cycle.