ANGELO STATE UNIVERSITY
The long term goal of this project is to develop therapeutics that will result in the elimination of human immunodeficiency virus. The objective of this particular application, which represents a first step toward the attainment of our long term goal, is the development of Trojan horse inhibitors (THIs) that are activated by HIV protease and serve to kill the cells in which the HIV is attempting to replicate. The specific aims of the project are to: 1. Determine the in vitro functionality of THIs, 2. Investigate the ability of the first generation THI to kill HIV infected cells and reduce virus production in tissue culture, and 3. Design the second and subsequent generations of THI. Expected outputs and outcomes: It is expected that the first generation Trojan Horse Inhibitor, THI-1, will be produced and evaluated for in vitro and in vivo activity.
The second generation THI, THI-2 has been designed but not cloned. It is expected that the THI-2 will be expressed and purified. Significant deliverables will include production and evaluation of THI-1 and production of THI-2. The genes for Trojan Horse Inhibitor 1 (THI-1) and Trojan Horse Inhibitor 2 (THI-2) were synthesized as fusion constructs with maltose binding protein (MBP). The MBP-THI-X fusion proteins expressed well in E. Coli. Initial purification was accomplished with dextrose columns. The resulting affinity purified protein was subjected to cyanogen bromide (CNBr) cleavage to release the THI-X.
Identification of the authentic THIs in the reaction mixtures was not achieved. It was discovered after some experimentation that the filters being used to remove particulate matter before injection onto the high performance liquid chromatograph (HPLC) were binding a variety of protein peaks. After that discovery, it was possible to isolate a several partially helical protein peaks from the mixtures. These were analyzed off-site by mass spectroscopy. None proved to the be the authentic THIs.
Both THI-1 and THI-2 were cloned into plasmids without the maltose binding protein fusion in hopes of eliminating the CNBr cleavage step. The THI-1 construct would not grow well enough to isolate colonies. Although it was possible to grow colonies of E. Coli containing the THI-2 plasmid, these cells would not grow after the THI-2 was induced. The THI-1 results suggest that the masking function of the extra helix of the three helix bundle is not performing as desired. The THI-2 results suggest that the toxicity is much reduced from the early design but that the peptide is still toxic at the expressed levels. It should be noted that the expressed levels can be quite high compared to the intended therapeutic levels. So the toxicity in these experiments is worrisome but not definitive.