The non-peptidic HIV protease inhibitor tipranavir and two synthetic peptidomimetics (TS98 and TS102) modulate Pneumocystis carinii growth and proteasome activity of HEL299 cell line.

THE clinical efficacy of highly active antiretroviral therapy (HAART) containing HIV protease inhibitors against opportunistic infections is now well known (Sepkowitz 1998). Widely used HIV protease inhibitors (including Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, Lopinavir, and Atazanavir) are compounds with structures that mimic the local topography around an amide bond. They are low MW peptidomimetics (MWo1,000Da) containing transition-state isostere (hydroxyethylamine and hydroxyethylene) that match the peptide backbone atom-for-atom and form important contacts with the HIV protease binding-site (Ripka and Rich 1998). We previously demonstrated that the majority of available HIV protease inhibitors partially and non-specifically reduced rat-derived Pneumocystis carinii growth in vitro when tested at clinically administered concentrations (Atzori et al. 2000, 2001; Mazza 2003). In this study we evaluated the anti-Pneumocystis activity of three other compounds, Tipranavir, TS98, and TS102. TS98 and TS102 are small peptidomimetics that are structurally and functionally correlated with clinical HIV protease inhibitors. TS98 and TS102 were designed by taking into account the binding site of HIV-1 and Candida albicans (Sap2) proteases (Tossi et al. 2003). Tipranavir is the first compound of a new class of non-peptidic HIV protease inhibitors based on novel templates which, though appearing unrelated to the original peptides, contain the necessary groups positioned on a non-peptidic scaffold that serves as a topographical mimetic. This kind of molecules represents an ideal peptidomimetic in that their non-peptidic nature makes them recalcitrant to proteolytic degradation and hence have increased bioavailability (Ripka and Rich 1998). Because of the lack of a continuous in vitro culture system for P. carinii, we assayed Tipranavir, TS98, and TS102 in a monoxenic culture system containing P. carinii isolated from infected rat lungs and human embryonic lung cells (HEL299) feeder cells. To understand if the effects observed on P. carinii proliferation in vitro were related to modulation of feeder cell proteasomes, we performed examined the effect of the three compounds on intact HEL299 cells and the 20S proteasome isolated from them (Groettrup et al. 1995). The 26S proteasome is a multifunctional proteolytic complex of approximately 2,000 kDa consisting of two subcomplexes. These include the 20S proteasome and the 19S particle that serve catalytic and regulatory functions, respectively. The 26S proteasome is able to degrade proteins with almost complete specificity for ubiquitin-conjugated proteins, and its catalytic activity depends on ATP. In vivo, the 20S proteasome is likely to exist as a single particle or in combination with the 19S regulator. It can also combine with another particle, 11S. 11S plays a regulatory role in producing the hybrid 19S–20S–11S particle, which requires ATP for its assembly and retains the ability to degrade ubiquitinated conjugates. Proteasomes purified from different tissues and species are approximately 700 kDa in size and are comprised of four staked rings, each containing multiple subunits. This particle designated as the 20S proteasome retains peptidase activity without requiring ATP and is unable by itself to degrade proteins combined with ubiquitin molecules (Piccinini, Mostert, and Rinaudo 2003).