Defined Viral and Cellular Pathways and Designed Host Cells

In a related system with the stably integrated Tat-responsive reporter cassette, the activity ofAZT was further characterized by transfection of an expression plasmid for Tat in the presence or absence of AZT [65]. Such a system where isolated viral events are probed in cells allows drug screens without the hazards ofviable virus, and this may be advantageous for more dangerous pathogens than HIV. However, as AZT interferes with reverse transcription the reporter signal strength was not affected. Only drugs that target the transactivation step of HIV can be detected in this particular set-up of the LTR-reporter system.

This apparent limitation provides a very important opportunity: the screen for drugs specifically targeting a defined node or phase in the infectious cycle. This is especially important for complex pathogens that frequently change appearance (due to high mutation rates or complicated sequence of generations, as in the eukaryotic parasite Plasmodium) or alternate between dormant states and active replication. HIV has a high mutation rate and spawns escape mutants in single-drug approaches. Furthermore, drugs that target early phases of the infectious cycle (from attachment to insertion of proviral DNA) are without effect on cells that already carry proviral inserts and continuously shed virus. Finally, although HIV is chronically produced in massive numbers (even in clinically latent phases of disease), the virus also remains in a truly latent state as inactive provirus in a subset of infected cells. Current therapeutic approaches therefore aim at a combination of drugs that specifically target different pathways of complex pathogens, and highly active anti-retroviral therapy (HAART) against AIDS is a very good example of the success of such an approach.

Whilst HAART has significantly increased the quality of life and extended the life expectancy of patients infected with HIV, it cannot yet provide a cure for AIDS. Hence, pharmaceutical research is still faced with the challenge to refine screens in order to identify complementary drugs that specifically target only defined aspects in the life cycle of HIV. Patients infected with hepatitis B virus (HBV), hepatitis C virus (HCV) or certain herpesviruses may also benefit from complementary chemotherapy; other pathogens that may be tackled by such approaches include parasites such Plasmodium, the cause of malaria. The principle behind drug screening against HIV therefore is instructive also for other diseases.

Rational drug design, molecular modeling, and enzymatic, cell-free systems each represent extremely powerful methods of developing new therapies. Moreover, designed cell-based systems provide a means of testing these drug candidates in a setting with a complexity which is just one step removed from animal experiments or actual clinical studies.

Indeed, a drug which was rationally designed to interfere with viral trans-activation of transcription (ALX 40-4C) was shown, in a cellular fusion assay, not to target the intended intracellular event but rather to interfere with viral entry [66, 67].

As discussed briefly above, the Tat protein is also a preferred target in complementary chemotherapy. Here, the inhibition of Tat prevents activation of the provirus and thus forces the virus in a dormant state to control further viremia; the T cells already infected may be rescued with Tat inhibitors. It is perhaps surprising today that a great variety of assays and a significant number of drug leads still has not produced a released compound against Tat. Nevertheless, the lessons learned from these experiments may be applicable to screens for inhibitors of other viral or cellular trans-activators of transcription.

When the Tat inhibitor Ro 5-3335 [68] was assayed in animal studies, some toxicity was encountered. Consequently, 400 analogues ofRo 5-3335 were synthesized and probed for interference with Tat activity by transient co-transfection of LTR-driven SEAP reporter plasmid with TAT expression plasmid. Among the promising analogues was Ro 24-7429. Interference with trans-activation was demonstrated using cells chronically infected with HIV and which had down-regulated levels of CD4 receptor so that reinfection with a released virus was inhibited. Thus, a decrease in released particles correlated with the interference of provirus activation. Virion production was quantified via release of p24 viral antigen and cell-associated RNA.

Tat is known to interact with several cellular proteins, and this interaction is essential for trans-activation. This constrains the number of available escape mutations. Indeed, a cell-based screen for the emergence of escape mutants was performed by passaging virus on acutely infected cells in the presence of inhibitor; the inhibitor dose was adjusted to 50% to 95% of depression of p24 antigen expression. Again, Ro 24-7429 showed much promise as it did not allow escape mutants to emerge even after 100 passages in this in-vitro assay.

Ro 24-7429 failed in clinical trials, however, as it was found to be toxic before antiviral concentrations could be reached. One explanation is that the availability of the drug was reduced by binding to human serum; in fact, cellular assays where cultivation was performed in the presence of human serum instead of fetal bovine serum suggested a severely reduced intracellular concentration of Ro 24-7429 [69, 70].

The latter result highlights an additional advantage of the complexity in cell-based systems as opposed to theoretical or enzymatic approaches: physiological inhibitors or modifications towards activation can be considered in the set-up and thus observed at a level well below those in actual animal or clinical studies. Choosing the adequate host cell for the assays will reveal whether this activation can be expected in the patient. For example, nucleoside analogue reverse transcriptase inhibitors (e.g., ddC and AZT) must be activated in the host cell by phosphorylation, but not all HIV-susceptible cells perform this modification [71].

The early cellular systems for medium- to high-throughput screens for interference with defined viral pathways have been further refined [72-75]. Among others, the CAT and lacZ reporter genes have been replaced with EGFP, SEAP or luciferase to provide fast readouts with minimal handling and development times. Another complication of HIV pathogenesis is a shift in the requirement for co-receptors on target cells: early in seroconversion, the predominant HIV-1 population in the patient is specific for the CCR5 co-receptor, whereas increasing numbers of CXCR4-specific viruses emerge in later stages [76, 77]. The patterns of co-receptor expression varies among T-cell populations [78], including the generally accepted immortalized Jurkat T cell used in some drug screens. The shift in co-receptor specificity is important for pathogenesis and epidemiology. Consequently, the indicator cells are designed or selected to express either or both co-receptors at high levels to assess specific or both types of viruses.

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