Transcriptase Inhibitors
1.4.4. Protease Inhibitors
Protease cleavage o f polyprotein precursors is another enzymatic function coded for by the virus, and has become one o f the two targets for clinically available anti-HIV drug therapy (the other being RT). Ease o f recombinant protein production and purification has facilitated extensive research into the dimeric enzyme and its inhibition. Reference to other retroviral proteases together with biochemical and crystallographic analysis confirmed that HIV-1 protease comprises two identical 99 amino acid subunits, and like human renin, is an aspartyl protease (Navia et al., 1989; Fitzgerald and Springer, 1990). Its natural substrates are nine dissimilar and variable motifs at the sites o f Pr55gag and Prl60gag-pol precursor cleavage. Failure to cleave these polyproteins leads to the release o f immature, non-infectious virions (Kohl et al., 1988; Freed, 1998). Combinations o f random screening and rational drug design led to the development o f the five currently licensed protease inhibitors (Pis - saquinavir, ritonavir, indinavir, nelfmavir and amprenavir). These are ‘peptidomimetic’ in that they mimic the short peptide substrates o f HIV-1 protease, binding the enzym e’s active site and competitively inhibiting the enzyme (Martin, 1992).
Significant side effects have been reported by patients on Pl-containing regimens. Most notable are gastrointestinal problems such as nausea, diarrhoea and vomiting, and metabolic problems such as abnormal processing o f lipids, dangerously elevated cholesterol levels, and hyperglycaemia. Future PI design must take these increasingly serious side effects o f long-term PI use into account (Graham, 2000).
1.4.5.
Other Antiretroviral Strategies
Unfortunately, o f all potential drug targets, only protease and RT have yet proven susceptible to clinically useful compounds. The utility o f potential compounds depends on many factors on top o f their antiretroviral effects. Oral bioavailability, pharmacokinetics, toxicity, target specificity, activity against viruses to existing drugs, efficacy against diverse strains o f HIV-1, compatibility with other therapies, ability to
cross the blood-brain barrier, and the cost o f continuous treatment must all be taken into account.
Much early interest was in the interaction between the viral envelope glycoproteins and CD4. Soluble forms o f CD4 (sCD4) were tested, but despite good in vitro blocking activity, early doses proved ineffective in vivo due to short half-lives, resistant clinical isolates and ultimately little effect on plasma viraemia. Elevated doses o f sCD4 conjugated to immunoglobulin G molecules showed improved pharmacokinetics but little clinical benefit (Hadden, 1998).
The discovery o f co-receptors led to renewed interest in virus entry as a target, and explained the mode o f action o f bicyclams - a class o f compounds now known to inhibit HIV-1 replication by interfering with CXCR-4 usage. To date, AM D3100 is the most promising, but low oral bioavailability, toxicity, and the rapid emergence in vitro o f resistant virus continue to hamper its usefulness in vivo (De Clercq, 2000).
A short peptide called T22 with anti-HIV properties was also found to act through CXCR-4 antagonism. Since its discovery in 1992, more potent and less cytotoxic derivatives with activity against bicyclam-resistant viruses have been developed. To date, viruses resistant to this class o f inhibitor have not been reported, despite determinants o f viral susceptibility having been located within the hypervariable V3 loop o f g p l2 0 - a region noted for its plasticity (LaBranche et al., 2001). Other developments towards fusion and entry inhibitors include an inhibitor o f CCR-5- mediated entry with in vitro anti-HIV activity, and 5-Helix, a promising synthetic fusion inhibitor mimicking a six-helix bundle found in gp41 essential for membrane fusion (Cammack, 2001).
The virally encoded enzyme integrase is essential to the viral life cycle and has no cellular homologue. Despite its catalytic domain having been crystallised, only weak inhibitors o f integrase have so far been discovered. It is hoped that these will act as ‘leads’ in the development o f more potent inhibitors (Pani and Marongiu, 2000). Interventions at other life-cycle stages are still in their infancy - licensed therapies for in vivo use remain distant. Drug-based approaches include a peptide mimic o f the
nucleocapsid protein (Druillennec et al., 1999b), and Vpr-binding compounds inhibiting nuclear import o f viral nucleic acids (Dubrovsky et al., 1995). Others are employing gene therapy vectors to deliver specific D N A sequences to infected cells. The range o f products that these could express includes antisense RNA designed to anneal to viral transcripts such as the TAR, decoy RNA to inhibit Rev, H IV -1-specific ribozymes, or mRNA to be translated into antibody fragments binding specific viral proteins (VandenDriessche et al., 1997).
In parallel to the design o f novel therapeutic compounds, a vast amount o f work is being done towards the development o f both preventative and therapeutic HIV-1 vaccines. Immunogenic variability, sequestration, infection o f many immune system cell types, inability to identify suitably protective immune mechanisms, and varied transmission routes all hinder advances in this field. The discovery o f HIV-1 specific immunity in persistently exposed but uninfected individuals, and the successful application o f new technologies to achieve protective immunity in primates have both been encouraging developments in this field. Many vaccine trials are currently being carried out, with systems as diverse as simple peptide stimulation through D N A vaccination, to bacterial and viral vector delivery systems (Hanke, 2001).