steps within the infected cell. Sometimes a non-specific enzyme (e.g.pyruvate kinase) can perform this
task. Mammalian cellular kinases are usually highly selective in their substrate requirements and this
greatly restricts the acceptability of potential chain terminator nucleosides. AZT has only a small struc-
tural modification and is a good substrate for the host kinase. Thus its triphosphate is available as a sub-
strate for the viral RT and so causes chain termination. Unfortunately, the high dose levels needed for AZT
(around 1 g per day) give rise to considerable host toxicity. This is probably because the triphosphate of
AZT is to some extent a substrate for the DNA polymerase of the host cell, and thus can contribute to the
observed toxic side effects of the drug, including bone marrow suppression.
A number of other nucleosides, including dideoxynucleoside analogues, such as 2 ,3-dideoxyinosine
(ddI, Figure 3.90) and 2 ,3-dideoxycytidine(ddC, Figure 3.90), are also approved for the treatment of HIV
infection. They share the same problems of toxicity and requirement for phosphorylation by host enzymes
with AZT. The use of nucleotide prodrugshas greatly improved the efficacy of these and other nucleo-
side analogues that are not good substrates for phosphorylation in vivo.^128 Such prodrugs are non-ionic, which
aidstheir cellular uptake, and they are converted enzymatically or spontaneously into their monophosphates
after entering the cell. Phosphorylation to the bioactive triphosphate forms then follows.
The use of combination therapyin the treatment of HIV is becoming increasingly common to combat
problems of drug resistance. This typically involves the use of AZT together with a second anti-HIV com-
pound such as lamivudine (Figure 3.90). The rationale for this approach is that the use of a combination of
drugs that are synergistic and have no overlapping toxicity can reduce toxicity, improve efficacy and pre-
vent drug resistance from arising.
The prolonged use of AZT in AIDS patients leads to the development of drug-resistant HIV strains because
the drug is not 100% effective in killing the virus and mutants resistant to AZT survive and proliferate. Single
mutations at residue-184 of the RT in HIV cause high-level resistance to 2,3-dideoxy-3-thiacytidine
(3TC, lamivudine, Figure 3.90) that is an important component of triple-drug anti-AIDS therapy. Such
mutations contribute to the failure of anti-AIDS combination therapy.
Considerable progress is being made in understanding the nature of drug resistance through analysis of
X-ray structures of wild-type and mutated HIV RT complexed with a nucleotide drug and DNA. Arnold^129
has determined crystal structures of the 3TC-resistant mutant HIV-1 RT (M184I) in both the presence and
absence of a DNA/DNA template-primer. In the absence of a DNA substrate, the wild-type and mutant struc-
tures are very similar. However, comparison of structures of M184I mutant and wild-type HIV-1 RT with and
without DNA shows that the template-primer is repositioned in the M184I/DNA binary complex and there
are other smaller changes in residues in the dNTP-binding site. These structural results support a model that
explains the ability of the 3TC-resistant mutant M184I to incorporate dNTPs but not the nucleotide analogue
3TCTP. The same model can also explain the 3TC resistance of analogous hepatitis B polymerase mutants.
3.7.2.1.2 Non-Nucleoside Reverse Transcriptase Inhibitors.^130 The structure of HIV-1 RT
has been solved by X-ray crystallography (Figure 10.30). The active form of the enzyme is a heterodimer
having one polymerase active site and one RNaseH active site. Several potent and specific inhibitors of
HIV-1 RT were discovered in the early 1990s that are not nucleosides (NNRTIs) and probably do not
require kinase metabolism to generate an active form (Figure 3.91). One of them, nevirapine (Figure
3.91c), has been co-crystallised with the transcriptase and its binding site on the enzyme is seen to be a
hydrophobic pocket guarded by two tyrosine residues close to the polymerase active site. Thus NNRTIs
result in allosteric inhibition of the enzyme rather than the competitive inhibition that results from the
nucleoside-based inhibitors binding to the active site and so binding of an NNRTI can inhibit reverse
transcription directly.
Although, the structures of these non-nucleoside inhibitors are very diverse, they are all believed to bind
in a similar (though not identical) location. They show little toxicity and have a very high anti-HIV activity
in cell culture. However, HIV-1 rapidly becomes resistant to these drugs, in most cases, owing to the selec-
tion of strains containing an RT mutated at one or both of the tyrosines, while retaining infectivity. In add-
ition to nevirapine, two other NNRTIs are also in clinical use are – delavirdine and efavirenz (Figure 3.91).
Nucleosides and Nucleotides 131