HIV reverse transcriptase is the primary target of AIDS drugs. It converts HIV's RNA code into DNA for viral replication. There are two classes of inhibitors: nucleoside analogs and non-nucleoside inhibitors (NNRTIs). NNRTIs bind to a site near the active site and prevent conformational changes needed for viral replication. Using composite binding sites from multiple structures allows rational drug design of new NNRTIs with higher inhibitory activity and lower toxicity than previous methods.

1. HIV Reverse Transcriptase; Target for AIDS Treatment AIDS, though an incurable disease, can be significantly slowed in its onset through HIV chemotherapy. The polymerase HIV-1 RT is the primary target of current drug design. This enzyme converts the RNA code contained in the HIV retrovirus into DNA that can be spliced into the host genome for viral replication. Two classes of HIV-1 RT inhibitors have emerged; these are nucleoside analogs and non-nucleoside inhibitors (NNRTIs). In the following presentation, the general structure of HIV-1 RT will be investigated, followed by a demonstration of the means by which these inhibitors halt transcriptase function in vivo . Furthermore, computational methods of drug design will be explored.

2. General Structure of HIV-1 RT The enzyme is a heterodimer of two subunits, p66 & p51. Both subunits are derived from a common peptide precursor, since mutations in the HIV genome replace aa’s in both subunits. 1 Both subunits (shown only for the p66 domain) have 4 well defined regions; Finger, Thumb, Palm and Connection. The p66 domain also has an additional RNase H domain, which cleaves the template RNA strand following its conversion into DNA. 2 The arrangement of subdomains in the two subunits is dramatically different; p66 is organized to form a cleft into which the RNA template;DNA primer bind. The 3’-OH of the primer terminus lies in proximity to a triad of conserved aspartic acid residues in the active site. There is only a single active site in each HIV-1 RT.

3. Crystal Structures Provide Different Insight Multiple crystal structures of HIV-1 RT have been obtained under different conditions, though perhaps the most revealing of the enzyme mode of action was recently obtained by the Verdine group. 2 This structure utilizes the formation of a disulfide bond formed between the protein and a DNA template strand (note that HIV-1 RT can polymerize DNA and RNA) to stall the enzyme immediately before the incorporation of a 4th dNTP into a growing DNA duplex. Binding of the template:primer and dNTP induce p66 conformation changes that shift the finger domain nearer to the palm region. This movement brings several new residues in contact with the bound dNTP. The dNTP site, well resolved in Verdine’s model (unable to be seen w/ a dNTP present in earlier structures), shows that the coordination of a lysine, arginine, and two magnesium ions form the active complex that incorporates the base into the growing duplex. Finger Region Palm Region ThumbRegion

4. Further Investigation of the Active Site and Conformational Changes A further image of the active site is shown to the right in which Mg (B) can be seen to possess an octahedral type geometry reminiscent of T7 DNA polymerases. To the left, Verdine’s structure (red) is superimposed on several other crystal structures such as the unligated (green) and NNRTI bound structure (blue) to demonstrate the conformational changes occurring in each case. Note that the finger domain in the Verdine structure is the most altered, indicative of its importance in the incorporation of additional dNTPs to the growing duplex.

5. Investigation of Drug Binding Sites in HIV-1 RT Due to the lack of a 3’-OH group, the strand cannot be further elongated, hence the complex is halted. Though effective, the nucleoside analogues are non-specific and can cause severe damage to host cells. 1 NNRTIs bind allosterically to a distinct NNIBP site nearly 10  away from the catalytically active site. It is framed by two tryptophan residues that swivel to incorporate the NNRTI drugs. The NNRTIs are thought to prevent the conformational changes demonstrated in Verdine’s structure to be necessary for elongation of the DNA strand. 3 As mentioned previously, the two types of HIV chemotherapy drugs, nucleoside analogs and NNRTIs, bind to two distinct sites in the HIV-1 RT complex. Nucleoside analogs bind to the dNTP site (here labeled dTTP), as shown above, and become directly incorporated into the growing duplex.

6. The following drugs strongly inhibit the action of HIV-1 RT, by either binding to the nucleoside binding site or the NNIBP site. Note that the nucleoside analogs all lack a 3’-OH, a requisite for their ability to halt polymerization. Note also that the NNRTIs all possess structures with large phenyl substituents. This common structural feature has been attributed to their binding through pi-stacking interactions with the swiveling tryptophan residues in the NNIBP site of HIV-1 RT. 1 Examples of Drugs Inhibiting HIV-1 RT Nucleoside Analogs NNRTIs

7. Recently, however, several groups have investigated the idea of using “composite binding pockets” in which several crystal structures have been hybridized to give an averaged consensus binding site that can be used to target new analogs. This idea is rational, given that evidence indicates that the NNIBP is quite flexible. This is demonstrated by its ability of to bind drugs that appear to be too large by initial analysis, but fit comfortably in the product crystal structures. The results of this “composite binding site” are shown above. The grid lines represent the Van der Waals surface, while the internal molecules show the superimposed structures of several NNRTIs from their crystal structures. 3 NNRTI Binding Site – Use of Rational Drug Design to Find New Leads Given that the nucleoside analogs are non-specific and generally toxic, developing new NNRTIs is of the foremost interest. Over the last 20 years, multiple crystal structures of HIV-1 RT complexed with various NNRTIs have supplied a wide range of models, all of which have significant differences in the demonstrated NNIBP. This fact makes rational drug design impossible to implement.

8. With this composite binding site, Mao et al. were able to identify several new drugs with IC-50 values higher than those found conventionally, with equal to or lower than typical cytotoxicity. 3 Demonstration of their binding to the composite binding site is also displayed to the left. Use of Rational Drug Design to Find New Leads, Continued…

9. As further evidence to the validity of the techniques used, Mao et al. superimposed the composite site into the active site residues of NNIBP (right). Those areas where a great deal of overlap occur indicate NNIBP site residues that are more flexible than others. Use of Rational Drug Design to Find New Leads, Continued… In addition, demonstration of the derivative, HI-244 is demonstrated in both the composite site and among the binding site residues (left) (Wing 1 and 2 represent domains of the NNIBP).

10. Conclusions; Targeting of HIV-1 RT by chemical agents has provided a way by which to slow the onset of AIDS. Though not a cure, these drugs provide individuals afflicted with the disease prolonged life spans, or in rare cases, near eradication of the virus. Use of nucleoside analogs together with NNRTIs provides a synergistic, two-front attack on the enzyme. This is possible given that these drugs act at distinct sites and by different mechanisms. Though direct use of HIV-1 RT crystal structures is not amenable to rational drug design, use of composite binding sites has been demonstrated to provide leads into potential therapeutic targets. References Tantillo, C.; Ding, J.; Jacob-Molina, A.; Nanni, R. G.; Boyer, P. L.; Hughes, S. H.; Pauwels, R.; Andries, K.; Janssen, P. A. J.; Arnold, E. J. Mol. Biol. 1994 , 243 , 369-387. Huang, H.; Chopra, R.; Verdine, G.; Harrison, S. C. Science . 1998 , 282 , 1669-1675. Mao, C.; Sudbeck, E. A.; Venkatachalam, T. K.; Uckun, F. M. Biochemical Pharmacology . 2000 , 60 , 1251-1265.