HIV-1 Reverse Transcriptase Still Remains a New Drug Target: Structure, Function, Classical Inhibitors, and New Inhibitors with Innovative Mechanisms of Actions

Abstract

During the retrotranscription process, characteristic of all retroviruses, the viral ssRNA genome is converted into integration-competent dsDNA. This process is accomplished by the virus-coded reverse transcriptase (RT) protein, which is a primary target in the current treatments for HIV-1 infection. In particular, in the approved therapeutic regimens two classes of drugs target RT, namely, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). Both classes inhibit the RT-associated polymerase activity: the NRTIs compete with the natural dNTP substrate and act as chain terminators, while the NNRTIs bind to an allosteric pocket and inhibit polymerization noncompetitively. In addition to these two classes, other RT inhibitors (RTIs) that target RT by distinct mechanisms have been identified and are currently under development. These include translocation-defective RTIs, delayed chain terminators RTIs, lethal mutagenesis RTIs, dinucleotide tetraphosphates, nucleotide-competing RTIs, pyrophosphate analogs, RT-associated RNase H function inhibitors, and dual activities inhibitors. This paper describes the HIV-1 RT function and molecular structure, illustrates the currently approved RTIs, and focuses on the mechanisms of action of the newer classes of RTIs.

Figure 1

HIV-1 reverse transcription process. Step 1: host cell tRNALys3 hybridizes to the PBS near the 5′-end of the (+)strand RNA genome (orange). (−)strand DNA (blue) synthesis starts using host tRNALys3 as a primer. DNA synthesis proceeds up to the 5′-end of the RNA genome. Step 2: RNase H hydrolysis of the RNA portion of the RNA:DNA hybrid product exposes the ssDNA product determining the (−)strand strong stop DNA. Step 3: strand transfer of the (−)strand DNA through its hybridization with the R region at the 3′-end of the ssRNA genome and further elongation of the (−)strand DNA. Step 4: DNA synthesis proceeds, and the RNase H function cleaves the RNA strand of the RNA:DNA at numerous points leaving intact two specific sequences (cPPT, 3′PPT) resistant to the RNase H cleavage. Step 5: (−)strand DNA synthesis (green) initiation using PPTs as primers. Step 6: RNase H hydrolysis of the PPT segments and the junction of the tRNA:DNA hybrid, freeing the PBS sequence of the (+)strand DNA. Step 8: strand transfer of the PBS sequence of the (+)strand DNA that anneals to the PBS on the (−)strand DNA. DNA synthesis then continues with strand displacement synthesis. Step 9: the product is a linear dsDNA with long terminal repeats (LTRs) at both ends.

Figure 2

Structure of HIV-1 RT. The enzyme has two domains: the p66 (colored) and the p51 (gray). The polymerase domain shows a characteristic highly conserved structure that resembles a right hand, consisting of fingers domain (magenta), palm domain (cyan), thumb domain (blue). The p66 subunit also comprises the connection domain (orange) and RNase H domain (yellow). The polymerase active site is located in the middle of palm, fingers, and thumb subdomains. The three catalytic aspartic acid residues (D110, D185 and D186) located in the palm subdomain of p66 that bind the cofactor divalent ions (Mg²⁺) are shown (red). The RNase H domain is located at C-terminus of the p66 subunit, 60 Å far from polymerase active site. The RNase H active site contains a DDE motif comprising the carboxylates residues D443, E478, D498, and D549 that can coordinate two divalent Mg²⁺.

Figure 3

Chemical structures of approved NRTIs.

Figure 4

Amino acid residues involved in RTI binding. RT two subunits are in green (p66) and in gray (p51). The catalytic residues of the polymerase active site and the RNase H active site are colored in yellow. NRTIs and NtRTIs interact with residues close to the polymerase active site (blue). NNRTIs bind in a hydrophobic pocket next to the polymerase active site (magenta). RHRTIs such as DKAs, N-hydroxyimides, N-hydroxy quinazolinediones and naphthyridine derivatives bind in the RNase H active site (in yellow on the right). Vinylogous ureas bind to a hydrophobic pocket at the interface between the RNase H domain and the p51 subunit (cyan). Hydrazone derivatives have been proposed to bind two different sites (red). One located between the polymerase active site and the NNRTI-binding pocket (sharing a few residues with it) and the second one located between the RNase H and the connection domain. Anthraquinone derivatives have been proposed to bind to the first hydrazone pocket next to the NNRTI-binding site.

Figure 5

Mechanism of action of RT inhibitors acting as chain terminators. The RT is represented as a pale green circle with the priming binding site in cyan (P) and the nucleotide binding site in white (N).  The RNA template is showed in blue and the (−)strand DNA in purple. The NRTI triphosphate (strong green) (1) competes for the binding with the natural dNTPs, it is incorporated into the growing DNA (2) and it blocks the further DNA elongation because it lacks the 3′-hydroxyl group (3).

Figure 6

Chemical structure of approved NtRTI.

Figure 7

Chemical structures of approved NNRTIs.

Figure 8

Chemical structures of new NRTIs acting as chain terminators.

Figure 9

Chemical structures of NRTIs with new mechanisms of action.

Figure 10

Mechanism of action of TDRTIs. The RT is represented as a pale green circle with the priming binding site in cyan (P) and the nucleotide binding site in white (N). The RNA template is shown in blue and the (−)strand DNA in purple. The TDRTI triphosphate (strong green) can be used as RT substrate (1) and is incorporated in the nucleic acid (2). The incorporated TDRTI blocks the further DNA synthesis since the enzyme is not able to efficiently translocate (3).

Figure 11

Mechanism of action of DCTRTIs. The RT is represented as a pale green circle with the priming binding site in cyan (P) and the nucleotide binding site in white (N). The RNA template is shown in blue and the (−)strand DNA in purple. DCTRTI triphosphate (strong green) is incorporated into the growing DNA chain (1). After further nucleotides addition, its presence blocks DNA elongation, probably through steric hindrance interference (yellow) between the RNA:DNA hybrid and the RT nucleic-acid-binding cleft (2). In addition, their incorporation can also block the synthesis of the (+)strand DNA affecting the base pairing (3).

Figure 12

Chemical structures of new NNRTIs.

Figure 13

Chemical structures of NcRTIs.

Figure 14

Chemical structures of metal chelating RHRTIs.

Figure 15

Chemical structures of dual RHRTI-INIs.

Figure 16

Chemical structures of nonmetal chelating RHRTIs.

Figure 17

Chemical structures of dual RNase H and polymerase inhibitors.

Figure 18

Chemical structures of DimRTIs.