Avoiding Drug Resistance in HIV Reverse Transcriptase

Abstract

HIV reverse transcriptase (RT) is an enzyme that plays a major role in the replication cycle of HIV and has been a key target of anti-HIV drug development efforts. Because of the high genetic diversity of the virus, mutations in RT can impart resistance to various RT inhibitors. As the prevalence of drug resistance mutations is on the rise, it is necessary to design strategies that will lead to drugs less susceptible to resistance. Here we provide an in-depth review of HIV reverse transcriptase, current RT inhibitors, novel RT inhibitors, and mechanisms of drug resistance. We also present novel strategies that can be useful to overcome RT's ability to escape therapies through drug resistance. While resistance may not be completely avoidable, designing drugs based on the strategies and principles discussed in this review could decrease the prevalence of drug resistance.

Figure 1.

Structure of HIV-1 RT in complex with dsDNA and dTTP substrates and binding locations of FDA-approved drugs targeting RT. HIV-1 RT (PDB 1RTD) is comprised of two subunits, p66 (multicolored cartoon) and p51 (gray cartoon). The enzymatically active p66 subunit has both RNA-dependent and DNA-dependent DNA polymerization activities and contains four subdomains: fingers (blue), palm (red), thumb (green), and connection (yellow), as well as an RNase H domain (purple). Template DNA is shown in light brown, primer DNA is shown in dark brown, and dTTP is shown as gray sticks at the polymerase active site (black dashed circle). Two Mg²⁺ ions in the polymerase active site are shown as spheres. The polymerase active site is also the location where NRTIs bind. The NNRTI binding pocket (NNIBP) is located in the p66 palm subdomain, near the base of the p66 thumb subdomain and adjacent to the polymerase active site (black dashed rectangle).

Figure 2.

Structure of the HIV-1 RT polymerase active site. dTTP (gray sticks) is bound at the polymerase active site (PDB 1RTD). Conserved active site residues D110, V111, D185, and D186 chelate two Mg²⁺ ions (light-green spheres), which also bind the alpha, beta, and gamma phosphates of the dTTP. Residue Y115 is located at the bottom of the active site. dTTP sits above Y115 and is additionally stabilized through hydrogen bond interactions between the alpha and beta phosphates and R72 in the β4 fingers, and the gamma phosphate and K65 in the β3−β4 fingers loop. The conserved YMDD loop is between β9 and β10, and the primer grip is located between β12 and β13 in the p66 palm subdomain. αH and αI of the p66 thumb subdomain interact with the nucleic acid substrate to help position it correctly in the polymerase active site.

Figure 3.

Reverse transcription of HIV-1. An HIV virion has two copies of the ssRNA genome. Once a host tRNA_Lys3 binds to the primer binding site (PBS), DNA synthesis starts forming the minus-strand strong-stop DNA. During DNA synthesis, RNase H digests RNA, exposing the minus-strand DNA that hybridizes to the R region of the 3′-end of either viral genome copy. The elongation of the minus strand continues until the polypurine tract (PPT), which is RNase H resistant and serves as a primer for plus-strand DNA synthesis. After plus- and minus-strand DNA syntheses are completed, the RNase H removes the tRNA primer and releases the PBS, thus facilitating the “second jump”. Strand displacement activity of RT to the PBS and PPT ends and/or DNA repair and ligation lead to dsDNA or a circular intermediate with two long terminal repeats (LTRs). Figure adapted with permission from ref . Copyright 2020 Elsevier. License number: 4943870547699.

Figure 4.

Chemical structures of approved NRTIs and NNRTIs.

Figure 5.

Structure of the HIV-1 RT NNIBP. Select residues of the NNIBP of HIV-1 RT (PDB 1VRT) are shown as sticks, with NVP shown as transparent orange sticks. These include L100, K101, K103, V106, V179, Y181, Y188, F227, W229, L234, and P236 of the p66 palm subdomain (pink sticks), Y318 of the p66 thumb subdomain (green sticks), and E138 of the p51 subunit (gray sticks). Additional residues in the NNIBP include T107, V108, V189, and G190, which are not shown for clarity.

Figure 6.

Structural changes occur in HIV-1 RT upon NNRTI binding, which affect the position of nucleic acid binding. HIV-1 RT covalently cross-linked to dsDNA (p66 palm is shown in red cartoon, p66 thumb is shown in green cartoon, primer is shown as dark-gray sticks; template has been removed for clarity) with NVP (purple sticks) bound at the NNRTI binding pocket and AZT-MP (dark gray sticks) at the 3′-end of the DNA primer strand in the P-site (PDB 3V81). The crystal structure of HIV-1 RT (p66 palm and p66 thumb in pink and light-green cartoon, respectively) covalently cross-linked to dsDNA (primer in light gray sticks; template not shown for clarity) with AZT-MP at the P-site and AZT-TP in the N-site (PDB 3V4I) was aligned to structure 3V81 by p66 residues 100−210. Binding of NVP at the NNIBP causes Y181 and Y188 in the NNIBP to flip 180° and also a shift in the position of W229 (pink vs red sticks before and after NVP binding). These changes lead to a cascade of structural rearrangements that include repositioning of the conserved YMDD loop at the polymerase active site, the primer grip, and the p66 thumb subdomain, which are important for nucleic acid binding (movements noted with black dashed arrows). The nucleic acid repositioning is noted by the black dashed arrow between the two AZT-MP molecules at the P-site.

Figure 7.

Second-generation NNRTIs have more intrinsic flexibility that enables binding and inhibition in the presence of several NNRTI resistance mutations. (A) Chemical structure of second-generation NNRTI rilpivirine (RPV), with labeled torsion angles demonstrating the flexibility of this molecule. (B) The terms “wiggling” and “jiggling” are used to describe how newer NNRTIs position themselves to adopt multiple conformations and adjust for the changing side chains in mutated NNIBPs. (C) Although structurally similar, diarylpyrimidine (DAPY) analogues R120393 (a), TMC120-R147681 (dapivirine) (b), TMC125-R165335 (etravirine) (c), and R185545 (d) are able to bind at the NNIBP assuming diverse conformations, thus avoiding mutated residues in the NNIBP. (B,C) Reproduced unmodified from ref . Copyright 2004 American Chemical Society.

Figure 8.

Immediate and delayed chain termination mechanisms of EFdA inhibition. Initial binding of EFdA-TP in the N-site (N, orange box), prior to incorporation (left). Once EFdA-MP is incorporated into the 3′-end of the primer, it may cause immediate chain termination (ICT) (middle) or delayed chain termination (DCT) (right). During DCT, after EFdA-MP incorporation at the 3′-end of the primer and translocation, which moves the EFdA-MP at the P-site (P, blue box), RT adds a single additional nucleotide before inhibition of further DNA synthesis. Figure based on ref .

Figure 9.

Mutation K65R in HIV-1 RT imparts resistance to NRTIs through discrimination. Structural studies revealed that K65R becomes resistant to NRTIs because 65R together with R72 form a platform (light-blue sticks and spheres) that helps discriminate dATP (A, magenta sticks and spheres, PDB 3JYT) from TFV-DP (B, gold sticks and spheres, PDB 3JSM) at the polymerase active site (red cartoon and pink sticks and spheres). Figure based on ref .

Figure 10.

Structural basis for excision-based AZT resistance: interactions of excision product with TAMs RT. HIV-1 RT is shown as cartoon (colored as in Figure 1) bound to dsDNA (template in light brown, primer in dark brown) with key residues shown as pink sticks. The product of AZT-MP excision, AZTppppA, is shown as cyan sticks at the N-site. AZT-resistance mutations (M41L, D67N, K70R, T215Y, K219Q; thymidine-associated mutations or TAMs; shown as magenta sticks) facilitate excision by forming the ATP substrate binding site (PDB 3KLE). Figure based on ref .

Figure 11.

TFV avoids resistance relationship between NRTI resistance and the substrate envelope. The natural substrate dATP (gray sticks; PDB 5TXL) defines the substrate envelope. FTC-TP (blue sticks; PDB 6UIR) protrudes from the substrate envelope, thus causing a steric clash with M184V (red dashed lines and explosion cartoon), resulting in >100-fold resistance. TFV-DP (light-cyan sticks; PDB 3JSM) binds within the substrate envelope and thus remains active against M184V/I. Structural alignments were based on RT residues 50−200.

Figure 12.

Interactions of EFdA-TP at the N-site of the HIV-1 RT polymerase active site. HIV-1 RT (PDB 5J2M) is colored as in Figures 1 and 2. The α-, β-, and γ-phosphates of EFdA-TP (gold sticks) participate in chelation of the Mg²⁺ ion along with conserved active site residues D110, the main chain of V111, and D185 (interactions shown as blue dashed lines). The 4′-ethynyl (4′-E) group of EFdA-TP sits in a hydrophobic pocket at the base of the polymerase active site composed of residues A114, Y115, F160, M184, and D185 (interactions shown as black dashed lines). The 3′-OH participates in hydrogen bond interactions with its β-phosphate (interaction shown as green dashed line). For clarity, we do not show additional interactions between the 3′-OH and the main chain of Y115 and water-mediated hydrogen bond interactions with A114 and F116. The 2-fluoro (2-F) group of EFdA-TP participates in water-mediated interactions with Y115 and the main chain nitrogen of G152 (interactions shown as red dashed lines).

Figure 13.

Chemical structures of pyrophosphate (PPi) and foscarnet (phosphonoformic acid, PFA).

Figure 14.

Interactions of RNase H inhibitors (RNHIs) at the RNase H active site of HIV-1 RT. (A) A 2-hydroxyisoquinoline-1,3-dione RNHI, YLC2–155 (orange sticks), chelates two Mn²⁺ ions (light-blue spheres) that are bound by conserved RNase H active site residues D443, E478, E498, and D549. YLC2–155 also forms hydrogen bond interactions with Q500 and H539 (PDB 5UV5). YLC2–155 can also bind to the RNase H active site in a different conformation, with the furan ring pointing toward H539 (not shown). Based on ref . (B) A hydroxypyridonecarboxylic acid RNHI, 10y (yellow sticks), chelates two Mg²⁺ ions (light-green spheres) that are bound by the conserved RNase H active site residues. 10y also forms hydrogen bond interactions with H539 and K540 (PDB 5J1E). Based on ref .

Figure 15.

RT interactions during the delayed chain termination (DCT) inhibition mechanism. Superposition of two RT structures with EFdA variants before and after translocation. Transparent light-brown sticks show the position of the DNA primer with ddG at the post-translocation P-site and EFdA-TP at the pretranslocation N-site (PDB 5J2M). After EFdA-MP is incorporated into the 3′-end of the primer and further elongated by a single nucleotide (RT/DNA_EFdA-MP^P_{• dTMP}^N), EFdA-MP is located at the P-site (PDB 5J2N). In this position, the 4′-ethynyl (4′-E) sterically clashes with the main chain carbonyl of Y183, and a large shift occurs only at the P-site of this primer to alleviate this negative interaction, as shown by the distance between the 4′ carbon atoms and the dihedral angles of the two structures. Based on ref .

Figure 16.

Several NNRTIs interact with residues in the NNIBP through main chain interactions. (A) EFV (orange sticks, PDB 1FK9) forms hydrogen bond interactions with the main chain C=O and N−H groups of K101. (B) NVP (green sticks, PDB 1VRT) interacts with the main chain C=O and N−H groups of K101 through water-mediated hydrogen bonds. (C) RPV (yellow sticks, PDB 2ZD1) forms hydrogen bonds with the C=O and N−H groups on the main chain of K101 and interacts with E138 in p51 through water-mediated hydrogen bonds with the main chain C=O group. (D) DOR (blue sticks, PDB 4NCG) has hydrophobic interactions with L100 and V106 and forms hydrogen bond interactions with the C=O and N−H groups on the main chain of K103.

Figure 17.

Covalent inhibitors targeting NNRTI-resistant HIV-1 RT. (A) Chemical structure of compound 1, which served as the basis for the design of covalent RT inhibitors (CRTIs). A chloride bound to a carbon (circled in green) in this compound was replaced with an electrophilic warhead to create various CRTIs that were designed to inhibit Y181C HIV-1 RT. (B) Crystal structure of compound 3 covalently bound to 181C in the NNIBP of Y181C HIV-1 RT (PDB 5VQX). Compound 3 (beige sticks) forms additional interactions with the main chain amide of K103.

Figure 18.

Fragment screening to identify potential binding sites for novel antivirals targeting HIV-1 RT. Fragment screening can be used to identify new binding sites for allosteric inhibitors. Shown are fragment binding sites on HIV-1 RT (orange spheres). Reproduced with permission from ref . Copyright 2013 American Chemical Society.

Figure 19.

Chemical structures of dinucleoside tetraphosphate analogues. ATP-based excision reaction of AZT-chain terminated template/primers leads to the production of AZTppppA dinucleoside tetraphosphate products. Analogues of such products are shown with substitutions of oxygen with sulfur atoms in the tetraphosphate moiety. These modifications ensure that the compounds cannot support DNA synthesis by RT. The design strategy aims for compounds that can be used as inhibitors of the polymerase and excision activities of RTs, especially those carrying excision mutations (TAMs). This is accomplished by binding at the N- and the ATP binding sites. Modified from 284. Copyright 2007 American Chemical Society.

Figure 20.

Chemical structures of EFdA, CdG, and remdesivir.

Figure 21.

Structural basis of HIV-1 RT inhibition by nucleotide competing RT inhibitor (NcRTI) INDOPY-1. (A) Chemical structure of INDOPY-1. (B) Structure of INDOPY-1 (orange sticks) in complex with HIV-1 RT (yellow sticks) and template/primer (pink sticks). (B) Reproduced modified from ref . Copyright 2019 American Chemical Society.