HIV‑1 integrase inhibitors targeting various DDE transposases: Retroviral integration versus RAG‑mediated recombination (Review)

Abstract

Transposases are ubiquitous mobile genetic elements responsible for genome development, driving rearrangements, such as insertions, deletions and translocations. Across species evolution, some transposases are tamed by their host and are made part of complex cellular systems. The proliferation of retroviruses is also dependent on transposase related enzymes termed integrases. Recombination‑activating gene protein (RAG)1 and metnase are just two examples of transposase domestication and together with retroviral integrases (INs), they belong to the DDE polynucleotidyl transferases superfamily. They share mechanistic and structural features linked to the RNase H‑like fold, harboring a DDE(D) metal dependent catalytic motif. Recent antiretroviral compounds target the catalytic domain of integrase, but they also have the potential of inhibiting other related enzymes. In this review, we report the activity of different classes of integrase inhibitors on various DDE transposases. Computational simulations are useful to predict the extent of off‑target activity and have been employed to study the interactions between RAG1 recombinase and compounds from three different pharmacologic classes. We demonstrate that strand‑transfer inhibitors display a higher affinity towards the RAG1 RNase H domain, as suggested by experimental data compared to allosteric inhibitors. While interference with RAG1 and 2 recombination is associated with a negative impact on immune function, the inhibition of metnase or HTLV‑1 integrase opens the way for the development of novel therapies for refractory cancers.

Figure 1.

Scheme illustrating the transposition reaction. In the first step the transposon is cut from its original place followed by pasting into another location in the genome (strand transfer). The initial cleavage occurs at each strand sequentially. The first strand is cleaved using a water molecule as a nucleophile is common for all DDE motif enzymes and releases an activated 3′ hydroxyl group on either the flanking DNA or on the signal end of the transposon (TIR). The second strand is cleaved by the activated OH resulting in a hairpin structure or by another water molecule. In the case of RAG recombinase, the hairpin is opened by NHEJ repair enzymes and the flanking ends are united, while the signal ends very rarely perform transposition and are instead united. In the case of retroviral IN, the vDNA is already independent and the activated OH proceeds directly to strand transfer. NHEJ, non-homologous end joining; RAG, recombination-activating gene protein.

Figure 2.

RAG-mediated recombination (left) and HIV-1 IN mediated retroviral integration (right) share a two-step transesterification mechanism. The RAG complex recognizes specific sequences named recognition signal sequences (RSS). RSS flank the segments (coding segments) to be recombined in the antigen receptor loci. IN recognizes sequences flanking the long terminal repeats of the viral cDNA (U5′LTR and U3′LTR). Both enzymes are active in the context of a paired complex with DNA: synaptic complex heterotetrameric (RAG1 and 2)₂ or intasome homotetrameric (2×IN)₂. In the first step RAG1 and 2 cleaves one DNA strand between the heptamers and the coding segments ends, with the release of a reactive 3′ hydroxyl on the coding end. Similarly, IN cleaves one DNA strand at the end of LTR, near the conserved CA dinucleotide, with the removal of the last two 3′ nucleotides of LTR ends and the release of a reactive 3′ hydroxyl (3′ processing). In the second step the viral cDNA reactive ends attack the double stranded target DNA and the 3′ viral DNA ends are united with 3′ target strand. By contrast, RAG generated reactive hydroxyl, attacks the second coding end strand forming a structure called hairpin and detaching the RSS ends (signal ends). Cellular repair enzymes open the hairpins and fuse together the coding segments and in a similar fashion they repair the gaps near the inserted viral DNA. In vivo RAG mediated transposition events of signal ends are highly uncommon, but they result in a 5-bp target site duplication (TSD) similarly to HIV-1 IN strand transfer product. The TSD arises from the 5 nucleotides in the target DNA separating the insertion sites of LTRs and RSS, respectively. NHEJ, non-homologous end joining; RAG, recombination-activating gene protein.

Figure 3.

RNase H-like fold-crystal structure of E. coli ribonuclease H [PDB accession code 1RNH (37)], structure of HIV-1 IN [PDB accession code 5U1C (90)] and RAG1 structure from the crystal RAG1 and 2 complex [PDB accession code 4WWX (18)]. HIV-1 IN and RAG1 share the RNase H-like fold (green) harboring the DDE catalytic motif (red). Note: The HIV-1 IN structure reported (90) has an E152Q mutation, within the DDE motif.

Figure 4.

Structure of IN inhibitors discussed in this review. (A) Diketo acid INSTIs, (B) styrylquinoline derivative FZ41, (C) allosteric inhibitors tert-butoxy-(4-phenyl-quinolin-3-yl)-acetic acid derivatives and chemically related compounds.

Figure 5.

Selected docking conformations for raltegravir (magenta). The binding pocket is defined by all the residues within 5 Å (blue). The top image illustrates docking conformations corresponding to cluster number 1 (top image) and cluster number 2 (bottom image). DDE motif residues are depicted in red and the RNase H fold is depicted in green.

Figure 6.

Selected docking conformations for elvitegravir (magenta, upper image) and DTG (magenta, bottom image). The binding pocket is defined by all the residues within 5 Å (blue). DDE motif residues are depicted in red and the RNase H fold is depicted in green.

Figure 7.

Selected docking conformations for FZ41 (magenta, upper image) and GSK1264 (magenta, bottom image). The binding pocket is defined by all the residues within 5 Å (blue). DDE motif residues are depicted in red and the RNase H fold is depicted in green.