Structural Biology of HIV Integrase Strand Transfer Inhibitors

Abstract

Integrase (IN) strand transfer inhibitors (INSTIs) are recent compounds in the antiretroviral arsenal used against HIV. INSTIs work by blocking retroviral integration; an essential step in the viral lifecycle that is catalyzed by the virally encoded IN protein within a nucleoprotein assembly called an intasome. Recent structures of lentiviral intasomes from simian immunodeficiency virus (SIV) and HIV have clarified the INSTI binding modes within the intasome active sites and helped elucidate an important mechanism of viral resistance. The structures provide an accurate depiction of interactions of intasomes and INSTIs to be leveraged for structure-based drug design. Here, we review these recent structural findings and contrast with earlier studies on prototype foamy virus intasomes. We also present and discuss examples of the latest chemical compounds that show promising inhibitory potential as INSTI candidates.

Keywords: HIV; antiretroviral therapy; drug resistance; integrase; structure-based drug design.

Figure 1.. Early INSTIs and molecular basis of their inhibitory activity.

(A) Extensive screening helped to identify first effective strand transfer inhibitors with β-diketo acid scaffold (marked in red) like compound L-731,988. (B) Chemical structures of the FDA-approved first-generation INSTIs, Raltegravir (RAL) and Elvitegravir (EVG). Magnesium chelating heteroatoms are in red and the halobenzyl group is in green. Central rings are numbered starting from the linker region that bridges the pharmacophore and halobenzyl groups. (C) Overall structure of PFV intasome showing differently colored IN subunits, viral DNA in grey, and active site regions with bound inhibitor Raltegravir (RAL) marked by arrows (PDB ID: 3OYA). (D) Close-up of the intasome active site in the apo form (PDB ID: 3OY9). The catalytic DDE residue triad, which is shown in red, consists of D128 (HIV D64), D185 (HIV D116), and E221 (HIV E152). For crystallography of this sample manganese (purple spheres) was used instead of magnesium to better resolve the ion positions within the intasome active site. (E) Zoom into the PFV IN active site, when bound to RAL (shown as magenta sticks). The RAL bound PFV intasome (PDB ID: 3OYA, vDNA, in grey). The catalytic DDE residue triad, which is shown in red, consists of D128 (HIV D64), D185 (HIV D116), and E221 (HIV E152). Three heteroatoms of the inhibitor’s pharmacophore bind the two Mg²⁺ ions (green spheres), and its fluorobenzyl group displaces the terminal adenine of vDNA (dA17) to occupy its original binding site. In consequence, the disrupted 3′-hydroxyl group is in its unreactive position (indicated by red arrow). The methyl-oxadiazole group of RAL makes a π–π stacking interaction with Y212 (corresponding to Y143 in HIV).

Figure 2.. Second-generation INSTIs and their binding characteristics.

(A) Chemical structures of selected second-generation INSTIs – MK2048, DTG, BIC and CAB. Magnesium chelating heteroatoms are in red and the halobenzyl groups are in green. Central rings are numbered starting from the linker region that bridges the pharmacophore and halobenzyl groups. (B) Superposition of BIC bound SIV_rcm intasome (PDB ID: 6RWM, green) and BIC bound HIV intasome (PDB ID: 6PUW, purple) shows extensive contacts between oxazepane ring of BIC (shown by red arrow) with N117 and G118 (in the IN β4-α2 loop) of the intasome. A10 Å zone of bound BIC is depicted. Residue labels follow the HIV/ SIV_rcm IN sequence order. Non-conserved residues in SIV_rcm vs HIV IN are shown in orange ball-and-stick representation. (C) Active site pocket of DTG (blue sticks) bound PFV intasome (PDB ID: 3S3M) superimposed with RAL (magenta sticks) bound PFV intasome (PDB ID: 3OYA) depicts the deeper penetrance of the halobenzyl substituent of DTG (depicted by the black arrow) when compared to RAL. (D) Structures of BIC bound SIV_rcm (PDB ID:6RWM, light green) and HIV (PDB ID:6PUW, purple) intasomes are superimposed. Selected water molecules are shown as small red spheres. (E) Structures of BIC bound G140S/Q148H SIV_rcm intasome (PDB ID: 6RWO, green) is shown overlaid with selected WT HIV intasome residues (PDB ID:6PUW, purple sticks), with small red spheres depicting water molecules. Upon introduction of G140S/Q148H changes, the W5 water (shown in panel D) providing secondary Mg²⁺ coordination shell is expelled from the active site (indicated by red arrow). An extended hydrogen bond network couples T138 to H148 forming a possible proton wire reinforcing the S140-H148 interaction. SIV_rcm residues I74 (L74 in HIV) and T97 are in close proximity to F121 which in turn is involved in van der Waals interactions with the carboxylate of D116. Readjustment of F121 side chain would result in perturbations to the metal-chelating cluster. Interatomic distances are given in Ångstroms and depicted with black dashed lines. In panels B-E the two Mg²⁺ ions are depicted as green spheres.

Figure 3.. Naphthyridine-based INSTI drug candidates.

(A) Chemical structures of 1,6-napthyridine-based (e.g. L-870,810) and 1,8-napthyridine-based compounds 4a, 4c, 4d, 4f, 6b and 6p. Heteroatoms chelating Mg²⁺ ions are depicted in red and blue. 4-amino substituent of the latter compounds is highlighted in purple. Fluorobenzyl groups are in green. Central rings are numbered starting from the linker region that bridges the pharmacophore and halobenzyl groups. (B) Superposition of 4d (PDB ID: 6PUY, light blue) and BIC (PDB ID: 6PUW, salmon) bound HIV intasome active site shows the compact pharmacophore of compound 4d allows it to bind closer to Mg²⁺ ions in comparison to BIC. (C) Superposition of 4d (PDB ID: 6PUY, light blue) and 4c (PDB ID: 6V3K, green) bound HIV intasomes with 4c bound PFV intasome (PDB 5FRN, grey) shows overall similar compound binding modes, with 6’ substituent of 4c exhibiting similar conformation to 4d while bound to HIV intasomes in comparison to distinct 4c orientation found previously in PFV intasome. Analogous IN and vDNA binding residues are labeled accordingly with HIV in green and PFV in black. 3′ terminal dA is hidden for clarity. (D) Comparison of compound 4f binding modes in PFV (PDB ID: 5FRO, light brown) and HIV intasomes (PDB ID: 6PUZ, pink). The 6’ substituent of compound 4f exhibits strikingly different conformation when bound to HIV intasome in comparison to PFV intasome. Analogous IN and vDNA binding residues are labeled accordingly with HIV in pink and PFV in brown. 3′ terminal dA is hidden for clarity. In panels B-D the two Mg²⁺ ions are depicted as green spheres.

Figure 4,. Key Figure. Summary of IN/DNA-INSTI interactions and representation of active site water networks based on two highest-resolution SIV_rcm and HIV intasome structures currently available in the PDB.

(A and B) Schemes showing ligand binding interactions (<5 Å away from the bound INSTI) for (A) SIV_rcm intasome bound to BIC (PDB ID: 6RWM) and (B) HIV intasome bound with compound 4d (PDB ID: 6PUY), presented using Maestro software (Schrödinger Release 2020-1: Maestro, Schrödinger, LLC, New York, NY, 2020). (C and D) Showing water molecules located <5 Å away from the bound inhibitor (shown as red spheres) in (C) SIV_rcm intasome bound to BIC (PDB ID: 6RWM, model in grey, BIC shown in orange) vs (D) HIV intasome bound to 4d (PDB ID: 6PUY, model in light blue, 4d shown in dark blue). In panels C and D the 3′-terminal dA is hidden for clarity, DDE motif residues are highlighted in bold and, Mg²⁺ ions and Cl⁻ are shown as green and blue spheres respectively.

Figure 5.. Chemical structures of developmental INSTI candidates.

1 and 2 were developed based on RAL [67], 3 represents a 3-hydroxypyrimidine-2,4-dione (HPD) scaffold [68] and 4 shows a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) scaffold [69]. Compounds 5 and 6 present a hydroxyquinoline tetracyclic (HQT) scaffold [70], 7 and 8 are the tricyclic 2-pyridinone aminal lead molecules [72], and 9 and 10 are bridged tricyclic pyrimidinone-carboxamide derivatives [73]. Chemical groups expected to hydrophobically interact with penultimate nucleotide from the 3′ end of vDNA are colored in green. Heteroatoms expected to chelate Mg²⁺ ions are depicted in red and blue. Central rings are numbered starting from the linker region that bridges the pharmacophore and hydrophobic groups involved in nucleotide interactions.

Box 1, Figure I.

(A) Schematic of intasome assembly. Under specific biochemical conditions, an oligomer of IN proteins (labeled IN_n) will assemble on the ends of vDNA to form the stable synaptic complex (SSC), which contains between 4 and 16 IN protomers. IN then cleaves two nucleotides from the 3′ ends of vDNA, forming the cleaved synaptic complex (CSC) that exposes the conserved free 3′-OH groups of the catalytically competent CA dinucleotides. CSC intasomes can capture target DNA (tDNA) to form the target capture complex (TCC), which will rapidly catalyze strand transfer to form the post-catalytic strand transfer complex (STC) in which the tDNA and the integrated vDNA are still bound to the intasome. INSTIs specifically bind to CSC intasomes and prevent the formation of the TCC, inhibiting catalysis. In the cell, the above steps occur within the larger context of the pre-integration complex (PIC). (B) Tetrameric PFV intasome (C) dodecameric HIV intasomes are shown. (D) Close-up of the intasome active site without and (E) with bound INSTI (red). The CIC is represented by surface and each protomer forming CIC is colored differently. Other protomers and DNA are in gray. Black arrows indicate the INSTI binding sites.

Box 2, Figure I.. Substrate envelope in HIV intasome.

(A) The substrate envelope of HIV intasome (shown as multicolor surface) comprises of van der Waals volumes of the post-3′-processed vDNA substrate from the HIV intasome (PDB 5U1C, dark red), a dinucleotide fragment of pre-3′-processed vDNA substrate from the PFV intasome (PDB 4E7I, light brown) and host target DNA substrate (PDB 5U1C, green). The intasome is colored by asymmetric unit (grey and dark grey). Panel A is reproduced from supplementary material of Passos et al. 2020 [22]. (B) Inset shows the zoom into the HIV intasome active site and fit of compounds 4d (PDB 6PUY, light blue) and 4f (PDB 6PUZ, pink) within the substrate envelope represented with the same surface/structure-color fashion as described in panel A. Within surface representation the corresponding DNA residues are shown as thin wire. The DDE motif residues are shown as sticks, and Mg²⁺ ions as green spheres.