The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir

Abstract

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents which potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into Phase 2a clinical trials. Previous cell culture based viral breakthrough assays identified the HIV-1_{(Y99H/A128T IN)} variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. While both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCD_Y99H/A128T + PIR complex. Consequently, full-length IN_Y99H/A128T was substantially less susceptible to the PIR induced hyper-multimerization than the WT protein, and HIV-1_{(Y99H/A128T IN)} conferred >150-fold resistance to the inhibitor compared to the WT virus. By rationally modifying PIR we have developed its analog EKC110, which readily induced hyper-multimerization of IN_Y99H/A128T in vitro and was ~14-fold more potent against HIV-1_{(Y99H/A128T IN)} than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.

Keywords: ALLINI; HIV-1 integrase; Pirmitegravir; antiretroviral drug.

FIG 1.

(A) The chemical structure of PIR. The separate functional groups are color-coded: carboxylate (red); tert-butoxyl (green); chlorophenyl (blue); core pyrrolopyridine and methylpyrazole rings (black). The 3-methyl group on the pyrrolopyridine ring is indicated. (B) Infectivity of WT and indicated mutant viruses.

FIG 2.

DLS analysis of PIR induced aberrant IN multimerization. 500 nM PIR was added to 200 nM full-length WT IN (A) or IN_Y99H/A128T (B) and DLS signals were recorded at indicated times (1–15 min). DMSO controls are shown after incubation of full-length IN proteins for 15 min to indicate that these proteins remained fully soluble in the absence of PIR.

FIG 3.

SPR analysis of PIR binding to WT CCD and CCD_Y99H/A128T. Representative sensorgrams for PIR binding to of WT CCD (A) vs CCD_Y99H/A128T (B). PIR concentrations are indicated. The K_D values for PIR + CCD (C) and PIR + CCD_Y99H/A128T (D) were determined using the Hill equation.

FIG 4.

Affinity pull-down assays to probe PIR induced CCD-CTD interactions. Lane 1: molecular weight markers; Lanes 2 – 4: loads of His₆-CCD (lane 2), His₆-CCD_Y99H/A128T (lane 3), and tag-less CTD (lane 4); Lanes 5 – 7: affinity pull-down using Ni beads of CTD alone (lane 5, control), His₆-CCD + CTD (lane 6), His₆- His₆-CCD_Y99H/A128T + CTD (lane 7) in the absence of PIR; Lanes 8 – 10: affinity pull-down using Ni beads of CTD + PIR (lane 8, control), His₆-CCD + CTD (lane 9), His₆- His₆-CCD_Y99H/A128T + PIR + CTD (lane 10).

FIG 5.

Structural analysis of PIR interactions with CCD vs CCD_Y99H/A128T. (A) Superimposed crystals structures of WT CCD (green) + PIR (magenta) and CCD_Y99H/A128T (cyan) + PIR (pale cyan). Distances were measured between the 3-methyl group of PIR ‘s pyrrolopyridine ring to the closest Cβ on either Ala128 or Thr128, as well as between the closest methyl group on PIR ‘s tert-butoxy to either Tyr99 or His99. (B) Van der Waals surface for indicated residues are shown in the structure of WT CTD-CCD + PIR. (C) The structure of CCD_Y99H/A128T + PIR superimposed onto the structure of WT CTD-CCD + PIR. Van der Waals surface for indicated residues reveals steric clashes observed by overlapping, shaded surfaces. PIR is not shown for clarity.

FIG 6.

Interactions of EKC110 with HIV-1 IN. (A) The chemical structure of EKC110. The separate functional groups are color-coded: carboxylate (red); tert-butoxyl (green); chlorophenyl (blue); core pyrrolopyridine and methylpyrazole rings (black). (B, C) DLS analysis of EKC110 induced aberrant IN multimerization. 500 nM EKC110 was added to 200 nM full-length WT IN (B) or IN_Y99H/A128T (C) and DLS signals were recorded at indicated times (1–15 min). DMSO controls are shown after incubation of full-length IN proteins for 15 min to indicate that these proteins remained fully soluble in the absence of EKC110.

FIG 7.

The structural analysis of EKC110 interactions with CCD and CTD-CCD. (A) For comparison the crystal structure of CCD + EKC110 is superimposed onto CCD + PIR, which reveals a noticeable tilt of the EKC110 pyrrolopyridine core toward A128 compared to PIR. C) For comparison the crystal structure of CTD-CCD + EKC110 is superimposed onto CTD-CCD + PIR to show repositioning (yellow arrow) of CTD in the presence of EKC110 compared to PIR. CTDs are shown in yellow and gray in EKC110 + CTD-CCD and PIR + CTD-CCD structures. PIR and EKC110 are in magenta and blue. The side chain of Ala128 in each structure is shown by sticks.

FIG 8.

MD simulation of CTD interactions with WT CCD vs CCD_Y99H/A128T in the complex with PIR (A) and EKC110 (B). Displacement of the CTD domain from the PIR + CCD_Y99H/A128T complex is measured with a center-of-mass displacement of d=4.85Å and rotations along the principal axes of inertia of θ=26.66°, Φ=23.84° and Ψ=11.81°. WT CCD and CCD_Y99H/A128T are colored cyan and green, respectively. CTDs interacting with WT CCD and CCD_Y99H/A128T are colored red and blue, respectively.