Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration

Abstract

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Keywords: fluoxetine; health insurance databases; macular degeneration; molecular modeling; retina.

Fig. 1.

In vitro binding analysis of fluoxetine with NLRP3. (A) CY-09, a small molecule inhibitor of NLRP3 (15), fluoxetine, and fluvoxamine all contain a (trifluoromethyl)phenyl moiety (highlighted in dashed circles). (B) Biotinylated fluoxetine was incubated with protein lysate collected from LPS-primed human THP-1 monocytes. Protein complexes were precipitated by streptavidin pull down. The input and pull-down fractions were immunoblotted for NLRP3 or GAPDH. Excess amounts of free (nonbiotinylated) fluoxetine were used as a competitor. Higher (H) and lower (L) exposures shown. (C and D) In vitro interaction of biotinylated fluoxetine (Btn-FLX, C) or biotinylated CY-09 (Btn-CY-09, D) with GST-NLRP3 protein analyzed by streptavidin pull down. The input and pull-down fractions were immunoblotted for GST (Top) and, for confirmation, NLRP3 (Bottom). Excess-free (nonbiotinylated) fluoxetine was used as a competitor. (E) Biotinylated fluoxetine was incubated with protein lysate collected from LPS-primed human THP-1 monocytes. Protein complexes were precipitated by streptavidin pull down. The input and pull-down fractions were immunoblotted for NLRP3. Excess-free (nonbiotinylated) fluvoxamine (FLV) was used as a competitor.

Fig. 2.

In silico–binding analysis of fluoxetine with NLRP3. (A–D) Binding modes of ADP and ATP to NLRP3 as a basis for fluoxetine binding. (A) The fully internal cavity in which ADP is bound to the NACHT domain of NLRP3 is shown in a Z-clipped cutaway, with the internal protein surface and several key residues depicted, including several mostly hydrophobic residues that lead to a narrowing of the cavity around the nucleotide 5′ carbon (Ile232, Leu411. and His520) and a cluster of basic residues (Arg235, His258, and Arg260) at the left. In the 6NPY PDB structure shown, there is a substantial void in the cavity near those basic residues. (B) A simple rotation of the γ dihedral angle (C4′–C5′ bond) of the ribose sugar of the bound ADP to the -sc orientation, along with the addition of a terminal phosphate group to make ATP, shows that a bound ATP in an otherwise identical binding mode as ADP could engage in electrostatic interactions between the terminal phosphate oxygens and the triad of basic residues mentioned above, partially filling the void in the cavity. This could represent an alternate mode of ATP binding prior to hydrolysis, possibly associated with an inactive state. (C) The CF₃ group of fluoxetine, with negative charges on fluorine, could act as a phosphate isostere. If it is overlapped with the terminal phosphate group as modeled in B, a low-energy extended conformation of fluoxetine can be fitted onto the molecule of ATP shown in B, with the second aromatic ring of fluoxetine occupying the space of the ribose moiety. In this preliminary manual docking (see Materials and Methods), the N-methylamine sidechain of S-fluoxetine projects toward the viewer, that is, the part of the cavity closest to the protein surface. (D) This initial manual docking of S-fluoxetine into the nucleotide-binding cavity of NLRP3 is shown with the ligand in yellow space fill rendition, showing that S-fluoxetine has the potential to fit well within the experimentally observed cavity. This hypothesis was then assessed using in silico docking methods. (E) Best docked pose of S-fluoxetine fully inside the nucleotide-binding cavity as identified by HADDOCK. The conformation of the complex shown is after an OPLS3 force field minimization. The viewpoint is slightly rotated around the y-axis relative to A for clarity in showing the interacting side chains. These include the triad of basic residues mentioned in A (R235, H258, and R260, using the numbering system of 6NPY.PDB), two nitrogenous aromatic amino acids (H520 and W414), and two residues that engage the S-fluoxetine amino group in H-bonding (E150) or Pi-cation bonding (F506). The binding energy of this complex was calculated as ΔG = –8.9 Kcal/mol, using the PRODIGY-LIGAND web server (19, 20). A complete schematic interaction diagram for this complex is shown as SI Appendix, Fig. S2A. (F) Alternate docked pose of S-fluoxetine only partially inside the nucleotide-binding cavity. HADDOCK yielded a marginally slightly higher ranking to a cluster best represented by this complex in which S-fluoxetine interacts with four of the same residues as the “fully inside” complex shown in E. These residues include E150, except the fluoxetine amino group forms a salt bridge to the glutamate side chain rather than H-bonding to the backbone carbonyl. Trp414, Phe505, and His520 all interact with S-fluoxetine but in different ways. This conformation could represent an intermediate state of entry of fluoxetine into the buried cavity. The third least highly ranked cluster was similar to this, except the drug molecule is inverted with the CF₃ group of fluoxetine protruding slightly from the protein. It had less favorable electrostatic binding energy than the pose shown here with the amino group interacting with the glutamate side chain.

Fig. 3.

Fluoxetine inhibits Alu/B2 RNA–induced NLRP3 inflammasome assembly and inflammasome activation. (A–C) Representative immunofluorescent images of ASC specks (red circles pointed to by white arrows) in wild-type mouse BMDMs that were mock transfected (A), transfected with Alu RNA (B), or transfected with Alu RNA and treated with fluoxetine (C). Cell nuclei stained blue by DAPI. (D) Bar graph of ASC speck quantification of A to C. n = 5 per group. (E) Representative Western blot images show that FLX inhibits caspase-1 cleavage induced by Alu RNA (Left) or B2 RNA (Right) in ARPE-19 cells (Top) and BMDMs (Bottom). Integrated densitometry values are shown below blots. n = 3 per group. (F–I) ELISA-based quantifications of cytokine release show that FLX inhibits IL-18 secretion by ARPE-19 cells (F and H) and IL-1β secretion by BMDMs (G and I) induced by Alu RNA (F and G) and B2 RNA (H and I). FLX, fluoxetine. n = 3 per group. All P values are for Alu/B2 RNA versus Alu/B2 RNA + FLX group comparisons using two-tailed Student’s t test. Mean and SEM values are presented.

Fig. 4.

Alu RNA–induced RPE degeneration is blocked by fluoxetine but not by other antidepressant drugs. (A and B) Subretinal administration of Alu RNA and intravitreous administration of selective serotonin reuptake inhibitor (SSRI) (A) and non-SSRI (B) antidepressant drugs in wild-type mice. (Top) Fundus photographs. (Bottom) Flat mounts stained for zonula occludens-1 (ZO-1; red). Degeneration outlined by white arrowheads. Binary (Healthy %) and morphometric (PM, polymegethism) (mean [SEM]) quantification of RPE degeneration is shown (Fisher’s exact test for binary; two-tailed Student’s t test for morphometry; **P < 0.001). Loss of regular hexagonal cellular boundaries in ZO-1–stained flat mounts is indicative of degenerated RPE. (Scale bars, 50 μm.)

Fig. 5.

Fluoxetine use is associated with reduced risk of developing dry AMD. (A and B) Kaplan–Meier survival curves showing the probability of not developing dry AMD (survival) over time for subjects in the Truven Marketscan (A) and PearlDiver Mariner (B) databases (baseline characteristics in SI Appendix, Tables S1 and S2) based on fluoxetine (FLX) exposure or nonexposure. Difference between FLX exposure or nonexposure groups was significant (P < 0.0001 by log-rank test). (C) HRs for developing dry AMD derived from propensity score–matched models (baseline characteristics in SI Appendix, Tables S3 and S4) adjusted for the confounding variables listed in Methods and Materials were estimated separately for the Truven Marketscan and PearlDiver Mariner databases. Adjusted HRs along with their 95% CIs are shown as black lines. Diamonds show the pooled estimate of the adjusted hazard ratio and the 95% CIs for meta-analyses using inverse variance–weighted random-effects and fixed-effect models. The broken vertical line represents an adjusted hazard ratio of 1, which denotes equal risk between FLX exposure and nonexposure. Horizontal bars denote 95% CIs. P values derived from z-statistics for individual databases are reported. The estimates of heterogeneity (χ²), results of the statistical test of heterogeneity using the χ² test statistic and its degrees of freedom (df), and posterior probabilities of a nonbeneficial effect for each model are shown below the plot. The Higgins I² statistic and its 95% CI are presented. The results of the statistical tests of overall effect, the z-test statistics, and corresponding P values are presented.