FDA-approved drug library screen identifies antidepressants, antimicrobials, anti-COPD, and anti-CVD agents as blockers of NLRP3 inflammasome and sepsis in a sex-dependent manner.

This study utilizes a phenotypic screen of an FDA-approved drug library to identify diverse repurposable agents, including antidepressants and antimicrobials, that selectively block NLRP3 inflammasome priming or assembly and demonstrate protective effects against sepsis in a sex-dependent manner.

The Gist

The Big Picture: Finding Old Keys for New Locks

Imagine your body's immune system is a highly trained security team. Its job is to spot intruders (like bacteria or viruses) and sound the alarm. One of the most powerful alarms in this system is called the NLRP3 inflammasome.

Think of the NLRP3 inflammasome as a giant, explosive firework inside your cells.

• When it works right: It goes off once to blast away a dangerous infection.
• When it goes wrong: It keeps exploding uncontrollably. This causes a "cytokine storm"—a massive flood of inflammation that damages your own organs. This happens in diseases like sepsis (blood poisoning), Alzheimer's, diabetes, and heart disease.

The Problem: Currently, we don't have a specific "off switch" for this firework that is approved by the FDA. We have drugs that try to clean up the mess after the explosion (like putting out the fire), but we need something that stops the firework from lighting in the first place.

The Solution: The researchers didn't try to invent a brand-new drug from scratch (which takes 10+ years). Instead, they went to a massive library of 190 drugs that are already approved and safe for humans (like antidepressants, heart meds, and antibiotics). They asked: "Do any of these old, familiar drugs accidentally happen to stop this firework?"

The Experiment: The "Firework" Test

The scientists used a special type of white blood cell (a THP-1 cell) that has a built-in camera.

1. The Setup: They added a trigger (LPS, which mimics bacteria) to the cells. This is the "Signal 1" that tells the firework to get ready.
2. The Explosion: Then they added a second trigger (Nigericin). This is "Signal 2" that actually lights the fuse.
3. The Visual: When the firework lights, the cells glow green and form bright dots (called "ASC specks").
4. The Screen: They dropped different FDA-approved drugs into the mix. If a drug worked, the green dots disappeared or the cells didn't glow.

The Winners: The "Unexpected Heroes"

They found a whole bunch of drugs that worked, even though they were originally made for completely different jobs. It's like finding that a toothbrush can also be used to scrub a floor.

The paper groups these "heroes" into two teams based on how they stop the firework:

Team 1: The "Gatekeepers" (Stop the Signal)

These drugs stop the cell from even hearing the alarm. They block the first signal (priming).

• The Drugs: Antidepressants (Fluoxetine/Prozac, Duloxetine), Antimalarials (Mefloquine), Antifungals (Ciclopirox), and some heart meds.
• The Analogy: Imagine the firework needs a specific radio frequency to receive the "Light Me" command. These drugs jam the radio signal. The cell never gets the message, so the firework never gets ready.
• The Result: In mice with severe sepsis (a life-threatening infection), these drugs saved lives. Interestingly, they worked differently in males and females, suggesting that biology is complex and sex matters in medicine.

Team 2: The "Cleanup Crew" (Stop the Assembly)

These drugs let the cell hear the alarm, but they stop the firework from actually being built.

• The Drugs: Diabetes meds (Rosiglitazone), Blood pressure meds (Irbesartan, Amlodipine), and HIV meds (Saquinavir).
• The Analogy: The firework is being assembled on an assembly line. These drugs act like a quality control inspector who pulls the plug on the conveyor belt. They also act like a garbage truck (autophagy) that sweeps away the broken, rusty parts of the cell (damaged mitochondria) that usually trigger the explosion.
• The Result: These drugs stopped the firework from forming, even when the cell was under attack.

Why This Matters

1. Speed: Because these drugs are already FDA-approved, we know they are safe for humans. If we can prove they work for inflammation, we could start using them for sepsis or autoimmune diseases much faster than inventing a new drug.
2. Repurposing: It shows that a drug made for depression might save a heart patient, or a drug for malaria might stop a deadly infection.
3. The "Sex" Factor: The study found that the drugs worked better in male mice than female mice (or vice versa, depending on the drug). This is a huge reminder that men and women react differently to medicine, and future treatments need to account for this.

The Bottom Line

The researchers found a treasure trove of "accidental" cures hidden in our existing medicine cabinets. By using old drugs to stop the NLRP3 "firework," we might soon have powerful new tools to fight sepsis, heart disease, and neurodegenerative disorders, all without the long wait for new drug development.

In short: They looked through a box of old keys and found several that fit a lock we didn't know they could open. Now, we just need to try them out in the real world.

Technical Summary

Title

FDA-approved drug library screen identifies antidepressants, antimicrobials, anti-COPD, and anti-CVD agents as blockers of NLRP3 inflammasome and sepsis in a sex-dependent manner.

1. Problem Statement

The NLRP3 inflammasome is a critical component of the innate immune system responsible for host defense. However, its dysregulated activation drives a wide spectrum of pathologies, including metabolic syndrome (diabetes, obesity, CVD), neurodegenerative diseases (Alzheimer's, Parkinson's), autoinflammatory conditions, and respiratory illnesses.

• Therapeutic Gap: While the NLRP3 pathway is a validated target, there are currently no FDA-approved small-molecule inhibitors that directly target NLRP3. Existing biologics (e.g., Anakinra, Canakinumab) only block downstream IL-1 activity, failing to prevent pyroptosis or the release of other inflammatory mediators.
• Challenge: Developing new drugs is slow and costly. There is an urgent need for "drug repurposing" strategies to identify existing, safe FDA-approved compounds that can selectively inhibit NLRP3 priming or assembly without broadly suppressing innate immunity.

2. Methodology

The authors employed a multi-stage phenotypic screening and validation approach:

• Primary Screen:
  • Library: Tocriscreen FDA-approved drug library (190 compounds).
  • Cell Model: THP-1 monocytes stably expressing ASC-GFP under an NF-κB-inducible promoter.
  • Assay Logic: Cells were primed with LPS (Signal 1) to induce ASC-GFP expression.
    • Category A (Priming Blockers): Drugs that reduced LPS-induced ASC-GFP expression (indicating inhibition of the priming step).
    • Category B (Assembly Blockers): Cells primed with LPS, then stimulated with Nigericin (Signal 2). Drugs were screened for the ability to inhibit ASC speck formation (inflammasome assembly) without reducing the expression of inflammasome components.
• In Vitro Validation:
  • Mechanistic Assays: NF-κB nuclear localization, LPS binding assays, mitochondrial membrane potential, mitochondrial ROS (mtROS) production, and autophagic flux (using LC3-II/p62 markers and AMPK phosphorylation).
  • Cytokine Profiling: Measurement of pro-inflammatory cytokines (IL-1β, IL-18, TNF-α, etc.) via ELISA and multiplex arrays.
• In Vivo Validation:
  • Models: C57BL/6J mice (both sexes).
  • Sepsis Model: High-dose LPS-induced lethality (25 mg/kg) to test survival rates.
  • Inflammasome Assembly Model: LPS + ATP injection to induce NLRP3 assembly, measuring IL-1β in peritoneal lavage.
  • Endpoints: Survival curves (Kaplan-Meier), organ damage markers (ALT, ALP, creatinine), and plasma cytokine profiles.

3. Key Contributions & Results

A. Identification of Two Distinct Drug Classes

The screen identified two mechanistic categories of NLRP3 inhibitors among FDA-approved drugs:

1. LPS Priming Blockers (Signal 1 Inhibitors)
These drugs inhibit the initial transcriptional upregulation of NLRP3 and pro-IL-1β, often by interfering with TLR4 signaling or LPS binding.

• Identified Drugs: Mefloquine (antimalarial), Fluoxetine (antidepressant), Ciclopirox (antifungal), Febuxostat (antidiabetic), Dasatinib (tyrosine kinase inhibitor), Amlodipine (antihypertensive), and others.
• Mechanism:
  • Mefloquine, Fluoxetine, Ciclopirox: Directly reduced LPS binding to the cell membrane.
  • Febuxostat: Reduced NF-κB nuclear localization without affecting LPS binding.
  • Dasatinib: Induced autophagy (unlike other priming blockers which tended to block it).
• In Vivo Efficacy: Pretreatment with Mefloquine, Fluoxetine, or Ciclopirox significantly improved survival in LPS-induced sepsis.
  • Sex-Dependent Effects: The protection was highly sex-specific. For example, Mefloquine and Ciclopirox provided robust protection in males but less so in females; Fluoxetine showed the opposite trend (better survival in females).
  • Cytokine Profile: Treated mice showed reduced pro-inflammatory cytokines (TNF-α, IL-1β, IL-17A) and, in females treated with Fluoxetine, elevated anti-inflammatory IL-10.

2. NLRP3 Assembly Blockers (Signal 2/ASC Speck Inhibitors)
These drugs allow LPS priming (normal expression of NLRP3/ASC) but prevent the oligomerization of NLRP3 and the formation of ASC specks.

• Identified Drugs: Rosiglitazone (antidiabetic), Irbesartan (antihypertensive), Felodipine (antihypertensive), Saquinavir (antiviral), Salmeterol (anti-COPD), Miconazole (antifungal).
• Mechanism:
  • Autophagy Induction: Rosiglitazone, Irbesartan, and Felodipine robustly induced autophagy via AMPK phosphorylation, leading to increased LC3-II and decreased p62.
  • Mitochondrial Protection: These drugs reduced LPS-induced mitochondrial ROS (mtROS) and preserved mitochondrial membrane potential, thereby removing a key trigger for NLRP3 activation.
  • Exception: Salmeterol increased mtROS, suggesting a distinct mechanism of action.
• In Vivo Efficacy: Saquinavir and Salmeterol significantly reduced IL-1β levels in peritoneal lavage and plasma pro-inflammatory cytokines in mice.

B. Specific Drug Insights

• Fluoxetine: Previously known to protect against sepsis via IL-10; this study adds that it blocks LPS binding to immune cells.
• Mefloquine: Identified as a potent blocker of LPS binding and a sex-dependent protector against sepsis.
• Ciclopirox: An iron chelator that blocks LPS binding and protects against sepsis, with efficacy varying by sex.
• Rosiglitazone/Irbesartan/Felodipine: Demonstrated that common cardiovascular and metabolic drugs can suppress NLRP3 via the AMPK-autophagy-mitochondria axis.

4. Significance

• Repurposing Potential: The study provides a strong rationale for repurposing a diverse array of existing drugs (antidepressants, antihypertensives, antimalarials, etc.) for treating NLRP3-driven inflammatory diseases (sepsis, metabolic syndrome, neurodegeneration).
• Mechanistic Diversity: It highlights that NLRP3 can be inhibited at multiple points: by blocking LPS binding, inhibiting NF-κB priming, inducing autophagy to clear damaged mitochondria, or directly preventing ASC speck assembly.
• Sex-Dependent Therapeutics: A critical finding is the sex-specific efficacy of these drugs in sepsis models. This underscores the necessity of including both sexes in preclinical trials and suggests that personalized medicine approaches may be required for inflammasome-targeted therapies.
• Clinical Translation: Since these are FDA-approved drugs with known safety profiles, they could rapidly move to clinical trials for conditions like sepsis, where current mortality rates remain high and specific NLRP3 inhibitors are lacking.

Conclusion

This study successfully leveraged a phenotypic screen of FDA-approved drugs to identify novel inhibitors of the NLRP3 inflammasome. By categorizing hits into "priming blockers" and "assembly blockers," the authors elucidated distinct mechanisms of action (including LPS binding inhibition and AMPK-mediated autophagy). The validation in mouse models, particularly the demonstration of sex-dependent survival benefits, offers a promising pathway for the rapid clinical translation of repurposed drugs to treat severe inflammatory conditions like sepsis.