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. 2024 Oct 7:15:1402032.
doi: 10.3389/fphar.2024.1402032. eCollection 2024.

Diacerein reduces inflammasome activation and SARS-CoV-2 virus replication: a proof-of-concept translational study

Helison R P Carmo #  1 , Alejandro Rossel Castillo #  1 , Isabella Bonilha  1 , Erica I L Gomes  1 , Joaquim Barreto  1 , Filipe A Moura  2   3 , Gustavo Gastão Davanzo  4 , Lauar de Brito Monteiro  4 , Stéfanie Primon Muraro  5 , Gabriela Fabiano de Souza  5 , Joseane Morari  6 , Flávia Elisa Galdino  7   8 , Natália S Brunetti  9 , Guilherme Reis-de-Oliveira  10 , Victor Corasolla Carregari  10 , Wilson Nadruz  1 , Daniel Martins-de-Souza  6   10   11   12 , Alessandro S Farias  6   9   12 , Licio A Velloso  6 , José Luiz Proenca-Modena  5   12 , Marcelo A Mori  6   12   13 , Watson Loh  7 , Deepak L Bhatt  14 , Derek M Yellon  15 , Sean M Davidson  15 , Pedro G De Oliveira  16   17 , Pedro M Moraes-Vieira  4   6   12 , Andrei C Sposito  1
Affiliations

Diacerein reduces inflammasome activation and SARS-CoV-2 virus replication: a proof-of-concept translational study

Helison R P Carmo et al. Front Pharmacol. .

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is linked to high mortality, primarily through an intense inflammatory response. Diacerein has emerged as a potential therapy for COVID-19 due to its potential impact in decreasing the inflammasome activation and coronavirus replication. This study aims to explore diacerein's influence in inhibiting both viral replication and the inflammatory response after SARS-CoV-2 infection.

Methods: Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and infected in vitro with SARS-CoV-2. Additionally, we carried out a pilot randomized, double-blind, placebo-controlled study with 14 participants allocated to diacerein (n = 7) or placebo (n = 7) therapies every 12 h for 10 days. The primary endpoint was change in plasma markers of inflammasome activation (NLRP3, caspase-1, and gasdermin-D).

Results: In vitro protocols have shown that rhein, diacerein's primary metabolite, decreased IL-1β secretion caused by SARS-CoV-2 infection in human PBMCs (p < 0.05), and suppressed viral replication when administered either before or after the virus incubation (p < 0.05). This later effect was, at least partially, attributed to its inhibitory effect on 3-chymotrypsin-like protease (SARS-CoV-2 3CLpro) and papain-like protease in the SARS-CoV-2 (SARS-CoV-2 PLpro) virus and in the phosphorylation of proteins related cytoskeleton network (p < 0.05). Diacerein-treated COVID-19 patients presented a smaller area under the curve for NLRP3, caspase-1 and GSDM-D measured on days 2, 5, and 10 after hospitalization compared to those receiving a placebo (p < 0.05).

Conclusion: The indicated mechanisms of action of diacerein/rhein can reduce viral replication and mitigate the inflammatory response related to SARS-CoV-2. These findings are preliminary and require confirmation in clinical trials.

Keywords: COVID-19; clinical trial; diacerein; pre-clinical; rhein.

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Conflict of interest statement

DB discloses the following relationships - Advisory Board: Angiowave, Bayer, Boehringer Ingelheim, CellProthera, Cereno Scientific, Elsevier Practice Update Cardiology, High Enroll, Janssen, Level Ex, McKinsey, Medscape Cardiology, Merck, MyoKardia, NirvaMed, Novo Nordisk, PhaseBio, PLx Pharma, Stasys; Board of Directors: American Heart Association New York City, Angiowave (stock options), Bristol Myers Squibb (stock), DRS.LINQ (stock options), High Enroll (stock); Consultant: Broadview Ventures, GlaxoSmithKline, Hims, SFJ, Youngene; Data Monitoring Committees: Acesion Pharma, Assistance Publique-Hô pitaux de Paris, Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute, for the PORTICO trial, funded by St. Jude Medical, now Abbott), Boston Scientific (Chair, PEITHO trial), Cleveland Clinic, Contego Medical (Chair, PERFORMANCE 2), Duke Clinical Research Institute, Mayo Clinic, Mount Sinai School of Medicine (for the ENVISAGE trial, funded by Daiichi Sankyo; for the ABILITY-DM trial, funded by Concept Medical; for ALLAY-HF, funded by Alleviant Medical), Novartis, Population Health Research Institute; Rutgers University (for the NIH-funded MINT Trial); Honoraria: American College of Cardiology (Senior Associate Editor, Clinical Trials and News, ACC.org; Chair, ACC Accreditation Oversight Committee), Arnold and Porter law firm (work related to Sanofi/Bristol-Myers Squibb clopidogrel litigation), Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute; RE-DUAL PCI clinical trial steering committee funded by Boehringer Ingelheim; AEGIS-II executive committee funded by CSL Behring), Belvoir Publications (Editor in Chief, Harvard Heart Letter), Canadian Medical and Surgical Knowledge Translation Research Group (clinical trial steering committees), CSL Behring (AHA lecture), Cowen and Company, Duke Clinical Research Institute (clinical trial steering committees, including for the PRONOUNCE trial, funded by Ferring Pharmaceuticals), HMP Global (Editor in Chief, Journal of Invasive Cardiology), Journal of the American College of Cardiology (Guest Editor; Associate Editor), K2P (Co-Chair, interdisciplinary curriculum), Level Ex, Medtelligence/ReachMD (CME steering committees), MJH Life Sciences, Oakstone CME (Course Director, Comprehensive Review of Interventional Cardiology), Piper Sandler, Population Health Research Institute (for the COMPASS operations committee, publications committee, steering committee, and United States national co-leader, funded by Bayer), WebMD (CME steering committees), (steering committee); Other: Clinical Cardiology (Deputy Editor); Patent: Sotagliflozin (named on a patent for sotagliflozin assigned to Brigham and Women' s Hospital who assigned to Lexicon; neither I nor Brigham and Women' s Hospital receive any income from this patent); Research Funding: Abbott, Acesion Pharma, Afimmune, Aker Biomarine, Alnylam, Amarin, Amgen, AstraZeneca, Bayer, Beren, Boehringer Ingelheim, Boston Scientific, Bristol-Myers Squibb, Cardax, CellProthera, Cereno Scientific, Chiesi, CinCor, Cleerly, CSL Behring, Eisai, Ethicon, Faraday Pharmaceuticals, Ferring Pharmaceuticals, Forest Laboratories, Fractyl, Garmin, HLS Therapeutics, Idorsia, Ironwood, Ischemix, Janssen, Javelin, Lexicon, Lilly, Medtronic, Merck, Moderna, MyoKardia, NirvaMed, Novartis, Novo Nordisk, Otsuka, Owkin, Pfizer, PhaseBio, PLx Pharma, Recardio, Regeneron, Reid Hoffman Foundation, Roche, Sanofi, Stasys, Synaptic, The Medicines Company, Youngene, 89Bio; Royalties: Elsevier (Editor, Braunwald’s Heart Disease); Site Co-Investigator: Abbott, Biotronik, Boston Scientific, CSI, Endotronix, St. Jude Medical (now Abbott), Philips, SpectraWAVE, Svelte, Vascular Solutions; Trustee: American College of Cardiology; Unfunded Research: FlowCo. AS discloses the following relationships - Research Funding: Amgen, AstraZeneca, National Council for Scientific and Technological Development (CNPq) and Fundaç and#227; o de Apoio and#224; Pesquisa do Estado de Sã o Paulo (FAPESP). Dr. Pedro Gonç alves de Oliveira is responsible for R&D activities at TRB Pharma Indú stria Quí mica e Farmace utica Ltda, SP, Brazil. TRB Pharma is the owner of the product ARTRODAR® a diacerein-based product for osteoarthritis treatment. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Non-toxic effects were verified in a cell viability assay. Human PBMCs were treated with different doses following 24-h treatment with rhein (A), VRT043198 (B), and emricasan (C).
FIGURE 2
FIGURE 2
Rhein treatment reduces IL-1β gene expression and protein secretion in human PBMCs. Human PBMCs obtained from healthy volunteers were infected in vitro for 1 h under continuous agitation and stimulated with rhein, VRT-043198, emricansan or vehicle (dimethyl sulfoxide, DMSO) for 24 h. The supernatant and protein extract were obtained at the end of the protocol to analyze for inflammatory markers. (A) timeline diagram of infection and drug stimulation. Relative mRNA expression of (B) NLRP3, (C) caspase-1, (D) ASC, (E) IL-18, (F) gasdermin-D, and (G) IL-1β. (H) Measurement of IL-1β secretion by ELISA. The data represent the mean ± SEM of at least two independent experiments performed in triplicate.
FIGURE 3
FIGURE 3
Rhein treatment under protocol of priming (or synthesis) stimulation and activation of NLRP3 inflammasome reduces IL-1β secretion in human PBMCs. Human PBMCs obtained from healthy volunteers were subjected to the NLRP3 inflammasome transcriptional and post transcription protocol also named priming and activation protocol and treated with rhein at two distinct time points. (A) timeline diagram of drug stimulation. (B) evaluation of IL-1β secretion under rhein treatment starts with lipopolysaccharide for 3 h of exposure plus nigericin for 1 h, or (ii) only with nigericin for 1 h. The positive control was the vehicle (dimethyl sulfoxide, DMSO) and the negative control was VRT-043198 (0.5 µM). The data represent the mean ± SEM of at least two independent experiments performed in triplicate.
FIGURE 4
FIGURE 4
Non-toxic and non-proliferation inducer effects were verified during the priming and activation protocol. Human PBMCs were intracellularly labeled and analyzed using flow cytometry to assess toxicity and proliferation capacity in the presence of rhein (60 µM) and the positive control VRT-043198 (0.5 µM).
FIGURE 5
FIGURE 5
Rhein treatment reduces the viral load of SARS-CoV-2 in infected human PBMCs in vitro. Human PBMCs obtained from healthy volunteers were infected in vitro for 1 h under continuous agitation and stimulated with rhein, VRT-043198, Emricasan, or vehicle (dimethyl sulfoxide, DMSO) for 24 h. (A) Timeline diagram of drug stimulation. (B) Evaluation of viral load with inhibitors at different dose concentrations plus Mock and vehicle groups. The data represent mean ± SEM of at least two independent experiments performed in triplicate.
FIGURE 6
FIGURE 6
Rhein treatment reduces the viral load in human PBMCs prior to in vitro infection with SARS-CoV-2. Human PBMCs obtained from healthy volunteers were treated with rhein at different doses before being infected in vitro for 1 h under continuous agitation and stimulation.
FIGURE 7
FIGURE 7
Rhein molecule has a low affinity for the S1 protein and significant pharmacological properties in reducing the activity of SARS-CoV-2 proteases 3CLPro and PLpro. The specific assay evaluated the interaction between rhein and the viral S1 protein revealed low binding energy between them. In parallel, rhein significantly reduced the activity of the proteases used by SARS-CoV-2 for genetic material replication. (A) baseline corrected raw isothermal titration calorimetry (ITC) data of rhein injection into S1 solution and rhein dilution in PBS. (B) binding isotherm of rhein-S1 after subtraction of the rhein heat of dilution. (C) Effect of rhein on the processing of non-structural virus proteins by 3CLpro-SARS-CoV-2, and (D) PLpro-SARS-CoV-2 with rhein, VRT-043198, emricansan or vehicle (dimethyl sulfoxide, DMSO). The data represent mean ± SEM of at least two independent experiments performed in triplicate.
FIGURE 8
FIGURE 8
Rhein treatment can change phosphorylation patterns of extracellular matrix-related proteins in human PBMCs infected with SARS-CoV-2. A mass spectrometry-based phosphoproteomics assay revealed an increase in non-phosphorylated compared to phosphorylated proteins in vitro. (A) heatmap of phosphopeptides from human PBMCs infected with SARS-CoV-2, control (no treatment), and rhein treatment. (B) volcano plot of phosphopeptides that were found to be deregulated in control relative to rhein treatment. (C) the enrichment analysis of biological pathways with phosphopeptides and their respective phosphorylation sites (upstream or downstream).
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. Funding for this study was provided by grants from the Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP, grant number: 2020/05430-7 to ACS, 2020/04558-0 to JLPM, 2018/17792-0 to HRPC, and 2020/12649-5 to ARC). This study was also supported by Fundo de Apoio ao Ensino, Pesquisa e Extensão of UNICAMP (FAEPEX-UNICAMP, grant number: 2391/20 to ACS; and 2266/20 to JLPM). National Council for Scientific and Technological Development (CNPq) supported (grant number: 304257/2021-4 to AS, and 305628/2020-8 to JP-M). In addition, TRB PHARMA’s support in donating diacerein and placebo capsules.

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