Abstract

 

High throughput screening identifies novel pharmacological inhibitors of interferon-gamma-induced nitric oxide production, alleviating ulcerative colitis and bacterial sepsis in mice

Avik Chattopadhyay, Joel Joseph, Sirisha Jagdish, Somak Chaudhuri, Nikita Ramteke, Aagosh Karhale, Uchenna Waturuocha, Deepak Saini, Dipankar Nandi

 

Author Affiliations

Indian Institute of Science, Bengaluru, India

 

Correspondence: Avik Chattopadhyay

avik95ac@gmail.com

 

IJIR. 2023;(7)1:A4

 

 Submitted: 8 November 2023, Published: 9 November 2023

Background: Interferon-gamma (IFN-γ) is a type II interferon primarily produced by T cells and natural killer cells. One of the key markers in IFN-γ signaling is the expression of NOS2 catalyzing the production of Nitric Oxide (NO). IFN-γ signaling and NO production combat infectious diseases like Mycobacterium tuberculosis and Salmonella Typhimurium infections. However, excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including ulcerative colitis, multiple sclerosis, systemic lupus erythematosus, and sepsis. Disease exacerbation in chronic inflammatory diseases is managed with steroidal medications; however, long-term use of corticosteroids often leads to unavoidable adverse effects. These problems necessitate identifying alternative non-steroidal anti-inflammatory drugs, potentially targeting IFN-γ-induced NO hyperproduction.

 

Previous studies conducted by the same research team showed that H6 mouse hepatoma cells produce NO and ROS upon IFN-γ-activation and undergo cell cycle arrest and apoptosis. This study aimed to identify novel non-steroidal anti-inflammatory compounds against IFN-γ-induced NO production using the in vitro H6-IFN-γ-activation model system.

 

Methods: The chemical library: LOPAC®1280, was screened in vitro on the H6 mouse hepatoma cell line to identify novel inhibitors. The lead compounds were studied for mechanism and tested across tumor cell lines and primary cells: mouse peritoneal macrophages. The compounds were assessed in vivo in two distinct pre-clinical mice inflammatory disease models: DSS-induced ulcerative colitis and Salmonella Typhimurium-induced bacterial sepsis.

 

Results: The lead compounds were identified: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC50 and goodness of fit analyses. Mechanistic investigations revealed that most lead compounds suppress the IFN-γ-induced transcription of NOS2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1, Socs1, and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced ROS amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in residents and thioglycolate-elicited peritoneal macrophages. In vivo, pentamidine and auranofin protected mice from DSS-induced ulcerative colitis and Salmonella Typhimurium-induced sepsis.

 

Conclusion: The lead compounds namely pentamidine, azithromycin, and auranofin are FDA-approved drugs. Roflumilast, a drug with a similar mechanism of action to rolipram, is FDA-approved. Therefore, the lead compounds are suitable for drug repurposing. The in vitro high throughput screening and validation model against IFN-γ-activation is suitable for screening other large chemical libraries and identifying novel anti-inflammatory compounds. Auranofin and pentamidine alleviate pathogenesis in two distinct inflammatory disease models and may serve as therapeutic options in alternative or combinatorial regimens to treat the chronic inflammatory conditions of inflammatory diseases.

 

Presented by: Avik Chattopadhyay