IDENTIFICATION OF IL-1Β CYTOKINE SECRETION MODULATORS TARGETED BY REPOSITIONED DRUGS

Scientific background: There is a clear and well-documented correlation between the aging process and the activation of the pro-inflammatory immune response that leads to chronic inflammation and accelerates various existing pathologies or favors the establishment of new ones. Inflammatory processes, either chronic or acute, underlie many pathologies and abnormalities associated with inflammation such as rheumatoid arthritis, atherosclerosis, cancer, auto-inflammatory diseases, type 2 diabetes and several neurodegenerative pathologies such as Alzheimer's and Parkinson's disease. This phenotype is largely due to excessive activation of macrophages resident in tissues or circulating in the bloodstream, which secrete pro-inflammatory mediators that support inflammation. Extensive research in the field has revealed complex signaling pathways and inflammation catalysts such as cyclooxygenase 1 and 2 (COX1/2), mitogen-activated kinases (MAPKs), Janus-type tyrosine kinases (JAKs), and also a large number of pro-inflammatory cytokines and regulatory proteins such as: IL-1, IL-6, TNFα, MIF, IFNγ, etc., which support inflammatory processes both in homeostatic and pathological conditions [2].

Generic anti-inflammatory therapies are based on non-steroidal anti-inflammatory drugs and inhibitors targeting prostaglandin metabolism, for which considerable side effects have recently been 
identified [3]. In this context, the identification of new molecules or the repositioning of drugs with anti-inflammatory activity and reduced side effects, in order to treat diseases associated with inflammation, 
is of major interest.

Interleukin 1β (IL-1β) is a member of the IL-1 cytokine family that plays a central role in regulating the immune and inflammatory response to infection or injury. IL-1β activation has been implicated in 
the activation of helper T cells, stimulation of prostaglandin or collagenase production, neutrophil chemotaxis, activation of acute-phase pathogen response proteins, and suppression of plasma iron levels.

IL-1β is synthesized by macrophages in the precursor form (proIL-1β), in response to the activation of intracellular signaling pathways following the binding of "danger molecules" 
(pathogen-associated molecular patterns (PAMPs) or damage- associated molecular patterns (DAMPs)) that activate the TLR (Toll like receptors)-NFkB pathway. A second signal that stimulates purinergic 
receptors on the surface of macrophages triggers the assembly of inflammasomes and the activation of caspase-1 that cleaves proIL-1β to its mature form, which is rapidly secreted into the extracellular 
environment to propagate the pro-inflammatory response. Because the process of synthesis and secretion of IL-1β is complex, recombinant systems cannot recapitulate all steps of this process, 
and primary cells, which can be multiplied in limited quantities, cannot be used for screening studies with the aim of discovering new molecules with anti-inflammatory potential.

Clinical relevance of the project: A clinical correlation between exacerbated secretion of IL-1β and chronic inflammation has been demonstrated for decades, but until now medical approaches to block
the secretion of IL-1β associated with inflammation are based on the ability of recombinant proteins (Ankinra) or antibodies (Canakinum) to block cytokine binding to extracellular receptors exposed on
target cells to limit the spread of inflammation. However, the effectiveness of these molecules has been shown to be reduced in clinical trials, and therefore the identification and validation of small
molecules that limit the inflammation caused by IL-1β hypersecretion are of great interest.

In recent years, sustained efforts have been directed towards the repositioning of drugs already approved for the treatment of various diseases. These have the advantage of having passed through
all stages of testing and most of the time only dose adjustment is necessary, and in this context the best example is aspirin (acetylsalicylic acid) which was initially approved as an analgesic and
later repositioned as an antiplatelet agent, and even more recent studies have shown that long-term aspirin treatment protects against the development of several forms of cancer [4].

In a previous project, funded through national competition, of demonstrative experimental type (PED 337 of 03/08/2020 -PN-III-P2-2.1-PED-2019-3297) we developed a reporter line for IL-1β secretion
using CRISPR/Cas9 knock-in technology that allows the addition of a small tag in the endogenous locus of the gene coding for interleukin-1β (IL-1β) so that the response of cells to physiological stimuli
is maintained at a level similar to that of primary macrophages . The reporter line was successfully used for the screening of two existing compound libraries within IBAR.

As a result of the high-performance screening of a drug library approved by the FDA (Food and Drug Administration-USA), carried out within the aforementioned project, we identified 4 drugs that have
anti-inflammatory activity by reducing the secretion of interleukin-1β, in addition to their already known pharmacological activity.

In this project we aim to validate these 4 drugs for their anti-inflammatory capacity using primary macrophage cells isolated from mice and an animal model of septicemia and to identify the molecular
targets of these drugs in the IL-1β secretion pathway.

At the end of this project we want to propose:

-one or two drugs that can be repositioned as anti-inflammatory agents

-mapping the targets of these drugs in the IL-1β cytokine secretion pathway, which contributes to the understanding of the molecular mechanism of inflammation.