Pre-clinical development of natural compound-based drugs, targeting IL-23-Th17 pathway in immune-mediated inflammatory diseases – IMIDRUG

Identification of Molecular Targets

Several approaches were used in parallel. The first involved validating a hypothesis based on the known effect of the compound on IL-23 production in macrophage cells. Since the transcription of the IL-23 p19 gene, the IL-23-specific subunit (with p40 being shared with IL-12), is controlled by the NFκB pathway, we used biochemical approaches to determine whether AL1 could act on one or more components of the NFκB activation pathway.

A second approach involved performing a chemical two-hybrid screen, which was conducted by the company Hybrigenics. Additionally, after grafting an alkyne group onto AL1 at a position that did not affect its pharmacological properties, we coupled the molecule to agarose beads. These beads were used to precipitate molecules expressed in the cytoplasm of LPS-stimulated THP-1 macrophage cells, either pre-incubated with or without AL1. Proteins specifically precipitated by the beads were identified using mass spectrometry. One candidate protein (hereafter referred to as Protein X) was subsequently studied in detail. Protein X was demonstrated to be the primary target of AL1, and its inhibition explained all the pharmacological effects observed.

Medicinal Chemistry

By modeling the AL1 interaction site on Protein X, we performed in silico docking of several thousand structurally related compounds to identify AL1 analogs. A detailed computational analysis of active and inactive compounds allowed us to identify a potential pharmacophore. Based on this pharmacophore, 11 additional compounds were identified, and their activity was validated in vitro and in vivo. We now have a family of compounds distinguished by their physicochemical properties and bioavailability. Five of these compounds have undergone further investigation in animal models of human diseases.

Additional Models

We confirmed the efficacy of AL1 in delaying the onset of neurological symptoms in a murine model of ALS (mSOD-1 mice) and in experimental autoimmune encephalomyelitis (a model of multiple sclerosis).

The company ALLSPIM also independently tested the efficacy of AL1 in additional models of human diseases, including autoimmune myocarditis, gout, wet age-related macular degeneration, uveitis, and MASH.

Molecular Targets

AL1 inhibits the kinase Ikkβ and stabilizes IκB in the NFκB pathway.

AL1 inhibits Protein X, which is involved in the signaling pathway downstream of danger signal receptors, particularly Toll-like receptor 4 (TLR4). Protein X controls both:

- Transcriptional activation pathways leading to the production of pro-inflammatory cytokines (such as IL-1β and IL-23), and

- The "priming" of the NLRP3 inflammasome.

The role of Protein X in these mechanisms has not been previously reported, making this a major discovery of our research program.

Medicinal Chemistry

After multiple iterations and in vitro and in vivo validations, we identified 10 compounds sharing the same effects, including one with potency comparable to AL1.

Independently, ALLSPIM, in collaboration with CROs,developed the synthesis of the molecule (Drug Substance, DS) and confirmed that its in vitro and in vivo properties matched those of the plant-purified compound. The preclinical development of the molecule, including the production of the Drug Product in its final formulation, is based on this DS.

Models of Human Diseases

In the mSOD-1 ALS mouse model, daily oral administration of AL1 significantly delays the onset of symptoms linked to the degeneration of motor neurons in the anterior horn of the spinal cord in animals carrying the mutated gene. Histological and biochemical studies of the spinal cords show:

- A reduction in inflammatory microglia infiltration (Iba1 staining), and

- A decrease in MMP9 metallo-protease expression, a disease marker, in treated mice.

- Motor neuron loss is also limited in treated subjects.

In the experimental autoimmune encephalomyelitis (EAE) model, preventive administration of AL1 inhibit, or even completely prevents (in a specific formulation), the onset of neurological disorders.

The scientific perspectives of the IMIDRUG project include a new understanding of the molecular mechanisms and signaling pathways governing innate immunity, as well as a better integration of this aspect of immunity in many human diseases.

From a patient benefit perspective, ALLSPIM will make every effort to ensure that this new pharmacological family of "innate immunity homeostasis modulators" reaches clinical trials and ultimately leads to a new category of treatments. At this stage, the potential and hopes are high, but so are the remaining challenges, one of the greatest being the unpredictable possibility of toxic effects arising during development.

We are developing a new class of antiinflammatory drugs dedicated to immune-mediated inflammatory diseases (IMID) a heterogeneous group of diseases characterized by acute or chronic inflammation affecting any organ system whose physiopathology includes imbalance of inflammatory cytokines. IMID cause high morbidity and represent a considerable burden on patients in terms of altered quality of life and on society. We discovered 2,4-D a natural anti-inflammatory compound targeting the IL-23/IL-17 axis and identified its cellular targets in vitro and its efficacy in preclinical models of IMID (creation of a startup, article submitted and patent filed). Our project aims to identify the molecular targets of 2,4-D, to use medicinal chemistry for R&D, and to complete the pre-clinical study in a flagship animal model of IMID, Crohn's disease, and in ALS a model of deadly neurodegenerative disease with a strong inflammatory component The study will speed up the timeliness for a phase I study.