We have carried out a structure-activity relationship study to identify highly potent analogues of kartogenin with chondrogenic and chondroprotective activities. Over 150 analogues were synthesized with structurally diverse elements and assessed for chondrogenic activity (ability to induce mesenchymal stem cells to differentiate into cartilage producing chondrocytes) on human and rodent mesenchymal stem cells. A number of highly potent lead compounds were identified which will next be assessed in chondroprotective assays, cell-based selectively and toxicity assays, pharmacokinetic assays and in vivo rodent efficacy models. At the same time a number of assays were developed and used to assess the chondrocyte protective effects, joint retention, and proliferative activity on human chondrocytes of the parent compound, kartogenin. Kartogenin was found to: (1) have long term human and rodent chondrogenic activity; (2) possess chondroprotective activity in bovine chondrocytes (i.e., protects against degradative activities in the joint); (3) minimally induce chondrocytes proliferation (an undesired activity that could lead to fibrotic and immune responses); (4) have good joint retention (compound retained in the intra-articular space at the site of action); and (5) is subject to rapid systemic clearance (a desirable property to minimize systemic adverse effects).
We also identified the mechanism by which the compound functions. In contrast to other drugs in development for osteoarthritis, kartogenin does not target extracellular enzymes involved in joint cartilage degradation. Rather it appears to act directly on an endogenous stem cell population and induce chondrocyte formation. The molecule binds selectively to an intracellular protein filamin A, a protein involved in regulating the cell’s cytoskeletal network (structural elements inside the cell). Rather than modulating the interaction of filamin A with other structural proteins, kartogenin blocks its interaction with the protein CBFβ (core binding factor β subunit, a subunit of a transcription complex with the runt-related transcription factor (RUNX) family). The result is an increase in CBFβ levels in the nucleus where it binds and activates transcription of RUNX dependent genes. In particular CBFβ activates RUNX1 dependent transcription of genes that play key roles in chondrogenesis. Thus this molecule acts by a novel mechanism directly and selectively on gene transcription to induce the selective differentiation of mesenchymal stem cells to chondrocytes. Importantly molecules that act by this method should complement the activity of drugs in clinical trials aimed at blocking degradative enzymes.