Development of new matrix metalloproteinase-13 inhibitors

Osteoarthritis (OA), the degeneration of cartilage, is the most prevalent chronic joint disease in the western world and ranks among the top five causes of physical disability. Despite intensive research over the past years, medical treatment options for OA are still rather limited and are mainly focused on relieving pain. There is an urgent need for the development of disease modifying agents to increase the quality of life for OA patients. The zinc-dependent matrix metalloproteinase-13 (MMP-13) was identified to be mainly responsible for the cleavage of type II collagen in OA, which leads to the destruction of articular cartilage. First-generation zinc-chelating inhibitors failed mostly due to dose limiting side effects collectively described as musculoskeletal syndrome. As a consequence compounds, which interact with secondary binding sites (exosites) of MMP-13 and lack zinc-chelating groups were extensively screened. Especially MMP-13 S1 subsite binding compounds exhibited promising inhibitor properties and selectivity profiles but none of these have successfully been employed in clinical trials. A high throughput screening (HTS) campaign using a newly developed triple-helical peptide (THP) fluorescence resonance energy transfer (FRET) assay, was carried out, based on the knowledge about the importance of subsite-binding properties and the resulting intention to identify new exosites which could be utilized for the design of next-generation inhibitors. THPs represent perfect substrates for the development of non-active site binding and selective MMP inhibition due to their distinct conformational features. THP assays are not limited to the identification MMP-13 inhibitors and therefore, in future studies, the drug design approach described within this proposal, will be extended toward the investigation of non-active site binding inhibitors targeting the remaining human collagenases (MMP-1, -8 and -14), which play important roles during tumor progression and metastasis. Based on the isolation of three, low micromolar MMP-13 inhibitors as a result of the HTS and crystallographic studies revealing that two molecules of the identified inhibitors are simultaneously bound in two distinct binding sites, one known and a second, previously unknown exosite, a structure- guided drug design approach will be launched. The origin of synthetic considerations is the design of fused inhibitor scaffolds by exploiting the advantages of known MMP-13 inhibitor binding modes. The synthetic planning represents a fragment-based approach, which allows the modular and fast preparation of a large number of potential drug candidates. The synthetic studies will be accompanied and clearly rationalized by structural analysis and molecular models and comprehensive in vitro SAR studies will validate the optimization process. The drug design approach elaborated within the proposal is focusing on the improvement of selectivity and inhibiting potency of the three initially identified MMP-13 inhibitors, in order to ultimately develop efficient anti-osteoarthritic drugs.

Osteoarthritis (OA), the degeneration of cartilage, is the most prevalent chronic joint disease in the western world and ranks among the top five causes of physical disability. Despite intensive research over the past years, medical treatment options for OA are still rather limited and are mainly focused on relieving pain. There is an urgent need for the development of disease modifying agents to increase the quality of life for OA patients. Matrix metalloproteinase 13 (MMP-13) is known to be mainly responsible for the degradation of collagen type 2 during the progress of OA. As a consequence articular cartilage, the mechanical stress absorber within human joints, is getting degraded, and OA patients suffer from severe pain. First-generation MMP-13 inhibitors were chelating the zinc ion located in the active site of the enzyme. The interaction with the metal ion induced non-selectivity within the MMP-family and all compounds, which entered clinical trials had to be withdrawn mainly due to dose limiting side effects, collectively described as musculoskeletal syndrome. Within this project we have developed selective, non-chelating MMP-13 inhibitors through a three- step process. Step one included a comparative structural analysis of the X-ray crystal structure of our lead compound in complex with MMP-13 with published structures of MMP-13-inhibitor complexes. In step two we utilized docking studies to design and further synthesize potent, but non-selective zinc-chelating MMP-13 inhibitors. After demonstrating that the pharmacophores of the chelating inhibitors were binding within the MMP-13 active site, the Zn2+ chelating unit was replaced with non- chelating polar residues that bridged over the Zn2+ binding site and reach into a solvent accessible area during step three to achieve selectivity within the MMP-family. In further course we used the physicochemcial as well as in vitro pharmacokinetic properties of our initial compounds as a starting point to perform a structure-activity relationship (SAR)/structure- property relationship (SPR) study and found a set of non-zinc chelating preclinical candidates, which show at least 500-fold selectivity versus other MMPs and IC50 values in the single digit nanomolar range. As MMP-13 is known to be one of the key enzymes in the progression of OA, the development of preclinical candidates, able to selectively inhibit this enzyme is the foundation for the implementation of a new drug against OA. The introduction of a MMP-13 inhibitor on the market would have the potential to improve OA patients quality of life tremendously.