Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase and an attractive anti-fibrotic target. To identify novel DDR1 inhibitors, we used an integrated lead-finding approach relying in parallel on structure-based hybrid design and a focused screening campaign. Combining structural elements from both approaches allowed us to quickly overcome several compound liabilities and optimize the hits to advanced lead compounds. Despite a very high sequence conservation between DDR1 and DDR2, we were able to identify potent DDR1 inhibitors with close to 1000-fold selectivity against DDR2 as well as good selectivity against the full kinome. Exploitation of a DDR1 selectivity pocket detected by structural bioinformatics was crucial in the optimization process. Compounds with very high selectivity suffered from poor metabolic stability in rodents but may serve as useful DDR1-selective in vitro tool molecules.

Optimal amidation conditions between hyaluronic acid (HA) and a methotrexate (MTX)-derived amine that gave rise to a HA-MTX conjugate having a molecular weight of similar to 2000 kDa have been established based on a model experiment using a tryptophan-derived amine in aqueous THF. This method is robust and provides a conjugate with properties of primary importance for efficacy-MW and amine binding ratio-that satisfy the criteria for a HA-MTX conjugate to be a good candidate.

We previously reported that a conjugate of hyaluronic acid (HA) and methotrexate (MTX) could be a prototype for future osteoarthritis drugs having the efficacy of the two clinically validated agents but with a reduced risk of the systemic side effects of MTX by using HA as the drug delivery carrier. To identify a clinical candidate, we attempted optimization of a lead, conjugate 1. Initially, in fragmentation experiments with cathepsins, we optimized the peptide part of HA-MTX conjugates to be simpler and more susceptible to enzymatic cleavage. Then we optimized the peptide, the linker, the molecular weight, and the binding ratio of the MTX of the conjugates to inhibit proliferation of human fibroblast-like syno-viocytes in vitro and knee swelling in rat antigen-induced monoarthritis in vivo. Consequently, we found conjugate 30 (DK226) to be a candidate drug for the treatment of osteoarthritis. (C) 2009 Elsevier Ltd. All rights reserved.

Hyaluronic acid (HA) provides synovial fluid viscoelasticity and has a lubricating effect. Injections of HA preparations into the knee joint are widely used as osteoarthritis therapy. The current HA products reduce pain but do not fully control inflammation. Oral methotrexate (MTX) has anti-inflammatory efficacy but is associated with severe adverse events. Based on the rationale that a conjugation of HA and MTX would combine the efficacy of the two clinically evaluated agents and avoid the risks of MTX alone, we designed HA-MTX conjugates in which the MTX connects with the HA through peptides susceptible to cleavage by lysosomal enzymes. Intra-articular injection of our HA-MTX conjugate (conjugate 4) produced a significant reduction of the knee swelling in antigen-induced arthritis rat, whereas free MTX, HA or a mixture of HA and MTX showed no or marginal effects on the model. The efficacy of conjugate 4 was almost the same as that of MTX oral treatment. Conjugate 4 has potential as a compound for the treatment of osteoarthritis.

A simplified procedure to isolateα-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrateα-connectin is introduced. Separation ofα-connectin fromβ-connectin is introduced. Separation ofα-connectin fromβ-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure ofα-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition ofα-connectin (MW, approximately 3×106) were similar to those of its proteolytic product,β-connectin (MW, approximately 2×106). Circular dichroic spectra suggested that bothα- andβ-connectin consist of 60%β-sheet and 30%β-turn. It thus appears that the whole elastic filament of connectin has a foldedβ-strand structure. Proteolysis ofα-connectin by calpain resulted in formation ofβ-connectin and smaller peptides. Theα-connectin interacted with both myosin and actin filaments similarly toβ-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted withα-connectin (titin 1, TI) but only weakly withβ-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears thatβ-connectin extends from the edge of M-line to the above epitope region in the I-band.

When rabbit skeletal muscle myofibrils were kept for 12 h at 4-degrees-C, alpha-connectin was partially degraded and 1,200 kDa peptide was newly formed [Takahashi, K. & Takai, H. (1988) Abst. 80th Jpn. Soc. Zootech. Sci. p-102]. The latter was isolated together with remaining alpha-connectin. Ultracentrifugation of the mixture at low ionic strength resulted in sedimentation of alpha-connectin, leaving the 1,200 kDa peptide in the supernatant. Physicochemical properties of the isolated 1,200 kDa peptide were investigated: UV absorption spectra, circular dichroism spectra, amino acid composition, and molecular size and shape. Polyclonal antibodies against the 1,200 kDa peptide [PcAb(1200)] bound the Z line and I-band. The position of the stripe in the I band near the N1 line due to the binding of PcAb(1200) moved both away from the Z lines and from the A band as sarcomeres were elongated. Therefore, it is considered that the 1,200 kDa portion of alpha-connectin is elastic.

Monoclonal antibodies, 3B9 and 4C9, specific to connectin (also called titin), 3000 kDa elastic filamentous protein of vertebrate skeletal muscle, crossreacted with a high molecular weight protein (500 kDa) of the nematode Caenorhabditis elegans. However, its crossreactivity was weak to that of the unc-22 gene deficient mutant. Immunofluorescence showed that the antibodies stained both bodywall and pharynx muscles in the wild type, but only pharynx muscle in the mutant. Immunoelectron microscopy revealed that the antibodies bound to the dense bodies of bodywall muscle cells of the wild type but not to those of the mutants. In the pharynx muscles the localization of the antibodies was not clear in both normal and mutant worms.

Nebulin, a high molecular weight (~700 kDa) protein of vertebrate skeletal muscle, was not detected in embryonic and adult cardiac muscles of the chicken by SDS-gel electrophoresis. No peptides reactive with anti-nebulin antiserum were found by immunoblot tests. Nebulin was detected in chicken embryonic skeletal muscles after 17 days of incubation. On the other hand, connectin, another giant protein, was present in both cardiac and skeletal muscles of the chicken embryos after 15 days of incubation.