Random high throughput sequencing of a human osteoclast cDNA library was employed to identify novel osteoclast-expressed genes. Of the 5475 ESTs obtained, approximately 4% encoded cathepsin K, a novel cysteine protease homologous to cathepsins S and L; ESTs for other cathepsins were rare. In addition, ESTs for cathepsin K were absent or at low frequency in cDNA libraries from numerous other tissues and cells. In situ hybridization in osteoclastoma and osteophyte confirmed that cathepsin K mRNA was highly expressed selectively in osteoclasts; cathepsins S, L, and B were not detectable. Cathepsin K was not detected by in situ hybridization in a panel of other tissues. Western blot of human osteoclastoma or fetal rat humerus demonstrated bands of 38 and 27 kDa, consistent with sizes predicted for pro-and mature cathepsin K. Immunolocalization in osteoclastoma and osteophyte showed intense punctate staining of cathepsin K exclusively in osteoclasts, with a polar distribution that was more intense at the bone surface. The abundant expression of cathepsin K selectively in osteoclasts strongly suggests that it plays a specialized role in bone resorption. Furthermore, the data suggest that random sequencing of ESTs from cDNA libraries is a valuable approach for identifying novel cell-selective genes.

We have studied two related proteins that contain a repeated amino acid motif homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (TSP1). Complete sequence analysis revealed no other similarities with TSP1, but identified unique signal sequences, as well as metalloprotease and disintegrin-like domains in the NH 2 termini. We named these proteins METH-1 and METH-2 due to the novel combination of metalloprotease and thrombospondin domains. Overall amino acid sequence identity between METH-1 and METH-2 is 51.7%, yet transcript distribution revealed non-overlapping patterns of expression in tissues and cultured cell lines. To characterize these proteins functionally, we isolated full-length cDNAs, produced recombinant protein, and generated antisera to the recombinant proteins. Both METH-1 and METH-2 represent single copy genes, which encode secreted and proteolytically processed proteins. METH proteins suppressed fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited vascular endothelial growth factor-induced angiogenesis in the chorioallantoic membrane assay. Suppression of vessel growth in both assays was considerably greater than that mediated by either thrombospondin-1 or endostatin on a molar basis. Consistent with an endothelial specific response, METH-1 and METH-2 were shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth. We propose that METH-1 and METH-2 represent a new family of proteins with metalloprotease, disintegrin, and thrombospondin domains. The distinct distribution of each gene product suggests that each has evolved distinct regulatory mechanisms that potentially allow for fine control of activity during distinct physiological and pathological states. Thrombospondin-1 (TSP1)1 is a modular protein that associates with the extracellular matrix and has the ability to inhibit angiogenesis in vivo (1, 2). Under culture conditions, TSP1 blocks capillary-like tube formation (3) and endothelial cell proliferation (4). The region responsible for the anti-angiogenic activity has been mapped to the 385-522 amino acid region of the protein, which contains three type 1 (properdin or TSP) repeats (2). Recombinant and proteolytic fragments containing these repeats exhibited angio-inhibitory activity in the rabbit corneal pocket and chorioallantoic membrane assays (2, 5, 6) and peptides derived from the second and third type 1 repeats of TSP1 inhibit endothelial cell chemotaxis and proliferation (7). Furthermore, peptides derived from the same region induce apoptosis of endothelial cells and suppress tumor growth when injected systemically (8, 9). Of the five known members of the TSP family, only TSP1 and TSP2 contain type 1 repeats, and are the only members that inhibit angiogenesis (10, 11). We hypothesized that TSP/type 1 repeats might be present in other yet unidentified proteins, in a context that enables activity as inhibitors of capillary growth. We therefore initiated a search for novel cDNAs using the anti-angio...

A cDNA clone for the serine proteinase inhibitor (serpin), neuroserpin, was isolated from a human whole brain cDNA library, and recombinant protein was expressed in insect cells. The purified protein is an efficient inhibitor of tissue type plasminogen activator (tPA), having an apparent second-order rate constant of 6.2 ؋ 10 5 M ؊1 s ؊1 for the two-chain form. However, unlike other known plasminogen activator inhibitors, neuroserpin is a more effective inactivator of tPA than of urokinase-type plasminogen activator. Neuroserpin also effectively inhibited trypsin and nerve growth factor-␥ but reacted only slowly with plasmin and thrombin. Northern blot analysis showed a 1.8 kilobase messenger RNA expressed predominantly in adult human brain and spinal cord, and immunohistochemical studies of normal mouse tissue detected strong staining primarily in neuronal cells with occasionally positive microglial cells. Staining was most prominent in the ependymal cells of the choroid plexus, Purkinje cells of the cerebellum, select neurons of the hypothalamus and hippocampus, and in the myelinated axons of the commissura. Expression of tPA within these regions is reported to be high and has previously been correlated with both motor learning and neuronal survival. Taken together, these data suggest that neuroserpin is likely to be a critical regulator of tPA activity in the central nervous system, and as such may play an important role in neuronal plasticity and/or maintenance.

Abstract. The Drosophila 36B muscle myosin heavy chain (MHC) gene has five sets of alternatively spliced exons that encode functionally important domains of the MHC protein and provide a combinatorial potential for expression of as many as 480 MHC isoforms . In this study, in situ hybridization analysis has been used to examine the complexity and muscle specificity of MHC isoform expression in the fibrillar indirect flight muscle (IFM), the tubular direct flight muscles (DFM) and tubular tergal depressor of the trochanter muscle (TDT), and the visceral esophageal muscle in the adult thorax . Our results show that alternative T HE myosin heavy chain (MHC)' protein is a major component of thick filaments of the skeletal muscle myofibrillar contractile apparatus . Biochemical and biophysical studies have shown that discrete domains of the MHC protein control its assembly into thick filaments as well as its function in binding to actin and regulatory contractile proteins during the contraction cycle (Harrington and Rodgers, 1984;Warrick and Spudich, 1987) . The aminoterminal region forms a globular head that includes domains that function in ATP binding, actin-activated ATP hydrolysis, binding of myosin light chain subunits, and actin binding . The carboxy-terminal region forms an a-helical coiled-coil rod structure involved in dimerization ofMHC subunits and in higher order interactions to assemble myosin dimer molecules into thick filaments . The coiled-coil structure of the rod is interrupted by a random coil "hinge" sequence domain that interrupts the coiled-coil structure of the rod and may be a site for molecular force production during contraction (Ueno and Harrington, 1986a,b) .Although biochemical studies have provided structure/ function information on the vertebrate skeletal muscle myosins, molecular genetic studies of myosin in Drosophila and the nematode, Caenorhabditis elegans, are identifying new

Recent clinical data provided proof-of-concept for selective B-Raf inhibitors in treatment of B-Raf(V600E) mutant melanoma. Pyrazolopyridine-type B-Raf inhibitors previously described by the authors are potent and selective but exhibit low solubility requiring the use of amorphous dispersion-based formulation for achieving efficacious drug exposures. Through structure-based design, we discovered a new class of highly potent aminopyrimidine-based B-Raf inhibitors with improved solubility and pharmacokinetic profiles. The hinge binding moiety possesses a basic center imparting high solubility at gastric pH, addressing the dissolution limitation observed with our previous series. In our search for an optimal linker-hinge binding moiety system, amide-linked thieno[3,2-d]pyrimidine analogues 32 and 35 (G945), molecules with desirable physicochemical properties, emerged as lead compounds with strong efficacy in a B-Raf(V600E) mutant mouse xenograft model. Synthesis, SAR, lead selection, and evaluation of key compounds in animal studies will be described.