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The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells. Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were identified as resulting from cleavages after ]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type. [Ser 139 ,Ala-163 ]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.Bone remodeling is a constant process that involves bone resorption and rebuilding (for review, see Ref. 1). The resorption phase of this process is carried out by osteoclasts, which adhere to the surface of bone leading to the creation of an extracellular compartment termed the resorption pit. The resorption pit is maintained at an acidic pH, causing the dissolution of the mineral components of the underlying bone and exposure of the proteinaceous matrix to the action of proteolytic enzymes (2-6). The rebuilding phase of the remodeling process involves the recruitment of osteoblasts to the sites of prior bone resorption, where the layering of a new proteinaceous matrix occurs and becomes mineralized.Cathepsin K, a member of the papain cysteine protease family, has recently been implicated in the resorption of the bone matrix (7-12). The cDNA encoding this protease was cloned from human, rabbit, and mouse osteoclast libraries and expressed in baculovirus-infected insect cells by several independent groups as an inactive secreted proenzyme (7-13). Bossard (14) and Brömme (13) have demonstrated activation of the recombinant proenzyme in vitro by proteolytic degradation of the N-terminal 99-amino acid propeptide; however, different mechanisms leading to its activation were implicated.Activation of procathepsin K in vivo is likely to occur in the low pH environment of the resorption pit, via two possible mechanisms. The propeptide may be cleaved by another protease, such as cathepsin D as suggested by Brömme et al. or by an autocatalytic process, which is more consistent with the data presented by Bossard et al. (14).To elucidate the mechanism of activation of cathepsin K, we constructed a mutant in which the presumed active site Cys at position 139 was changed to Ser. The kinetics of activation of mutant and wild-type cathepsin K were studied in vitro.In this report we provide the following evidence for an autocatalytic activation mechanism. First, in vitro self-activation of wild-type procathepsin K occurs spontaneously at 4°C, pH 4 and is catalyzed by mature cathepsin K. Second, unlike wildtype enzyme, t...

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Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have been reported to be efficacious but in many cases present hypoglycaemia risk due to activation of the enzyme at low glucose levels in the pancreas, leading to inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic control with reduced hypoglycemia risk. Herein, we report structure-activity studies on a carboxylic acid containing series of glucokinase activators with preferential activity in hepatocytes versus pancreatic β-cells. These activators were designed to have low passive permeability thereby minimizing distribution into extrahepatic tissues; concurrently, they were also optimized as substrates for active liver uptake via members of the organic anion transporting polypeptide (OATP) family. These studies lead to the identification of 19 as a potent glucokinase activator with a greater than 50-fold liver-to-pancreas ratio of tissue distribution in rodent and non-rodent species. In preclinical diabetic animals, 19 was found to robustly lower fasting and postprandial glucose with no hypoglycemia, leading to its selection as a clinical development candidate for treating type 2 diabetes.

Inhibitors of the Hedgehog signaling pathway have generated a great deal of interest in the oncology area due to the mounting evidence of their potential to provide promising therapeutic options for patients. Herein, we describe the discovery strategy to overcome the issues inherent in lead structure 1 that resulted in the identification of 26), which has been advanced to human clinical studies.

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Glucokinase is a key regulator of glucose homeostasis and small molecule activators of this enzyme represent a promising opportunity for the treatment of Type 2 diabetes. Several glucokinase activators have advanced to clinical studies and demonstrated promising efficacy; however, many of these early candidates also revealed hypoglycemia as a key risk. In an effort to mitigate this hypoglycemia risk while maintaining the promising efficacy of this mechanism, we have investigated a series of substituted 2-methylbenzofurans as ''partial activators'' of the glucokinase enzyme leading to the identification of N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)-carbamoyl)benzofuran-4-yloxy)pyrimidine-2carboxamide as an early development candidate.