Cynomolgus Monkey (Macaca fascicularis) Cathepsin K: Cloning, Expression, Purification, and Activation

McQueney, Michael S.; Feild, John; Hanning, Charles R.; Brun, Kimberly A.; Kannan, Ravindran; Connor, J.T.H.; Drake, Fred H.; Jones, Christopher S.; Amegadzie, Bernard Y.

doi:10.1006/prep.1998.0965

Cited by 27 publications

(11 citation statements)

References 24 publications

(17 reference statements)

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“…The yields obtained in our system are similar to those seen with other secreted constructs [41–43] (reviewed in [44], but see [45]). We used shaker culture at the 8‐ to 12‐L scale to obtain sufficient quantities of protein, an approach that has several advantages relative to spinner or fermenter systems: relatively low cost, simplicity of construction leading to very low frequency of contamination, and, compared to fermenters, the ability to perform successive infections without interruption for cleaning and sterilization of equipment.…”

Section: Discussionsupporting

confidence: 79%

Large‐scale expression and thermodynamic characterization of a glutamate receptor agonist‐binding domain

Madden

Abele

Andersson

et al. 2000

European Journal of Biochemistry

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The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10-40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal 'X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable.

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Section: Discussionsupporting

confidence: 79%

Large‐scale expression and thermodynamic characterization of a glutamate receptor agonist‐binding domain

Madden

Abele

Andersson

et al. 2000

European Journal of Biochemistry

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“…Extremely potent human cathepsin K inhibitors are significantly less potent against rat cathepsin K. Therefore, to test the effect of these molecules on bone resorption in vivo, a model using a species in which the sequence of the active enzyme is similar to human was required. The amino acid sequence of active cathepsin K in cynomolgus macaques ( Macaca fascicularis ) was cloned, expressed, and shown to be identical to the human form (13) . Furthermore, activity of the human and monkey orthologues is quite similar using small peptide substrates (T. Tomaszek, unpublished observation, 1999).…”

Section: Introductionmentioning

confidence: 99%

Potent and Selective Inhibition of Human Cathepsin K Leads to Inhibition of Bone Resorption In Vivo in a Nonhuman Primate

Stroup

Lark

Veber

et al. 2001

Journal of Bone and Mineral Research

147

111

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“…The oligonucleotide primers 5Ј-TGC-TGG-ATC-CAT-GCC-GGA-GGA-AAC-GCT-G-3Ј (containing a BamHI restriction site, underlined) and 5Ј-CTG-GTC-TAG-ATG-AAT-CAC-ATC-TTG-GGG-3Ј (containing an XbaI restriction site, underlined) were used for amplification of a 1-kb fragment. The PCR product was subcloned into the baculovirus vector, pBlueBac4.5, to create the plasmid pBlueBac-RCK (McQueney et al, 1998). To test the pBlueBac-RCK composite viruses for the expression of rat cathepsin K, Sf9 cells were infected at varying multiplicities of infection with pBlueBac-RCK, and the cell lysate and medium were collected every 24 to 96 h (Mitsudo et al, 2003).…”

Section: Methodsmentioning

confidence: 99%

An Orally Active Cathepsin K Inhibitor, Furan-2-Carboxylic Acid, 1-{1-[4-Fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide (OST-4077), Inhibits Osteoclast Activity in Vitro and Bone Loss in Ovariectomized Rats

Kim

Kim²,

Park³

et al. 2006

J Pharmacol Exp Ther

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Human cathepsin K, a cysteine proteinase of the papain family, has been recognized as a potential drug target for the treatment of osteoporosis. The predominant expression of cathepsin K in osteoclasts has rendered the enzyme into a major target for the development of novel antiresorptive drugs. Now, we report the pharmacological properties of OST-4077 [furan-2-carboxylic acid (1-{1-[4-fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide] as a novel selective cathepsin K inhibitor. Human and rat cathepsin K were inhibited in vitro by OST-4077 with the IC 50 values of 11 and 427 nM, respectively. OST-4077 suppressed bone resorption induced by rabbit osteoclasts (IC 50 , 37 nM) but did not affect bone mineralization or cellular alkaline phosphatase activity in MC3T3-E1 cells. Parathyroid hormone-induced bone resorption was inhibited in a dosedependent manner in thyroparathyroidectomized rats gavaged with a single dose of OST-4077 (ED 50 , 69 mg/kg). When given orally twice daily for 4 weeks to 3-month-old ovariectomized (OVX) rats, OST-4077 dose-dependently prevented bone loss, as monitored by bone densitometry, ash content, and urinary excretion of deoxypyridinoline. No change in serum osteocalcin in the OVX rats by OST-4077 suggested that bone formation might not be affected by the agent. In summary, OST-4077 selectively inhibited bone resorbing activities of osteoclasts and prevented bone loss induced by estrogen deficiency but did not affect bone formation.

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Cynomolgus Monkey (Macaca fascicularis) Cathepsin K: Cloning, Expression, Purification, and Activation

Cited by 27 publications

References 24 publications

Large‐scale expression and thermodynamic characterization of a glutamate receptor agonist‐binding domain

Large‐scale expression and thermodynamic characterization of a glutamate receptor agonist‐binding domain

Potent and Selective Inhibition of Human Cathepsin K Leads to Inhibition of Bone Resorption In Vivo in a Nonhuman Primate

An Orally Active Cathepsin K Inhibitor, Furan-2-Carboxylic Acid, 1-{1-[4-Fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide (OST-4077), Inhibits Osteoclast Activity in Vitro and Bone Loss in Ovariectomized Rats

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