An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells

Falgueyret, Jean‐Pierre; Black, W. Cameron; Cromlish, Wanda; Desmarais, Sylvie; Lamontagne, Sonia; Mellon, Christophe; Riendeau, Denis; Rodan, Sevgi B.; Tawa, Paul; Wesolowski, Gregg; Bass, Kathryn E.; Shankar, Viswanathan; Percival, M. David

doi:10.1016/j.ab.2004.09.005

Cited by 51 publications

(45 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Next, we compared the occupancy of cathepsin C active sites present in EcoM-G with the percentage of NE inhibition, over a range of concentrations of cathepsin C inhibitors. Active site probes such as Ala-4-125 I-Phe-DMK have been used to detect cysteine, serine, and proteosome proteases in complex tissue extracts or living cells (Liu et al, 1999;Patricelli et al, 2001;Falgueyret et al, 2004; reviewed by Fonović and Bogyo, 2007). As with cell-surface receptor occupancy studies, a tracer amount of probe should be used to minimize the number of sites occupied by the probe itself and prevent competition with the reversible inhibitor.…”

Section: Resultsmentioning

confidence: 99%

In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

Méthot

Guay²,

Rubin³

et al. 2008

Molecular Pharmacology

Self Cite

View full text Add to dashboard Cite

Inhibition of cathepsin C, a dipeptidyl peptidase that activates many serine proteases, represents an attractive therapeutic strategy for inflammatory diseases with a high neutrophil burden. We recently showed the feasibility of blocking the activation of neutrophil elastase, cathepsin G, and proteinase-3 with a single cathepsin C selective inhibitor in cultured cells. Here we measured the fractional inhibition of cathepsin C that is required for blockade of downstream serine protease processing, in cell-based assays and in vivo. Using a radiolabeled active site probe and U937 cells, a 50% reduction of cathepsin G processing required ϳ50% of cathepsin C active sites to be occupied by an inhibitor. In EcoM-G cells, inhibition of 50% of neutrophil elastase activity required ϳ80% occupancy. Both of these serine proteases were fully inhibited at full cathepsin C active site occupancy, whereas granzyme B processing in TALL-104 cells was partially inhibited, despite complete occupancy. In vivo, leukocytes from cathepsin C ϩ/Ϫ mice exhibited comparable levels of neutrophil elastase activity to wild-type animals, even though their cathepsin C activity was reduced by half. The long-term administration of a cathepsin C inhibitor to rats, at doses that resulted in the nearly complete blockade of cathepsin C active sites in bone marrow, caused significant reductions of neutrophil elastase, cathepsin G and proteinase-3 activities. Our results demonstrate that the inhibition of cathepsin C leads to a decrease of activity of multiple serine proteases involved in inflammation but also suggest that high fractional inhibition is necessary to reach therapeutically significant effects.

show abstract

Section: Resultsmentioning

confidence: 99%

In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

Méthot

Guay²,

Rubin³

et al. 2008

Molecular Pharmacology

Self Cite

View full text Add to dashboard Cite

show abstract

“…The protease substrates were as follows: Z-Arg-Arg-AMC (Calbiochem, San Diego, CA), Z-Leu-Arg-AMC (Novabiochem, Laufelfingen, Switzerland), Ac-Glu-Asp(EDANS)-Lys-Pro-Ile-Leu-Phe-Phe-ArgLeu-Gly-Lys(DABCYL)-Glu-NH 2 (Bachem), N-methyl umbelliferyl-N-acetyl-␤-D-glucosamidine (Sigma-Aldrich), p-nitrophenyl phosphate (Sigma-Aldrich), and p-iodonitrotetrazolium violet (Sigma-Aldrich). Cathepsin inhibitors and the activity based probe 125 I-BIL-DMK were prepared by the Medicinal Chemistry Department at Merck Frosst Canada (Falgueyret et al, 2004). Protease inhibitors E-64 and Ca-074 were from Sigma-Aldrich and Bachem (Torrance, CA), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…These basic compounds concentrate in acidic subcellular organelles of isolated cells and in rat tissues with high lysosome content, such as the lung, liver, kidney, and spleen. Members of this class of Cat K inhibitors show significantly increased potencies in cell-based assays against both Cat K and the off-target family members Cat B, L, and S, compared with potencies against isolated enzymes (Falgueyret et al, 2004(Falgueyret et al, , 2005Black and Percival, 2006). In contrast, nonbasic ␣-aminoacetonitrile Cat K inhibitors do not accumulate in lysosomes, and their potencies are generally similar, or weaker, in whole cells compared with purified enzyme assays.…”

mentioning

confidence: 99%

Effect of Cathepsin K Inhibitor Basicity on in Vivo Off-Target Activities

Desmarais

Black²,

Oballa³

et al. 2007

Mol Pharmacol

Self Cite

View full text Add to dashboard Cite

Cathepsin K is a lysosomal cysteine protease that is a pharmacological target for the treatment of osteoporosis. Previous studies showed that basic, lipophilic cathepsin K inhibitors are lysosomotropic and have greater activities in cell-based assays against cathepsin K, as well as the physiologically important lysosomal cysteine cathepsins B, L, and S, than expected based on their potencies against these isolated enzymes. Long-term administration of the basic cathepsin K inhibitors N- (1-(((cyanomethyl) -006235) and balicatib to rats at a supratherapeutic dose of 500 mg/kg/day for 4 weeks resulted in increased tissue protein levels of cathepsin B and L but had no effect on cathepsin B and L message. This is attributed to the inhibitor engagement of these off-target enzymes and their stabilization to proteolytic degradation. No such increase in these tissue cathepsins was detected at the same dose of, a potent nonbasic cathepsin K inhibitor with a similar off-target profile, although all three inhibitors provided similar plasma exposures. Using an activity-based probe, 125 I-BIL-DMK, in vivo inhibition of cathepsins B, L, and S was detected in tissues of mice given a single oral dose of L-006235 and balicatib, but not in mice given L-873724. In each case, similar tissue levels were achieved by all three compounds, thereby demonstrating the in vivo cathepsin selectivity of L-873724. In conclusion, basic cathepsin K inhibitors demonstrate increased off-target cysteine cathepsin activities than their nonbasic analogs and potentially have a greater risk of adverse effects associated with inhibition of these cathepsins.The CA1 family of human papain-like cysteine proteases comprises 11 members. These enzymes are collectively known as cathepsins, a name which is derived from the Greek kathepsein, to digest. Being largely lysosomal enzymes, cathepsins have acidic pH activity and stability optima. These enzymes are synthesized as preproenzymes, the mature proteins sharing between 25 and 80% sequence identity (Lecaille et al., 2002;Turk et al., 2003). Cathepsin K (Cat K) is highly and somewhat specifically expressed in osteoclasts, the multinucleated giant cells of hematopoietic origin that are responsible for the normal physiological process of bone resorption. Cat K destroys the organic fraction of bone through its potent collagenase activity, this process taking place in the acidic pit between the osteoclast and the bone surface and also intracellularly within lysosomes of the osteoclast (Saftig et al., 1998). A large volume of genetic and pharmacological data points to a pivotal role for Cat K in bone resorption, and Cat K inhibitors are presently being evaluated in clinical trials as a treatment of osteoporosis, a disease characterized by an imbalance of bone resorption over bone formation (performed by osteoblasts) (Deaton and Tavares, 2005;Grabowskal et al., 2005;Yasuda et al., 2005;Boyce et al., 2006;Close et al., 2006). Both physiological and pathological roles have been identified for the remaining 10 ...

show abstract

“…Assays for human cathepsins B, F, K, L, S, and V and mouse cathepsins B and S were performed as described previously (30,33,34).…”

Section: Figmentioning

confidence: 99%

Reversible Cysteine Protease Inhibitors Show Promise for a Chagas Disease Cure

Ndao

Beaulieu

Black

et al. 2014

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile "warhead" were prepared and demonstrated 50% inhibitory concentrations (IC 50 s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 M IC 50 range; however, trypomastigote production from the amastigote form was ϳ90 to 95% inhibited at 2 M. Two key compounds, Cz007 and Cz008, with IC 50 s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg.

show abstract

An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells

Cited by 51 publications

References 24 publications

In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

Effect of Cathepsin K Inhibitor Basicity on in Vivo Off-Target Activities

Reversible Cysteine Protease Inhibitors Show Promise for a Chagas Disease Cure

Contact Info

Product

Resources

About