Novel, nonpeptidic cyanamides as potent and reversible inhibitors of human cathepsins K and L.
Compounds containing a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compound 1 inhibits cathepsins K and L with IC(50) values of 0. 37 and 0.45 M, respectively. Modification of compound 1 by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compound 2 was found to be a potent inhibitor of cathepsins K and L with a K(i) value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compound 2 by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chemical reactivity of the compound toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to determine the association and dissociation rate constants with human cathepsin K. The kinetic data obtained showed that the increase of potency observed between different 1-cyanopyrrolidinyl inhibitors is due to an increase of k(on) values and that the association of the compound with the enzyme fits an apparent one-step mechanism. (13)C NMR experiments performed with the enzyme papain showed that compound 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic analysis, the addition of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as observed by (13)C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compound 2 inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC(50) value of 0.7 M. 1-Cyanopyrrolidine represents a new class of nonpeptidic compounds that inhibit cathepsin K and L activity and proteolysis of bone collagen.
Falgueyret
J P
JP
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Highway, Kirkland, Quebec H9H 3L1, Canada.
Oballa
R M
RM
Okamoto
O
O
Wesolowski
G
G
Aubin
Y
Y
Rydzewski
R M
RM
Prasit
P
P
Riendeau
D
D
Rodan
S B
SB
Percival
M D
MD
D000818:Animals; D001862:Bone Resorption / Q000473:pathology; D001842:Bone and Bones / Q000187:drug effects / Q000378:metabolism; D020134:Catalytic Domain; D056657:Cathepsin K; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002417:Cattle; D003094:Collagen / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D010450:Endopeptidases; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009570:Nitriles / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism; D011759:Pyrrolidines / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D000096926:Benzenesulfonamides
D056657:Cathepsin K; D056668:Cathepsin L; D002403:Cathepsins; D003094:Collagen; D003545:Cysteine; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D010450:Endopeptidases; D005978:Glutathione; D009570:Nitriles; D011759:Pyrrolidines; D013449:Sulfonamides; C419405:N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide; D000096926:Benzenesulfonamides; C534281:CTSL protein, human; C534280:Ctsl protein, rat; C534130:CTSK protein, human; C534133:Ctsk protein, rat