title: The consequences of lysosomotropism on the design of selective cathepsin K inhibitors.
abstract: Many drug candidates contain a basic functional group that results in lysosomotropism--the accumulation of drug in the acidic lysosomes of a cell. When evaluating inhibitors of lysosomal enzymes, such as the cathepsins, this physical property can have a dramatic impact on the functional selectivity of the test compounds. A basic P3 substituent in cathepsin K inhibitors provides a means of achieving potent and selective enzyme inhibition. To evaluate the whole-cell selectivity of the basic cathepsin K inhibitor L-006235, we identified the irreversible pan-selective cathepsin probe BIL-DMK and used it to design whole-cell enzyme-occupancy assays. These cell-based assays showed a dramatic reduction in selectivity against cathepsins B, L, and S relative to the selectivities observed in enzyme assays. Two-photon confocal fluorescence microscopy showed punctated subcellular localization of L-006235, which colocalized with BODIPY-labelled Lysotracker, consistent with compound lysosomotropism. To address this potential problem, a series of potent cathepsin K inhibitors was developed by replacing the P2--P3 amide bond with a metabolically stable trifluoroethylamine moiety. X-ray crystallography has identified the binding of this functional group to active-site residues in cathepsin K. This modification resulted in increased potency and selectivity that allowed the removal of the basic P3 substituent. The resulting nonbasic inhibitor L-873724 is a 0.2 nM inhibitor of cathepsin K with cathepsin B, L, and S potencies that were not shifted between purified enzyme and whole-cell assays; thus indicating that this compound is not lysosomotropic. L-873724 exhibits excellent pharmacokinetics and is orally active in a monkey model of osteoporosis at 3 mg kg(-1) q.d.
authors.affiliation: Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Québec, Canada. blackc@merck.com
authors.forename: W Cameron
authors.initials: WC
authors.lastname: Black
authors.forename: M David
authors.initials: MD
authors.lastname: Percival
mesh_terms: D000818:Animals; D001549:Benzamides / Q000737:chemistry / Q000378:metabolism; D001713:Biphenyl Compounds / Q000737:chemistry / Q000378:metabolism; D056657:Cathepsin K; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D015195:Drug Design; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism
chemical_list: D001549:Benzamides; D001713:Biphenyl Compounds; C507712:CRA 013783; D015853:Cysteine Proteinase Inhibitors; C503724:N-((1S)-1-(((1-(2-diazoacetyl)butyl)amino)carbonyl)-3-methylbutyl)-4'-iodo-(1,1'-biphenyl)-4-carboxamide; D010455:Peptides; D013844:Thiazoles; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534130:CTSK protein, human; D056657:Cathepsin K; D007930:Leucine