Structure-based design of cathepsin K inhibitors containing a benzyloxy-substituted benzoyl peptidomimetic.

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.

Autocatalytic activation of human cathepsin K.

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 Glu19, Ser98, and Glu110. The mature enzyme was composed of mixture of enzymes with N termini of Gly113, Arg114, and Ala115 with varying ratios depending on the preparation. Molecular weight determinations were consistent with the absence of carbohydrate in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide linkage, and that the protein had Met329 as the C-terminal residue. A mutant was constructed in which the active site Cys139 was changed to Ser. [Ser139,Ala163]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. [Ser139,Ala163]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.

Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification.

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F.H., Dodds, R., James I., Connor J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M. (1996) J. Biol., Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not for by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (kobs/[I] = 273,000 m(-1).s(-1)). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies.

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.

Solubility properties of neuronal tubulin: evidence for labile and stable microtubules.

The solubility properties of tubulin and microtubules in pure cultures of sympathetic neurons were examined by electron microscopic and biochemical techniques. For morphological analyses, neurons were extracted with Triton X-100 in the presence or absence of 1 mM CaCl2, and the resulting detergent-extracted residues were examined for microtubules. In parallel experiments, the solubility of tubulin was determined under various solution conditions. Detergent-extracted residues of neurons prepared without Ca2+ contained many microtubules. Neurite residues prepared in the presence of Ca2+ also contained microtubules, but at substantially lower numbers than in residues prepared without Ca2+. The biochemical data parallel the morphological observations. Following detergent-extraction under microtubule stabilizing conditions, 30% of the tubulin was detergent-soluble (i.e. unpolymerized), while 70% was detergent-insoluble (i.e. polymerized). A more detailed examination of the solubility properties of tubulin indicated that 62% was detergent-insoluble but soluble in buffers containing mM CaCl2, while 5-8% was detergent and Ca2+-insoluble. A variety of control experiments indicated that non-specific adsorption of tubulin onto detergent-insoluble components of the cultures, assembly of tubulin onto pre-existing microtubules, and incomplete extraction of tubulin from cells contributed minimally to the levels of Ca2+-soluble and insoluble tubulin obtained with the extraction conditions used. These results indicate that (a) the majority of neuronal tubulin is assembled into microtubules which disassemble upon treatment with Ca2+ and (b) a portion of the neuronal tubulin is assembled into microtubules which show the unusual property of Ca2+-stability.

Post-translational modification of tubulin in cultured neurons.

Isoelectricfocusing x SDS polyacrylamide gel electrophoresis of cultured sympathetic neurons revealed two alpha- and two beta-subunits of tubulin. In pulse-chase experiments, the spot morphology of the beta-subunits synthesized during a 10 min pulse did not change appreciably during the chase period. However, the appearance of the labeled alpha-subunits changed during the chase period. With chase periods of less than 4 h, the labeled alpha-subunits focused as discrete spots, while after chase periods of greater than 6 h, they focused as short streaks.

Microtubule-associated proteins of neurons.

Microtubule-associated proteins (MAP) have been identified in cultures of rat sympathetic neurons. In all of the experiments performed here, the cultures consisted of greater than 97% neurons. 26 proteins were identified in these neuronal cultures that (a) remained associated with cytoskeletons prepared with a Triton X-100-containing microtubule-stabilizing buffer, (b) were released from such cytoskeletons by incubation in microtubule-depolymerizing buffers, (c) were not detected in cytoskeletons prepared from cultures depleted of microtubules by treatment with podophyllotoxin, and (d) co-cycled with rat brain microtubule proteins. We conclude that these 26 proteins are associated with microtubules in sympathetic neurons. Two of these proteins have molecular weights of approximately 30,000 and isoelectric points of approximately 6.2; the rest of the proteins range in molecular weight from 60,000 to 76,000 and isoelectric point from 6.3 to 6.9. This latter group of MAPs was heat labile. Several other proteins in the neuronal cultures had the solubility properties and drug-lability expected of MAP. All of these proteins had apparent molecular weights greater than 200,000; one of these putative MAP co-migrated with rat brain MAP-1. We did not detect any putative MAP in these cultures that co-migrated with rat brain MAP-2. In isoelectric focusing-SDS PAGE, the 24 MAP with molecular weights of 60,000-76,000 appeared to comprise four distinct molecular weight classes. Each molecular weight class was in turn composed of several proteins that varied in isoelectric point. In peptide mapping experiments, the isoelectric variants of each molecular weight class gave rise to very similar peptide maps. These observations suggest that each molecular weight class consists of several closely related proteins. It was also determined that all except the most basic member of the four MAP classes could be phosphorylated in vivo, raising the possibility that differential phosphorylation contributed to the variation in the isoelectric points of the members of each MAP class. We performed pulse-chase experiments to further evaluate the contribution of posttranslational modification to the generation of the complex population of MAP in the molecular weight range of 60,000 to 76,000. In cultures labeled for 20 min, only the more basic members of each MAP class were detectably labeled, while in cultures labeled for 20 min and then chased for 220 min the more acidic members of the MAP classes became labeled.(ABSTRACT TRUNCATED AT 400 WORDS)