Small Molecules Regulate Bone Resorption And Enzyme Activity In Osseous Cells

The first one is centered on the effects of azadipeptide nitrile cathepsin Kinhibitors (CKIs) on bone formation mediated by osteoblasts and on bone resorptioncontrolled by osteoclasts. Our laboratory synthetized the cathepsin K inhibitors as ithas been reported in the experimental part. However the organic synthesis has beenpublished, therefore I did not include the organic synthesis of CKIs in the Resultsection. The second part is focused on the development of an alkaline phosphataseactivity assay in matrix vesicles by infrared spectroscopy. The third part is theextension of the infrared assay of the kinetics of phosphate-release from naturalsubstrates in living cells.1. Effects of Cat K inhibitors on mineralization and bone resorptionIn order to provide a proof of concept of the potential use of our CKIs in drugtreatments for osteoporosis, as well as to consolidate the efficiency/biologicalevaluation of these compounds, prior to preclinical trials, we compared the effects oftwo inhibitors, CKI-8and CKI-13with those of the commercial inhibitor E64oncell viability, osteoblast-induced mineralization and osteoclast-induced boneresorption. The objective is to determine whether CKIs (CKI-8and CKI-13) canaffect mineralization using osteoblast cells and to evaluate their bone resorptionability on osteoclasts. Cell viability assay is performed on osteoblasts and onosteoclasts to check the toxicity of CKIs before mineralization and resorptiontreatment (CKI-8and CKI-13showed no toxicity up to1000nM on osteoblast-likeSaos-2cells and on osteoclast RAW264.7cells. However, they were toxic whentheir concentrations exceeded100nM in primary osteoclasts. This suggested that the maximum concentration in cells without producing any cytoxicity was around100nM for the candidate molecules). The effect of CKIs on mineralization is evaluatedon osteoblasts (human osteoblast-like Saos-2cells and primary osteoblast frommurine calvaria) which underwent the entire osteoblastic differentiation programfrom proliferation to mineralization. Mineralization is monitored by detectingcalcium nodules with Alizarin Red-S and by measuring TNAP activity (CKI-13slowed down significantly the mineralization process induced by the Saos-2cell line,as well as inhibiting slightly the TNAP activity.). Inhibition of Cat K inosteoclast-derived intracellular medium is evaluated by a gelatin zymographymethod (The IC50values determined by zymography: CKI-8,51±20nM andCKI-13,29±11nM), which could be used as a screening test of inhibitors beforechecking their effects on bone resorption. Bone resorption assay was assayed on thesurface of an inorganic crystal coating surface (Corning Bone assay surface) and onbovine bone slices (Bone resorption on bovine slices decreased significantly at10nM CKI-13, while100nM CKI-8and100nM of the commercial inhibitor E64were necessary to significantly decrease bone resorption.).2. Development of a new Infrared assay to monitor pyrophosphate hydrolysisby Matrix VesiclesThe aim of our work was to develop an assay of the enzymatic activity in thehydrolysis TNAP as PPia natural substrate for MVs activity assay. We demonstratethe feasibility of the infrared assay by measuring the TNAP activity in MVs that wasaround1.4μmol min-1mg-1, while we observed a significant inhibition of PPihydrolysis in the presence of5mM levamisole (a TNAP inhibitor) from343±112nmol min-1mg-1confirming that the pyrophosphatase activity originated mostlyfrom TNAP. The main advantages of the IR assay lie in the possibility to determinedirectly in situ hydrolytic activity of TNAP and to obtain IC50of inhibitors usingnatural substrates in an almost continuous manner at physiological pH. In thisrespect, IR is insensitive to light scattering effects caused by the turbidity of thesamples and relatively large concentrations of protein can be employed. However, for the IR assay, this technique presents some limitations related to the accuracy ofdetermination of the base line but also in the thickness of the spacer. We estimatedthat the sensitivity based on a30-min assay is around0.2mg cell proteins mL-1in a5-10μL sample containing50mM PPi. Lower protein concentration could be usedwith longer incubation times. The other limitation concerns the minimal amount ofPior PPiconcentrations which should be at least around1mM to be reliabilydetected.3. An infrared assay of the kinetics of phosphate-release from physiologicalsubstrates in living cellsMVs are released from chondrocyte as well as from osteoblasts. Havingdemonstrating that the infrared assay can monitor phosphatase activity in MVs.Here,we developed a continuous infrared (IR) assay to determine phosphatase activity inSaos-2cells and in primary osteoblasts from exogenous substrates such as PPi, AMP,ADP, ATP, UTP, β-glycerophosphate or α-D-glucose-1-phosphate andpara-nitrophenylphosphate (p-NPP). We obtained an apparent specific phosphataseactivity in Saos-2cells of around116±113nmol min-1mg-1. Quantitativedeterminations were also obtained for AMP, G-1-P, β-GP, p-NPP using both Pibands at990and1070-1080cm-1. The IR assay can be employed as a cost-effective label-freeapproach to determine the phosphatase activity from extracellular physiologicalsubstrates in whole cells. It may serve to screen the phosphatase inhibitors inTNAP-enriched cells. At the concentrations of5mM, levamisole almost completelyinhibited the PPihydrolysis by Saos-2cells (IC50=1.16±0.03mM).