Specific Recognition And Enzymatic Inhibition: Chemical And Biochemical Aspects Of Mineralization Mechanisms

Although specific recognition of biologically important substrates by enzymes and other biological macromolecules is well known, the detailed molecular mechanisms involved in these specific interactions are only partially elucidated. My Ph D thesis is focused on distinct topics starting from molecular recognition, especially on chiral molecules interacting specifically with proteins such as bovine serum abumin (BSA). Such property was also observed in the case of alkaline phosphatase, which could have medical applications. One application of the ligand recognition by enzymes is the search for their new inhibitors. Tissue non-specific alkaline phosphatase (TNAP), a marker in mineral formation, is enriched in matrix vesicles (MVs) implicated in the initiation of mineral formation. Molecular recognition was exploited by searching inhibitors that can interact selectively with enzymes implicated in the mineralization process. More precisely, our aim was to find inhibitors which can inhibit pathological mineralization by acting in different ways, for example, by altering the enzyme TNAP activity, or by inhibiting HA formation, or by suppressing the calcium and phosphate fluxes. Such specific inhibitors could serve as therapeutical options for curing osteoarthritis.Firstly, during the molecular recognition research, dansyl-D-phenylalanine, dansyl- D-tryptophan, and dansyl-D-serine were successful synthesized (because L-isomers are commercially available). The specific recognition of three amino acid derivatives by BSA indicated that BSA could interact selectively with stereoisomers. The enantioselective ratios (KL/KD) were determined by fluorescence titration spectrum. The findings confirmed that the complex binding was controlled by both the size and the aromaticity of side groups in the process of chiral recognition.Further more, the amino acids derivetives for investigating the molecular recognition as well as a library of over 130 benzothiophenes were screened for their inhibition effects on alkaline phosphatase. Amone them, dansyl-L-phenylalanine was found to be an inhibitor which can selectively interacte with calf intestinal AP. We found also that benzothiophene derivative tetramisoles are water soluble specific inhibitors of TNAP. Both of them were determined as uncompetitive inhibitors.To search inhibitors such as PPi which can directly inhibit HA formation, and to elucidate the inhibition mechanism of mineralization, a simple biological model which can produce HA as MVs was developed by addition of DMSO (4% v/v) in synthetic cartilage lymph (SCL) medium containing calcium and inorganic phosphate at pH 7.6 and 37°C. Inhibitors of HA formation were screened, providing the evidence that several nucleotides are inhibitors of HA formation and the inhibition mechanism was elucidated.In addition, MVs which initiate calcification in osseous tissues undergoing both physiological and pathological calcifications served to determine the effects of Chinese drugs on TNAP activity and mineralization process. We demonstrated that the anti-rheumatic Chinese medicine sinomenine having no effect on TNAP, sinomenine and theophylline (a TNAP inhibitor), both slowed down the HA formation by interfering probably with Pi or Ca2+ transports. It was concluded that Sinomenine, by delaying HA formation could indirectly boost its anti-inflammatory effect. Although the mineralization models do not address cellular issues, they presented great potential to screen putative drugs to cure osteoarthritis.