The Development of New Purple Acid Phosphatase Inhibitors: A Light into Anti-osteoporotic Drugs

Abstract Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of phosphorylated substrates under acidic to neutral conditions. Mammalian PAPs are believed to be involved in bone resorption and bone remodelling. Elevated serum concentrations of human PAP are observed in patients suffering from osteoporosis. To date, PAP is the only enzyme with an established direct link to bone resorption. This makes PAP an ideal target for the development of novel therapeutic agents to treat osteoporosis. Two classes of potential inhibitors – acetohydrazide derivatives and α-alkoxy-substituted naphthylmethylphosphonic acid derivatives as shown below – have been identified by the computer docking program Molegro Virtul Docker (MVD, Molegro ApS) as potential high affinity binders to human PAP. The idea for the elongatation of the alkyl chains in both classes of compounds arose due to the presence of a long hydrophobic groove located in proximity to the metal centres in the active sites of mammalian PAPs. Docking studies suggested that the long alkyl chains may exhibit hydrophobic interactions with the residues forming the groove and thus increase the binding affinities of these two classes of compounds. These compounds were synthesised in good yields. Their inhibition potencies (IC50 or Ki values) were determined for pig and red kidney bean (rkb) PAPs. The α-alkoxy-substituted naphthylmethylphosphonic acids were very potent inhibitors against both enzymes, with most compounds within the series exhibiting Ki values in the nanomolar range. The most potent inhibitor within this series against pig PAP was the C18 derivative with a Ki value of 203 nM, and the most potent inhibitor against rkbPAP was the C14 derivative with a Ki value of 186 nM. These are the most potent inhibitors against PAPs to date. Fragment-based screening was also utilised in order to identify possible starting points for the development of alternative PAP inhibitors. A library of five hundred compounds was screened with pig and rkbPAP. The four most potent fragments are shown below and their Ki values against pig and rkbPAP were determined. The most potent fragment against pig PAP exhibited a Ki value of 37 μM, and the most potent fragment against rkbPAP had a Ki value of 32 μM. rkbPAP was extracted and purified for co-crystallisation with the inhibitors. A crystal structure (2.18 Å resolution) of the C18 derivative from the α-alkoxy-substituted naphthylmethylphosphonic acid series bound to the active site of rkbPAP was solved, and the electron density map indicates that while the inhibitor does not directly coordinate to the metal ions it is involved in an extensive network of electrostatic, hydrophobic and hydrogen bonding interactions that lead to its very tight binding. This is a very important result as no crystal structure of a PAP with a medicinally interesting inhibitor bound to the active site was available previously. The structure will thus aim as an ideal guide to further improve both the potency and specificity of future generation PAP inhibitors