Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

Abstract

Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K i values in the 30-60 lM range. Ligand efficiency values are 0.39-0.44 kcal ⁄ mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.Key words: X-ray, crystallography, drug design, fragment screening, purple acid phosphatase, osteoporosis Purple acid phosphatases (PAP) are metalloenzymes that hydrolyse phosphate esters and anhydrides, generally at low pH values (1). The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III) (2,3). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II ⁄ III) in mammals (4,5) or a Zn(II) or Mn(II) in plants (6,7). Mammalian PAP is a 35 kDa monomeric protein also known as tartrate-resistant acid phosphatase (TRAP or TRAcP). In contrast, plant PAP is a 110 kDa homodimer, with each subunit consisting of two domains, an N-terminal one whose function is unknown and a catalytic C-terminal domain that strongly resembles the overall structure of the mammalian enzyme (1). Crystal structures of human (8), pig (9), rat (10,11) and plant PAPs (12,13) have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.A number of biological roles for PAP have been proposed. These include (i) the transport of iron from the mother to the developing foetus during gestation (14); (ii) bone resorption in osteoclasts (evidence for this activity is derived from the effect of PAP on osteoclasts that were cultured on cortical bone slices (15) and from t...

Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-containing Active Site

“…The closest overall structural match was to red kidney bean PAP (PDB 4DHL) (25) with a DALI Z -score of 28.6 and a main chain root mean square deviation relative to PhoD of 2.47 Å. The similarity between the two protein families is illustrated in Fig.…”

The Binding Mode of an ADP Analogue to a Metallohydrolase Mimics the Likely Transition State

“…The proposed oxyphosphorane transition state for the reaction with ADP is also shown. [26] The structures weres olved by differenceF ourier calculations by using the coordinates from ap reviously solved rkbPAP structure (PDB ID:4 DHL [27] )a ss tarting model. [2,5] [a] D. Feder sequently,c rystals were soaked in as olution containing 0.1 m sodium citrate (pH 5.0), 0.1 m lithium chloride, 25 %p oly(ethylene glycol) 3350, 20 %i sopropyl alcohol, 10 %g lycerol, 5mm sodium vanadate, and 5mm adenosine.…”

“…Vanadate is known to form stable, five-coordinate, trigonal bipyramidal complexes, thus mimicking the oxyphosphorane species (Figure 1). [23,27] The focus of this study is on subunit A, the subunit with the highestquality electron density,t hough the structural features in each of the other three subunits, as far as observable, are similar ( Figure S1 in the Supporting Information). X-ray diffraction data were collected at the Australian Syn-chrotronM X1 beam-line and weres caled andm erged by using XDS.…”

The Inhibitory Effect of Metals and Other Ions on Acid Phosphatase Activity FromVigna aconitifoliaSeeds

“…[17] Few investigations have been carried out regarding inhibitors of acid phosphatase that seem to be promising in controlling bone resorption. [18,19] Our present attempt explores the effect of various metals and other ions on catalytic activity of acid phosphates isolated from Vigna aconitifolia seeds. Also, the mode of inhibition by these effectors was analyzed.…”