Drug discovery using rational structure based design approaches: The purple acid phosphatases

Abstract

Purple acid phosphatases (PAPs) are metalloenzymes that catalyse the hydrolysis of phosphorylated esters and anhydrides under acidic conditions. The active site of PAPs contains a catalytically essential FeIIIFeII metal centre in mammals and a FeIIIZnII or FeIIIMnII centre in plants. In humans, elevated PAP levels in serum strongly correlate with the progression of osteoporosis and metabolic bone malignancies, as well as other diseases that are associated with elevated levels of this enzyme. Consequently, PAP has emerged as a target suitable for the development of chemotherapeutics to combat bone ailments including osteoporosis. The most compelling evidence for the involvement of PAP in osteoporosis comes from transgenic studies in mice, which have demonstrated a direct association between the level of expression of PAP and the progression of this debilitating disease. Thus, PAP has emerged as a promising target for the development of novel therapeutic agents to treat osteoporosis (Chapter 1).nIn Chapter 2, the identification of three novel PAP inhibitors (CC24201, CC27209 and MO07123, shown below) from the MaybridgeTM library by fragment-based screening is described. The crystal structures of these fragments in complex with red kidney bean PAP were solved and the binding modes of the fragments to human PAP were predicted using computational docking simulations. These structures represent the first examples of a PAP with drug-like molecules bound to the active site. The structures of the fragments were then employed as a scaffold for rational structure-based design of new derivatives (1a-f, 4a-f below, synthesised by Dr. Waleed Hussein), resulting in the diversification of this library; while no significant improvements in inhibitory potency have been achieved, these fragments do nonetheless provide promising starting points for the the development of novel drug leads. The strongest inhibitor from this series- 4f was soaked into rkbPAP crystals. This crystal structure shows the first example of a rationally designed inhibitor bound to a PAP.nIn a parallel study (Chapter 3), a-alkoxy-substituted naphthylmethylphosphonic acid derivatives were designed computationally based on a pre-existing scaffold (shown below). My docking simulations predicted that these molecules would bind tightly to the active site of human PAP. The rational for the elongation of the alkyl chains in these compounds arose due to the presence of a long hydrophobic groove located under the repression loop in the active sites of mammalian PAPs. The inhibitors were synthesised by Dr. Meng Wei Kan and the most potent inhibitor from these (4) has a Ki of ~200 nM against pig PAP. Crystal structures of (R)-1 and- (S)-4 in complex with red kidney bean PAP have been solved and show that the dodecyl derivative is a transition state analogue and the octadecyl derivative mimics the binding mode of an incoming substrate. Furthermore, the length of the alkyl chain appears to influence the ability of the phosphonate moiety to interact directly with the bimetallic centre in the active site. The crystal structure of red kidney bean PAP in complex with (S)-4 represents the first structure of a PAP in complex with a potent, medicinally relevant inhibitor.nI also crystallised rkbPAP in the presence of vanadate and either adenosine or ADP to create a transition state (ADV, Chapter 4) or substrate (ADPV, Work in progress 8) analogue respectively. Importantly, one of the vanadate moieties in ADV coordinates to the active site of rkbPAP in a tripodal, trigonal bipyramidal configuration, thus mimicking the anticipated oxyphosphorane transition state of a metallophosphatase (shown below). In addition to providing unprecedented insight into the precise catalytic mechanism of PAPs in general, this structure also represents the first illustration of a metallohydrolase-substrate complex captured in the transition state. Employing the rkbPAP-ADPV structure, I then performed a comprehensive computational analysis of PAPs from different, agriculturally relevant crops and found that plant PAPs fall into three major categories based on their substrate preference: i.e. ATP and ADPases, phosphoenol pyruvatases (PEPases), and phytases. These results may guide future bioengineering studies aiming to incorporate the genes of plant PAPs into the genomes of common crops in order to enhance phosphate uptake from the soil and thus reduce or eliminate the need for fertilisers.n