Early studies of cyclotides [7][8][9][13][14][15] were based on discovery and characterization efforts at the peptide level. The general approach for these studies involved either bioassay-guided or mass-guided fractionation of plant extracts, followed by peptide isolation and purification, amino acid analysis, Edman sequencing and chemical, NMR 9 or mass spectrometry-based 10 approaches to deduce disulfide connectivity. The first step in many of the early discovery programs was a pre-screening procedure to determine if a given plant tissue might contain cyclotides (Figure 3). Various protocols 13,15,57,69,116,123 have been reported for quickly screening if a plant might be cyclotide-positive, with the simplest based on extraction (using dichloromethane/methanol, aqueous acetonitrile, or aqueous buffer) and purification on a C18 reverse phase column followed by a main-screen. The latter typically involves (A) examination of a reversed phase HPLC-MS profile for late-eluting peaks that are (B) in the mass range 2800-4000 Da and (C) contain six Cys residues as judged by a mass shift of 348 Da after reduction and S-alkylation, as shown in Figure 3. 116 The rationale for the target HPLC elution range was that all of the cyclotides discovered in early studies were somewhat hydrophobic and eluted relatively late on HPLC. Similarly, many of the initial cyclotides were in the mass range 2500-4000 Da and it was assumed that new cyclotides would fall within a similar range. Finally, by definition, a cyclotide must contain six Cys residues so the mass shift test in step C of the screen provided a simple way of testing for that criterion. An example of the 348 Da mass shift after reduction and alkylation is illustrated for the identification of the cyclotide Cter A from Clitoria ternatea (butterfly pea) seeds in Figure 3. Chassalia chartacea chassatides 235 Chassalia curviflora chacur c Chassalia discolor CD-1 c Chassalia parvifolia circulins 7,22,406 Hedyotis biflora hedyotides 207,213,280,350 H. centranthoides hcf-1 c H. terminalis htf-1 c Oldenlandia affinis kalata peptides 1-5,9 Palicourea condensata palicoureins 25 P. rigida parigidin-br1 236,329,345 P. tetragona paltet 1 c Psychotria brachiata psybra 1 c Psychotria brachyceras Psyleio and psybry peptides c Psychotria leiocarpa Psyleio peptides 338 Psychotria leptothyrsa psyle peptides 157,158 Psychotria longipes cyclopsychotride A 8 Psychotria poeppigiana psypoe 1 Psychotria solitudinum psysol 2 305 Psychotria suterella PS-1 c Cucurbitaceae Momordica clarkeana TI peptides 315 These trypsin inhibitor cyclotides are also referred to as cyclic knottins; typically, these peptides are found only in seeds. M. cochinchinensis MCoTI peptides 24 M. denticulate TI peptides 315 M. diocia Modi peptides 424 M. gilgiana TI peptides 315 M. macrophylla TI peptides 315 M. subangulata TI peptides 315 Fabaceae Clitoria ternatea Cter peptides; 204 cliotides 206 These cyclotides are biosynthetically processed from 'hijacked' genes (i.e. from albumin genes in which an ancestral albumin subun...

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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...

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