Inhibition of eukaryote protein kinases and of a cyclic nucleotide-binding phosphatase by prenylated xanthones

Chemico-Biological Interactions 114 (1998) 121 – 140

Inhibition of eukaryote protein kinases and of a cyclic nucleotide-binding phosphatase by prenylated xanthones Zhe Xiong Lu a, Mery Hasmeda a, Wilawan Mahabusarakam b, Bela Ternai c, Prapaipit Chamsuksai Ternai c, Gideon M. Polya a,* b

a School of Biochemistry, La Trobe Uni6ersity, Bundoora, Victoria 3083, Australia Chemistry Department, Prince of Songkhla Uni6ersity, Hat Yai, Songkhla, Thailand 90110, Thailand c Chemistry Department, Chulalongkorn Uni6ersity, Patumwan, Bangkok, Thailand 10330, Thailand

Received 5 December 1997; received in revised form 28 April 1998; accepted 4 May 1998

Abstract A series of prenylated xanthones are variously potent inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA), rat brain Ca2 + and phospholipid-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK), wheat embryo Ca2 + -dependent protein kinase (CDPK) and potato tuber cyclic nucleotidebinding phosphatase (Pase). The prenylated xanthones examined are mostly derivatives of a-mangostin in which the 3-hydroxyl and 6-hydroxyl are variously substituted with groups R or R%, respectively, or derivatives of 3-isomangostin (mangostanol) in which the 9-hydroxyl is substituted with groups R% or the prenyl side chain is modified. The most potent inhibitors of cAK have non-protonatable and relatively small R% and R groups. Conversely, the most potent inhibitors of PKC and MLCK have bulkier and basic R% groups. Some prenylated xanthones are also potent inhibitors of CDPK. PKC and cAK are competitively inhibited by particular prenylated xanthones whereas the compounds that are the most potent inhibitors of MLCK and CDPK are non-competitive inhibitors. Prenylated xanthones having relatively small and non-protonatable R% and R groups inhibit a high-affinity cyclic nucleotide binding Pase in a non-competitive fashion. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Protein kinases; Prenylated xanthones; Phosphatase

* Corresponding author. Tel.: +61 3 94792157; fax: + 61 3 94792467. 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(98)00049-0

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1. Introduction A wide variety of secondary metabolites are elaborated by plants for defensive purposes [1] and such bioactive compounds are the basis of herbal medicinal efficacy [2,3]. However the molecular mechanisms of action of most isolated plant secondary metabolites have yet to be determined. In recent years it has become clear that representatives of a number classes of plant defensive secondary metabolites are potent inhibitors of eukaryote serine/threonine-specific protein kinases and in particular of the catalytic subunit (cAK) of cyclic AMP-dependent protein kinase (PKA). Such inhibitors include particular flavones [4–6], isoflavones [7], coumarins [7], anthraquinones [8], xanthones [9], condensed tannins [10,11], hydrolysable tannins [12], amphiphilic triterpenoids [13], isoquinoline and oxazine alkaloids [14,15], carotenoids [13], sesquiterpenoids [5], aromatic organic acid derivatives [16] and stilbenes [17]. While cAK is inhibited by representatives of all these classes of plant defensive compounds, other protein kinases inhibited by particular plant defensive compounds include Ca2 + - and phospholipid-dependent protein kinase C (PKC) [6,8], calmodulin (CaM)-dependent myosin light chain kinase (MLCK) [4,18], and protein tyrosine kinases [19]. PKA is apparently absent from higher plants [20,21], but second messenger-mediated signalling can occur in plants via Ca2 + -calmodulin- and Ca2 + -dependent protein kinases (CDPKs) [22]. It is notable that in many instances plant defensive PKA inhibitors are inactive or relatively ineffective as inhibitors of plant CDPK [4,7,12 – 14,16]. Since PKA is involved in mediating hunger responses in eukaryotes that consume plants, this pattern is consistent with specific inhibition of animal and fungal PKA-mediated signalling as a significant mechanism of plant defence [7]. However some plant PKA inhibitors do inhibit CDPK, including some condensed and hydrolysable tannins [10,12], flavonoids [4] and xanthones [9]. Naturally-occurring and synthetic compounds that inhibit CDPK are of pharmacological interest because this type of protein kinase is present in the malaria-causing Plasmodium falciparum as well as in plants while being apparently absent from all other eukaryotes [23]. The xanthones a-mangostin and g-mangostin are major bioactive compounds from the hull of the fruit of the mangosteen tree Garcinia mangostana [9,24–26]. Preparations of the hull are used in traditional medicine in South East Asia for anti-inflammatory, anti-diarrhoea, anti-ulcer and antiseptic purposes [2,24]. a-Mangostin and g-mangostin variously have anti-inflammatory, antimicrobial, amoebocidal and anti-ulcer activity [2,24,25]. g-Mangostin is a potent inhibitor of animal cAK and of plant CDPK (IC50 values 2 and 6 mM, respectively) [9]. a-Mangostin is a less effective inhibitor of cAK and CDPK (IC50 values 13 and 21 mM, respectively) [9], but is a potent inhibitor of rabbit skeletal muscle sarcoplasmic reticulum Ca2 + -ATPase (IC50 5 mM) [27]. 3-Isomangostin (mangostanol), a-mangostin and g-mangostin are relatively weak inhibitors of cyclic AMP phosphodiesterase (IC50 values 47, 24 and 50 mM, respectively) [24]. However a-mangostin and g-mangostin are potent antagonists of serotonin and histamine receptors, respectively [25,26]. These naturally-occurring compounds may serve as useful lead

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compounds for the synthesis of bioactive derivatives. The present paper describes the differential interactions of a variety of a-mangostin and 3-isomangostin derivatives with eukaryote signal regulated protein kinases and a cyclic nucleotide-binding phosphatase.

2. Materials and Methods

2.1. Materials [g-32P]ATP (specific activity 4000 Ci/mmol) was obtained from Bresatec, Adelaide, Australia. Kemptide (LRRASLG), epidermal growth factor receptor-derived synthetic peptide (EGFRP; VRKRTLRRL-NH2) and myosin light chain-based synthetic peptide (MLCP; KKRAARATSNVFA-NH2) were obtained from Auspep, Melbourne, Australia. Prenylated xanthone derivatives were synthesised from a-mangostin (series A) or from 3-isomangostin (series B and C) (Fig. 1) by procedures to be described elsewhere. Calmodulin was purified from wheat embryo as previously described [4].

2.2. Protein kinase isolation and assay Rat brain Ca2 + - and phospholipid-dependent protein kinase C (PKC) (specific activity 0.6 mmol/min per mg protein with 3.5 mM EGFRP as substrate), chicken gizzard Ca2 + -calmodulin (CaM)-dependent myosin light chain kinase (MLCK) (specific activity 0.05 mmol/min per mg protein with 20 mM MLCP as substrate), rat liver cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK) (specific activity 0.2 mmol/min per mg protein with 20 mM kemptide as substrate) and wheat embryo Ca2 + -dependent protein kinase (CDPK) (specific activity 0.01 mmol/min per mg protein with 1 mg/ml histone III-S as substrate) were partially purified and assayed radiochemically as described previously [4,8]. In examining the effects of prenylated xanthones on protein kinase activity, test compounds were added to protein kinase assays dissolved in methanol to give 20% (v/v) final methanol concentration in cAK and CDPK assays and 17% (v/v) final methanol concentration in PKC and MLCK assays. IC50 values (concentrations for 50% inhibition) were determined from plots of protein kinase activity versus inhibitor concentration. Control protein kinase activity (no inhibitor added but with the assay containing the appropriate methanol concentration) was routinely determined in sextuplet and assays containing test inhibitors were conducted in duplicate. Standard deviations associated with control assays were routinely about 10% of mean values. To avoid possible interactions of test compounds with protein substrates [16], synthetic peptide substrates were routinely employed in particular protein kinase assays. PKC and cAK were assayed using VRKRTLRRL-NH2 (EGFRP) (3 mM) and LRRASLG (kemptide) (20 mM) as substrates, respectively and MLCK was assayed using KKRAARATSNVFA-NH2 (MLCP) (18 mM).

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Fig. 1.

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CDPK was assayed with 0.8 mg/ml III-S histone preparation as substrate since it had been shown previously that the IC50 values for a-mangostin and g-mangostin are the same when CDPK is assayed with the peptide substrate MLCP or with III-S histone [9]. PKC was assayed in a reaction medium (120 ml) containing 33 mM Tris (Cl – , pH 8.0), 0.7 mM MgCl2, 7 mM dithiothreitol, 0.2 mM EGTA, 0.7 mM CaCl2, 3 mM EGFRP, 0.04 mg/ml phosphatidylserine-rich brain extract, 17% (v/v) methanol, PKC and 17 mM ATP (specific activity of [g-32P] ATP about 30 Ci/mol). The cAK assays were performed in a reaction medium (100 ml) containing 40 mM Tris (Cl – , pH 8.0), 8 mM MgCl2, 8 mM dithiothreitol, 20 mM kemptide, 20% (v/v) methanol, cAK and 20 mM ATP (specific activity of [g-32P] ATP about 30 Ci/mol). MLCK was assayed in a reaction medium (120 ml) containing 6.4 mM Hepes (Na + , pH 7.0), 0.8 mM Mg acetate, 0.1 mM CaCl2, 0.17 mg/ml bovine serum albumin, 0.02% tween-80, 0.16 mM calmodulin, 18 mM MLCP, 0.4 mM potassium phosphate, 10 mM NaCl, 0.04 mM dithiothreitol, 17% (v/v) methanol, MLCK and 17 mM ATP (specific activity of [g-32P] ATP about 30 Ci/mol). CDPK was assayed in a reaction medium (100 ml) containing 40 mM Tris (Cl – , pH 8.0), 8 mM MgCl2, 8 mM dithiothreitol, 0.7 mM MgCl2, 0.2 mM EGTA, 0.8 mM CaCl2, 0.8 mg/ml III-S histone, 20% (v/v) methanol, CDPK and 20 mM ATP (specific activity of [g-32P] ATP about 30 Ci/mol). All assays were terminated by spotting 80 ml aliquots of reaction mixtures onto 4 cm2 squares of phosphocellulose paper (Whatman P-81) which were subsequently washed in 500 ml 75 mM H3PO4 (five times) and twice in EtOH before drying and using Cerenkov counting to determine the amount of [32P] phosphopeptide formed.

2.3. Phosphatase isolation and assay Potato tuber high-affinity cyclic nucleotide-binding phosphatase (Pase) was purified to homogeneity as previously described [28]. The Pase was assayed at 30°C in a reaction medium (500 ml) containing 0.1 M acetate (Na + , pH 5.0), 4 mM MgCl2, 1 mM p-nitrophenylphosphate (PNP), enzyme and 10% (v/v) methanol. Test prenylated xanthones were added to the reactions in methanol. Reactions were terminated by the addition of 0.5 ml 0.2 M NaOH and the liberated p-nitrophenolate measured from absorbance at 400 nm [28].

2.4. Antifungal testing Alternaria brassicicola (Schw) was isolated from Brassica oleracea L. var gemmifera DC. Fusarium oxysporum Schlecht was isolated from Atriplex sp. and Chalara elegans was isolated from tobacco root. Fungi were grown on potato dextrose agar (PDA) plates prepared as described below. Potato tubers (100 g) were washed, peeled, cut into small pieces, added to 500 ml H2O and heated at 100°C for 15 min. After immediate filtration, 10 g of dextrose and 10 g agar were added and Fig. 1. Structures of the prenylated xanthones studied. The A series involve different R and R% substitutions (R%= R= H, a-mangostin). The B series involve R% substitutions (R%=H, 3-isomangostin).

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dissolved using a microwave oven at maximum power (850 W) for 2 min. The PDA solution was autoclaved for 40 min and dispensed into 9 cm diameter Petri dishes in aseptic conditions. Fungal cultures were spread onto Petri dishes and allowed to grow at 23°C towards 6 mm diameter Whatman 1 disks upon which 50 ml of 1 mM test compound had been deposited. Fungal growth inhibition was assessed qualitatively by observing growth inhibition in the vicinity of active test compounds.

3. Results and Discussion

3.1. Inhibition of cAK by prenylated xanthones The prenylated xanthone derivatives examined were of three classes that we will refer to as A, B and C for clarity. Compounds in group A are all derivatives of the polyoxygenated prenylated xanthone a-mangostin (Fig. 1) in which the 3-hydroxyl and 6-hydroxyl are variously substituted with groups R and R%, respectively (A, Fig. 1, Table 1). The group B compounds are derivatives of 3-isomangostin in which the 9-hydroxyl is substituted with various groups R% (B, Fig. 1, Table 1). Group C contains two compounds in which the 7-prenyl group has been modified (compounds C1 and C2, Fig. 1). A range of A series compounds are potent and relatively selective inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA) (Fig. 2, Table 1). The most potent cAK inhibitors found with IC50 values of 4 mM or less have relatively small and uncharged R% and R substituents, namely A1 (R% = H, R=CH2CH(OH)CH2OH; IC50 2 mM), A2 (R% = COCH3, R= H; IC50 3 mM), A3 (R% = CH2CH2CH2CN, R= H; IC50 4 mM), A4 (R% = CH3, R =H; IC50 4 mM). However A11 (R% = CH3, R = CH3; IC50 35 mM) is an exception to this pattern. While A1 (R% =H, R=CH2CH(OH)CH2OH) is the most potent inhibitor found, all of the other compounds with relatively bulky R% or R substituents are poorer inhibitors than compounds A1 – A4 (Table 1). This is clearly seen in the series of compounds differing from A1 only in the nature of R%, namely A1 (R% =H, IC50 2 mM), A7 (R% = CH2CH(OH)CH2OH, IC50 10 mM) and A15 (R% = CH2CH2CH2N(CH3)2, only 28% inhibition at 200 mM). In the set of A series compounds in which R= H, relatively bulky and protonatable R% substituents decrease inhibitory effectiveness (compounds A5, A9, A12, A13 and A14). However A5 and A9, both having an hydroxyl on the R% substituent are much more inhibitory (IC50 values 7 and 17 mM, respectively) than compound A12 (IC50 58 mM) and compounds A13 and A14 (51 and 48% inhibition at 200 mM, Fig. 2. Inhibition of cAK by a-mangostin derivatives. cAK was measured in the standard assay conditions in the presence of increasing concentrations of test compounds. Protein kinase activity is expressed as percentage of control (no added inhibitor). (A) - -, A1; --, A2; --, A3; -"-, A4; (B) --, A5; --, A6; - -, A7; - -, A8; (C) --, A9; - ×-, A10; -*-, A11; -“-, A12.

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Fig. 2.

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CH2CH2CH2CN CH2CH(OH)CH2N(CH2CH3)2 CH2CH2CH2N(CH3)2 CH2CH2N(CH3)2 CH2CH2N(CH2CH3)2 CH2CH(OH)CH2N(CH3)2

Series B B1 B2 B3 B4 B5 B6

CH2CH(OH)CH2OH H H H H CH2CH2N(CH2CH3)2 CH2CH(OH)CH2(OH) CH2CH2CH2CN H CH2CH(OH)CH2NHCH(CH3)2 CH3 H H H CH2CH(OH)CH2(OH)

6 6

8 62 [34] [43] [64] [72]

2 3 4 4 7 9 10 15 17 26 35 58 [49] [52] [72]

[83] [47]

[156] 11 8 24 [53] 11

80 [65] [71] 43 11 13 17 [113] 20 6 [159] 50 76 104 15

[66] [6 79]

[93] 6 6 15 [54] 6

[47] [72] [78] 13 4 9 [48] [67] 6 1 [85] 12 [72] [278] 9

MLCK [%]

[174] [60]

[109] [48] 37 [45] 84 [83]

[97] [80] [81] [48] 0.2 [91] [84] [59] [57] 6 [97] [65] [39] [359] 70

CDPK [%]

[98] [80]

22 [97] [85] [105] [106] [100]

[96] 9 8 2 [79] [105] [86] [82] [105] [103] 43 [103] [96] [88] [104]

Pase [%]

Protein kinase and phosphatase were assayed in the standard assay conditions described in Section 2 in the presence of increasing concentrations of test compounds. IC50 values (concentrations for 50% inhibition) were determined from plots of enzyme activity versus inhibitor concentration. In those situations in which compounds were relatively poor inhibitors, enzyme activity in the presence of 100 mM inhibitor (Pase assay), 167 mM inhibitor (PKC and MLCK) or 200 mM inhibitor (cAK and CDPK assays) are presented in parentheses as percentage of control activity (no added inhibitor).

Series C C1 C2

H COCH3 CH2CH2CH2CN CH3 CH2CH(OH)CH2NHCH(CH3)2 CH2CH2N(CH2CH3)2 CH2CH(OH)CH2(OH) CH2CH2CH2CN CH2CH(OH)CH2N(CH3)2 CH2CH(OH)CH2NHCH(CH3)2 CH3 CH2CH2CH2N(CH3)2 CH2CH2N(CH3)2 CH2CH2N(CH2CH3)2 CH2CH2CH2N(CH3)2

PKC [%]

cAK [%]

R%

R

IC50 (mM) or (% control)

Substituents

Series A A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15

Compound

Table 1 Inhibition of protein kinases and phosphatase by prenylated xanthones

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respectively) (Table 1). Compound A10, having relatively bulky and protonatable R% and R groups which are both hydroxylated, is still an effective cAK inhibitor (IC50 26 mM) (Table 1). Increasing the bulkiness of the R group in series A compounds also decreases inhibitory effectiveness with respect to cAK. Thus one can compare pairs of compounds differing only in the R substituent: A3 (R= H, IC50 4 mM) and A8 (R = CH2CH2CH2CN, IC50 15 mM); A4 (R= H, IC50 4 mM) and A11 (R=CH3, 35 mM); A5 (R= H, IC50 7 mM) and A10 (R= IC50 CH2CH(OH)CH2NHCH(CH3)2, IC50 26 mM); A12 (R= H, IC50 58 mM) and A15 (R = CH2CH(OH)CH2OH; only 28% inhibition at 200 mM) (Table 1). The pattern of inhibition found with the A series of compounds is also found with the related compounds C1 and C2 and the series B compounds. Thus the most potent inhibitors are C1 (IC50 6 mM), C2 (IC50 6 mM) and B1 (R% = CH2CH2CH2CN, IC50 8 mM) (Fig. 3, Table 1). Compounds B2–B6 have relatively bulky and protonatable R% substituents and are relatively poor inhibitors of cAK (Table 1).

Fig. 3. Inhibition of cAK by 3-isomangostin derivatives. Other details as for the legend to Fig. 2. (A) - -, B1; --, B2; (B) --, C1; - -, C2.

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Fig. 4. Inhibition of PKC by a-mangostin derivatives. PKC was measured in the standard assay conditions in the presence of increasing concentrations of test compounds. Protein kinase activity is expressed as percentage control (no added inhibitor). (A) - -, A1; --, A4; --, A5; -"-, A6; -“-, A12; (B) - -, A9; --, A10; --, A12; (C) - -, A13; --, A14; - × -, A15.

3.2. Inhibition of protein kinase C and myosin light chain kinase by prenylated xanthones The pattern of inhibition observed with cAK is reversed with rat brain protein kinase C (PKC) (Figs. 4 and 5, Table 1) and chicken gizzard myosin light chain

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kinase (MLCK) (Fig. 6, Table 1). This is most clearly seen with the series B compounds and compounds C1 and C2. C1, C2 and B1 (R% = CH2CH2CH2CN) (Fig. 1) are potent inhibitors of cAK but are inactive or poor inhibitors of PKC and MLCK (Table 1). Similarly compounds B2, B3, B4 and B6 have relatively bulky and protonatable R% groups and are potent inhibitors of PKC (Fig. 5) and MLCK (Fig. 6) while being very poor inhibitors of cAK (Table 1). By way of exception, B5 (R% = CH2CH2N(CH2CH3)2) is equally ineffective as an inhibitor of cAK, PKC and MLCK (Table 1). With the series A compounds there is also a generally obverse relationship between inhibition of PKC and MLCK on the one hand and inhibition of cAK on the other. Thus A series compounds having both R% and R substituents that are non-protonatable (A1 – A4, A7, A8 and A11) are good inhibitors of cAK (IC50 values in the range 2 – 35 mM) (Table 1). However with the exceptions of A4 (IC50 values for PKC and MLCK 43 and 13 mM, respectively) and A7 (IC50 value for PKC 17 mM), these compounds are ineffective or poor inhibitors of PKC and MLCK (Figs. 4 and 6, Table 1). While bulkier and/or protonatable R% and R substituents tend to decrease inhibitory effectiveness against cAK, the opposite is generally true in relation to inhibition of PKC and MLCK. Thus A5, A6, A9, A10, A12 and A15 all have relatively bulky and protonatable R% groups (and with A6 and A10 such R groups also), all of these compounds being good inhibitors of PKC and MLCK. Compounds A13 and A14 have relatively bulky R% groups (although R=H in both cases) but these compounds are poor or ineffective as inhibitors of cAK and MLCK while inhibiting PKC (IC50 values 76 and 104 mM, respectively) (Table 1). The increased effectiveness of compounds with bulkier R% groups is clearly seen when one compares the efficacy of otherwise identical series A compounds having R =CH2CH(OH)CH2OH, the following order of effectiveness as PKC inhibitors being observed (R% groups and IC50 values in parenthesis): A15

Fig. 5. Inhibition of PKC by 3-isomangostin derivatives. Other details as for the legend to Fig. 4. - -, B2; --, B3; --, B4; -"-, B6.

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Fig. 6. Inhibition of MLCK by derivatives of a-mangostin and 3-isomangostin. MLCK was measured in the standard assay conditions in the presence of increasing concentrations of test compounds. Protein kinase activity is expressed as percentage of control (no added inhibitor). (A) - -, A4; --, A5; --, A6; -"-, A9; (B) -“-, A10; - -, A12; --, A15; (C) --, B2; - -, B3; --, B4; - ×-, B6.

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(CH2CH2CH2N(CH3)2; 15 mM) \A7 (CH2CH(OH)CH2OH); 17 mM)\ A1 (H; 80 mM). A similar relationship is observed with respect to MLCK inhibition with this same series of compounds: A15 (R% = CH2CH2CH2N(CH3)2; IC50 9 mM)\A7 (R% = CH2CH(OH)CH2OH) and A1 (R% = H) (both yielding about 50% inhibition at 167 mM). However with the series of A compounds with R = H this is also observed (with some exceptions), the following order of effectiveness as PKC inhibitors being observed (R% groups and IC50 values in parenthesis): A5 (CH2CH(OH)CH2NHCH(CH3)2; 11 mM)\A9 (CH2CH(OH)CH2N(CH3)2; 20 mM)\ A4 (CH3; 43 mM) \A12 (CH2CH2CH2N(CH3)2; 50 mM)\A13 (CH2CH2N(CH3)2; 76 mM) \A14 (CH2CH2N(CH2CH3)2; 104 mM)\ A2 (COCH3; 35% inhibition at 167 mM)\ A3 (CH2CH2CH2CN; 29% inhibition at 167 mM). In relation to inhibition of MLCK, a similar loose relationship relating to R% size is observed (with some exceptions): A5 (R% = CH2CH(OH)CH2NHCH(CH3)2; IC50 4 mM) \ A9 (CH2CH(OH)CH2N(CH3)2; 6 mM)\A12 (CH2CH2CH2N(CH3)2; 12 mM)\ A4 (CH3; 13 mM) \A1 (H), A2 (COCH3), A13 (CH2CH2N(CH3)2), A3 (CH2CH2CH2CN) (all of these being poor inhibitors)\ A14 (CH2CH2N(CH2CH3)2; no inhibition at 167 mM). A similar effect of the R group on PKC and MLCK inhibition can be seen by comparing pairs of series A compounds having the same R% group. Thus with A6 and A14 (R% = CH2CH2N(CH2CH3)2), A6 (R= CH2CH2N(CH2CH3)2) is a more effective inhibitors of PKC and MLCK (IC50 values 13 and 9 mM, respectively) than A14 (R=H) (IC50 value for PKC 104 mM; no inhibition of MLCK at 167 mM). With A5 and A10 (R% = CH2CH(OH)CH2NHCH(CH3)2), A10 (R = CH2CH(OH)CH2NHCH(CH3)2) is a better inhibitor of PKC and MLCK (IC50 values 6 and 1 mM, respectively) than A5 (R=H) (IC50 values 11 and 4 mM, respectively). Furthermore, with A12 and A15 (R% =CH2CH2CH2N(CH3)2), A15 (R = CH2CH(OH)CH2OH) is more effective as an inhibitor of PKC (IC50 value 15 mM) than A12 (R= H) (IC50 value 50 mM). However with A4 and A11 (R% = CH3), A4 (R = H) is a more effective inhibitor of PKC and MLCK (IC50 values 43 and 13 mM, respectively) than A11 (R=CH3), which is not inhibitory. A3 and A8 (R% = CH2CH2CH2CN) are ineffective or poor inhibitors of PKC and MLCK whether R= H (A3) or R= CH2CH2CH2CN (A8) (Table 1).

3.3. Inhibition of CDPK by prenylated xanthones Only two of the prenylated xanthones tested are potent inhibitors of wheat embryo Ca2 + -dependent protein kinase (CDPK), namely the series A compounds A5 (R% = CH2CH(OH)CH2NHCH(CH3)2; R=H; IC50 0.2 mM) and A10 (R% = R= CH2CH(OH)CH2NHCH(CH3)2); IC50 6 mM) (Fig. 7, Table 1). A larger protonatable R% group is common to both of these inhibitors but other series A compounds with relatively bulky and protonatable R% groups and variously large or small R substituents are inactive or relatively ineffective as CDPK inhibitors. The only other effective A series inhibitor is A15 (R% = CH2CH2CH2N(CH3)2; R= CH2CH(OH)CH2OH; IC50 70 mM) (Fig. 7, Table 1).

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Fig. 7. Inhibition of CDPK by prenylated xanthones. CDPK was measured in the standard assay conditions in the presence of increasing concentrations of test compounds. Protein kinase activity is expressed as percentage control (no added inhibitor). (A) - -, A5; --, A10; --, A15; (B) -"-, B3; -“-, B5.

The importance of the R substituent for efficacy of prenylated xanthones as CDPK inhibitors is borne out by the series B compound B6 which has the same R% substituent (CH2CH(OH)CH2N(CH3)2) as the potent series A inhibitors A5 and A10 but is ineffective as an inhibitor of CDPK (Table 1). B6 differs from A5 and A10 in having a distal cyclic ether ring system (Fig. 1) The R% substituent importance is borne out by the fact that B3 and B5 (R% =CH2CH2CH2N(CH3)2 and R% =CH2CH2N(CH2CH3)2, respectively) are CDPK inhibitors (IC50 values 37 and 84 mM, respectively) (Fig. 7, Table 1) whereas the other related compounds having different R% substituents including C1 and C2, are ineffective (Table 1).

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3.4. Inhibition of a high-affinity cyclic nucleotide-binding phosphatase by prenylated xanthones A small set of the prenylated xanthones examined inhibit a high-affinity cyclic AMP-binding phosphatase (Pase) (Fig. 8, Table 1). The common feature of the series A inhibitors is that the R% substituent is not protonatable and the R substituent is relatively small. Thus, in order of effectiveness as inhibitors of the Pase, A4 (R% =CH3, R =H; IC50 2 mM)\ A3 (R% = CH2CH2CH2CN, R= H; IC50 8 mM) \A2 (R% = COCH3, R= H; IC50 9 mM)\ A11 (R% = CH3, R=CH3; IC50 43 mM) (Fig. 8, Table 1). Clearly, substitution of R= CH3 (in A11) for R= H (in A4) severely decreases inhibitory effectiveness. Similarly, replacement of R= H in the potent inhibitor A3 (R% =CH2CH2CH2CN, IC50 8 mM) for R = CH2CH2CH2CN yields A8 which is inactive as an inhibitor of the Pase. Furthermore, the B series Pase inhibitor B1 that also has R% = CH2CH2CH2CN is less effective than A3 (Fig. 8, Table 1), B1 (IC50 22 mM) differing from A3 (IC50 8 mM) in having a distal ether ring system (Fig. 1). The most effective inhibitors are compounds in which R =H. However the A series compounds in which R = H but R% is a relatively bulky and protonatable group (A5, A9, A12, A13 and A14) are not inhibitory. Finally, compounds C1 and C2 that have the same basic structure as the B series compounds but have a 9-hydroxy and also have the prenyl group substituted with a carboxyl and a hydroxy, respectively are inactive. It is therefore possible that a polar prenylated ring obviates inhibition of the Pase. Thus A1 (R% = H, R = CH2CH(OH)CH2OH) is ineffective as a Pase inhibitor (Table 1).

Fig. 8. Inhibition of Pase by prenylated xanthones. Pase was measured in the standard assay conditions in the presence of increasing concentrations of test compounds. Pase activity is expressed as percentage control (no added inhibitor). - -, A2; --, A3; --, A4; -"-, A11; -“-, B1.

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Fig. 9. Competitive inhibition of cAK by A1. cAK was assayed in the standard conditions but with increasing concentrations of ATP (A) or kemptide (B) and in the absence of added inhibitor (-“-) or the presence of 2 mM A1 (- -) or 4 mM A1 (--). Double reciprocal plots of kinetic data are presented. v − 1 [(initial rate) − 1] is in arbitrary units.

3.5. Mechanism of inhibition of protein kinases and the cyclic nucleotide-binding phosphatase by prenylated xanthones Representative examples of prenylated xanthone protein kinases and phosphatase inhibitors were studied in enzyme kinetic experiments to determine the mode of inhibition of these enzymes by such compounds. Lineweaver-Burk double reciprocal plots of vo− 1 ([initial velocity] − 1) versus [substrate] − 1 from enzyme kinetic data obtained in the presence or absence of various concentrations of A1 show that this compound inhibits cAK in a fashion that is competitive with respect to both ATP and the synthetic peptide substrate kemptide (Fig. 9). The Ki value of 3.3 9 1.9 mM (mean 9 S.D. from three determinations) is similar to the IC50 value of 2 mM for A1 (Table 1). Compounds B1 and C1 also inhibit cAK in a fashion that is

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Fig. 10. Non-competitive inhibition of the Pase by B1 and A4. The Pase was assayed in the standard conditions but with increasing concentrations of PNP in the absence of added inhibitor (-“-) or the presence of 2 mM B1 (A) or of 25 mM A4 (B). Double reciprocal plots of kinetic data are presented. v −1 [(initial rate) − 1] is in arbitrary units.

competitive with respect to both ATP and substrate with Ki values of 4.7 93.1 mM (mean 9 S.D. from three determinations) and 5.79 2.7 mM (mean9 S.D. from four determinations) respectively, (data not shown). While A10 inhibits PKC in a competitive fashion with respect to ATP and EGFRP (Ki 5.6 9 2.4 mM, mean9 S.D. from four determinations), A10 inhibits MLCK non-competitively with respect to both ATP and the peptide substrate MLCP (Ki 6.3 9 2.2 mM, mean 9 S.D. from three determinations). The most potent inhibitor found for CDPK, A5 (IC50 0.2 mM), also inhibits CDPK in a fashion that is non-competitive with respect to both ATP and the protein substrate histone III-S (Ki 0.8 9 0.5 mM, mean9 S.D. from three determinations) (data not shown). The most potent prenylated xanthone inhibitor of the cyclic nucleotide-binding Pase

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found in this study, namely A4, is a non-competitive inhibitor of the enzyme (Fig. 10), the Ki value of 1.5 mM being in good agreement with the observed IC50 value 2 mM (Table 1). Similarly, the weaker inhibitor B1 also inhibits the Pase non-competitively (Fig. 10) with a Ki value of 43 mM as compared to the IC50 value of 22 mM (Table 1).

3.6. Antifungal acti6ity of prenyl xanthones Possible biological activity of the prenyl xanthone derivatives was investigated by examining the effect of these compounds on the growth on agar of various fungi, namely C. elegans, A. brassicicola and Fusarium oxysporum. The only effective inhibitors of the growth of C. elegans are A13 (R% = CH2CH2N(CH3)2, R= H), a very poor inhibitor of all enzymes tested, and A10 (R% =R= CH2CH(OH)CH2NHCH(CH3)2), a potent inhibitor of PKC, MLCK and CDPK (Table 1). Similarly, the only compounds inhibiting the growth of A. brassicicola are A14 (R% = CH2CH2N(CH2CH3)2, R = H), a very poor protein kinase inhibitor, and B6 (R% =CH2CH(OH)CH2N(CH3)2), a potent inhibitor of PKC and MLCK (Table 1). A14 was the only compound found to inhibit the growth of F. oxysporum. The only common feature of these compounds is a relatively bulky and protonatable R% substituent. The derivatives studied here mostly involve R and R% substituents that are straight chain alkyl groups. Clearly other types of substituents could be the subject of further structure/activity studies noting that protein kinase inhibitors can have a variety of pharmacological effects, e.g. the selective cytotoxicity of DNA-binding compounds that are also PKC and/or MLCK inhibitors [8,19,29], the anti-malarial activity of the cAK inhibitor halofantrine [30], the antiprotozoal activity of the cAK and CDPK inhibitor g-mangostin [31] and the anti-inflammatory properties of a variety of cAK inhibitors such as acidic triterpenoids [13] and a-mangostin [2,24,25].

Acknowledgements This work was supported in part by a grant from the Australian Research Council. M. Hasmeda was supported by an AusAid postgraduate scholarship.

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