Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 27 © 2002 Elsevier Science B.V. All rights reserved. 819

MODULATION OF PROTEIN PHOSPHORYLATION BY NATURAL PRODUCTS

SALVADOR MANEZ* and MARIA DEL CARMEN RECIO

Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Av, V. A, Estelles s/n, 46100 Burjassot (Spain)

ABSTRACT: Studies carried out to determine the influence of phosphorylation and dephosphorylation of proteins in a variety of physiological events are of increasing interest. The activity of kinases and phosphatases and their respective inhibition by endogenous mediators and by pharmacological agents regulates a huge number of biochemical pathways involved in cellular proliferation, apoptosis, inflanunation, hormonal activity, and gene transcription, among other processes. This article focuses on the recently described natural products able to interfere negatively with the activity of serine/threonine and tyrosine kinases. These agents are classified, according to their biosynthetic origin and chemical properties in phenolics, terpenoids, alkaloids and miscellaneous substances. The nucleus of the review is preceded by a general overview on kinase activity, followed by a chapter devoted to naturally occurring kinase activators. Finally, a section concerning the advances in phosphatase inhibition research is included. The main sources of novel phenolic kinase inhibitors are tannins, coumarins, polycyclic isopentenyl isoflavonoids, and phloroglucinols. Other phenolics like flavonols or simple isoflavones are also reported, together with some reputed plant active principles such as curcumin, hypericin or resveratrol. Among the terpenoids, the effects of wortmannin, and those of certain triterpenoids like ginsenoside Rbi or Rhi should be mentioned. The alkaloids comprise two main groups of inhibitors: the indole alkaloids, headed by staurosporine and its derivatives, which are potent, selective inhibitors of protein kinase C, and the isoquinoline alkaloids, subdivided into aporphines, benzophenanthridines and naphtylisoquinolines. The scientific panorama regarding the inhibition of phosphatases is dominated by the polyether and cyclic polypeptide environmental toxins, although some new agents such as indole and isoquinoline alkaloids have been described.

PREFACE

The introduction or removal of a phosphate group in an organic molecule may be the most decisive reaction that occurs in biochemical processes. Many substances of importance for living cells are transformed into their respective phosphates as a previous step to their metabolism, transport, energy transfer, or incorporation into larger macro-structures. Phosphate performs extremely different dynamic and static functions, such as utilisation and storage of glucose, regulation of cell signalling or linkage of nucleotides within nucleic acid chains. Under the broad subject of

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phosphorylation, the phosphorylation of proteins by specific kinases has obviously been an object of extensive research, because of the muhiplicity of ways in which proteins regulate all life events.

Although the inhibition of protein kinases by natural products isolated fi-om either higher plants or microorganisms has been known for years, and some kinase natural inhibitors and activators are widely used as laboratory tools in pharmacology and allied sciences, not many reviews have been published to the present, and certainly none have brought together all the recent advances in both kinase and phosphatase interactions by natural products. Our review covers the major research in these fields produced in the 1993-1999 period, although some particularly interesting papers published before 1993 and others in the year 2000 are included. In order to make it clear that the present work is closely related to those published previously we should mention some reviews that in our opinion are of obligatory reading, such as those of Hu [1] on selective protein kinase C inhibitors, and Chang and Geahlen [2], and Levitzki and Gazit [3], on protein tyrosine kinase inhibitory drugs. The works of Cohen [4], and by Hunter [5] are essential if we want to see the entire panorama of the intervention of kinases and phosphatases in cellular signalling.

BIOCHEMICAL BASIS OF PROTEIN PHOSPHORYLATION

Some essential concepts

The term phosphorylation is applied to a reaction by means of which a phosphate group, any of the anions derived form or/o-phosphoric acid (H3PO4), is covalently integrated into the structure of a given molecule to convert it into a phosphate ester, phosphate-anhydride or phosphate-amide. In most cases, the donor of this phosphate group is adenosine 5'-triphosphate (ATP), although other nucleotides such as cytidine 5'-triphosphate and uridine 5'-triphosphate participate in certain biochemical pathways. In the process of releasing one phosphate group fi-om the ATP molecule, the rupture of the terminal phosphate needs 418 kJ/mol, but if this phosphate is transferred to one water molecule, or in other words, if hydrolysis is associated with this release, 31 kJ/mol are generated. (AG° = -31 kJ/mol). Consequently, in this kind of processes phosphorylation becomes thermodynamically possible if the theoretical reaction coupled to the ATP hydrolysis has a AG° not higher than 31 kJ/mol. A long list of substances of biological interest are substrates for the transfer of a

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phosphate from a nucleoside triphosphate, and the individual enzymes regulating this reaction receive the name of kinases, or protein kinases when the target is a protein.

One of the best-known roles of protein phosphorylation is the regulation of enzymatic activity, given that a lot of proteins acquire their catalytic properties only after phosphorylation. Such enzymes coexist under two forms, a (active) and b (not active or less active), and the passage from one to another is catalysed by other enzymes that are usually also activated by phosphorylation. The conformational changes induced by this process lead to changes not only in the catalytic activity itself but also in the sensitivity to allosteric effectors. The sort of cascade of reactions in which a phosphorylated protein product is the active enzyme (kinase) necessary for subsequent phosphorylation of another inactive enzyme is frequent in primary metabolism, and is found in the glycogen breakdown, yielding glucose-1-phosphate, and also in the signal transduction systems dependent on the activation of G-protein-coupled receptors and cytokine/growth factor receptors linked to tyrosine kinase activity [6].

Structural and mechanistic features of kinases

How does an enzyme become active by phosphorylation? What changes occur to facilitate the catalytic activity?. Questions of this kind began to be answered in detail by crystallographic studies on the glycogen phosphorylase (GP) molecule. This is a dimer that acquires its active form by phosphorylation of a serine (mammalian GP) or a threonine (yeast GP) near the NH2 terminal residue. In mammalian GP the presence of a phosphate causes, by ionic phosphate/arginine interactions, the two subunits of the enzyme to approach the phosphorylating serine, which then moves the subunits away to open the catalytic crevice. In the similar yeast GP an analogous "mechanic" effect occurs, though it does not depend on an ionic binding, but on a disturbance of the lipophilic interactions between non-polar residues in the vicinity of both the phosphate acceptor and catalytic sites [7].

The catalytic cores of protein kinases present in eukaryotic beings share the same fimdamental structure, best defined for cAMP-dependent protein kinase (PKA) and characterised by an amino terminal P-sheet-rich nucleotide-binding domain and a larger, helical peptide-binding domain. In the nucleotide-binding domain there is a flexible glycine-rich loop, absent in prokaryote kinases, that wraps the nucleotide and whose phosphate-

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interacting tip is only stable when a peptide is bound with high affinity. Another characteristic of the nucleotide-binding domain is that the majority of the known natural and synthetic kinase inhibitors act by recognising this site [8]. In spite of the similarities reported in recent studies on protein sequence and conformation affecting phosphorylation processes, there is a huge variety of structures for protein kinases, and this implies different ways of regulation [9].

For many years ago, scientists have known that protein kinases only catalyse the phosphorylation of some of the aminoacids among the "free" positions existing in a peptide sequence. As an example, only one (Ser " ) out of 64 serine or threonine hydroxyl residues of GP is replaced by the action of GP-kinase. In this context, the substrate aminoacid sequence in the neighbourhood of the hydroxyl group is of critical importance because of its tendency to undergo phosphoiylation by PKAs. The major and simplest positive influence was determined by the presence of one, or preferably two, arginine residues two positions before the phosphate acceptor serine (Ser*): Arg-X-Ser* or Arg-Arg-X-Ser*, where X is a variable aminoacid residue. Among some of the other best-known kinases, miosin light-chain kinase (MLCK) from smooth muscle recognises in the susbtrate the closely-related motif Lys-Lys-Arg-X-X-Arg-X-X-Ser*-X and multifunctional calmodulin-dependent kinases (CaMKs) act on the sequence Arg-X-X-Ser*. A slight variation represents the favourite substrate sequence for the classical forms of protein kinase C (PKC) : X-Arg-X-X-Ser*-X-Arg. This sequence is mimicked in a regulatory part of the enzyme, the "pseudosubstrate site", and repeats the same motif with the exception of alanine instead of serine. Enzyme specificities on the substrate primary structure are based on sequences of native proteins and synthetic peptides and should be considered in non-exclusive terms, because other, often quite different, sites may be recognised by kinases [10].

Basis of phosphorylation regulation

The inverse pathway, through which a compound, e.g. a protein, loses a phosphate group, is termed dephosphorylation and is catalised by the phosphatases. Thus, protein kinases and protein phosphatases have general opposite effects, and when operating on the same reaction the preponderant activity of each produces a displacement of the equilibrium between phosphorylated and non-phosphorylated forms that can therefore

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determine the efficacy of a physiological system. This phenomenon, called reversible phosphorylation, is not the only way to regulate cellular events dependent on phosphate transfer. In fact, both kinds of enzymes possess, in certain cases, targeting subunits responsible for recognising the target locus (a subcellular structure or a substance dissolved in the cytosol) and therefore also responsible for carrying the catalytic subunit into contact with the substrate. In the case of PKA, there are two regulatory subunits that function as inhibitory targeting subunits because they maintain the well-known inactive tetrameric form, but other true (positive) targeting subunits of PKA bound to neuronal organelles have been described. This binding is mediated by the presence of the so-called A-kinase anchor proteins (AKAPs). An ulterior level of complexity in such a per se complex scenario is revealed by the fact that the activity of protein phosphatase 1 is modulated by phosphorylation of a targeting subunit [11].

TYPES OF KINASES

Introduction

Enviromental changes can be perceived by cells through extracellular signals. In addition, cells can communicate with each other because they also produce signals. The extracellular signals can be either physical (light, temperature, etc.) or chemical (food, hormones and neurotransmitters). In this sense, two groups of chemical signals can be distinguished: a) membrane permeable signals such as steroid hormones (estrogens, progesterone and androgens), which can directly regulate gene expression, b) the membrane impermeable signal molecules which include acetylcholine, growth factors, extracellular matrix components, thrombin, lysophosphatidic acid, etc. They are recognised by receptors which are locaUsed in the plasme membrane of the cell. The receptors are specific for one particular signal molecule or a family of closely related signal molecules. If a ligand binds to an ion channel receptor it can directly lead to its altered opening, which results in a charged membrane potential. Binding of a ligand may stimulate an intrinsic enzymatic activity of its receptor or the modulation of a transducing protein. Activity modulation can be achieved by covalent modification at the molecular level. The most common and important modifications are phosphorylation and dephosphorylation of serine, threonine or tyrosine residues [12].

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Receptors with enzymatic activity

Receptor tyrosine kinases

Epidermal growth factor (EGF), platelet derived growth factor, and insulin are extracellular signal molecules that bind to receptor tyrosine kinases (RTKs). Upon ligand binding, RTK auto-phosphorylates to give phosphotyrosine residues (PY). These receptors acts as highly selective binding sites for the so-called Src homology domain 2 (SH2)-containing proteins, which transduce the signal by changing the enzymatic activity of other recruited proteins. Examples of SH2-containing proteins are the Ras-GTPase activating protein (GAP) that activates phospholipase C-y (PLC-y), which in turn hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 releases Ca ^ from intracellular stores and DAG activates PKC.

Receptor serine/threonine kinases

These receptors have a cysteine-rich extracellular domain and a cytoplasmic serine/threonine kinase activity. Transforming growth factor-p (TGF-P) superfamily induces growth arrest in epithelial cells. In addition to the three TGF-P isoforms, this family comprises activins, bone morphogenetic proteins and other secreted factors.

The two component regulatory system: histidine kinases

This system is employed by prokaryotic organisms, and homologous pathways have recently been identified in eukaryotes. The prototypical two-component pathway consists of two proteins: A protein histidine kinase (sensor kinase) and a response regulator. Histidine kinases are very distinct from the superfamily of conventional protein serine/threonine and tyrosine kinases. The histidine kinases auto-phosphorylate on histidine residues and are involved in the phosphorylation of aspartate amino acids and their targets.

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Receptors without enzymatic activity

Cytokine receptors

The activation of cytokine receptors after ligands binding, provokes tyrosine phosphorylations by non-covalenty associated protein tyrosine kinases (PTKs): the Janus kinases (JAKs), a family that comprises JAKl, JAK 2, JAK 3 and TYR2.

Integrins

Integrins are the major type of cell surface receptors and are formed by a heterodimer with a and (3 subunits. Most integrins bind extracellular matrix components like fibronectin, collagen or intronectin. It seems that integrins activate PTKs clustering.

G-protein coupled receptors

The G-protein coupled receptor group (GPCRs) includes adrenergic, muscarinic, serotonin, dopamine, adenosine, angiotensin II, and thrombin receptors. Upon binding of its ligand, a GPCR interacts with a heterotrimeric guanine-nucleotide binding protein (G-protein). GPCRs, like p2-adrenergic receptors, can be desensitised by decoupling from their G-proteins and intemalisation. Endocytosis of GPCR is necessary for the P2-adrenergic receptor-dependent activation of mitogen activated protein kinases (MAPKs).

Nine cloned mammalian adenylylcyclases (ACs) can be activated by stimulatory a subunits (Gas) and several are modulated by inhibitory a subunits (Gai) and/or Gp/y complexes. cAMP can activate the PKA, which in turn phosphorylates a wide range of substrates, such as the cAMP responsive element binding protein (CREB). When PKA translocates to the nucleus and phosphorylates CREB, the latter is stimulated to regulate gene transcription. There are three mammalian phospholipase C (PLC) isoforms families: PLC-p, PLC-y and PLC-5. The first is activated by serpentine receptors, while the second is stimulated by RTKs.

CytosoUc Ca ^ can modulate the activity of serine/threonine CaMKs via calmodulin. For instance, neuronal CaMK stimulates phosphorylation and activates tyrosine hydroxylase, the limiting enzyme in the synthesis of catecholamine neurotransmitters. Ca ^ also regulates the activation of the conventional PKC isoforms. Increased Ca ^ promotes binding of Ca ^ to

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inactive PKC in the cytosol. It leads to membrane association of PKC, which then binds DAG.

Classification

The superfamily of protein kinases can be classified by the nature of their substrate in two groups: kinases that phosphorylate serine or/and threonine residues, and kinases that phosphorylate tyrosine aminoacids. The serine-and threonine-specific protein kinases can be further classified by the nature of their activators. For example, cAMP-dependent, cGMP-dependent, Ca^Vcalmodulin-dependent and Ca V phospholipid-dependent protein kinases have been identified. Many tyrosine protein kinases are intrinsic parts of the cytoplasmic domains of growth factor receptors.

The activity of the protein kinases are regulated allosterically. The serine- and threonine-specific protein kinases have a regulatory domain which, in the resting state, keeps the catalytic part of the enzyme inactive. When a second messenger or activator (cAMP, cGMP, Ca ^ or DAG) binds to the regulatory domain, the enzyme is activated.

However, it seems that the catalytic domains of these enzymes have many common features and a basically identical mode of action, although serine/threonine and tyrosine kinases differ in the residues to which they transfer phosphate.

The eukaryotic protein kinases can be divided into five groups according to Hanks and Hunter [13]. The members of each group show similarities in the catalytic domain and frequently display an analogous mode of regulation and substrate specifities. These groups of protein kinases are: 1) The AGC group. The main characteristics are the following: The AGC

group are serine/threonine kinases. These kinases are basic amino acid-directed enzymes, and many of them are activated upon the release of second messengers. The group includes nine subgroups, among which are the cyclic nucleotide regulated protein kinase (PKA, PKG) family, the diacylglycerol-activated/phospholipid-dependent protein kinase (PKC) family, the "RAC" (PKB/AK) family; the CPCR kinase family and the ribosomal SG protein kinase family.

2) The CaMK group: This group is also made up at serine/threonine kinases. These kinases are basic amino-acid directed enzymes. Regulation via second messenger pathways is also common and Ca ^ is the main component responsible for the regulation of CaMKs. It

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includes the CaMK group comprised by kinases regulated by the Ca^Vcalmodulin and SNFl/AMPK families, MLCKS and the plant Ca^^-dependent PK. An important member of the CaMK group is MAPK activated protein kinase 2 (MAPKAP2).

3) The CMGC group: Another type of serine/threonine kinase, but that is not regulated by second messengers. This group includes cyclin-dependent kinases (CDKs) and the closely related MAPK/ERK family, the glycogen synthase kinase 3 (GSK3) family and the CIK (Ccd-like kinase) family. The members of the CDK and MAPK/ERK families are proline-directed, while kinases belonging to the GSK-3 group are acidophilic. The GSK-3 family contains the GSK-3 and caseinkinase II isoforms (CK2). CK2 phosphorylates more than 160 substrates among which are several transcriptional factors.

4) The PTK group or "Conventional" protein-tyrosine kinases. This group differs from all the other groups in that the kinases only phosphorylate tyrosine residues. PTKs are often involved in the transduction of growth and differentiation signals in metazoa.

5) Other protein kinases (OPK): These kinases fall outside of the major groups. They include the MEK/Ste7p family, p21-activated kinase (PAKySte 20p family, MEKK/Stellp family, the Raf family, the activin/TGpp receptor family, the flowering plant putative receptor kinase family, the casein kinase I family and the LIM kinases (LIMKs). The members of the first four families function in the MAPK family

protein kinase cascades. CKl has been implicated in the pathogenesis of Alhzeimer's disease through hyperphosphorylation of the x protein. In lower eukaryotes, individual CKl isoforms are involved in the regulation of repair pathway cell proliferation and morphogenesis.

Protein Kinase C

PKC has diverse functions in the control of membrane processes, growth, and differentiation. The PKC family includes 11 isoforms. All PKC isoforms contain four conserved (C1-C4) and five variable (V1-V5) regions. CI and C2 are localised in the regulatory domain, while the C3 and C4 regions are situated in the catalytic domain. The regulatory domain contains a pseudosubstrate site, which is involved in blocking the kinase. The association between the pseudo-substrate site and the C4 region resuks in an inactive PKC conformation. The CI region binds compounds such as DAG or 12-tetradecanoylphorbol-13-0-acetate (TPA) which

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activate the kinase. The C2 region is the site for binding Ca , and it is absent in Ca^^-independent PKC isoforms. In this region it is found the so-called "pseudo-anchoring site" which is involved in the regulation of PKC binding to receptors for activated C-kinase (RACKs). The C3 region contains the ATP binding site, while the C4 region is responsible for substrate binding.

PKC is rapidly activated by a a transient rise in DAG levels resulting from PLC stimulation. Phospholipase D (PLD) causes phosphatidyl­choline hydrolysis, which results in an increase in DAG and PKC activity. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), an other lipid metabolite, can activate PKC.

There is a functional difference between PKC activated through different pathways. The different PKC isoenzymes are also believed to have distinct biological functions.

On the basis of structural elements and activational characteristics, the PKC family has been divided into three subfamilies:

a) Classical or conventional PKCs (cPKCs): Phospholipid-acid Ca^^-dependent protein kinase: PKC-a, -pi, -pll and -y.

b) Non-classical or novel PKCs (nPKCs): PKC5, 8, r| and 9 isoforms.

c) PKC-|Li: It is an isoform between novel PKCs and atypical PKCs.

d) Atypical PKCs (aPKCs): They are independent of DAG and Ca^^ PKC-C and -xlX can be activated by PIP3.

Mitogen Activated Protein Kinases

MAPKs are a family of well-conserved serine/threonine kinases that have a central role in a wide variety of protein kinase cascades. These cascades consist of three kinases, a mitogen activated protein kinase (MAPK), a MAPK kinase (MAPKK) and a MAPK kinase kinase (MAPKKK), which modulate each other in a chain reaction. In other words, MAPKKK activates MAPKK which in turn activates MAPK.

The MAPK family comprises three subfamilies: 1. Extracellular signal regulated kinase (ERK) 2. Stress-activated PK/cJun N-terminal kinase (SAPK/JNK) 3. The p3 8 subfamily.

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The best studied subfamily is the one that includes ERKl and ERK2, which are involved in cascades consisting of Raf, MEKla/MEK2 and ERK1/ERK2 isoforais. Raf is in turn activated by a dynamic combination of phosphorylation (by PKC and/or other protein kinases) and interactions with Ras-GTP and 14-3-3 proteins. The Raf-MEK-MAPK/ERK pathway has effects on non-proliferating cells, but mitogenic signals especially stimulate the pathway. Proliferation can be blocked by inhibiting it.

Activated ERKl and ERK2 can phosphorylate a large number of proteins, among which we find transcription factors and other nuclear proteins. In fibroblasts, ERKl and ERK2 promote entry into the cell cycle. Other substrates for these ERKs are upstream proteins of the MAPK cascade (such as the EGF receptor and Raf-1). Finally, an important group of substrates for this kind of kinase are cytoskeletal elements (such as MAP-1 and MAP-2). Usually, ERKs in activated cells are bound to the cytoskeleton and a basal ERK activity is required for the maintenance of cell-matrix interaction in preference to cell-cell contacts. These findings suggest that ERKs are involved in cytoskeletal reorganisation.

Mitogens and many types of stress (cycloheximide, UV radiation, and heat shock can stimulate SAPK/JNK subfamily kinases. This subfamily is activate by SEK-1 (also named MKK4 for MAPK kinase 4) and MAPK kinase 7 (MKK7). SEKl can in turn be phosphorylated and activated by MEKKs and other kinases. The transcription factors cJun, EDC-l and ATF-2 serve as physiological substrates for the SAPKs.

ERK and SAPK/JNK ofl:en have opposite roles in the regulation of apoptosis. In some cells, such as PC 12 cells, ERK is protective while SAPK/JNK is facilitative. The effects of ERK, SAPK/JNK on p38 modulation appear to be largely dependent on the context in which these components act. Apoptosis is the resuh of a critical balance between several MAPK pathways.

NATURALLY OCCURRING PROTEIN KINASE ACTIVATORS

The human genome encodes over 2000 protein kinases that are finely turned off and on by different signals. For instance, the MAPK are activated through complex but controlled kinase cascades in response to a number of factors, including stress and cytokines. Experimental protein kinase activation can be performed by exogenous agents of plant or marine origin such as phorbols or bryostatins, respectively.

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Despite the large number of protein kinases types, studies on natural activators focus mainly on PKC activation. This kinase, which plays a decisive role in many cellular responses, is generally activated in the cell by lipid second messengers, predominantly DAG, in response to various extracellular agonists (hormones, neurotransmitters, growth factors and cytokines). We now turn our attention to some of the best known PKC activators together with other novel compounds.

Phorbol esters and related diterpenes

The Euphorbiaceae produce a range of toxic diterpenes belonging to a number of structural types. Esters of tigliane, daphnane and ingenane alcohols, possess diflferent biological effects including tumour promotion and cell proliferation, inflammation, degranulation of neutrophils, etc. Phorbol belongs to the tigliane group, and its esters have been found in the genera Croton, Sapium and Euphorbia (Euphorbiaceae) [14]. One of these, TPA (1), is very potent and is used in many studies as a pharmacological tool [15,16]. Diterpene esters are potent activators of PKC [17]. For instance, TPA is able to activate all PKC isoenzymes except the -C, and -X forms [18,19]. 12-Deoxyphorbol-13-0-phenylacetate -20-acetate (dPPA) and thymeleatoxin are selective activators for PKC-pI (dPPA) and PKC-a, -P and -y but not PKC-5 or -s (thymeleatoxin) in vitro. In intact cells it was observed that these phorbol derivatives translocate and down-regulate PKC isoenzymes, including -5 and -s [18].

H3C(CH2),200

CH2OH

ITPA

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OCOCgHsj HO CH2OH

2 gnidimacrin

The daphnane-type diterpene gnidimacrin (2), isolated from the root of Stellera chamaejasme (Thymelaeaceae), inhibited the cell growth of human leukaemias, stomach cancers and non-small cell lung cancers in vitro and showed significant antitumoral activity against murine leukaemias and soUd tumours in vivo. It inhibited phorbol-12,13-dibutyrate (PDBu) binding to K562 cells and directly stimulated PKC activity in the cells in a dose-dependent manner (3-100 nM) [20]. It has recently been demonstrated that the antitumoral mechanism of gnidimacrin is related to PKC activation: gnidimacrin binds to K562 cells three times more than to HLE cells. Immunoblot analyses revealed pronounced PKC-PII expression in gnidimacrin-sensitive cell lines including K562 cells, while refractory HLE cells strongly expressed PKC-a, but not PKC-pII. At the growth-inhibitory concentration of 0.0005 (ig/ml, the Gl phase was arrested and inhibition of cdk2 kinase activity was found. Therefore, one of the main determinants of the ability of cells to respond to gnidimacrin is PKC-PII and the antitumoral action might be associated with cell-cycle regulation through suppression of cdk2 activity [21].

The bryostatin family

The bryostatins are naturally occurring macrocyclic lactones isolated from marine bryozoa that have antineoplastic activity. These macrolactones exhibit high affinities for PKC isoenzymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. They do not act as tumour promoters. The bryostatins are a class of PKC activators that induce only a subset of the typical phorbol ester responses

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and antagonise those phorbol ester-mediated responses that they themselves fail to induce. These compounds are isolated from natural sources in a limited manner, and their synthesis is complex. Several synthetic analogues have been determined that bound strongly to a mixture of PKC isoenzymes and exhibited significant levels of in vitro growth inhibitory activity against human cancer cell lines [22].

3 Bryostatin 1

Bryostatin 1, Fig. (3), showed lower affinity for PKC-pI and -y than for PKC-a, -5, -s and -^ [23]. Similar results have been found by Keenan et al in a novel in vivo assay valid for identifying PKC isoform-specific activators and inhibitors: The fission yeast Schizosaccharomyces pombe under a thiamine-repressible promoter expresses either a conventional isoform of PKC (PKC-y) or a novel isoform PKC-5. When the authors compared the effects of bryostatin 1 on the growth of the PKC-y and PKC-5 transformants with those of TPA, they observed that bryostatin 1

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activated PKC-5 but inhibited PKC-y activity [24]. In contrast with long-term TPA treatment, which induces neuronal differentiation through downregulation of PKC activity, it has been demonstrated that bryostatin 1, under similar conditions, increased the levels of PKC-s. This isoform is implicated in phosphorylation of the microtubule-associated protein tau and in neuritogenesis. Treatment with TPA induces neuritogenesis, while treatment with bryostatin 1 for 72 h increases tau phosphorylation and inhibits neuritogenesis [25]. Other differences between phorbol esters and bryostatin 1 have been observed in intestinal transport and barrier function. Bryostatin 1 reduced CI" secretion, Na -K -Cl" cotransporter, and cotransport mRNA expression. Unlike phorbol esters, these effects were largely transient. The barrier function was not affected by bryostatin 1 in contrast with what happened with phorbol esters. These differences imply that bryostatin 1 and phorbol esters affect PKC isoforms involved in junctional regulation and that epithelial transport and barrier function may be regulated by different PKC isoforms [26].

Other activators

Daphnoretin (4) is a dicoumarin isolated from Thymelaceous plants, {Wikstroemia indica. Daphne mezereum and D, cannabind) that directly activates PKC, which in turn elicits the respiratory burst in rat neutrophils.

MeO

4 Daphnoretin

This effect was greatly reduced by the PKC inhibitor staurosporine, which means that PKC plays a major role in daphnoretin-induced respiratory burst. Daphnoretin, like TPA, increased the membrane associated PKC activity in neutrophil suspension. It is possible that daphnoretin, like TPA, may bypass the membrane receptor and act as a PKC activator. Daphnoretin reduced the [ H]PDBu binding to PKC in a

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concentration-dependent manner, which implies that daphnoretin may bind to the phorbol ester binding site in the regulatory domain of PKC and lead to its direct activation. The specificity of the dicoumarin for certain PKC isoforms is not yet known [27].

Kazinol B (5), a natural isoprenylated flavan isolated from Broussonetia papyrifera (Moraceae), induces the stimulation of respiratory burst in rat neutrophils. This effect is probably mediated by the synergism of PKC activation and [Ca ]i elevation in rat neutrophils. The membrane-associated PKC-a and PKC-0 were increased following the stimulation of neutrophils with kazinol B. The conventional and novel PKC isoforms might contribute to the PKC activation upon exposure of cells to kazinol B. In addition, the novel PKC isoforms may play the major role since these are more sensitive and more rapidly activated by kazinol B than the conventional isoforms [28].

5 Kazinol B

6 Decursin

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From the roots of Angelica gigas and A. decursiva (Umbelliferae) the pyranocoumarin decursin (6) was isolated. This compound showed cytotoxic activity against several human cancer cell lines and in vitro PKC activation. The PKC from rat brain used in this assay was mainly composed of conventional isoenzymes (PKC-a, -p and -y). 10 |ag/ml of decursin activated in vitro PKC activity in presence of intrinsic activators such as 10 mM Ca ^ and 10 |ig/ml phosphatidylserine [29].

The MeOH extract from the rhizomes of Iris tectorum (Iridaceae) gave a spiroiridal triterpenoid, 28-deacetylbelamcandal (7). This substance stimulated differentiation of human promyelocytic leukaemia (HL-60) cells. It inhibited the specific [ H]PDBu binding to PKC, similarly to TPA in a dose-dependent fashion. In addition, 28-deacetylbelamcandal directly enhanced PKC activity [30].

7 28-Deacetylbelamcandal

The effect of nicotine, the main principle of tobacco, on PKC activity was measured as a function of time. At a concentration of 100 nmol/1, nicotine caused an increase in PKC activity in endothelial cells from human adult CNS. The increase in PKC activity was significant in 30 s and attained maximum levels at 2 min. In order to assess the significance of the nicotine-induced PKC activation in the observed increase in plasminogen activator inhibitor-1 (PAI-1) production, the effect of nicotine was measured in the presence of the PKC inhibitor GF-109203-X. In these conditions, nicotine had no effect on PAI-1 mRNA levels. Similar results were obtained with another PKC inhibitor (calphostin C), demonstrating that in CNS-endothelial cells PAI-1 mRNA expression and protein production are dependent on the activation of PKC [31].

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NATURAL INHIBITORS OF PROTEIN KINASES

Introduction

There are different sites in protein kinases that may be considered pharmacological targets for selective inhibitors. a) The binding sites for the activator molecules of the serine and

threonine protein kinases. b) The region involved in the inactivation of enzyme in absence of

activator molecules. c) The ATP site on the catalytic domain of the protein kinases. This ATP

binding site and much of the rest of the catalytic domain of protein kinase C shows striking homology with ATP binding sites of the other serine and threonine-specific kinases and even of the tyrosine-specific kinases. This means that selective inhibitors at this level are unlikely to be found.

Although the biochemical functions and the stereochemistry of the enzyme active sites are completely established, and many natural and synthetic compounds have been assayed for their inhibitory activity, there is no an unique or preferred structural type for the highest effectiveness. In this review we have classified the active compounds in phenolics, terpenoids, alkaloids, and other principles.

Phenolic compounds

Phenylpropanoid and phenylethanoid glycosides

One of the most widespread examples of phenylethanoid glycosides is verbascoside, also called acteoside, which has been obtained form sources belonging to different plant families. This compound, isolated from Lantana camara (Verbenaceae), was characterised as an inhibitor of rat brain PKC, with an IC50 value of 25 |LIM. This effect was abolished by adding ATP, which indicated a competitive interaction with the nucleotide. The inhibition was non-competitive with respect to the phosphate acceptor, histones type HIS in this case, but other kinases, such as PTK from a lymphoma cell line or PKA, were not inhibited. In order to translate the biochemical effect to a related cellular event, the ability of verbascoside to reduce the proliferation of the lymphocytic mouse leukaemia L-1210 cells was examined. This compound showed an IC50 value of 13 |LIM [32].

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A number of closely-related phenylethanoid glycosides isolated from Digitalis purpurea and Penstemon linarioi (Scrophulariaceae) were studied for their inhibitory activity against recombinant human PKCa, using glycogen synthase peptide as a substrate. An IC50 of 9.3 |iM was reported for verbascoside itself, whereas calceolariosides A (8) and B, and forsythiaside, from D. purpurea, were more potent, with IC50 ranging from 0.6 to 4.6 |LiM. Other phenylethanoids oiPenstemon, like leucosceptoside and poliumoside, showed lower potency [33].

8 Calceolarioside A

Other PKC-inhibitory phenolic saccharide conjugates, which are not true glycosides and contain cinnamic moieties, are vanicosides A (IC50 = 44 |LiM) and B (IC50 = 32 |LIM), from Polygonum pensylvanicum (Polygonaeceae). These are mono-feruloyl-tri-/7-coumaroyl-esters of sucrose, differing only in that vanicoside B presents an acetyl group on glucose C-2 [34].

Oligomeric catechins and tannins

In a study by Polya and Foo, twelve polyphenols of increasing complexity, sharing the catechin or epicatechin structure, were evaluated for their effect on the activity of PKC, PKA-catalytic subunit and MLCK. In general, rat brain PKC was the most sensitive to polyphenol interaction, given that eight of the inhibitors had IC50 values below 10 |jM. The compounds that showed the highest potency were the procyanidins obtained from the bark of Pseudotsuga menziesii (Pinaceae), and among these, the one possessing the highest degree of polymerisation was a tetramer of epicatechin with repeated 4-8 linkages (9) (IC50 = 0.6 |LIM).

838

OH

OH

9 An epicatechin tetramer from Pseudotsuga meinziesii

This compound behaved as a non-competitive inhibitor with respect to ATP and to the substrate, EGF-derived synthetic peptide. Other quite potent agents were two single 5-deoxy-6,8-dihydroxyepicatechins differing in the orientation of the 4-hydroxyl group, which were isolated from Acacia melanoxylon (Leguminosae) heartwood, and two catechin esters of 3-hydroxy-5-(3,4-dihydroxyphenyl) pentanoic acid from the cladodes of Phyllocladus trichomanoides (Podocarpaceae). Most of the assayed substances were effective inhibitors of bovine heart PKA-catalytic subunit, and again the highest potency corresponded to compound 9. According to the authors, it seems that the presence of a voluminous substitution embracing C-7 and C-8, or at C-3 or C-4 is detrimental to the inhibitory activity of monomers [35]. In order to identify the mechanism underlying the antiproliferative effect of some naturally occurring polyphenols on

839

vascular smooth muscle cells, epigallocatechin (10) was evaluated for its PKC and PTK inhibitory activities by using cultured rat aorta A7r5 cells. This substance, one of the major constituents of green tea, exerted at 10" M a faint inhibition (19.3 %) of membrane-associated PTK activity, but this effect disappeared at 10" M. Epigallocatechin was found to be inactive against cytosolic PKC activity both in presence and absence of TPA. However, it managed to reduce substantially the serum-stimulated expression of the protooncogene c-jun, measured by the corresponding mRNA level, which is an important indicator of growth factor stimulation. Furthermore, epigallocatechin acts as an inhibitor of JNK. For that reason, activity of the c-Jun protein itself is limited, and the antiproliferative effect is partially explained [36].

OH

OH

H O ^ , ^ ^ , ^ . ^ ^ , ^ ^ ^ ^ , ^ " ^ " ^ ^ * O H

10 Epigallocatechin

The effect produced by catechin structurally-related tannins (condensed tannins) was determined for eighteen plant tannin preparations. When the results on PKC, PKA and MLCK were considered globally, the most active among the screened extracts were those obtained from the leaves of Grevillea robusta (Proteaceae), Ribes sanguineum and K rubrum (Grossulariaceae), the fruits of Vaccinium corymbosum (Ericaceae) and from Lotus corniculatus (Leguminosae). For each of the tannins studied the potency diminished according to the sequence PKA > PKC > MLCK. It was further observed, with few exceptions, that the potency as PKA inhibitors (IC50 between 9 and 70 nM) correlates positively with the proportion of trans forms in C-2/C-3 and also with the proportion of prodelphinidin units. The most potent product, Ribes rubrum tannin, acts as a competitive inhibitor of the rat liver PKA-catalytic subunit with respect to ATP and non-competitive with respect to the substrate peptide

840

(kemptide). The inhibitory effect on rat brain PKC was expressed by IC50 values below 1 |LIM, with a maximum potency attributed to Phoenix canariensis (Palmae) frond and Ribes nigrum leaf tannins, IC50 = 0.3 |LIM [37].

On the basis of the studies concerning the activity of the hydrolysable tannins from Phyllanthus amarus (Euphorbiaceae), it seems that their effectiveness against common protein kinases is lower than that of condensed tannins. In fact, IC50 values ranged from 0.2 to 1.7 |LIM for the rat liver PKA-catalytic subunit, and was 26 |iM for geraniin (11) and other closely-related compounds against rat brain PKC. Other tannins of the same plant inhibited PKC at the high fixed dose of 167 |LIM [38].

HO OH OH OH

HO HO

11 Geraniin

Some experiments have demonstrated that tannic acid, also called simply "tannin", a natural mixture of gallic esters of glucose classically obtained from Rhus species (Anacardiaceae), interferes with cellular activation of PKC. It inhibited, for example, the phosphorylation catalysed

841

by membrane-bound PKC and DNA synthesis, both of which events are induced by TPA in NIH 3T3 cells. It did not compete with PDBu for binding with PKC and did not affect membrane PKC translocation [39]. Tannin obtained from cotton bracts was able to inhibit histone III phosphorylation in membranes of canine airway cells and to reduce CI" secretion induced by TPA in a tracheal epithelium mucosal preparation, which suggested that inhalation of tannin by cotton manipulators may produce its deleterious effect (byssinosis) trhough PKC inhibition [40].

Flavonoids

In this category we include phenolic compounds based on the structure of 2-phenyl-4H-l-benzopyran-4-one (ftavones, flavonols and related dihydro analogues), the isomeric 3-phenyl derivatives (isoflavonoids, including isoflavans) and other non-pyronic C6-C3-C6 plant pigments such as chalcones, aurones and anthocyanidins.

One of the most frequent flavonols found in plants is quercetin (3,5,7,3',4'-pentahydroxyflavone), for which interesting inhibitory properties affecting serine/threonine and tyrosine protein kinases were reported in several studies. In addition to the effects on many other kinases, quercetin was characterised as an inhibitor of the oncogene product pp60 ' , in an ATP-competitive fashion [41]. In another study done prior to the time period covered by the present review, it was found that myricetin (3,5,7,3',4',5'-hexahydroxyflavone) exerted similar effects with even higher potency. Some of the tyrosine protein kinases tested were not affected by the presence of myricetin, namely PTK from platelet particulate fraction. Other ones, such as the oncogene product ppno^"", were competitively inhibited by myricetin with respect to ATP {Ki =1.8 |LiM), and in other cases myricetin inhibited without competing with ATP (rat liver insulin receptor, Ki = 2.6 |LIM). The inhibition of serine/threonine protein kinases was competitive for MLCK and CKI and CKII {Ki =1.7, 9.0 and 0.6 |LIM, respectively). Myricetin also inhibited PKC and PKA, though at Ki values over 10 |iM. Comparison of the potency of seven flavones and flavonols with increasing degrees of hydroxylation, from flavone itself to myricetin, revealed that the number of free hydroxyl groups correlated closely with the potency against the susceptible tyrosine protein kinases, whereas this correlation was not entirely applicable to the inhibition of serine/threonine protein kinases [42]. When the inhibition of

842

pSe ' -PTK activity was studied for the flavonoid aglycones and glycosides isolated from Koelreuteria henryi (Sapindaceae), no clear correspondence between hydroxylation and potency was found, although glycosidic forms were the least potent ones [43].

In a study on the effects on rat brain PKC, fisetin, the 5-deoxy analogue of quercetin, was found to be the most active among 15 flavonoids tested at 50 |LiM. Some chemical features such as the presence of ortho-dihydroxyl groups and a 2-3 double bond, and the absence of methoxyl groups and sugars were recognised to be essential for the activity. The inhibitory effect of fisetin was dose-dependent (IC50 = 10 |j,M) and competitive with respect to ATP {Ki ^4.6 |iM) [44].

The flavone apigenin (5,7,4'-trihydroxyflavone) was studied for its effect against several kinases in mouse NIH 3T3 cells. It competed with ATP to inhibit PKC (IC50 = 10 |LIM). In addition, it inhibited the PTK activity of the fibroblast growth factor with half the potency (IC50 = 20 |LiM), but was not active on the phosphorylation mediated by pp60''" ' '' (IC50 > 200 |LiM). Apigenin also inhibited the expression of c-fos and c-jun induced by TPA in the same cellular system [45].

In a study on human promyelocytic leukaemia HL-60 cells, quercetin reduced cellular growth dose-dependently, and abolished it at a concentration of 80 |LIM. This effect was attributed to the concomitant inhibition of membrane, but not cytosolic, PTK activity, determined on poly Glu-Tyr (4 :1) as a substrate (IC50 = 20.1 fxM). Inversely, quercetin inhibited cytosolic, but not membrane, PKC activity. As a possible result of the inhibition of phosphatidyl-inositol kinases, the production of phosphoinositides in intact HL-60 cells was greatly diminished by quercetin [46].

Activation of PKC is supposed to be a necessary prerequisite for neutrophil respiratory burst, because it mediates the phosphorylation of the proteins involved in the assembly of NADPH oxidase, which at last produces superoxide anion (O2") from external oxygen. The isoprenylflavone cycloheterophyllin (12) isolated from Artocarpus heterophyllus (Moraceae) was known to inhibit that respiratory burst, and its interaction with PKC was therefore studied.

Enzyme preparations obtained from rat neutrophil cytosol were inhibited very slightly by cycloheterophyllin, whereas those obtained from rat brain were moderately dose-dependently inhibited by the same compound at doses between 6 and 60 |LIM. Some degree of competition

843

with PDBu for PKC binding (30 % inhibition) was observed with cycloheterophyllin at 60 |LIM, but this did not affect translocation to the membrane [47].

Me>^ ^ M e

OH

12 Cycloheterophyllin

Although strictly speaking phosphatidylinositol 3-kinase (PI3K) is not covered by the present review, it deserves particular attention because after stimulation by growth factors, it contributes to the generation of PIP2 and PIP3, which are resistant to the hydrolysis by PLC, and its increased levels are associated with cellular transformation induced by diverse oncogenes. Given that the modulation of other kinases by flavonoids was already known and also presumably involved in their antitumoral properties, the interactions with PI3K were investigated. Quercetin was reported to be a potent inhibitor of bovine brain PI3K (IC50 =1.3 |jM), whereas different chemical modifications on the flavonoid structure led to a decrease in activity [48]. At a fixed concentration of 60 |iM, a short series of structurally dissimilar flavonoids were tested for their inhibition of PI3K fi-om human platelets, and other kinases. As quercetin and luteolin (5,7,3',4'-tetrahydroxyflavone) produced the highest inhibition (near 90 %) of PI3K, whereas catechin, genistein (13) (an isoflavone) and hesperetin (a flavanone) were inactive, the study was broadened to include other flavones and flavonols. Myricetin practically abolished PI3K activity, while galangin and chrysin, both lacking hydroxyl groups in the phenyl radical, had no effect, and apigenin and diosmetin (4'-methylluteolin) showed significant inhibition of the enzyme activity. On a dose-response

844

analysis, myricetin showed an IC50 value of 1.8 |iM and luteolin of 8 |LIM. An analogous profile was observed for the inhibition of bovine brain PKC, with an inversion in the magnitude of the effects of apigenin and kaempferol (3,5,7,4'-flavone).

However, when the flavonoids were assayed on A431 carcinoma cells for PTK activity associated with EGF overexpression, genistein (13) (as expected) and kaempferol were the most active compounds, ahead of myricetin and diosmetin [49, 50].

One of the most typical chemical characteristics in the Leguminosae is the production of isoflavones and related compounds, which often confer pharmacological or toxicological properties on plants of this family. Species of the genus Derris, for example, have insecticidal activity because of their rotenone content. One of them, D. scandens, is a good source of several prenylisoflavones, which were studied together with other coumarins and simple isoflavones as inhibitors of certain kinases. The most sensitive of the enzymes tested was PKA (catalytic subunit), as it was potently inhibited by warangalone (14) (IC50 = 3.5 |LIM). The isomers 8-and 3-dimethylallylwighteone showed IC50 values below 30 JLIM. Nallanin (15) was the most potent of the isoflavones against PKC (IC50 =120 |LIM).

Robustic acid (16) was the only coumarin that potently inhibited a kinase activity. Its IC50 for PKA inhibition was 10 |j.M, whereas other coumarins showed IC50 values higher than 100 |LIM for inhibition of any of the kinases tested [51].

Genistein (13) is a compound massively present in some foods, like soybeans, and well known for its estrogenic and PTK inhibitory properties. For these two reasons, genistein is considered to prevent hormone-dependent breast cancer and strongly inhibit the mitogenesis induced by growth factors like EGF and TGF-P, with positive implications in the amelioration of osteoporosis and haemorrhagic telangiectasia. However,

845

the range of concentrations at which this isoflavone impairs the growth of tumoral cells is substantially lower than the concentration at which it inhibits PTK activity. This suggests that although the activity of tyrosine protein kinases is involved in the cellular effects of genistein, it does not occur by, or at least not only by, a direct inhibitory mechanism [52]. In a study on the proliferative effects of angiotensin II on rat aorta smooth muscle cells, genistein inhibited the incorporation of [ HJthymidine to cells, which is an indicator of DNA synthesis inhibition. Moreover, genistein was a weak inhibitor of MAPK [53].

Mes ,Me

®v»

e >r°v

y

k^°\ kAAyA

o

14 W ara ngalone

15 Nallanin

846

OMe OH

16 Robustic acid

OMe

Glabridin (17) is an isoflavane derivative present in liquorice root that has been investigated for its ability to modify the chemical degradation of low density lipoprotein (LDL) by macrophages, an event strongly linked to the genesis and progression of atherosclerosis. After incubation with 20 laM glabridin, it is incorporated into the murine peritoneal macrophages and there produces a pronounced inhibition of both cytosoHc and, especially, membrane PKC. This PKC inhibition is thought to be a crucial step in the inhibition of the assembly of NADPH oxidase, an enzyme responsible for the oxidation of LDL by macrophages. Methylation of the two free hydroxyl groups of glabridin leads to a loss of the effect [54].

OH

17 Glabridin

Butein is a simple chalcone formed by the union of a phloroglucinol ring with a catechol one through a propenone chain. It has been characterised as a PTK specific inhibitor, because it reduced the extent of the EGF-induced phosphorylation of the EGF receptor localised in human hepatocellular carcinoma cells, and the activity of the soluble EGF receptor and of p60''"'" . In contrast v^th other flavonoids, butein was ineffective against serine/threonine protein kinases [55].

847

Coumarins

Among the coumarins isolated from species of Artemisia (Asteraceae), esculetin (6,7-dihydroxycoumarin) and its dimethyl-ether scoparone were known to be antiproliferative on vascular smooth muscle cells. This activity was further found in some very simple mono-substituted, coumarins, which were even more potent than esculetin, although less effective. In an attempt to verify its mechanism of action, esculetin was tested for interactions with PTK and PKC. The induction of membrane PTK activity by either foetal calf serum or platelet-derived growth factor (PDGF) was moderately reduced by esculetin, whereas no effect was observed against PKC [56].

Daphnoretin (4) was characterised as an inhibitor of PTK. It managed to inhibit the tyrosine kinase activity of the EGF receptor from A431 human epidermoid carcinoma cells, with an IC50 value of 97.5 |LIM. However, it showed cytotoxicity on four different proliferating cell lines. The authors were cautious in ascribing a particular value to daphnoretin as tumour chemopreventive [57]. The resuks obtained for daphnetin (7,8-dihydroxycoumarin) on the same activity on soluble EGF receptor (IC50 = 7.7 |a.M) were of great interest because no inhibition of the EGF-induced phosphorylation of this receptor in hepatocellular carcinoma cells was observed. The inhibition of PTK activity was competitive with respect to ATP and non competitive with respect to the synthetic substrate. Daphnetin was also quite potent against PKA (IC50 = 9.3 \xM) and PKC (IC50 = 25 laM), practically abolishing their activities at 200 |iM. In the same study, the paucity of the effects of esculin and three simple coumarin monohydroxyl derivatives was demonstrated, and this made the importance of the 7,8-dihydroxyl substitution clear [58].

Dicoumarol is an artefactual coumarin formed by putrefaction of ensiled sweet clover and has been employed as an anticoagulant because it inhibits quinone reductase activity and, therefore, the function of vitamin K in the way of synthesising coagulation factors. Given that quinones are involved in redox systems affecting mitogenic kinase cascades, dicoumarol was studied for its interaction with some of these enzymes. In fact dicoumarol prevented the activation of SAPK and of nuclear factor-KB (NF-KB) [59].

848

Xanthones

The first natural xanthones described as kinase inhibitors were mangostin and y-mangostin, two diisoprenyl derivatives isolated fi*om the fiaiits of Garcinia mangostana (Guttiferae) and endowed with inhibitory activity against PKA fi-om rat liver in vitro, with IC50 values of 13 |iM and 2 [iM, respectively [60]. Much more extensive is the research on norathyriol (18), which was obtained fi-om the aerial parts of Tripterospermum lanceolatum (Gentianaceae) and later characterised as a vasorelaxant, anti-aggregant and anti-inflammatory agent. Norathyriol was demonstrated to inhibit TPA-induced neutrophil respiratory burst and aggregation, because of its PKC inhibitory activity in these cells. The xanthone reduced in a dose-dependent manner the activity of rat brain and neutrophil cytosolic PKC, and in the latter had this effect on both complete and trypsin-treated (deprived of its regulatory domain) forms. Although the IC50 values were not calculated, an inhibition of nearly 50 % was observed on the trypsin-treated preparation at 100 |LIM. Norathyriol acted non competitively with ATP and was unable to affect PKC-P translocation to the membrane or PDBu binding [61]. Further studies with cultured rat heart endothelial cells revealed microscopically that norathyriol induces notorious changes in the serotonin-induced effects on the localisation of several PKC isoenzymes, particularly a and 0 forms. In addition, it decreased serotonin-induced translocation of PKC-a to the membrane [62].

OH

18 Norathyriol

849

QH OH = COOMe MeOOC V"

19 Secalonic acid

Secalonic acid (19) is a fungal phenolic metabolite that can be formally considered a bis-tetrahydroxanthone and is found in Claviceps purpurea and Penicillium oxalicum. Due to its demonstrated teratogenic properties, this substance may make foods infested by the mentioned funguses toxic. In a survey of well-known natural food- and environment-related teratogens for their serine/threonine kinase inhibitory activity, Wang and Polya [63] found that secalonic acid was a potent inhibitor of rat brain PKC (ICso = 15 MM) and of the catalytic subunit of rat liver PKA (IC50 = 12 |a,M), and that it was also active against MLCK (IC50 = 60 |LIM). The authors suggested that by analogy with other kinase inhibitors, these effects could be related to the teratogeny, but the link between the two events must be demonstrated.

Quinones and related compounds

In this chapter, we include certain natural products, such as typical anthraquinones and naphthoquinones, which are present, along with condensed dimeric structural drivatives such as dianthrones, in many medicinal plants. In addition, we deal with the much more infrequent or///o-quinones.

Emodin (l,6,8-trihydroxy-3-methylanthraquinone), the active principle of^ Polygonum cuspidatum (Polygonaceae), was reported to be an inhibitor of the p56 '' -PTK activity from bovine thymus, with an IC50 of 18.5 piM. When the hydroxyl functions at C-6 or C-8 were blocked by methylation or glycosylation, respectively, the effect disappeared. The inhibition was competitive with respect to ATP and non competitive with respect to the substrate [64]. In a bioassay-guided separation of the anthraquinones found in rhizomes of another Polygonaceae species, rhubarb {Rheum

850

palmatum), emodin was again the main active principle when tested against the activity of several serine/threonine protein kinases, such as PKA, PKC, cyclin B/cdc2 kinase (cdc2), CKI and CKIL Emodin was highly potent and selective for CKI and CKII, with IC50 values of 7 |LIM and 2 |jM, respectively. Rhein, the carboxyl superior homologue of emodin, showed an IC50 value of 7 \JM for its CKII inhibition. Considering that CKII accumulates in the nuclei of growing cells and seems to be crucial for both normal and oncogenic proliferation, the inhibitory effects of emodin may correlate with its mouse antileukaemic and the traditional anticancer use of rhubarb in Asia [65].

Hypericin (20) is a natural phenolic naphtodianthrone pigment found in Hypericum species and known for its photosensitising, antiviral and inhibitory properties of both serine/threonine and tyrosine kinases. Hypericin inhibited irreversibly the autophosphorylation of EGF receptor in a time-dependent, oxygen-independent manner. Irradiation with fluorescent light caused a large increase in the potency of hypericin (IC50 = 0.044 versus 0.37 |iM). On the other hand, hypericin inhibited PKC, with an IC50 value of 3.4 |LIM, and was not active against other serine/threonine kinases [66]. Further studies by the same authors using fluorescent light irradiation established the selectivity of hypericin against membrane-bound PTKs (EGF and insulin receptors) versus cytosolic PTKs (Lyn, Fgr, c-Src kinase), and also the high potency of this quinone in inhibiting the serine/threonine kinases CKII (IC50 =" 6 nM) and MAPK (IC50 = 4 nM). Unlike what it was described for the perylene quinone calphostin C, a classical specific PKC inhibitor of similar structure, the activity of hypericin was not affected by singlet oxygen quenchers such as 1,4-diazabicyclo[2,2,2]-octane or 2,5-dimethylfuran. For this reason it was postulated that light-induced inhibition is not mediated through singlet oxygen generation [67]. Nevertheless, it should be pointed out that the oxygen-quenching interaction with calphostin C was tested using other substances such as lycopene, p-carotene or a-tocopherol [68]. The relationship between photo-irradiation and enzyme inhibitory activity was also examined by other authors, who measured the light-induced increase in the potency of hypericin as a brain PKC activity inhibitor. Light exposure did indeed influence positively the ability of hypericin to interfere with cellular activities dependent on PKC such as the generation of superoxide radical by guinea pig neutrophils [69]. In a study designed to search for the structure-activity relationships of hypericin derivatives, it

851

was demonstrated that di- or tetra-bromination of hypericin decreased its potency as a kinase inhibitor, and that exhaustive methylation augmented its potency and selectivity against PKC, but not against other serine/threonine kinases [70].

20 Hypericin

Lapachol, an alcohol that can be converted into p-lapachone (21), a prenyl-naphto-l,2-quinone known for its anti-tumoral, pro-apoptopic and topoisomerase I-inhibitory properties was obtained from the lapacho tree, Tabebuia avellanadae (Bignoniaceae). Although an inhibition of PTKs has not, to our knowledge, been reported, P-lapachone inhibited the activation of JNK and MEK by tumour necrosis factor (TNF). These effects may account for the observed inhibition of the NF-KB-mediated gene expression and the mentioned biological activity [71].

r r - ^ ^

21 p-Lapachone

852

Diarylalkanoids

In this section we report on the activity of certain diarylheptanoids, perhaps one of the most recently described type of kinase-inhibitory phenolics. Natural diarylheptanoids, like difemloylmethane (curcumin), are formed by two simple or/Zio-hydroxy substituted aromatic rings, bridged by a seven-carbon linear chain. Curcumin (22) is the main principle of Curcuma aromatica, C. longa, and C. xanthrorrhiza (Zingiberaceae) and possesses anti-tumoral, anti-inflammatory, and free radical-scavenger properties. In a study concerning PKC activity in a purified preparation from NIH 3T3 fibroblasts, curcumin caused little inhibition of enzyme activity, although it induced translocation to the membrane. The inhibition of the activity in the particulate fraction was stronger, 69 % after 30 min incubation with 15 |iM curcumin [72]. When it was tested at 100 iM for inhibitory activity against several kinases of different kinds and origin, curcumin blocked selectively and non competitively the in vitro activity of phosphorylase kinase. A dose-dependent inhibition of pp60''"''' '' PTK, culminating an almost complete inhibition at 0.6 mM, was also observed, but other serine/threonine kinases like PKC, PKA and cytosolic protamine kinase were only mildly inhibited or not inhibited at all [73]. However, it was later reported that curcumin inhibited the PKA-catalytic subunit and PKC, with IC50 values of 4.8 and 15 |iM, respectively, and the inhibition was in both cases competitive with ATP and the peptide substrate [74]. An interesting review of the biological effects of curcumin, apigenin and other phenolics in close relationship with dietary cancer prevention has been published [75].

o o MeO^ ^^s^ ^y-S^ JL JL ^^V^ ^ VW ^OMe

22 Curcumin

853

HO

HO

23 Hirsutenone

Other active diarylheptanoids found in nature were the five obtained from the stems of Pinus flexilis (Pinaceae). They have no unsaturations in the linear chain, except hirsutenone (23), and were potent inhibitors of recombinant human PKCa, with IC50 values ranging from 1.4 to 8.6 [xM [76].

Other phenolic compounds

Neolignans dijffer from true lignans in that a bonding between two phenylpropanoid moieties affects positions other than the two p-carbons of the lateral chain. A plain example of neolignans is provided by the structure of magnolol (24), which is known to be present in Magnolia officinalis (Magnoliaceae). This compound possesses a slight activity as an inhibitor of PKC from rat brain and neutrophils, without affecting PKC translocation or PDBu binding [77].

24 Magnolol

Rottlerin (25) is a chromene based on a prenyl-phloroglucinol structure and isolated from the ancient vermifuge drug kamala {Mallotus philippinensis, Euphorbiaceae). It demonstrated efficacy in inhibiting PKC activity competitively with ATP, although the potency varied notably depending on the isoforms. The IC50 value was 3 jiiM for the inhibition of

854

porcine spleen PKC-6; 30, 40 and 42 |iM for PKC-a, y and P from baculovirus-infected Sf9 insect cells, respectively, and higher than 80 |LIM for the atypical PKC s, rjand Q. Calmodulin kinase III (CAMK-III), an enzyme that catalyses the phosphorylation of the elongation factor-2 (EF-2), was also potently inhibited by rottlerin (IC50 = 5.3 [a,M), and not by the PKC inhibitor staurosporine at equivalent doses [78, 79]. It must be noted that EF-2 is insensitive to phosphorylation by kinases other than CAMK-n, participates in the GTP-mediated ribosomal translocation of the peptidyl-tRNA and is therefore a key element for protein synthesis in eukaryotes.

25 Rottlerin

More recently, it has been shown that rottlerin inhibits the migration towards the membrane of PKC-a and -5, diminishes the binding to DNA of the transcription factors AP-1 and NF-KB, and subsequently affects cytokine production by monocytes [80].

Some simple stilbenes isolated from the roots of Polygonum cuspidatum (Polygonaceae) showed moderate inhibitory activity of the bovine thymus P56 '' tyrosine kinase activity, when angiotensin 1 was used as a substrate. The most potent were resveratrol (26) and its cis form obtained by photochemical isomerisation. Both compounds showed similar potency against rat brain PKC, whereas resveratrol monoglucosides were less active [81].

855

HO

OH

OH

26 Resveratrol

Other active stilbenes of greater chemical complexity were the oligomers (tri- and tetramers) obtained from the roots of Canagana sinica (Leguminosae). They were tested for their in vitro inhibitory activity against different PKC isotypes. (+)-a-Viniferin was the most potent compound on a, y, and 6 isoforms, whereas miyabenol C was the strongest on P and s isoforms; the minimal IC50 values for each enzyme ranged from 7 to 37 |j,M for the stilbenes, and 0.02-0.06 for staurosporine. When cellular functions that are closely linked to PKC activation, like whole blood respiratory burst, neutrophil superoxide generation, and lymphocyte proliferation were studied, (+)-a-viniferin manifested the highest potency of the three active oligomers [82].

Gossypol (2,2'-bi [8 - formyl - 1,6,7 - trihydroxy - 5 - isopropyl - 3 -methylnaphtalene]), a compound known for years for its spermatocidal activity, is present in some species of Malvaceae, such as Gossypium herbaceum (cotton) and Thespesia populnea, a plant which is employed as an anti-inflammatory for external use. Gossypol produces apoptosis of cultured rat spermatocytes in the same range of concentrations as that producing inhibition of PKC. As PDBu protected the cells from gossypol-induced DNA degradation, it is thought that the enzyme preserves cell integrity [83].

Terpenoids

Sesquiterpenoids

The ethyl acetate extract of the sponge Aka coralliphagum was fractionated in order to isolate the components able to inhibit PKC activity. The responsible substances were identified as a mixture of two diastereomeric spirosesquiterpene aldehydes, corallydictyals A (27) and B. The IC50 for inhibition of PKC was 28 |LIM, while the corresponding

856

methyl ethers were inactive. Using four purified recombinant human isoforms of PKC (a, 8, r| and Q it was possible to show that the inhibitor was selective for inhibition of the a isoform (IC50 = 30 |LIM) [84].

Me

HO 0

<^J^CHO

^ ^ ^ ^ ] 0 J '"'Me

^ \ H "Me

27 Corallidictyal A

Stimulation of macrophages by lipopolysaccharide (LPS) results in activation of members of MAPKs, ERKl and ERK2. The main sesquiterpene lactone fi'om Tanacetum parthenium (Asteraceae), parthenolide (28), suppressed LPS-stimulated tyrosine phosphorylation of various proteins in RAW 264.7 cells. Of these proteins the MAPKs exhibited the most dramatic inhibition in response to parthenolide.

28 Parthenolide

Tyrosine phosphorylation of three MAPK subgroups (ERKl, ERK2 and P38) stimulated by LPS was inhibited by 28 in a dose dependent manner. It has been proposed that tyrosine kinase inhibition may occur through conjugation between the a-methylenebutyrolactone in parthenolide and the -SH group of target proteins [85].

857

Diterpenoids

The antineoplastic effect of the diterpene taxol (29) is know to be directly linked to its ability to stabilise microtubule structure and arrest cells in the G2/M phase of the cell cycle. Recently it has been demonstrated that taxol selectively inhibits TPA-induced NF-KB activation via inhibition of phosphorylation and subsequent degradation of an inhibitory protein named iKBa,. Furthermore, the PKC activity induced by TPA was partially inhibited in presence of taxol. Many studies have shown that TPA provokes changes in the microtubule cytoskeleton by activating PKC. It seems that the TPA pathway that leads to phosphorylation/degradation of iKBa and activation of the NF-KB dependent gene is comprised of a PKC-mediated MAPK-dependent pathway. Taxol stabilisation of microtubules may be sufficient to prevent TPA-stimulated iKBa phosphorylation thus maintaining NF-KB in a latent cytoplasmic state. On the other hand, taxol may modulate PKC activity directly; TPA stimulation gives the activation of a number of PKC isoforms, among which are PKC-a and PKC-C, which appear to be important in NF-KB induction. Since a reduction in PKC activity was observed in taxol treated cells, this result may indicate a specific decrease in the activity of taxol sensitive PKCs and not an attenuation of all PKC family members [86].

OCOMe Q

OCOMe

29 Taxol

Treatment of human leukaemic U937 cells with 20 nM of taxol for 24 h resulted in an 80% growth inhibition three days later. Kinetic studies of the cell cycle progress revealed that taxol accelerates the progression of

858

the cell cycle, which facilitates the process of apoptosis. This acceleration may result from the transient activation of p42/44 MAP kinase, because inhibition of upstream MEK by a protein kinase inhibitor such as PD98059 reverses this effect [87].

MeCOOi,

MeOCHj

30 Wortmannin

Wortmannin (30) is a fungal metabolite isolated from the strain Talaromyces wortmannii, and is a potent specific inhibitor of MLCK. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. It seems that wortmannin acts at or near the catalytic site of the enzyme. However, this compound has no inhibitory effect on PKA and CaMK II, and has little effect on PKC activity [88].

Triterpenoids

The biological effects of triterpenoids include cytotoxic, anti-tumour and anti-inflammatory activities. It may be that the triterpenoids act by interacting with signal-regulated protein kinases, thus producing these pharmacological activities. In order to study the inhibition of protein kinase by triterpenoids, four eukaryote protein kinases — wheat embryo Ca^^-dependent protein kinase (CDPK), avian gizzard calmodulin-dependent MLCK, rat liver PKA, and rat brain PKC —, were screened. The compounds proved to be potent, and selective inhibitors of PKA-catalytic subunit. The plant-derived 18a- (31) and 18p glycyrrhetinic acid, ursolic acid, oleanolic acid and betulin were inhibitors of PKA-catalytic subunit, with IC50 values in the 4-20 |iM range. However, lithocholic acid (32), an animal bile-derived steroid, was the most potent inhibitor in this study (IC50 = 4.2 |LiM). The active triterpenoids also inhibit PKC but with

859

ICso values 10-20 times greater than for PKA-catalytic subunit. The common feature of these amphiphilic triterpenoids is a 3-hydroxy group, a polar distal residue (e.g. a carboxyl group) and a non-polar, quasi-planar triterpenoid nucleus [89]. However, these structural features are not essential for potent inhibition of PKA-catalytic subunit.

HO

COOH

Me''' Me

31 18-a-Glycyrrhetinic acid

HO"'

COOH

32 Litocholic acid

The hydrophobic triterpenoids a-amyrin and lupeol (both having a 3-hydroxy as the only polar group) and their palmitate and linoleate esters are similarly potent and relatively selective inhibitors (IC50 = 4-9 |LIM).

These triterpenoids are in vivo anti-inflammatory agents, and their relatively selective PKA inhibition activity suggests that PKA could be involved in the inflammatory process ahhough the responsible mechanisms are not yet clear [90].

The effect of the major saponin from Panax ginseng (Araliaceae), ginsenoside Rbi (33), was investigated on rat liver protein phosphorylation. It was observed that 118, 63 and 34 kDa proteins were

860

phosphorylated in liver homogenates prepared from CCU-administered rats, while these protein phosphorylations were inhibited in the homogenate prepared from the ginsenoside Rbi-treated group. Ginsenoside Rbi is involved in the inhibition of Ca ^ accumulation and glycogen reduction induced by the in vivo treatment of CCI4. It seems that ginsenoside Rbi inhibits the CCU-induced protein phosphorylation by modulating CaMK rather than PKC. However, it is not yet clear whether the action of ginsenoside Rbi on the CCU-induced phosphorylation of 34 kDa is mediated by phosphoprotein phosphatase or CaMK [91].

33 Ginsenoside Rbi

Protein phosphorylation is necessary for the initiation of cell proliferation. Myristoylated alanine-rich C kinase substrate (MARCKS) protein is one of the major PKC substrates. MARCKS phosphorylation may contribute to the morphological changes requiring the rearrangement

861

of cytoskeleton, including the regulation of the cell cycle. It was found that ginsenosides Rhi and Rh2 inhibited cellular proliferation in NIH 3T3 fibroblasts. Both ginsenosides reduced PLC activity, with the subsequent decrease in the level of DAG. It was also observed that treatment of cells with Rhi or Rh2 reduced the PKC activity. In addition, it was shown that phosphorylation of MARCKS was inhibited by the ginsenosides. Although these mechanisms could explain the antiproliferative effect of ginsenosides, the elucidation of the biochemical mechanism by which this occurs demands further investigation [92].

Na O3SO

OSOs'Na

34 Halistanol trisulphate

In a systematic screening for protein tyrosine kinase inhibitors from marine organisms [93], an extract fi*om a Topsentia sponge showed potent activity against pp60''' ' '', an oncogenic protein tyrosine kinase. The active component was identified as halistanol trisulphate (34), a sulphated steroid with an IC50 of about 4 |iM against pp60''"'" . Acid hydrolysis to remove the sulphate groups yields the inactive tris-alcohol, halistanol. Kinetic studies of inhibition revealed that halistanol trisulphate is a competitive inhibitor with respect to the peptide substrate [Val^]-angiotensin II, and a mixed inhibitor with respect to ATP.

From the finit of Crataegus pinnatifida var. psilosa (Rosaceae) several triterpenoids were isolated, among them corosolic acid (35). This compound displayed potent cytotoxic activity similar to that of ursolic acid against several human cancer cell lines. The ED50 of corosolic acid was 0.4-5.0 |ig/ml depending on the cell line. The cytotoxic effect of corosolic acid was probably due in part to the inhibition of PKC activity because it

862

displayed an antagonistic effect on the morphological change in k-562 human myelogenous leukaemic cells induced by phorbol esters. An in vitro PKC assay showed that corosolic acid inhibited PKC activity with dose-dependent pattern, thus indicating that its cytotoxicity could be strongly related to PKC inhibition [94].

Me

COOH HO„

HO Me'" ^Me

35 Corosolic acid

Alkaloids

Indole alkaloids

Staurosporine (36), an indole carbazol alkaloid isolated from Streptomyces staurosporeus was considered the most potent protein kinase inhibitor until the discovery of balanol. Staurosporine is not a selective inhibitor because it also inhibits PKA, PKG and tyrosine kinases at similar concentrations [1]. This compound has significant cytotoxic and antiproliferative effects in vitro and several of its related analogues show antitumour activity in animal models. In addition, staurosporine and derivatives have been used to explore the role of PKC in cell functions. For instance, Jordan et al [95] studied the ability of staurosporine and other PKC inhibitors to affect TNFa and interleukin-la (IL-la)-induced chemokine gene expression and protein production in synovial fibroblasts. In these circumstances, staurosporine enhanced IL-la-induced chemokine mRNA production. A possible explanation for this result is that the mechanisms of gene expression could be negatively regulated by different isoforms of PKC. [95]. Previously it had been observed that staurosporine

863

was able to double the secretion of IL-8 protein in the U937 monocytic cell line [96] and to enhance PGE2 production in synovial fibroblasts [97].

NHMe

36 Staurosporine

On the other hand, staurosporine analogues do not exhibit specificity for particular PKC isoenzymes, but they inhibit cPKC isoenzymes more potenly than n- and aPKCs. [98]. UCN-01 (a 7-hydroxylated metabolite of staurosporine) blocks cells in the Gl phase by promoting accumulation of dephosphorylated retinoblastoma protein as a consequence of the inhibition of the activity of certain CDKs, downregulation of their partner cyclins and an increase in the expression of CDK inhibitor proteins [99].

In order to understand in detail the mode of inhibition and the parameters of high affinity binding of staurosporine to protein kinases, the molecule was cocrystallised with the catalytic subunit of PKA. The study of the crystal structure of this complex allowed to detect the catalytic subunit with staurosporine molecule bound to the adenosine pocket leading to notable induced-fit structural changes of the enzyme and to an open conformation [100].

Isoquinoline alkaloids

The benzophenanthridine alkaloid chelerythrine (37) isolated fi*om Zanthoxylum simulans (Rutaceae) is a potent, selective antagonist of PKC fi-om rat brain (IC50 = 0.66 |LIM). Inhibition was competitive with respect to the phosphate acceptor (histone Ills) and non-competitive with respect

864

to ATP [101]. In addition to phosphorylation inhibition, translocation of PKC from cytosol to membrane is recognised to be an essential process in the subcellular signalling pathway. Translocation from cytosol to the membrane of PKC-a and PKC-P in myenteric synaptosomes stimulated with TPA significantly decreased in presence of chelerythrine in a way similar to that of staurosporine. Inhibition was concentration-dependent. However, the concentration necessary for this was higher than that required to inhibit PKC phosphorylation. According to the results obtained, it seems that there could be an additional mechanism for the inhibition of PKC by chelerythrine. [102].

MeO''

OMe

37 Chelerythrine

However, contradictory data have recently been reported for this alkaloid and a related benzophenanthridine alkaloid namely angoline. In searching for novel natural cancer chemopreventive agents, this alkaloid was obtained from the stem extract of Macleaya cor data (Papaveraceae). This extract had previously been shown to antagonise the interaction of PDBu with PKC. In these conditions neither substance was an effective or selective inhibitor of partially purified calf brain PKC. In fact, to the contrary, when chelerythrine was evaluated with the cytosol fractions derived from rat and mouse brain, this alkaloid enhanced PKC activity. Angoline and chelerythrine were evaluated for their potential to facilitate translocation of PKC a, -p and -y with cultured HL-60 cells, but no significant effects were observed. However, with the cultured ME 308 cell system, chelerythrine stimulated TPA-induced ornithine decarboxylase (ODC) activity at lower concentrations, whereas angoline was inactive at lower concentrations but reduced activity by approximately 50% at a test concentration of 1 |i,g/ml. Inhibitors of ODC activity are of interest because this enzyme is a key component regulating intracellular

865

polyamines, which play essential roles in normal cell proliferation and differentiation and are overexpressed in various cancer cells [103].

The regulatory amino acid taurine in the retina requires an efficient uptake system to maintain the high physiological concentration of taurine in the retina. Stimulation and inhibition of PKC activity with TPA and with staurosporine, respectively, produced no significant effect on taurine uptake. On the other hand, chelerythrine significantly inhibited the taurine uptake systems, presumably through a PKC-independent mechanism [104].

Me OH

X^ ^Y^ OH \M

" II ^ OH

rWr OMe OH ^ ^ . X ^

HO,, ^^ 1 M

1 1

KX OH

38 Michelh imineC

OMe

^ 1

^^^ Me

X . ^®

^ ^ N H

Me

The dimeric stereoisomer alkaloids michellamines A, B and C (38) were obtained From the liane Ancistrocladus korupensis (Ancistrocladaceae). On the basis of their structural similarity to other PKC inhibitors, they have been studied for this activity. Michellamines inhibited rat brain PKC, with IC50 values in the 15-35 |aM range. Michellamine B was a non-competitive PKC inhibitor with respect to ATP, whereas mixed-type inhibition was observed when the peptide concentration varied. The results indicate that the dimeric alkaloids bind to the PKC kinase domain and not to its regulatory domain. All three michellamines blocked both the ATP and the

866

peptide substrate subsites through the binding of alkaloids in the active sit( cleft of the PKC kinase domain [105].

MeO Me

MeO

OH

39 Boldine

MeO

40 Bulbocapnine

A range of alkaloids including isoquinoline, indolizidine, benzazepine oxazine, quinoline and indole alkaloids were examined as potenti^ inhibitors of eukaryote protein kinases such as PKC, MLCK and PKA Only three oxazine alkaloids and four isoquinoline-based alkaloids ar inhibitors of the protein kinases tested. A narrow structural and protei kinase target specificity was observed. (+)-Boldine (39) and bulbocapnin (40) are specific for MLCK, while apomorphine (41) and sanguinarin (42)areforPKA.

867

HO

HO

Me

41 Apomorphine

Apomorphine was the most potent of the protein kinase inhibitors (IC50 for PKA-catalytic subunit 1 |LIM). However, the methylated aporphine alkaloid analogues of apomorphine such as bulbocapnine, isocorydine, glaucine and (+)-boldine were either inactive or poor inhibitors of this enzyme.

The benzophenantridine isoquinoline-based alkaloid sanguinarine (42) inhibited PKA-catalytic subunit (IC50 = 6 |LIM), but the methylated analogues were ineffective. Thus chelerythrine (37), a dimethylated analogue of sanguinarine, was a potent inhibitor of PKC (IC50 = 0.7 |LIM) but a very poor inhibitor of PKA-catalytic subunit (IC50 =170 |iM). Other alkaloids with structural similarities to sanguinarine, namely emetine, palmatine and berberine, were inactive. According to these resuhs, it seems that a planar nucleus is required for isoquinoline alkaloids to be effective as protein kinase inhibitors. It should emphasised that apormorphine, (+)-boldine and the oxazine alkaloids examined were also calmodulin antagonists [106].

\^i 42 Sanguinarine

868

The antioxidant alkaloids boldine and its dimethoxy analogue glaucine (43) inhibit TPA-induced down-regulation of gap junctional intercellular communication in WB-F344 rat liver epithelial cells in a dose-dependent manner. Analysis of the mechanism of action of these agents revealed that boldine and glaucine at 10 |iM totally inhibited the TPA-induced accumulation of intracellular oxidants. In addition, these alkaloids at 50 |LiM inhibited TPA-induced translocation of PKC to the particulate fraction of the cells [107].

MeO

MeO

MeO

OMe

43 Glaucine

Other nitrogenated compounds

Balanol (44), a hexahydroazepine amide alkaloid isolated from the fungus Verticillium balanoides, is a potent competitive inhibitor of ATP binding to several serine/threonine protein kinases. This inhibition occurs kinetically by competitive interaction with ATP on the catalytic domains of PKC and PKA with an aflBnity 3000 times that of ATP. Setyawan et al. [108] tested the capacity of balanol to inhibit representative serine-and threonine-specific protein kinases that share a common conserved catalytic core with PKA. The major subgroups of these kinases are the AGC, CaMK and CMGC groups. Balanol is a potent inhibitor of the AGC group (^i values between 1.6 and 6.4 nM). For protein kinases of the CaMK and CMGC subgroups, the effects of balanol vary. In the CaMK group, the K\ value toward CaMKII was 74 nM, and balanol did not inhibit the activity of phosphorylase kinase or smooth muscle MLCK. For the CMGC subgroup the K, values ranged from 30 nM for p34''' '' to 742 nM for

869

MAPK (ERKl). Minor modification of the balanol structure in ring D produces congeners (14" or 10"-deoxy) that show specificity toward PKA overPKC.

OH

44 Balanol

The effects of balanol and 10"-deoxybalanol on intact cells have been exmined to determine whether these compounds cross the cell membrane or if the potency and specificity observed in vitro are present in vivo. Western analysis showed that both compounds reduced phosphorylation of CREB in isoproterenol-stimulated A431 cells (IC50 = 3 |iM), although only balanol inhibits phosphorylation of MARCKS protein in phorbol ester-stimulated A431 cells (IC50 = 7 |aM) [109].

Phosphorylation of proteins on tyrosine constitutes less than 0.01% of the total intracellular phosphorylation. Enhanced activity of tyrosine kinases has been implicated in many cancers and other proliferative diseases as well as nonmalignant proliferative diseases such as atherosclerosis and psoriasis and in a large number of inflammatory responses such as septic shock [3]. A potent PTK inhibitor is lavendustin A (45), an fungal metabolite isolated from Streptomyces griseolavendus. It inhibited EGF receptor tyrosine kinase, with an IC50 of 4.4 ng/ml. It did not affect PKC or PKA but weakly inhibited phosphatdylinositol kinase. Kinetic studies by Lineweaver-Burk plotting indicated that lavendustin A inhibits the tyrosine kinase competitively with ATP and non-competitively

870

with the peptide substrate. It seems that the 2,5-dihydroxybenzyl group is essential for the inhibitory activity [110].

COOH

45 Lavendustin A

Lavendustin A and derivatives have been used to study the pathophysiological role of protein kinases. For instance, lavendustin A was used to study the potential role of tyrosine kinases in the regulation of wall stretch-induced atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) secretion. Because lavendustin A is selective for tyrosine kinases, the data obtained under these conditions showed that this kind of kinase is implicated in regulating cardiac hormone secretion. However, it is still unclear which tyrosine kinase is responsible for wall stretch-induced cardiac hormone secretion [111].

Miscellaneous compounds

The petroleum ether extract of Panax ginseng has antiproliferative effects on various cancer cell lines (murine sarcoma, murine leukaemia, human colon carcinoma, etc.). Polyacetylenic compounds from this extract such as panaxynol, panaxydol (46) and panaxytriol have been reported to be responsible for these effects [112, 113]. In order to identify the molecular mechanism of the polyacetylene compounds, one of the major cytotoxic compounds, panaxydol, was examined to see if it is able to disrupt cell cycle-related events. It was observed that panaxydol induces cell cycle arrest at Gi-S transition in SK-MEL-1 cells. This effect is related to the decreased Cdk2 activity, which seems to be caused by the increased level of p27^^^ protein expression. The protein synthesis inhibitor cycloheximide reversed the growth suppression induced by panaxydol (80 |Lig/ml) by

871

about 36%. In other words, growth inhibition by panaxydol requires new protein synthesis although other mechanisms are probably involved because cycloheximide did not reverse the growth inhibition caused by panaxynol completely [114].

CH2 = CHCH(C=C)2CH2CH CH(CH2)6CH3

OH

46 Panaxydol

47 Brefeldin A

Brefeldin A (47), a macrocyclic lactone isolated from fungal species of Dendryphion and Cylindrocarpon, is a powerful tool for investigating membrane traffic in eukaryotic cells. Brefeldin A added to cells causes rapid, reversible dissociation of a Golgi-associated peripheral membrane protein, implying that this substance prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In fact, brefeldin A inhibits guanine nucleotide exchange for ADP-ribosylation factor, an essential component of Golgi vesicle trafficking [115]. It was later observed that brefeldin A is able to activate the sphyngomyelin cycle, a signal transduction pathway that is involved in the control of cell growth, cell proliferation and apoptosis [116]. Oxysterol binding protein is a high affinity receptor for 25-hydroxycholesterol that is located in a Golgi/vesicular compartment. Phophorylation of rabbit oxysterol binding protein stably overexpressed in CHO-Kl cells was altered by staurosporine and okadaic acid, while other protein kinase activators and inhibitors had no effect. Treatment of these cells with brefeldin A caused dephosphorylation of oxysterol binding protein that coincided with disruption of the Golgi apparatus. Through immunoprecipitation experiments it was observed that brefeldin A inhibited phosphorylation of

872

oxysterol binding protein, but not its rate of dephosphorylation. It seems that rapid phosphorylation/dephosphorylation of oxysterol binding protein requires interaction with the Golgi apparatus and an associated kinase [117]. The activation of PLD by insulin and the subsequent generation of phosphatidic acid is linked to the activation of the MAPK cascade. Brefeldin A inhibited insulin-dependent activation of PLD and MAPK phosphorylation. The process by which insulin induced Raf-1 translocation to cell membranes was inhibited by brefeldin A and the addition of phosphatidic acid reversed the inhibition of MAPK [118].

Bromelain is a mixture of cysteine proteases obtained from pineapple stems (Ananas comosus, Bromeliaceae) that has been used therapeutically for the treatment of inflammation and trauma [119]. / ? vitro, it has varied stimulatory effects on leukocyte populations, increases CD2-mediated T cell activation, enhances Ag-independent binding to monocytes, etc. The effects of bromelain have previously been attributed to its degradative action at cell surfaces. However, it also acts independent of the removal of cell surface molecules [120]. In order to investigate the possible hormone­like effects of bromelain on intracellular signalling, its effects on TCR/CD3 signalling and IL-2 production were studied. It was observed that bromelain inhibits ERK-2 activation in ThO cells stimulated via the TCR, or with combined TPA plus calcium ionophore. In addtion, bromelain decreased IL-2, EFN-y, and IL-4 mRNA accumulation in ThO cells stimulated with TPA plus calcium ionophore, while the cytokine mRNA accumulation in cells stimulated via TCR was not affected. It seems that bromelain does not act on ERK-2 directly; but also inhibits p2r''^ activation, an effector molecule upstream from ERK-2 in the Raf-1/MEKl/ERK kinase cascade. Since p2r''^ is an effector for multiple MAPK pathways, it is likely that bromelain affects other MAPK signalling cascades, such as the INK pathway or p38 MAPK pathway [121].

PROTEm PHOSPHATASES

Just as there are two classes of protein kinases, there are also two main classes of protein phosphatases (PPs), the enzymes catalysing the hydrolysis of phosphate from a peptide structure. According to whether the phosphorylated aminoacid hydroxyl group is aUphatic or aromatic, serine/threonine PPs and tyrosine PPs are recognised.

Serine/threonine PPs comprise the phylogenetically-defined families I and II. Family I includes PPl, which preferentially dephosphorylates the P

873

subunit of phosphorylase kinase, and two forms of PP2, which preferentially dephosphorylate the a subunit of phosphorylase kinase: PP2A (spontaneously active) and PP2B (Ca " dependent). Family II encompasses PP2C, a monomeric enzyme characterised by its magnesium dependence. Apart from these initially considered enzymes, some other newer PPs such as mitochondrial PPs, PP>., PPG, PPX (or PP4), etc., have been reported.

PPl consists of a catalytic core bound to at least one subunit that functions as a carrier of the whole protein to reach the appropriate subcellular structures and increase the activity for a given substrate. Diverse forms of PPl participate in liver and muscle glycogen metabolism, calcium movements in sarcoplasmic reticulum and mitosis. PP2A is also formed by a rather dynamic association of two or three different subunits, and the trimeric form is activated by ceramide, which is in turn liberated from sphingomyelin after TNF-a receptor activation. PP2A is very potently inhibited by okadaic acid, and loses most of its activity through phosphorylation mediated by several PTKs. The importance of PP2A arises from its role in the control of the cell cycle, in relationship with the inactivation of the cyclinB-cdkl and cyclinB-cdk2 complexes, and of certain kinases of the ERK/MAPK pathway [122,123]. PP2B, also known as calcineurin, is a calmodulin-binding protein formed by two subunits: calcineurin A (catalytic) and calcineurin B (regulatory). Because of its activation of NF-KB and NF-AT it mediates IL-2 synthesis, and therefore T lymphocyte activation, a process that is blocked by the immunosuppressors cyclosporin and FK506. Calcineurin also participates in cAMP-mediated gene expression, as does PP2A, and in the control of corticotropine secretion [122]. PPl and PP2A-B have a large degree of homology (40-50 %) in their catalytic domains, which contain two a helices flanked by P strands to conform a (3-a-P-a-(3 structure. The active site contains two metallic ions (Ml and M2) that are essential for the enzyme function, each of which is coordinated by five ligands. In the PPl catalytic core. Ml coordinates with Asp 64, His 66, Asp 92 and two water molecules, and M2 with Asp 92, Asn 124, His 173, His 248 and one water molecule. Ml and M2 can also act as a kind of hook for the substrate phosphate group [124].

Tyrosine PPs are classified in four famiUes, depending on their biological function and aminoacid sequence: tyrosine-specific phosphatases (TSPs), vaccinia virus HI (VHl)-like phosphatases, cdc25 phosphatases

874

and low molecular weight phosphatases. Although the four main groups are quite dissimilar in structure, some fundamental features in their catalytic core are maintained: the active site contains a Cys-X-X-X-X-X-Arg sequence; in most cases cysteine is preceded by histidine, and arginine is followed by serine or threonine. Cysteine is essential for the phosphatase activity because of the formation of an infrequent phosphocysteine intermediate, and when cysteine is supplanted by serine the hydrolytic effect is lost. Arginine retains the phosphate group by hydrogen bonding, although the geometry of the alkyl-guanidino chain seems to be more important than its positive charge, because of the similar slowing in the catalysis observed when arginine is substituted by either lysine or alanine.

In addition, it should be stated that tyrosine PPs do not require metallic cations for their activity, are sensitive to sodium vanadate, and are localised in different subcellular structures, notably in the nucleus. Moreover, as it occurs with tyrosine kinases, some TSPs adopt the form of a membrane receptor [125]. Receptor-like protein tyrosine phosphatases (RPTPs) contain a variable extracellular domain that, depending on the RPTP subtype, exhibits immunoglobulin and fibronectin-like regions and other sequence motifs involved in cell-cell adhesion. The exact repercussion of ligand binding to RPTPs is currently being studied. The following subtypes have been defined: a) Subtype I RPTP CD45: Involved in the activation of B- and T-

lymphocytes. It stimulates kinases by dephosphorylation. b) Subtype II RPTP|a and %. Modulates cell-cell interaction. c) Subtype IV RPTPa: It binds to Grb2 [12].

For a recent review on the major biological aspects on tyrosine PPs see the work by Li and Dixon [126].

Protein phosphatase inhibitors

According to Mackinstosh and Mackintosh [127], the phosphatase inhibitors are classified in two main groups: Those inhibiting PP1/PP2A and those inhibiting PP2B. PP1/PP2A inhibitors are usually high molecular weigth substances produced by aquatic lower organisms as a mean of ecological defence and characterised by either long polyhydroxylated alkylic or cyclic polypeptide chains. The most important inhibitors of PP2B are cyclosporin A and tacrolymus (FK506) which will not be further an object of this study because of their well established therapeutic application as immunosuppressors.

875

Non-peptide polyether toxins

As it was mentioned above, okadaic acid (48) is surely the best characterised phosphatase inhibitor. It is a carboxylic acid based on a 38-C chain with multiple internal ether pyrane and furane cycles, whose isolation from sponges of the genus Halichondria was reported in 1981. In fact, it is produced by other organisms, such as certain species of Prorocentrum and Dinophysis, dinoflagellates that are ordinary food for marine invertebrates like sponges and edible molluscs. Okadaic acid makes the food in which it is present, even in the range of |Lig amounts, highly toxic, producing diarrhoea, vomiting and abdominal pain [128].The major mechanism behind its biological activity and toxicity is the interaction with serine/threonine PPs, particularly PPl, PP2A and PP3, which were inhibited with respective IC50 values of 49.0, 0.28 and 3.91 nm. If the carboxyl group of okadaic acid is esterified by a methyl radical or is reduced to the alcohol okadaol, potency is severely reduced and the activity against PPl disappears. Other chemical transformations that lead to a loss of the inhibitory effect on each of the three phosphatases are the reduction to 1-nor-okadaone and tetra-acetylation [129].

48 Okadaic acid

Okadaic acid has been demonstrated to enhance the activity of the kinases related to PKA and PKC (RAC-PKs), also termed PKB/Akt alluding to the viral oncogene v-aAt, which is related to the human RAC-a gene. These kinases are activated through a multi-stage phosphorylation process assessed by the parallel decrease in electrophoretic mobility of immunoprecipitated RAC-PK from Swiss 3T3 murine cells. The effect of okadaic acid lies in its inhibition of PP2A, which exerts control over RAC kinase activity by dephosphorylation. The process does not depend on the receptor-PTK-dependent PI3K activation because it is insensitive to 200 nM wortmannin [130]. Using MCF7 cells, it was demonstrated that

876

okadaic acid penetrates the membrane and binds strongly to the catalytic subunit of PP2A (PP2Ac), causing a decrease in the phosphatase activity of the soluble cellular fraction. The fact that okadaic acid provokes demethylation of the carboxymethyl terminal group of Leu ^ in a protein synthesis-independent manner may relate to PPA2 activity regulation, for this reaction was not affected by the presence of either cycloheximide or puromycin [131].

At present okadaic acid is a widespread laboratory tool used to detect the participation of PPs, or more strictly of PPA2, in many physiological processes. For that reason an increasing number of research studies deal more or less specifically with the activity of this toxin and related ones, such as calyculin A and others. For the purpose of the present review, a selection was made of recent papers focusing on those aspects with major pathological incidence.

One field in which phosphorylation and dephosphorylation play an important role is the transcriptional enhancement of inflammatory proteins brought about by NF-KB. This factor is inactivated by the IKB proteins, which retain it in the cytoplasm. This kind of protein is phosphorylated, prior to degradation in the 26S proteasome by a kinase (IKK), whose activity is enhanced by a series of physical, chemical and microbiological stimuli. Consequently, this mechanism is an indirect way of amplifying NF-KB signalling. Okadaic acid was reported to increase, at concentrations below 5 nM, the phosphorylation of iKBa [132]. Although it was unable to affect the activity of IKK itself isolated from HeLa cells challenged with TNF, it blocked the inhibitory effect that PP2A has on iKBa phosphorylation. In this way it cooperated in the cytokine-induced inflammatory response [133].

Cell death by apoptosis, a phenomenon that is increasingly considered the ultimate cause of many diseases, is also one of the patent toxic manifestations of okadaic acid. Micromorphological studies have demonstrated a parallel between the hyperphosphorylation of neuronal cytoskeletal proteins in Alzheimer disease and that produced by a treatment of cultured rat neurones with okadaic acid, which is accompanied by rounding of the cellular bodies and neurite degeneration. DNA fragmentation and neuronal death are inhibited by the presence of cycloheximide and by the caspase inhibitor benzoyloxycarbonyl-Val-Ala-Asp-0-methoxy-fluoromethyl ketone (ZVAD) [134, 135]. Okadaic acid was reported to be an apoptopic agent in rat thymocytes, because it causes

877

phosphorylation of histones HI and H2A/H3. This was demonstrated by detecting [^^P]-phosphate incorporation into proteins near 15 kDa on gel electrophoresis, and ulterior identification of histones by acetic acid-urea-Triton X-100 electrophoresis. It is suggested that after discrete phosphorylation, these histones may suffer structural changes affecting chromatin fibers, which results in a greater accessibility of DNA to DNAses, in this case the caspase-activated DNAse that mediates DNA breakdown in apoptosis [136]. When the cellular toxicity of okadaic acid against human myeloid leukaemia K562 cells was studied, it was proven that interruption of mitosis and apoptosis are in fact two synchronised PPA2-related events but generated by distinct mechanisms [137]. On the other hand, in HIT hamster pancreatic p-cells it was shown that the resistance to okadaic acid-induced apoptosis, which even led to define a particular cellular subline, was not dependent on changes in phosphatase activity, but possibly on the multidrug resistance phenotype [138].

Concerning involvement of phosphatase inhibition in hormonal activity, important modifications in intracellular trafficking of the glucocorticoid receptor (GR) have been detected. Okadaic acid aflfects the usual recycling of GR out of the nucleus and impairs ulterior activation upon hormone binding. Under habitual conditions, a phosphatase sensitive to okadaic acid regulates the dissociation of the GR binding to heat-shock protein 90 (hsp90) and formation of the hormone-receptor complex (HRC) that then enters the nucleus. As the inhibitory effect of this mechanism by okadaic acid in NIH/3T3 mouse embryo fibroblasts is lost when microtubules are disrupted by colcemide, it has been suggested that the transport into the nucleus normally requires cytoskeletal machinery, in absence of which HRC enters the nucleus by passive diffusion [139].

Calyculin A (49) was discovered to be a toxic compound from the sponge Discodermia calyx. Part of the molecule corresponds, as in okadaic acid, to a long chain (C-26 in this case) polycyclic ether, although instead of a carboxyl it has a nitrile group, four conjugated double bonds, and one phosphate group. Another part of the molecule can be theoretically derived from an oxazole alkylamide. Calyculin A is approximately equipotent as an inhibitor of PP2A and PPl (IC50 = 0.3 and 0.4 nM respectively, referred to phosphorylase-a dephosphorylation) and consequently two degrees of magnitude more potent than okadaic acid as an PPl inhibitor [129]. As was reported before for okadaic acid, calyculin A produces demethylation of the carboxymethyl terminal group of Leu ^

878

in PP2Ac but, unlike okadaic acid, it decreases immunoreactivity of the carboxyl end of the catalytic subunit of PPl (PPlc), possibly by proteolysis [131].

49 Calyculin A Ri = H, R2 = CN, R3 = H, R4= H 50 Clavosine A Ri = H, R2 = CONH2, R3 = Me, R4= rhamnose

When studying the influence of PP activity on the inhibitory effect of adenosine on neutrophil function, it was noted that a pre-incubation with 10 nM calyculin A deprived the adenosine agonist 5'-iV-ethylcarboxamidoadenosine (NECA) of its inhibitory activity on neutrophil superoxide generation. The fact that okadaic acid was ineffective at 10 |iM led researchers to attribute a principal role in the regulation of adenosine activity to PPl [140]. The direct effect of calyculin A on neutrophils was time-dependent. With a short pre-treatment, a theoretically unexpected induction of tyrosine phosphorylation of the signalling proteins Cbl and Syk was observed, whereas a pre-treatment longer than 5 min produced inhibition of tyrosine phosphorylation and an increase in the serine/threonine phosphorylation of Cbl and superoxide formation after cross-linking of CD32, a neutrophil low-affinity IgG receptor. Okadaic acid showed similar effects with slower kinetics [141].

After Calyculin A, many other calyculins, as well as the related calyculinamides, in which the nitrile group is hydrated to give an amide.

879

have been discovered. Two of the recently identified calyculinamides are clavosines A (50) and B, fi-om the sponge Myriastra clavosa, which share most of the structure of calyculin A but differ in that both are amides, C-31 methylated, and present a permethyl-0-rhamnose moiety at C-21. The C2-C3 double bond has a cis geometry in clavosine A and a trans one in clavosine B [142]. Clavosine A was as potent as calyculin A in inhibiting the y isoforms of PPlc (PPlcy) and PP2Ac (IC50 in the 0.5-0.7 nM), whereas clavosine B showed IC5o= 13 nM and 1.2 nM for the inhibition of PPlcy and PP2Ac, respectively. Studies with mutated PPlcy proteins showed that substitution of the 134-Tyr residue by Phe (hydrophobic residue) enhanced the inhibitory activity by the three calyculins, and that when the 134-Tyr was substituted by a 134-Asp (negatively charged residue) the inhibitory potency was seriously diminished [143].

Tautomycin was obtained fi-om the terrestrial microorganism Streptomyces spiroverticillatus and is chemically characterised as an ester of a carboxylic acid derived fi^om a dialkyl maleic anhydride, with a long chain (26-C) polyol containing two cyclic ethers. In a simultaneous study of the best-known natural inhibitory compounds of PP2A, in which the non-radioactive malachite green assay was applied, tautomycin was the least potent compound [144]. This is consistent with the findings reported by Honkanen et al. [129] and other authors. Its primary site of phosphatase inhibition is PPlc, as was reported in a comparative study with okadaic acid and calyculin A [131].

Cyclic polypeptide inhibitors

The most important of the peptidic phosphatase inhibitors are the microcystins and nodularin. Mycrocystins are heptapeptides characterised by the sequence cyclo(D-Ala-X-D-er);^/iro-P-methylisoAsp-Y-Adda-D-isoGlu-A^-methyldehydroAla), where X and Y are different L-aminoacids, and Adda is the abbreviation of the p-aminoacid [25',3iS',8iS',95]-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid. In the most common microcystin, namely microcystin-LR, X is Leu and Y is Arg. This kind of compounds was considered to be the highly hepatotoxic principle of the cyanobacterial genera Microcystis, Anabaena and Oscillatoria. Apart fi'om the variations represented by X and Y, other differences arising fi-om the demethylation of aminoacids, lead to the existence to more than fifty microcystins. The rare acid Adda is also

880

present in nodularin, from the cyanobacteria Nodularia spumigena, which contains the pentapeptide sequence cyclo(D-p-methylisoAsp-L-Arg-Adda-D-isoGlu-A^-methyldehydrobutyrin) [128, 129]. When the polar residue L-Arg of nodularin is replaced by L-Val, the result is motuporin, a compound that was isolated from the marine sponge Theonella swinhoei, and was reported to be a potent PPl inhibitor and cytoctoxic substance against several human cancer lines [145].

Microcystins show the highest potency among the known inhibitors of PP2A, ranging from 0.04 to 2 nM according to different authors and assay methods [144].The inhibition of PPl and PP2A activity by microcystins occurs through two steps: an immediate blockade of the catalytic site by non-covalent forces and a subsequent adduct covalent formation. With regard to this, it has been demonstrated that in the presence of each of the microcystins LR, RR and YR, the affinity of the substrate phosphorylase a for the complexed PP2A was four times lower than that for the unbound PP2A, indicating a partial reduction of substrate binding, which is compatible with a total abolition of the enzyme activity [146].

A rather close relationship between the apoptosis induced by microcystin-LR and caspase-3 activity was observed. Hepatocytes are extremely sensitive to microcystins and nodularin, as can be deduced from the quick apparition of cellular apoptotic changes such as superficial budding and shrinkage of cytosol and nucleus. Analogous findings were obtained in other kinds of cells, like Swiss 3T3 fibroblasts or promyelocytic IPC.81 cells, provided that the toxins were microinjected. However, in caspase-3 deficient MCF-7 cells, apoptosis developed slowly and was independent of the ZVAD, a compound that ordinarily inhibited apoptosis without affecting the hyperphosphorylation caused by PP inhibition [147].

Alkaloids

From the dichloromethane extract of the stems of Rollinia ulei (Annonaceae), three aporphine alkaloids were isolated as the principles responsible for the inhibitory activity on yeast recombinant CD45 tyrosine phosphatase. The most potent alkaloid was nomuciferine (51), with an IC5o=5.3^M[148].

881

MeO'

51 Nomuciferine

The bisindole brominated alkaloid, dragmacidin D (52), was isolated from a sponge of the genus Spongosorites found off the South Australian coast at a depth of 90 m. This compound was characterised as a selective inhibitor of PPl, while its isomer dragmacidin E also inhibited PP2A [149]. Another example of brominated alkaloid obtained from a sponge is discorhabdin P (53), which was isolated from a species of Batzella collected in the western Great Bahama Bank, 141 m below sea level. This alkaloid can be considered a condensed monoindole derivative and behaved as an inhibitor of bovine brain PP2B, with an IC50 of 1.1 |iM.

52 Dragmacidin D

882

o H

.A^"-

53 Discorhabdin P

Miscellaneous compounds

Cantharidin (54) is the vesicant substance obtained from some species of blister beetles, among them Mylabrisphalerata.M. cichorii and Cantharis vesicatoria. Known since ancient times as a strong rubefacient and used as a wart remover and aphrodisiac drug, it is now arousing new interest for its inhibitory activity on PPs. It was reported to inhibit PPl and PP2A with respective IC50 of 0.16 and 1.7 \xM, in phosphohistone dephosphorylation [150]. In bovine arterial tissue, cantharidin produced a smooth muscle contraction that was independent of cytosolic Ca ^ levels, but was, in contrast, closely linked with the phosphorylation of contractile proteins by inhibition of PPl and PP2A, both of which were detected together with their mRNA in the aortic tissues [151]. The same authors reported that cantharidin inhibited PPl and PP2A activity in bovine aorta endothelial cells and that it was determinant for the strong increase produced in albumin permeability in endothelial monolayers [152].

54 Cantharidin

883

HO Me

CH2OH

OPO3OH2

55 Fostriecin

Fostriecin (55) is a phosphoalkyl-pyrone antibiotic obtained from Streptomyces pulveraceus ssp. fostreus that was described as a phosphatase inhibitor, particulariy of PP2A (IC50 = 3.2 nM), and anti­neoplastic drug. Although it is less potent than okadaic acid, its selectivity towards PP2A is much higher [153] and it showed similar potency in inhibiting PP4 from porcine testes, a phosphatase that has a predominant presence in the nucleus and features in common with PP2 A.

ABBREVIATIONS

AGC = Cyclic nucleotide regulated protein kinase family ACs = Adenylyl cyclases AKAPs = A-kinase anchor proteins aPKCs = Atypical PKCs ANP = Atrial natriuretic peptide ATP = Adenosine 5'-triphosphate BNP = Brain natriuretic peptide CaMKs = Ca^Vcalmodulin-dependent kinases CDKs = Cyclin-dependent kinases CDPK = Ca ' -dependent protein kinase CKI = Casein kinase I CKII = Casein kinase II CIK = Dcd-like kinase CMGC = Cyclin-dependent kinases and close relatives family cPKCs = Classical or conventional PKCs CREB = cAMP responsive element binding protein DAG = Diacylglycerol DPP A = 12-Deoxyphorbol-13-O-phenylacetate-20-acetate EGF = Epidermal growth factor

884

EF-2 = Elongation factor-2 ERK = Extracellular signal regulated kinase GAP = Ras-GTPase activating protein GP = Glycogen phosphorylase GPCRs = G-protein coupled receptor group GR = Glucocorticoid receptor GSK3 = Glycogen synthase kinase 3 GTP = Guanosine 5'-triphosphate HRC = Hormone-receptor complex JAKs = Janus kinases JNK = c-Jun N-terminal kinase IL-\a = Interleukin-la IP3 = Inositol 1,4,5-trisphosphate LIMKs = LIM kinases LPS = Lipopolysaccharide MAPK = Mitogen activated protein kinase MAPKK = MAPK kinase MAPKKK = MAPK kinase kinase MAPKAP2 = MAPK activated protein kinase 2 MARCKS = Myristoylated alanine-rich C kinase substrate MEK = MAPK/ERK kinase MLCK = Miosin light-chain kinase nPKC = novel PKCs OPK = Other protein kinases PAI-1 = Plasminogen activator inhibitor-1 PAK = p21-activated kinase PDBu = Phorbol-12,13-dibutyrate PKA = cAMP-dependent protein kinase PI3K = Phosphatidylinositol 3-kinase PIP2 = Phosphatidylinositol 4,5-bisphosphate PIP3 = Phosphatidylinositol 3,4,5-trisphosphate PLC = Phospholipase C PLC-p = Phospholipase C-p PLC-5 = Phospholipase C-5 PLC-y = Phospholipase C-y PLD = Phospholipase D PPs = Protein phosphatases PTKs = Protein-tyrosine kinases

885

PKC = Protein kinase C RPTPs = Receptor-like protein tyrosine phosphatases RTKs = Receptor tyrosine kinases SAPK/JNK = Stress-activated PK/cJun N-terminal kinase SEKl = SAPK/ERK kinase 1 SH2 = Src homology domain 2 TGF-p = Transforming growth factor-p TNF = Tumour necrosis factor TPA = 12-0-tetradecanoylphorbol-13-acetate TSPs = Tyrosine specific phosphatases

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