Inhibitors of the Platelet-Derived Growth Factor Receptor Tyrosine Kinase

Inhibitors of the Platelet-Derived Growth Factor Receptor Tyrosine Kinase

Glenda E. Bilder and Camilo J . Rojas

Department of Carrlior~rr.sc.icltrr Biolog!. Rh6i1e Poirlenc Rorer Ceiitrol Research, Colleger~ille, P A , USA

Key Words: Atherosclerosis-PDGF-receptor tyrosine kinase-Phenolate anion-Phenylamino- pyrimidine--Quinoline-Quinoxaline-Restenosis-Tyrosine kinase inhibitor.

INTRODUCTION

Protein tyrosine phosphorylation is essential in key cellular activities such as division, phenotypic modulation, differentiation. migration, and apoptosis (56) . At the subcellular level. protein tyrosine phosphorylation drives enzymatic activation, protein-protein interaction, cytoskeletal reshuffling. and intracellular trafficking of molecules (80). Under- standing the role of protein tyrosine phosphorylation in the normal state and the discovery that certain viral oncogenes express tyrosine kinase activity have encouraged research efforts toward identification of protein tyrosine kinase inhibitors (TKls). In proportion to the growing interest in the biological role played by protein tyrosine phosphorylation, there are excellent comprehensive reviews on TKls ( 14.32.5 1,891. Other reviews focus on a specific receptor TKIs such as epidermal growth factor (EGF) receptor TKls (7,95) and insulin receptor TKls (91).

Among the membrane receptor tyrosine kinases. the platelet-derived growth factor receptors (PDGFr) are thought to play a pivotal role in the development of atherosclerosis and restenosis as well as certain malignancies (35.76). The development of PDGFr TKIs as potential new therapeutic modalities against these disorders is, therefore, a reasonable goal. This review will focus on the progress toward that end.

PDGF LIGANDS/RECEPTORS

PDGF is a pleiotropic growth factor that initiates cell-signalling by binding to its membrane tyrosine kinase receptors (16.35). PDGF ligands are hetero- or homodimers of protein chains designated A and B. The A B . BB. and AA ligands activate the PDGFr subunits termed alpha (a) and beta (p) by binding and dimerizing the subunits in a ligand-specific manner (33.83). In tissue culture, neuronal. vascular, and immune cells

Address correspondence and reprint requests to: Dr. Glenda Bilder. Department of Cardiovascular Biology. Rhone Poulenc Rorer Central Research. 500 Arcola Road. Collepevillc. PA 194?6-0107. USA. Fax: (610)- 454-8740

380

TYROSINE KINASE INHIBITORS 381

produce and secrete PDGF. Under physiological and pathological conditions, PDGFr on mesenchymal cells are present in sufficient concentrations to allow signalling by autocrine, paracrine (35), and possibly intracrine (54) mechanisms.

The PDGF-receptors share with thirteen other membrane receptor protein kinases a conserved extracellular binding domain, a transmembrane domain, and an intracellular catalytic domain that performs the phosphate transfer from ATP to tyrosine of the receptors or cytosolic signalling molecules (100). Also conserved is the ATP binding motif, which includes an essential lysine residue whose deletion abrogates tyrosine kinase catalytic activity (23).

Structural details relevant to the PDGFr are summarized in Table 1. The PDGFr and several close relatives, including colony stimulating factor- 1 -receptor (CSF- lr), steel cell factor (SCFrkkit), and Flk2 (KDWFlt3) contain a unique nonkinase sequence interrupting the kinase catalytic domain and distinguishing cysteine clusters in the extracellular domain (100). Although the (Y and f3 PDGFr subunits are somewhat homologous (27-87%) (18), differences exist in the intracellular domains that account for variations in cytoplasmic substrate specificity and may translate into differences in cellular functionality (22 , 34,79,99).

Oligomerization of the membrane tyrosine kinase receptor permits diversity in signal transduction and plays an important function in providing the structural basis for signal transmission by growth factors (81). Binding of the PDGF ligands is well characterized with dissociation constants in the nanomolar range (47). As dictated by the ligand-specific binding framework noted above, PDGF-AA will dimerize (Y subunits, PDGF-AB will dimerize (Y(Y and (YP subunits and PDGF-BB will produce dimers of all three possible

TABLE 1. Characteristics of PDGF @-receptor ~

AA Comparison to Domain length Characteristics Function a receptor

Ligand binding 499 5 immunoglobulin- like domains; cysteine-rich regions

Transmembrane 25 Hydrophobic residues Juxtamembrane 48

Catalytic 347 Split kinase; ATP binding site; multiple phosphorylation sites

Cytoplasmic tail 155

Ligand specificity-binds PDGF-B chain

Anchor; dimerization Src phosphorylation

site Signal transduction

and SH2 recruitment of substrates

Ubiquitization and degradation of receptors; negative regulation of signalling

a receptor binds A and B chain; 30% identity

48% identity 83% identity

Sites on a-receptor revealed with P-receptor binding; different substrate affinities; 87% identity tyrosine kinase 1; 74% identity tyrosine kinase 2

27% identity

From refs. 15, 17, 18, 34, 79, 106.

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382 G . E . BILDER AND C. J . ROJAS

combinations. Alternative models have been proposed that allow for spontaneous oligomerization without ligand in cases of elevated receptor numbers (36).

With dimerization, conformational changes ensue that can be detected with immuno- reactivity methods (6,46). Little is known, however, of the molecular mechanisms in which conformational changes activate receptor autophosphorylation or of specific con- tributions made by cytoplasmic domains such as the membrane spanning domain (106). Studies using dominant negative receptor mutants determined that autophosphorylation of the receptor proceeds by an intermolecular (trans) mechanism in which one receptor subunit cross-phosphorylates the other (47).

Peptide mapping, overexpression of chimeric receptor constructs or deletionlsite- directed mutants and synthetic peptide substrates have led to identification of multiple phosphorylation sites on the receptor (16,43,96). Both the phosphorylated tyrosine and flanking amino acids determine the specificity of the PDGFr signalling by providing high affinity regions which facilitate physical association of cytoplasmic proteins containing specific amino acid sequences referred to as SH2 (src homology 2) domains (88). SH2 domain-containing protein substrates are mainly adaptor molecules or enzyme substrates for the receptor kinase.

In some cases, such as phospholipase C-y, tyrosine phosphorylation serves to increase enzyme activity (65); in the case of nonenzymatic substrates, tyrosine phosphorylation may induce conformational changes leading to activation of other substrates (87). Table 2 summarizes the known substrates and their binding loci on the PDGFPr. Inhibitors of the PDGFr tyrosine kinase would be expected to reduce or eliminate association of some or all of these potential substrates.

Signal transduction converges beyond this point. The details from ras-raf-MEK 1 - MAP-kinase-phosphorylated transcription factors have been reviewed (43,56,88). Speci- ficity of PDGFr signalling, therefore, appears to reside in tyrosine phosphorylation sites and flanking amino acids. The specificity results not only from the primary sequence of

TABLE 2. PDCF @-receptor substrates

Mol wt Binding Substrate (kDa) location* Function Ref.

Src, Fyn, Yes Grb2

60 579,581 27 716

Shc 46.52,66 579,740.

P85/phosphoinositol 3-kinase 85 740,751

Nck 75 1 GTPase activating protein 125 77 1

751.771

(GAP)

Transformation 8,62 Linker to nuclear exchange 2

factor (Sos), Ras regulation Linker. binding sites for Grb2

Linker, protein shorting, 24,41

Transformation? 66 Negative regulation of RAS 24,45,41

109

mitogenesis, chemotaxis

Phosphotyrosine phosphatase 1 D 65 1009 Phosphatase, binding site for 44.50 Grb2 ~ ~~

Phospholipase C-gamma 145 1021,1009 Phosphoinositol production, 74,49,61 mitogenesis, chemotaxis

JAK1, JAK2, Tyk2 I20 ? Ubiquitinization, sorting 101 STATla 84.91 ? Translocation to nucleus 108

* Numbers of binding location corresponded to amino acid location in human PDGF P-receptor

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substrate and receptor but also in substrate recognition determined by peculiarities in secondary, tertiary and quaternary structures. Intracellular localization of potential substrates and receptor-associated phosphatase activity are also important in regulating signal transduction via activation of the PDGFr (88).

MECHANISMS OF RECEPTOR TYROSINE KINASE ACTIVITY

Both autophosphorylation and kinase activity involve transfer of a phosphate group from ATP to a protein tyrosine residue. The phosphate acceptor could be the receptor itself (autophosphorylation) or another protein (kinase activity).

The amino acid sequences of the catalytic domains of fourteen receptor protein-tyrosine kinases were aligned recently (100). The list included PDGFPr, insulin receptor (INr), and EGFr. The homologous alignment and information on the crystal structure of cyclic AMP-dependent protein kinase, a serineithreonine kinase, were used to postulate the likely role of conserved residues. Conserved residues may affect ATP binding, metal ion chelation, the chemical steps of phosphotransfer, peptide accessibility, substrate recognition, and the formation of ion bridges between the two structural lobes that form the catalytic site (94).

Phosphate transfer in the PDGFr-catalyzed reaction is postulated to require proton abstraction from phenol in the tyrosine residue by asparate 826 and nucleophilic attack of the resulting phenolate anion on tetracoordinate y-phosphorus of ATP to form a penta- coordinate transition state (or intermediate) followed by formation of phosphorylated tyrosine and ADP. A protonated side chain, arginine 830, could be involved in electrostatic stabilization of the intermediate (Fig. 1). These features are consistent with the proposed mechanism of phosphorylation proposed for the EGFr (102). The pH depen- dence of the EGFr-catalyzed reaction and thermodynamic considerations support the involvement of aspartate and arginine (aspartate 813 and arginine 817 in EGF-r); the purported pKa values are 6.3 for aspartate and 9.1 for arginine (102). The involvement of these residues in catalysis is also supported by the x-ray structure of the INr (aspartate 1132 and arginine 1136 in the INr) (37).

The ionization of the tyrosyl hydroxyl group on both chemical and enzymatic reactivities is important. For example, in a phosphorylation model reaction of acetylation of tyrosine and fluorotyrosine by N-acetylimidazole, tyrosine is seventeen times more reactive than fluorotyrosine and the reactivity is directly correlated to the relative concentration of the phenoxilate. Comparison of reactivities of fluorotyrosine- and tyrosine- containing peptides as substrates for the INr show higher V,,, and K, values for phosphorylation of the tyrosine peptide than for phosphorylation of fluorotyrosine peptide. The results indicate that phosphorylation is favored in environments that enable efficient deprotonation of the tyrosine residue (58).

Electrostatic interactions of manganese and/or magnesium play a pivotal role in PDGFr tyrosine phosphorylation. Early nuclear magnetic resonance studies of solutions of metal ions and ATP indicate binding of the manganese ion to oxygen atoms on the a-, p- and y-phosphates of ATP and binding of magnesium to only the p- and y-phosphates (9). Metal ion chelation of oxygen neutralizes the negative charge on ionized phosphate groups to facilitate the approach of the phenolate ion. Dissociation of the chelated species is favored over dissociation of the highly ionized pyrophosphate (9).

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384 G . E . BILDER AND C . J . ROJAS

Protein of Polypepttde Substrate

I

PDGF-r . Asp&%- COO---’

Prolein or Polypeptide Substrate

v Protein or Polypeplide Subslrate

0 ’ 0-Adenosine /y- o* / o o.

I p\ 0- 0-

Me2+

FIG. 1. Possible catalytic mechanism for PDGFr tyrosine kinase. Ionization of phenol is facilitated by car- boxylate of aspartate 826. Metal ion (Me*+) chelation of oxygen neutralizes the negative charges on ionized phosphate groups to facilitate the approach of the phenolate anion. Phenol ionization and attack on y-phosphate could be concerted or in a stepwise process. Bond makmg and breaking occurs through the assistance of the mine of arginine 830 (middle structure); the postulated electrostatic interactions illustrated here could also play a part during binding of substrates or before dissociation of products from the kinase active site. Products of the reaction, phosphorylated tyrosine and ADP, are shown in the last step.



PDGF-RECEPTOR TYROSINE KINASE INHIBITORS

Clinical Need

Since PDGF is the cellular homologue (c-sis) of the transforming v-sis oncogene of the Simian sarcoma virus (103) and is a potent inducer of cell proliferation, migration, phenotypic change, and transformation, inappropriate coexpression of ligands and receptor subunits may be an important mechanism underlying fibroproliferative disorders such as atherosclerosis, restenosis following balloon catheter angioplasty , malignancy, pulmo- nary fibrosis, and glomerulonephritis (72). Theoretically, specific inhibitors of the PDGFr tyrosine kinase would disrupt PDGF-dependent signal transduction and retard or block disease/disorder progression. The evidence that PDGF ligands/receptors are involved in atherosclerosis and restenosis is reviewed.

Atherosclerosis

Atherosclerosis is a complicated disease process contributing to coronary heart disease, a major cause of death i.n our society. Vascular lesions have been described histologically and range from fatty streaks containing a small number of lipid-filled, monocyte-derived macrophages and some T lymphocytes to complex lesions composed of increased numbers of smooth muscle cells, macrophages, and T lymphocytes and varying amounts of extracellular matrix, thrombi, and related degradation products, lipids, and calcium de- posits (75,92). The fatty streak may be the precursor to the more complex lesion that culminates in lumen narrowing, occlusion or rupture (75).

As determined by immunocytochemical staining, Northern analysis, and in situ hybridization, the mEWA and protein of the PDGF B-chain are upregulated in complex atherosclerotic lesions of humans and cholesterol-fed nonhuman primates (3,77,105) and are found in association with lesion macrophages (77) and intimal cells, referred to as intimal mesenchymal cells or modified smooth muscle cells (104,105). In addition, the transcript and protein for PDGFPr have been detected in advanced lesions (78,109, localized to modified smooth muscle cells of the lesions (78,105). The patterns of expression of PDGFPr and PDGF-B ligand are consistent with the proposal that autocrine and paracrine networks initiate PDGF-dependent activities in intimal smooth muscle cells to assist with development of the atherosclerotic plaque. In addition, PDGF B-protein is present in macrophages of some but not all fatty streaks from human aorta, suggesting that PDGF-BB ligand may participate in the initiation of atherosclerosis (42).

PDGF-A chain transcripts are also expressed in modified intimal smooth muscle cells of complex atherosclerotic lesions of the human carotid artery at levels higher than in normal arteries (104,105). This, however, has not been a consistent finding. Recently, the technique of competitive reverse transcription-polymerase chain reaction (RT-PCR) was applied to autopsy samples of three advanced atherosclerotic plaques of the aorta and ten normal aortic sections to measure PDGF A-chain mRNA (63). PDGF A-chain transcripts were approximately 100 times lower in sections from atherosclerotic tissue than in sections from normal tissues, and transcript levels in lesions were independent of the extent of cell proliferation as determined by staining with proliferating cell nuclear antigen. The discrepancy between these findings and those reported earlier may result from the dif- ference in tissue site (aorta and carotid) and/or the number of analyzed samples (three


G . E . BILDER AND C . J . ROJAS

versus greater than thirty). Although the results generated from the competitive RT-PCR analysis question the mitogenic role of the PDGF A-ligand in atherosclerosis, it is noted that the extent of proliferation at any one time in the atherosclerotic plaque may be variable and indeed, different proliferative rates have been published (67,70).

Although the role of PDGF A-ligand remains unclear and expression level of the PDGFar in lesions is yet to be determined, the detection of the PDGF ligands and the Q-receptor is an important first step in defining their influence in atherosclerosis. Given the pleiotropic nature of PDGF, it is likely that PDGF may stimulate one or more of its known activities to initiate and maintain plaque development. With identification of highly selective PDGFr TKIs, this hypothesis may soon be tested.

Restenosis

This is the clinical condition characterized by lumen narrowing (restenosis) of a dis- eased (stenosed) artery approximately 6 m to 1 y following successful dilation with an angioplasty balloon, stent, or atherectomy device. Since postangioplasty repair occurs on top of and within the atherosclerotic plaque, histology of restenotic lesions is as variable and complex as the atherosclerotic plaque. Arterial wall changes following restenosis have been attributed to combinations of vascular cell migration and proliferation, matrix deposition, vasoconstriction, and remodelling (59). There is currently no successful therapy to prevent restenosis following angioplasty .

Analysis of human coronary artery restenotic lesions from 2 d to 6 m following angioplasty revealed that the PDGFQr and PDGF B-ligand are expressed in regions of repair, in association with macrophages and a-actin negative spindle cells, most likely modified smooth muscle cells (93). These findings show, for the first time, coexpression of a compatible ligand and receptor combination following coronary angioplasty and signify that in human restenotic lesions the PDGF/PDGFr circuit is upregulated and may be responsible for vascular changes following balloon angioplasty.

Results of studies in animal models of balloon catheter denudation (BCD) injury indicate increased expression of PDGF A- and B-chain transcripts in rat aortic artery (60), rabbit iliac artery (20), and baboon brachial artery (68). The status of the PDGFr was not determined in these models. The BCD-induced injury of the rat carotid artery is the most intensely studied animal model for this disorder. Results in this model show that PDGF A-chain transcripts, as determined by Northern analysis, are transiently expressed within 6 h after BCD but without concomitant increase in PDGFar mRNA (57). Two weeks following BCD, PDGF A-chain and PDGFQr mRNA, measured by in situ hybridization, are evident in discrete regions of the intima (57). Although these results suggested that a paracrine or autocrine network within the intima was unlikely since the PDGF-A ligand does not activate the PDGFPr (33,83), subsequent findings using rat-specific RNA probes and enface tissue preparation showed coexpression of the PDGF B-chain and PDGFPr in luminal smooth muscle cells at several time points throughout the course of intimal formation following BCD (52). These latter findings support the hypothesis that PDGFr activation is involved in catheter-induced intimal growth.

Several studies in rat carotid and rabbit femoral BCD models in which infusions of PDGF or antibodies to PDGF or platelets were used to manipulate the PDGF/PDGF-r loop, have concluded that the main function of PDGF is to stimulate smooth muscle cell

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migration (25,26,27,40). Recent findings support this proposal by demonstrating injury- induced increase in transcript and protein expression of PDGF B- and A-chains in the leading endothelial edge of a denuded region of the rat carotid artery simultaneous with constant expression of PDGFPr in exposed smooth muscle cells (53). To understand further the in vivo functions of PDGF, the gene for the PDGF-B chain was transferred into the porcine iliac artery (64). Transfer of this gene without overt catheter stretching produced an increase in the number of intimal smooth muscle cells and a lesion that was histologically similar to that of the human restenotic lesion (64). These observations suggest that PDGF is able to initiate intimal hyperplasia in vivo.

A major limitation of the aforementioned animal models is that the vascular injury, whether in a carotid, iliac, or aortic artery, culminates in a stenotic lesion in a normo- cholesterolemic animal. Although it is possible that balloon injury to a normal artery and to an atherosclerotic artery may stimulate the same basic mechanisms, expression of ligands/receptors and effects of manipulation of ligandslreceptors in a balloon dilated atherosclerotic artery are lacking. Results from these models would add significantly to our understanding of restenosis and provide models in which to evaluate new therapies including PDGFr TKIs.

Development

A systematic scheme for discovery of TKIs has been described in relation to identification of EGFr TKIs with anticancer activity (7,28). Issues of potency, selectivity, cell permeability, animal bioavailability, and lack of toxicity are emphasized as central to TKI discovery (7,28,51). In addition, it is important to evaluate efficacy of potent TKIs on in situ receptor autophosphorylation and on protein tyrosine phosphorylation of known substrates in cell cultures. Subsequent determination of cellular activities of interest, for example, DNA synthesis, cell growth, migration, and phenotypic change can then be meaningfully assessed. Since there is no consensus as to the number of kinases, tyrosine, serine, and threonine, against which demonstrated inactivity confers a label of selectivity and since there is no consensus as to whether enzymatic, cellular, or in vivo selectivity provides the most meaningful information, evaluation of selectivity represents, in its purest form, a herculean task.

A postulated role for cellular and membrane receptor tyrosine kinases in tumor development prompted the initial search for TKIs (35,38). Natural products comprise the group of original inhibitors (Fig. 2). These compounds, extracted from microbial broths or plants, are inhibitors of the EGFr tyrosine kinase, p60 Src, or p40 kinase, display IC,, values ranging from 0.01 to 100 pM and share structurally overlapping features (14). Although generally more active against tyrosine kinases than threoninekerine kinases, the natural products in Fig. 2 are not selective TKIs. Methyl 2,5 dihydrocinnamamate (Fig. 3A), a stable analogue of erbstatin and one proposed to be a PDGFr TKI, is an example of a nonselective PDGFr TKI. Methyl 2,5-dihydrocinnamamate inhibits PDGF-dependent proliferation of rat aortic smooth muscle cells without reducing in situ PDGFr autophosphorylation (84). Its mode of action is unknown, although actin fiber polymerization and protein tyrosine phosphorylation of several low molecular weight cytoplasmic proteins are inhibited (84).



Natural Products Name

Q u e rcet i n

Erbstatin

Genistein

Herbimycin

Lavendustin A

Piceatannol

StructurdSource

cH Cyclic AMP-independent *& protein kinase; -1 00 pM

ar Fruits, vegetables, teas W O

EGF-receptor kinase; 0.55 pglrnl

1 Streptomyces o*

ra

EGF-receptor, p6Ov-src, p l l0gag-fes kinase; 6-8 pglml

a*

Pseudornonas sp. 0

ngc0 L, L, L., Streptomyces

EGF-receptor kinase; 4.4 ng/ml

I

Streptornyces griseolavendus o*

no \

o(

Euphorbia lagascae

FIG. 2. TKI natural products. From refs. 1, 30, 31, 69, 97, 98

The fust postulated “selective” PDGFr TKIs (Fig, 3B) belong to the dicyanobenzylidine series (5 ) . Some members of this chemical series were previously identified as potent substrate competitive EGFr antagonists, termed tyrphostins (107). Tyrphostins were designed to be competitive for the protein substrate, to be selective for EGFr tyrosine

Cardiovascular Drug Reviews, Yo/. 14. No. 4 , 1996


A. 9"

A RG 50872

S Q CH3

ST 638

RG 13022

RG 13291 I

FIG. 3. Representative PDGFr TKIs, dihydrocinnamamate (A), dicyanobenzilidine (B), and modified dicy- anobenziline series (C,D,E).

kinase compared to INr tyrosine kinase, and to be soluble in water and mildly hydrophobic solvents (29).

As PDGFr TKIs, members of the dicyanobenzylidine series inhibited the cell-free PDGFr kinase activity, in situ PDGFr autophosphorylation, PDGF-dependent DNA synthesis and PDGF-induction of c-fos transcripts at low micromolar concentrations (0.01-8 FM) in vascular smooth muscle cells (5). This series did not disrupt binding of ligand to


390 G. E . BILDER AND C . J . ROJAS

receptor and inhibitory activity was reversible ( 5 ) . Although some compounds from this series were 100- to 1000-fold more selective for EGFr than INr tyrosine kinase (29), the separation between inhibition of PDGFr tyrosine kinase and EGFr tyrosine kinase measured in the same cell type was a modest 20-fold ( 5 ) . Members of this series did not inhibit the enzymatic activity of PKC and PKA. Modification of the dicyanobenzylidene series by substitution of the hydroxyls with an indole did not improve potency for inhibition of in siru PDGFr autophosphorylation, DNA synthesis in fibroblasts, or selectivity for PDGFr compared to EGFr (10). Compounds of the dicyanobenzylidine series penetrated cells adequately but were chemically unstable (55,73). RG 50872, in particular, has been cited for its toxic damage to mitochondria1 membranes and time-related induction in cellular ATP reduction (13). It is noted that these activities were evident at a concentration (2 pM) which is 50 times above the IC,, value for previously reported inhibition of PDGFr-associated activities (5 ) .

A 4-hydroxycinnamamide derivative (ST 638) (Fig. 3C) has recently been shown to inhibit interleukin- 1-induction of intimal hyperplasia and autocoid-dependent vascular changes in the pig coronary artery (39). Although the vascular changes in this model are mediated largely by PDGF (39,71), ST 638 is not selective for the PDGFr tyrosine kinase and inhibits several tyrosine kinases (85,86). Additionally, it is interesting that ST 638 was administered topically to the coronary artery, achieving local concentrations in the arterial wall in the low mM range (39). It seems possible that derivatives of the cin- namamide series may be of use in local delivery systems. The ability of the hydrogel angioplasty catheter to absorb and release RG 50872 at concentrations that inhibit PDGF- dependent smooth muscle proliferation in culture has been demonstrated (19).

Replacement of a single cyano group with a phenyl or indole moiety did little to improve potency or selectivity. The resultant chemical entities were potentially more metabolically stable, however, and were reasonable candidates for in vivo efficacy evaluation. RG 13022 (2-[3-pyridyl1-3-[ 3,4-dimethoxyphenyl]-2-propenenitrile) (Fig. 3D) significantly decreased the growth of a human squamous cell carcinoma, an EGFr overexpressing tumor, in nude mice ( 1 10). RG 13291 (2-[3-indolyl-3-~3,4-dimethoxyphenyl]- 2-propenenitrile) (Fig. 3E) inhibited cuff-induced increase in arterial PDGFr tyrosine kinase activity in the rat carotid artery (4). Both compounds were administered intra- peritoneally.

Using RG 13022 as a structural base, Maguire et al. ( 5 3 , described the approach of constraining the acrylonitrile and generating a series of 3-substituted quinolines. With elimination of the potentially undesirable reactive cyano group of the modified tyrphostin RG 13022 and RG 13291, cishrans isomerization is removed, a basic center is created, and the compound is no longer a Michael acceptor. These changes resulted in a 250-fold separation in selectivity between PDGFr tyrosine kinase and EGFr tyrosine kinase and nanomolar potency for inhibition of the cell-free PDGFr autophosphorylation (Fig. 4). Substitutions at the 3 position, the 5-8 position, and the basic moiety influenced kinase activity. As illustrated, potency increased to 1-20 nM with 3-aromatic substitutions. Removal of the nitrogen resulted in loss of activity and potency decreased with removal of the 5,7 methoxy or methyl (or equivalent methoxy, methyl at 6, 7) substitutions or replacement with 8-substitutions (Fig. 4). Analysis of 3 ,4-pyridinyl) substituted quinoline (Fig. 5A) demonstrated that a potent TKl can be competitive for ATP (Ki = 14 nM) and still retain a favorable degree of selectivity for PDGFr tyrosine kinase compared with

Cardiovascular Drug Rer,iens. Vol. 14. N o . 4. 1996


RS

Quinoline PDGFr TKI

H OCH, OCH, H 3-thienyl 0.001 -0.02

H OCH, OCH, H NO2 >50

H OCH, OCH, H NH2 >50

H OCH, OCH, H OH >50

H OCH, OCH, H 3-pyridinyl 0.1-0.8 ~~

H OCH, OCH, H P-styryl 0.07-0.15

CH, H H CH, 3-thienyl >50

H 0-CH2-0 H 3-thienyl 0.2-0.6

H H H H 3-thienyl 0.2-0.5

OCH, H OCH, H 3-thienyl 0.001 -0.02

FIG. 4. Structure activity relationships of quinoline-based PDGFr TKIs. From ref. 55.

EGFr, erB2r, p56LCK tyrosine kinase and serine and threonine kinases PKC and PKA

Substituted quinoxalines also inhibit PDGF-dependent tyrosine kinase activity and DNA synthesis (Swiss 3T3 fibroblasts and porcine endothelial cells) with potency similar to the quinoline series. The most potent compound, AG1296 (Fig. 5B), inhibits SCFr but has less effect on EGFr autophosphorylation and DNA synthesis stimulated by insulin, fibroblast growth factor (FGF), EGF, and no effect on KDR-receptor (KDRr) autophosphorylation and VEGF-dependent tyrosine kinase activity and DNA synthesis. At higher concentrations, AG1296 reversed the phenotype of v-sis-transformed fibroblasts but not the phenotype of src-transformed cells (48). The authors comment on the need to improve selectivity of the quinoxaline series between PDGFr and SCFr tyrosine kinase but em- phasize that the absence of effect on KDRr tyrosine kinase makes AG1296 an attractive candidate for treatment of restenosis where inhibition of PDGFr-dependent stimulation of smooth muscle cell migration and proliferation without blockage of VEGF-dependent endothelial proliferation is a therapeutically desirable combination (48). Thus compounds

(21).



A.

3-(4-Pyridinyl) quinoline

6.

CH30

AG 1296

CGP 53716

I D.

Q Y N D 0

CGP 57148 FIG. 5. Representative PDGFr TKls, quinoline (A) , quinoxaline (B), and phenylaminopyrimidine series (C,D,E).

of the substituted quinolines and quinoxalines series represent potent inhibitors of the PDGFr tyrosine kinase with a moderate degree of selectivity.

A 2-phenylaminopyrimidine (CGP 53716) (Fig. 5C) was recently reported to be a highly selective inhibitor of the PDGFr tyrosine kinase. Selectivity was evaluated against more than twenty tyrosine kinases including EGFr, IGFr, INr, Src, Lyn, Fgr, Csk, and

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TPK (11), and among this series of kinases, only inhibition of Abl kinase was apparent. As a competitive antagonist for ATP (112), CGP 53716 inhibited cell-free and in situ PDGFr autophosphorylation with an IC5, value of 100 nM. CGP 53716 was also the first PDGFr TKI with demonstrated oral efficacy. Administration of CGP 53716 decreased tumor size in nude mice injected with v-sis-transformed BALB/c 3T3 cells, PDGFr- overexpressing cells, at doses of 6.3-50 mg/kg in a dose-dependent manner. In contrast, CGP 537 16 was ineffective in mice injected with the human epidermoid carcinoma A43 1, EGFr-overexpressing cells. Similarly, CGP 57148 (Fig. 5D), a potent inhibitor of Abl kinase (35 nM), also inhibited PDGFr tyrosine kinase and tumors of v-sis transformed BALBk3T3 cells (12) and displayed a selectivity profile similar to CGP 53719. These results support an interesting speculation that there exist similarities between the cytosolic Abl kinase and the membrane PDGFr kinase in the regions of the ATP binding cleft and surrounding amino acids that interact with the phenylaminopyrimidine structure (1 2).

Mechanistic Considerations In retrospect, the catalytic mechanism can explain, as a first approximation, the inhi-

bition by some EGFr and PDGFr TKIs. The main interactions during phosphate transfer must include the ATP y-phosphate, tyrosyl hydroxyl, and the tyrosyl aromatic ring (102). An effective inhibitor, in addition to resembling the natural substrates, must exhibit tighter binding and lower reactivity than the substrates. Reactivity is associated with the nucleophilicity of the tyrosyl anion, and, therefore, anion mimic-based inhibitors should exhibit lower nucleophilicity (1 11).

Detailed kinetic analysis of PDGFr TKIs is lacking. Of the known PDGFr TKI structures, only the quinolines have been shown to be competitive with respect to ATP (21). Extensive kinetic analysis using anilinoquinazolines as EGFr TKIs, however, indicates competitive kinetics with respect to ATP and non-competitive kinetics with respect to peptide substrate, suggesting anilinoquinazolines behave as analogs of ATP. Further- more, anilinoquinazolines include structural features that resemble aspects of both ATP and peptide substrate: quinazoline nitrogens mimic oxygens on the y-phosphate, the anilino nitrogen mimics the tyrosine hydroxyl oxygen, and the aromatic ring of the anilino function mimics the tyrosine aromatic ring (102).

Since the quinoline and quinoxaline PDGFr TKIs share with the quinazolines common features of a heterocyclic nitrogen atom and a lipophilic substitution (90), some compar- isons are possible. It is suggested that quinoline derivatives with substitution at the 3 position and methoxy groups at the 6 and 7 positions (Fig. 4) (55) can be considered bisubstrate analogs: methoxy groups mimic the potential tyrosyl anion while the nitrogen on the quinoline moiety mimics the oxygens of the y-phosphate of ATP. The aromatic substituent is most likely involved in interactions with the ATP binding site.

Consistent with this hypothesis are the results of extensive structure activity relation- ship studies that show that the nitrogen on the quinoline moiety and a lipophilic or aromatic substituent at the 3 position are required for inhibitory activity (55). Interest- ingly, inhibitory activity is lost by placement of a hydrophilic substituent at the 3 position (e.g., NO,, NH,, or OH), and recovered by addition of a benzyl group to the hydrophilic substituent. This underscores the importance of the nature of the substituent; since it is a site involved in binding, hydrophilic substituents must bring about looser binding.

CGP 53716 (Fig. 4C) also incorporates the general features of the TKIs mentioned

Cardiovascular Drug Reviews, Vol. 14, No. 4 , I996


above. The structure could be classified as a bisubstrate analog: it contains a peptide bond to which two benzene rings are attached; one of the benzene rings (possibly the tyrosine ring mimic) is bonded to a methyl group and to a nitrogen that could play the role of phenolate anion with reduced reactivity. The rest of the molecule includes two heterocyclic rings with nitrogens that resemble phosphate anions and/or the adenosine of ATP.

As noted (102), unbound ATP exists as a metal chelate allowing a greater degree of freedom than the anilinoquinazoline (or the quinoline) derivatives. As a result, binding to the receptor results in greater entropy loss for ATP binding than for inhibitor binding. ATP dissociation is favored over inhibitor dissociation because ATP is more polar. An additional possibility that favors inhibition over reaction with ATP could be tighter binding of inhibitors than ATP to the ground state thus preventing progress of reaction to the transition state (102). Kinetic analysis of PDGFr TKIs are necessary to support these suggestions.

CONCLUSIONS Crystallographic studies of cyclic AMP-dependent protein kinase and INr kinase, com-

puter modeling, and in v i m enzyme kinetics on EGFr TKIs have yielded an understanding of the tyrosine kinase reaction at the molecular level. This has assisted in part with the development of inhibitor molecules effective against the PDGFr tyrosine kinase. It is clear that knowledge of the crystal structure of both the activated and unactivated PDGFr and enzyme kinetic analyses with inhibitors would add immensely to the current understanding of mechanism(s) of inhibitor action. There are currently several substituted quinoline, quinoxaline, and phenylaminopyrimidine compounds that inhibit PDGFr tyrosine kinase with nanomolar potency. The structures of these compounds suggest that they incorporate features of both ATP and tyrosyl-containing protein substrate. Kinetic studies indicate that some of the series are competitive with respect to ATP but their interaction with substrate is yet to be defined. On the other hand, EGFr TKIs that display the general bisubstrate characteristics are competitive with respect to ATP and noncompetitive with respect to polypeptide substrate.

Although there have been attempts to define the specificity of PDGFr TKIs, information on the extent of inhibition of other tyrosine kinases and nontyrosine kinases is scant. Use of PDGFr TKIs as effective research tools to elucidate the involvement of PDGFr in normal and abnormal functions will require extensive selectivity studies. However, based on the consideration that fibroproliferative disorders may result from activation of several tyrosine kinases that synergize or potentiate PDGFr tyrosine kinase activity (75,82), however, so selectivity remains important but less critical an issue. Disease etiology and pathology may dictate the requirement for selectivity. In this regard, PDGF-dependent tumors might be treated with a selective PDGFr TKI, while atherosclerosis and restenosis, which are inadequately understood and driven by several growth factors, might respond more favorably to a relatively less selective PDGFr TKI.

PDGFr TKIs inhibit PDGF-dependent functions in cells but generally at higher concentrations than those required to inhibit the kinase in vitro. The discrepancy in potencies may be related to limits of membrane permeability, assay conditions, or cellular ATP concentrations. In animal studies, efficacy following oral administration of a PDGFr TKI, at least in mice, has been demonstrated. Whether this can be repeated in other species, especially in humans and in other pathological conditions, remains a future challenge.

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Inhibitors of the Platelet-Derived Growth Factor Receptor Tyrosine Kinase

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