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The Src pathway as a therapeutic strategy
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Drug Discovery Today: Therapeutic Strategies Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Cancer
The Src pathway as a therapeutic strategy Allison P. Belsches-Jablonski1,2,z, Michelle L. Demory2,z, J. Thomas Parsons2, Sarah J. Parsons2,* 1 2
Biology Program, Lynchburg College, 1501 Lakeside Drive, Lynchburg, VA 24501-3199, USA Department of Microbiology and Cancer Center, Box 800734, University of Virginia Health, System, Charlottesville, VA 22908-0734, USA
The protein and activity levels of the non-receptor tyrosine kinase Src are elevated in many cancers. In addition, Src has been shown to be involved in the regulation of important cellular processes including migration, survival and proliferation. Given the participation of Src in many diverse cell functions and the data implicating Src in tumor formation, there has been significant effort to develop Src inhibitors. Here, we review the cell culture, animal and clinical studies that document the use and specificity of Src inhibitors. Introduction Src is a 60 kDa non-receptor, membrane-associated TYROSINE (see Glossary). It is the cellular homologue of v-Src, a protein encoded by Rous Sarcoma virus, a retrovirus that causes solid tumors in chickens. Src is expressed in many cells and tissues, most notably in brain, platelets and osteoclasts [1]. The major defect in Src / knockout mice occurs in osteoclasts [2]. Therefore, many studies regarding Src have been performed in tissues (and cell lines derived from them) that express high levels of Src. The structure of Src is conserved among the Src family of kinases (Src, Lck, Hck, Fyn, Blk, Lyn, Fgr, Yes and Yrk). Src contains a MYRISTOYLATION (see Glossary) site at the N-terminus, followed by an Unique region, SH3 (see Glossary) domain, SH2 (see Glossary) domain, kinase domain and a short, C-terminal autoinhibitory domain (Fig. 1). The kinase domain is inactive when tyrosine KINASE
*Corresponding author: S.J. Parsons ([email protected]) z These authors have contributed equally to this work. 1740-6773/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2005.11.005
Section Editors: Tona Gilmer – GlaxoSmithKline, NC, USA Mike Luzzio – Pfizer Global Research and Development, USA 527, which resides within the C-terminal autoinhibitory domain, is phosphorylated by the Src negative regulator, C-terminal src kinase (Csk), and binds the SH2 domain. Dephosphorylation of tyrosine 527 or binding of cellular proteins, such as focal adhesion kinase (FAK), to the SH2 and/or SH3 domains results in a conformational change, allowing trans-phosphorylation on tyrosine 416 and kinase activity [3,4] (Fig. 1).
Significance of Src in targeted therapies Elevated protein levels and catalytic activity of Src have been detected in many cancers including breast, colon, lung, pancreatic, skin and head and neck. Src itself is only weakly oncogenic; therefore, it is hypothesized that it works in association with other proteins to regulate and/or promote multiple cellular processes [5]. Src has been shown to associate with tyrosine kinase receptors, cytokine receptors, steroid receptors, integrin receptors and G-protein coupled receptors. By associating with these molecules, Src is involved in the downstream pathways that, in combination, modulate a variety of cellular processes including growth, survival, adhesion and migration [5,2,6]. One example of Src involvement in cellular processes is its interactions with the epidermal growth factor receptor (EGFR). Src has been shown to phosphorylate tyrosine 845 www.drugdiscoverytoday.com
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Glossary Endocytosis: a process by which plasma membrane proteins are internalized into vesicles, which migrate into the cytoplasm of the cell. There are several types of endocytosis including, clathrin-mediated (discussed in this paper) and caveolin-mediated (not discussed). Several proteins regulate the clathrin mediated endocytic pathway, including, but not limited to, clathrin and dynamin. IC50: the molar concentration of receptor ligand in a competition experiment between two ligands at which one half of the agonist is bound to the receptor. Myristoylation: a protein modification that occurs through a cotranslational process. A 14-carbon saturated fatty acid, myristate, is covalently attached through an amide bond to an N-terminal glycine residue. The myristoylation of Src allows for its association with the plasma membrane. SH2: Src homology domain 2. This domain specifically binds to phosphotyrosine residues and their flanking sequences. In the case of Src, the SH2 domain participates in intramolecular interactions that maintain the protein in an inactive conformation. SH3: Src homology domain 3. This domain is involved in protein– protein interactions. Most simply, it associates with proline rich regions with the consensus sequence PxxP. Tyrosine kinase: an enzyme that transfers a phosphate group from ATP to a target tyrosine of a protein substrate. Ubiquitination: the process of transferring ubiquitin to a protein. This covalent modification has several functions, including degradation and sub-cellular localization of the modified protein.
of the EGFR, which is required for mitogenic and cellular survival signals emanating from the receptor [5]. Src phosphorylation can also modulate the activities of Akt, PLCg, PKCd and PP2A downstream of EGFR [7,8]. The endocytic components in the cytoplasm, clathrin and dynamin, are also targets of Src, which when phosphorylated, facilitate receptor internalization and continued signaling. By contrast,
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phosphorylation of Cbl by Src enhances UBIQUITINATION (see Glossary) and downregulation of EGFR [5] (Fig. 2). Through its involvement in cellular migration, Src might also play a role in metastasis of primary tumors. Indeed, Srctransformed cells exhibit decreased cell–extracellular matrix (ECM) interactions and loss of actin organization. Molecular mechanisms regulating these processes are dependent upon the interaction of Src with FAK. Binding of integrin receptors to ECM promotes integrin clustering and the phosphorylation and activation of FAK. Subsequently, FAK acts as a scaffold to recruit and maintain additional proteins in focal adhesions [8]. One of the recruited proteins is Src, which subsequently phosphorylates p130Cas, paxillin, vinculin, tensin, ezrin and cortactin [8] (Fig. 3).
An array of inhibitors The literature points to Src as a prime target for cancer therapy for two major reasons: (1) the key roles it plays in mediating cellular mitogenic, survival and migration processes; and (2) its increased protein levels and activation state in a broad spectrum of human tumors. Recently, numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. Src inhibitor studies can be divided into three general categories: in vitro studies only; preclinical studies in animal models; and clinical trials. Within these studies, three different types of inhibitors have been tested: ATP competitive inhibitors; molecules that disrupt protein–protein interaction with Src; and small molecule inhibitors that result in destabilization of Src. Tables 1 and 2 present a summary of recent studies describing Src inhibitors, grouped by their mechanism of inhibition.
Figure 1. Structure of Src. Src is a membrane-associated nonreceptor tyrosine kinase involved in several cell signaling pathways involved in cancer. It is composed of several domains, and can assume an active or an inactive configuration based on phosphorylation of a C-terminal tyrosine. Reproduced, with permission, from Ref. [41].
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Figure 2. Src phosphorylation and EGFR signaling. Src can phosphorylate the EGFR itself, downstream targets of EGFR signaling and/or mediators of EGFR ENDOCYTOSIS and downregulation.
In vitro studies 4-Anilinoquinazolines In 2001, Tian et al. [9] investigated several members of a class of protein kinase inhibitors, the 4-anilinoquinazolines (see Fig. 4 for backbone structure of Src inhibitors). Src kinase activity was tested in vitro using spectrophotometry, and one
such compound with a bulky 4-position group [4-(40 -phenoxyanilino-6,7-dimethoxyquinazoline, compound 4] exhibits specific inhibition of Src at 15 nM potency. Based on computer modeling of Src and the inhibitor, the authors suggested that this inhibitor might be a dual site inhibitor, with the inhibitor preventing binding of ATP and the peptide
Figure 3. (a) Src mechanism at focal adhesions. Src phosphorylation propagates adhesion and migration signals received by integrin receptors at focal adhesions. (b) The role of Hsp90. The chaperone protein Hsp90 aids newly synthesized proteins, including Src, in their maturation, stability and appropriate subcellular localization.
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Table 1. Src inhibitor compounds with clinical or preclinical data Inhibitor compound
Pros
Cons
Developments
Who is working on this compound
Refs
AP22408
Promising novel Src SH2amediated therapy for osteoporosis and bonerelated diseases, metastasis
No further advances since 2000
Binding studies of compound with Src or Lck SH2; shows decrease of bone resorption in bone culture assays and preclinical rat model
Ariad Pharmaceuticals http://www.ariad.com/
[28]
AZD0530
Promising antitumor results in preclinical rodent models of different cancers
Not yet tested in clinical trials of cancer patients
Clinical trials (Phase I) determined safety, tolerability, pharmacokinetics, biomarkers of bone resorption in healthy volunteers. Preclinical mouse and rat xenograft models of prostate cancer, pancreatic cancer tumors reduced in presence of AZD0530
AstraZeneca-US http://www.astrazeneca-us.com
[35,34,15,32, 29,33,30,31]
AZM475271
Effective treatment for pancreatic cancer
Requires gemcitabine co-treatment for greatest effects
Preclinical mouse models of human prostate or pancreatic cancer. Cell-based assays of cell death, migration and proliferation
AstraZeneca-UK http://www.astrazeneca.co.uk
[15,16]
BMS-354825
ABLb-specific inhibitor; very effective antitumor activity in mouse models of imatinib-resistant CMLc; low toxicity in clinical trial
No objective GISTd tumor responses by CTe in small Phase I trial (14 patients)
Clinical trial (Phase I) in patients with GIST; determined MTDf and toxicity; no objective tumor responses by CT. Preclinical studies of mouse B-cell or CML tumor growth show prolonged survival
Bristol Myers Squibb http://www.bms.com
[37,36]
CGP77675
Effectively reduces bone
Reduces tyrosine phosphorylation of Src substrates, but not Src itself
Preclinical study of bone resorption in rats shows decrease in bone resorption and loss after treatment
Novartis http://www.novartis.com/
[26,27]
resorption in rats 17-AAG
Targeted to peripheral blood mononuclear cells; one trial showed induction of HSP70g, an indicator that HSP90h is affected
No objective tumor responses in 66 patients over 2 trials
Clinical trials (Phase I) of patients with advanced cancer to determine MTD, toxicity, bioavailability, changes in Hsp90 levels; no tumor responses
Kosan Biosciences http://www.kosan.com
[39,40,38]
PP2
Allows gemcitabineresistant pancreatic cancer to become treatment sensitive
Works best in combination with other chemotherapeutic agents (gemcitabine, NI-5.12)
Preclinical studies in mouse xenografts of human pancreatic adenocarcinoma cells show decrease in tumor growth and inhibition of metastasis. PP2 pretreatment of mice prevents acute lung injury mediated by LPSi airway challenge
Pfizer http://www.pfizer.com
[24,22,25,21]
SKI-606
Dual inhibitor of Src and ABL; limited toxicity in animal models
Less obvious effects in cell culture than other compounds already used in clinic
Preclinical mouse xenografts of human colon cancer and CML tumors in nude mice show growth inhibition or complete regression
Wyeth Research http://www.wyeth.com/
[18,19,17]
SU6656
Potent inhibitor of Src family kinases in PDGFjmediated signalling
Similar effects as PP2 and tyrphostin A1; difficult to determine JAKk vs. Src specificity
SU6656 pretreatment of mice prevents acute lung injury mediated by LPS airway challenge
SUGEN/Pfizer http://www.pfizer.com
[21,10,20]
a
Src homology domain 2. Abelson murine leukemia viral oncogene. Chronic myelogenous leukaemia. d Gastrointestinal stromal tumor. e Computed tomography. f Maximum tolerated dose. g Heat shock protein 70 kDa. h Heat shock protein 90 kDa. i Lipopolysaccharide. j Platelet-derived growth factor. k Janus kinase. b c
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Table 2. Src inhibitors with test-tube or cell-based data only Inhibitor compound
Pros
Cons
Developments
Who is working on this compound
Refs
4-Anilinoquinazolines
Dual specificity for ATP binding site and peptide substrate binding site
Very preliminary work, no cell-based system
Test-tube assays of kinase activity; computer modeling of binding shows compound competitive with both ATP and peptide
GlaxoSmithKline http://www.gsk.com/index.htm
[9]
PD173952
Shows significant effects on downstream targets of Src pathway
Signaling in normal epithelial cells not necessarily indicative of malignant or transformed cells
Cell culture system (NHKa cells), compound inhibits cell proliferation and migration
Parke-Davis/Pfizer http://www.pfizer.com
[10]
PD173955
Strong antimitotic activity in cancer cell lines; competition not readily reversible
Inhibitor more effective for Yes than Src
Treatment of multiple cancer cell lines shows decrease in cellular Src and Yes kinase activity and block at G2/M phase of cell cycle
Parke-Davis/Pfizer http://www.pfizer.com
[11]
Terphenylquinone
Highly specific, IC50 = 3.9 mmol
Preliminary in vitro work, no cell-based system
Inhibitors from Stilbella sp. show significant inhibition of Src by test-tube kinase assays
Boehringer Ingelheim Pharma http://www.boehringer-ingelheim.com
[12]
UCS15A
Novel mechanism; shows significant effects on downstream targets of Src pathway
Not specific for Src, but for many SH3b-mediated protein–protein interactions
In vitro studies of compound disruption of SH3-mediated protein–protein interactions via proline-rich domains
Pharmaceutical Research Laboratories; Kyowa Hakko Kygyo Co., Ltd., Japan http://www.kyowa.co.jp
[13,14]
a b
Normal human keratinocytes. Src homology domain 3.
substrate. Other anilinoquinazolines such as AZM475271 and AZD0530 have been tested in preclinical and clinical models (see below).
PD173952 The pyrido-pyrimidine inhibitor PD173952 was examined for effects on Src activity in normal human keratinocytes
(NHK) [10]. In this system, PD173952 (as well as SU6656 and PP1) inhibits EGF-stimulated EGFR-Y1148 phosphorylation (with no decrease in EGFR kinase activity), ERK phosphorylation, cell proliferation, colony formation and migration. However, apoptosis is not seen in treated cells. Reduction of phosphorylation of EGFR-Y845 was evidence that Src kinase activity itself is inhibited. We suggest that
Figure 4. Heterocyclic templates used to design Src tyrosine kinase inhibitors. As reviewed in Sawyer et al. [42], below are the chemical structures of various starting compounds used in synthesis of Src inhibitors. Reproduced, with permission, from Ref. [42].
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inhibition of Src kinase activity affects erbB-family-driven growth and migration.
PD173955 This pyrido-pyrimidine is an ATP-competitor and tyrosine kinase inhibitor selective for Src and Yes, a Src family member. PD173955 effectively inhibits Src and Yes kinase activities in test-tube kinase assays using enolase as a synthetic substrate (IC50 = 22 nM; see Glossary), and in a study of eight breast cancer cell lines, it was found to block cell cycle progression, with the cells arresting at G2/M [11].
Terphenylquinone Identified in a natural product extract, terphenylquinone compound 4 (a benzoquinone) was isolated from the fungus Stilbella sp. strain 1586 and it exhibited inhibition of Src kinase activity (IC50 = 3.9 mM) [12]. Src kinase activity was measured by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA).
UCS15A Originally identified as an antibiotic produced by Streptomyces sp., UCS15A has been studied as a completely novel small-molecule inhibitor of Src [13]. It is not specific for Src and does not inhibit Src kinase activity, yet studies by Oneyama et al. [14] in HCT-116 colorectal carcinoma cells suggest that UCS15A disrupts SH3-mediated protein interactions by directly interacting with proline-rich (P-xx-P) domains in selective proteins. This mechanism is borne out by studies in HCT116 cells in which UCS15A prevents Src association with and phosphorylation of Sam68 and cortactin, substrates of Src. Further studies in a mouse calvaria organ culture system have shown that inhibition of Src by USC15A results in a dose-dependent inhibition of bone resorption, with no osteoclast toxicity [13].
Preclinical studies AZM475271 AZM475271 is a novel anilinoquinazoline, ATP-competitive inhibitor of Src. In a promising study by Yang et al. [15], mouse xenografts of LNCap prostate carcinoma cells engineered to express gastrin-releasing peptide (GRP) that were grown in cell culture or soft agar in the absence of androgen. AZM475271 shows significant inhibition of colony formation, suggesting that the compound might have therapeutic benefit for patients with androgen-independent prostate cancer. In a more complete study of AZM475271, Yezhelyev et al. [16] studied the response of nude mice with orthotopic L3.6pl human pancreatic cancer tumors to oral treatment with this compound alone and in conjunction with gemcitabine. With AZM475271 alone, primary pancreatic tumor volume 318
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is reduced by 40%, and together with gemcitabine, this was reduced by 90%, with no metastasis to lymph nodes. Cellbased studies noted that AZM475271 treatment (alone or with gemcitabine) reduces Src, Lck and Yes kinase activity (with IC50 = 0.01, 0.03 and 0.08 mM, respectively), reduces L3.6pl cell proliferation and cell migration and increases the rate of apoptosis.
SKI-606 SKI-606 (also known as compound 77) is a small-molecule ATP-competitive inhibitor, classified as a 4-anilino-3-quinolincarbonitrile, and is a potent dual inhibitor of Src and BCRABL kinase activity. Thaimattam et al. [17] have performed three-dimensional quantitative, structure–activity relationship (3D-QSAR) analyses and docking analyses of SKI-606 with Src and BCR-ABL kinases and have shown that SKI606 docks in the active site of Src and Abl. A cell culture model studied by Golas et al. [18] using the human CML cell lines, K562 and KU812, shows that SKI-606 inhibits autophosphorylation of Src family kinases Lyn and Hck, blocks phosphorylation of BCR-ABL and ablates phosphorylation of STAT5. Using test-tube Src kinase assays, an IC50 = 1.2 nM was measured [18]. In the colon carcinoma cell line HT-29, SKI-606 decreases phosphorylation of FAK and inhibits HT-29 colony formation in soft agar [19]. This group also studied preclinical in vivo mouse models of HT-29 and K562 xenografts. After oral administration of SKI-606 to nude mice, xenograft tumors of both origins either fully regress or fail to form. No toxicity is seen, even after prolonged administration [18,19].
SU6656 As a small molecule, indolinone-based inhibitor of Src family kinases (Src, Fyn, Yes, Lyn), SU6656 has been studied in a variety of cell types. Blake et al. [20] have used this inhibitor to probe signaling pathways of the PDGF receptor because SU6656 does not inhibit this receptor but does inhibit Src (IC50 = 0.28 mM), a mediator of PDGF signaling. From their findings they conclude that in NIH3T3 cells, SU6656 inhibits PDGF and Src-driven mitogenesis and also inhibits PDGFdriven c-Myc induction. Downstream Src substrates also exhibit decreased or ablated tyrosine phosphorylation after treatment with SU6656. Severgnini et al. [21] examined the role of Src in a lung injury model in mice, which involved a lethal lipopolysaccharide (LPS) challenge in the airway. In mice pretreated with SU6656, lung homogenates were examined by western blotting for phosphorylated Src; SU6656 sharply decreases Src phosphorylation to basal levels. Pretreatment with SU6656 (or PP2) decreases lung injury (neutrophil infiltration, hemorrhage, endothelial and epithelial damage, respiratory distress and cytokine production) after LPS treatment. Mortality from LPS challenge is also prevented.
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PP2 PP1 and PP2 were identified in 1995 as novel, pyrazolopyrimidine compounds that are selective inhibitors of Src family kinases [22]. Later studies suggested that PP1 was not as selective as first believed because it also inhibits the PDGFb receptor kinase (IC50 for Src = 0.7 mM; IC50 for PDGFb = 3 mM) [23]. Boyd et al. [24] examined the effects of PP2 in human colon cancer cells (HT-29; SW480). PP2 causes a dose-dependent decrease in urokinase-type plasminogen activator receptor (u-PAR) gene expression, u-PAR protein expression, cell invasiveness and Src kinase activity. Combined with NI-5.12 (u-PAR antagonist), PP2 treatment leads to a synergistic inhibition of invasiveness of cells. We noted that u-PAR expression can however be controlled by factors other than Src. Duxbury et al. [25] tested a panel of pancreatic adenocarcinoma cell lines for Src expression, Src phosphorylation and kinase activity after treatment with PP2 alone or in combination with gemcitabine. They reported that a decrease in Src Y419 phosphorylation is associated with a decrease in gemcitabine resistance, suggesting that Src is an important player in acquisition of gemcitabine resistance. In addition, PP2 augments apoptosis, decreases tumor growth and inhibits metastasis in nude mice xenografts with PNAC1 pancreatic adenocarcinoma cells.
CGP77675 Missbach et al. [26,27] examined several members of a class of di-phenyl-pyrrolo-pyrimidines for test-tube inhibition of Src. CGP77675 was the most promising of these compounds, inhibiting Src autophosphorylation and phosphorylation of FAK and paxillin (IC50 = 0.2–5.0 mM) in IC8.1 cells. In vivo studies of CGP77675 in ovariectomized rats show prevention of osteoclast-driven bone loss, bone resorption and bone architectural changes.
AP22408 Phosphotyrosine mimic AP22408 binds the Src and/or Lck SH2 domain, as shown by molecular modeling, X-ray crystallography and test-tube binding assays [28]. The compound was designed to be bone-specific and was shown through hydroxyapatite chromatography to specifically associate with bone. AP22408 inhibits rabbit osteoclast bone resorption (IC50 = 1.6 mM) and demonstrates bone antiresorptive activity in a rat model. We suggest that AP22408 will prove useful in treatment of bone cancer metastasis, osteoporosis and other bone diseases.
Clinical studies AZD0530 AZD0530 is a highly selective, dual-specificity small molecule inhibitor of Src and BCR-ABL. It is another anilinoquinazoline that has recently been studied primarily in cell culture
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models and preclinical animal models, but at least two Phase I clinical trials in healthy volunteers have been published as abstracts. In tissue culture, it shows promise as an inhibitor in breast cancer cell lines and in models of osteoclastic bone resorption, so it is a natural therapeutic candidate for clinical trials in metastatic cancers involving bone. AZD0530 inhibits migration of MDA-MB-231 breast carcinoma cells and decreases tyrosine phosphorylation of Src, FAK and paxillin in wild-type MCF7 breast carcinoma cells and MCF7 cells expressing an estrogen receptor-a mutant [29]. AZD0530 also blocks MCF7 cell proliferation and invasion stimulated by estrogen [30]. In wild-type and tamoxifenresistant MCF7 cells, AZD0530 reduces motility, FAK Y861 phosphorylation, integrin expression and cell attachment [31]. Preclinical animal models have shown that AZD0530 completely prevents growth of Src-expressing 3T3 xenografts in nude rats, and significantly increases survival rate in a rat orthotopic model of human pancreatic cancer (BxPC-3 cells) [29]. In a separate rat tumor model with Src-expressing 3T3 xenografts, AZD0530 inhibits tumor growth in a dose-related fashion, with >90% of tumor growth inhibition at 6 and 10 mg/kg [32]. Tissue distribution and pharmacokinetics were also determined in this study. In a preclinical model of bone dynamics by Mullender et al. [33], AZD0530 reduced rabbit osteoclastic bone resorption area, specifically the number and average area of resorption pits in a pit-formation assay. The authors concluded that AZD0530 can show therapeutic benefit in metastatic bone disease. In a Phase I clinical study designed to examine effects of AZD0530 on bone resorption in healthy male volunteers, Eastell et al. [34] reported inhibition of osteoclast-mediated bone resorption at a 1000 mg dose, as assayed by markers of serum crosslinked C telopeptide of type I collagen (sCTX) and urinary crosslinked N telopeptide (NTX). These authors also noted the therapeutic potential of AZD0530 for metastatic bone disease and osteoporosis. In a Phase I clinical trial to determine safety, tolerability and pharmacokinetics of AZD0530 in healthy male volunteers, Lockton et al. [35] reported that a once-daily oral dosing of 250 mg is well tolerated with mild adverse effects. The plasma concentrations of AZD0530 remain above the IC50 for Src kinase in these patients.
BMS-354825 BMS-354825 (a methyl-pyrimidine carboxamide) is an orally active, ATP-competitive, potent inhibitor of several kinase molecules including Src family members, Kit, PDGFR and BCR-ABL. Much of the preclinical work using BMS-354825 has been done in mouse models of imatinib-resistant CML, examining the signaling of BCR-ABL rather than Src. (Imatinib is a specific BCR-ABL inhibitor used as front-line therapy for CML.) In preclinical studies by Shah et al. [36], SCID mice www.drugdiscoverytoday.com
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were injected with mouse B-cells (Ba/F3) expressing BCR-ABL (either wild-type or an imatinib-resistant mutant). The authors report a reduction of tumor burden and prolonged survival with BMS-354825, regardless of the BCR-ABL isoform expressed. In addition, BMS-354825 inhibits growth of bone progenitor cells from CML patients but does not affect bone cell growth in healthy volunteers. A Phase I dose-escalation study of BMS-354825 in treatment-resistant gastrointestinal stromal tumors (GIST) was recently published as an abstract at the American Society of Clinical Oncology (ASCO; http://www.asco.org). Evans et al. [37] reported that BMS-354825 can be safely administered at doses of up to 70 mg BID on a 5-days on, 2-days off, weekly schedule. No objective tumor responses are seen by CT analysis, although the abstract suggests that the clinical benefits noted in a subset of patients are encouraging.
impact on human disease is disappointing. Among the challenges to investigators is defining how inhibition of Src activity impacts normal cell function as well as tumor cell homeostasis. For this, standard assays for the Src-dependent signaling pathways need to be evaluated. Fortunately, the characterization of two such pathways, EGF receptor signaling and integrin signaling, provide ample opportunity to carefully assess the efficacy of Src inhibitors both in cells in culture and in model systems. Application of these assays to well characterized preclinical animal models and human tumors where the relevant pathways are functional might speed the understanding of how Src inhibitors work. It is clear, however, that the careful characterization of compound activity and specificity is essential if new Src inhibitors are to be successful drugs in the clinic.
17-AAG/geldanamycin
Acknowledgements
17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an analog of geldanamycin and specifically binds to the cytosolic chaperone protein HSP90. HSP90 binds to Src, HER2/ neu, and other signaling molecules, and has been shown to aid in their maturation, stability and appropriate subcellular localization [38]. Presumably, 17-AAG disrupts this interaction between its target and HSP90, resulting in increased rates of degradation and inhibition of signaling activity (reviewed in Ref. [38]). Ramanathan et al. [39] reported a Phase I study of 17-AAG in 45 patients with refractory advanced cancers of many different origins. Doses of 17-AAG between 10 and 295 mg/ m2 have been found to be safe and of limited toxicity, given on a weekly basis. However, no objective tumor responses are observed. A similar Phase I trial published by Goetz et al. [40] determined that the maximum tolerated dose of 17-AAG in 21 patients with advanced cancer is 308 mg/m2, with no tumor response.
Conclusions Src and Src family kinases represent an attractive target for inhibitor development because of their roles in the regulation of cellular processes and the link to cancer. As noted above, several inhibitors have been identified and are in different stages of development, some showing promise in early clinical studies. Researchers and clinicians face a daunting task when deciding on potentially therapeutic compounds to move to clinical trials. Currently, compounds active against enzymes such as Src are identified using classic test-tube based assays, subsequently tested in cell culture systems, and if efficacious, the studies are moved into animal models of tumorigenesis. Finally, human trials are conducted. Given the thorough, time-consuming research and planning, the correlation between test-tube inhibitory activity, inhibitory activity in cell culture and antitumor activity in animals and 320
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We gratefully acknowledge the support of the following granting agencies: A.P.B-J. is supported by Lynchburg College and NIH-NCI grants CA104237 and CA71449, awarded to S.J.P. M.L.D. is a recipient of a Dissertation Research Award from the Susan G. Komen Breast Cancer Foundation, #DISS0402970, awarded to S.J.P. J.T.P. and S.J.P. acknowledge support from the NIH-NCI, CA40042 and CA29243 to J.T.P. and CA104237 and CA71449 to S.J.P.
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Yang, J.C. et al. (2005) Src kinase inhibition of neuropeptide-induced androgen-independent prostate cancer. 96th Annual Mtg. of Amer. Assoc. Cancer Res. (A3180) Yezhelyev, M.V. et al. (2004) Inhibition of src tyrosine kinase as treatment for human pancreatic cancer growing orthotopically in nude mice. Clin. Cancer Res. 10, 8028–8036 Thaimattam, R. et al. (2005) 3D-QSAR studies on c-Src kinase inhibitors and docking analyses of a potent dual kinase inhibitor of c-Src and c-Abl kinases. Bioorg. Med. Chem. 13, 4704–4712 Golas, J.M. et al. (2003) SKI-606, a 4-anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases, is a potent antiproliferative agent against chronic myelogenous leukemia cells in culture and causes regression of K562 xenografts in nude mice. Cancer Res. 63, 375–381 Golas, J.M. et al. (2005) SKI-606, a Src/Abl inhibitor with in vivo activity in colon tumor xenograft models. Cancer Res. 65, 5358–5364 Blake, R.A. et al. (2000) SU6656, a selective Src family kinase inhibitor, used to probe growth factor signaling. Mol. Cell Biol. 20, 9018–9027 Severgnini, M. et al. (2005) Inhibition of the Src and JAK kinases protects against lipopolysaccharide-induced acute lung injury. Am. J. Respir. Crit. Care Med. 171, 858–867 Hanke, J.H. et al. (1996) Discovery of a novel, potent and Src familyselective tyrosine kinase inhibitor. J. Biol. Chem. 271, 695–701 Waltenberger, J. et al. (1999) A dual inhibitor of platelet-derived growth factor b-receptor and src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. Circ. Res. 85, 12–22 Boyd, D.D. et al. (2004) Combination of an src kinase inhibitor with a novel pharmacological antagonist of the urokinase receptor diminishes in vitro colon cancer invasiveness. Clin. Cancer Res. 10, 1545–1555 Duxbury, M.S. et al. (2004) Inhibition of src tyrosine kinase impairs inherent and acquired gemcitabine resistance in human pancreatic adenocarcinoma cells. Clin. Cancer Res. 10, 2307–2318 Missbach, M. et al. (1999) A novel inhibitor of the tyrosine kinase Src suppressed phosphorylation of its major cellular substrates and reduces bone resorption in vitro and in rodent models in vivo. Bone 24, 437–449 Missbach, M. et al. (2000) Substituted 5,7-dephenylpyrrolo[2,3d]pyrimindines: potent inhibitors of the tyrosine kinase c-Src. Bioorg. Med. Chem. Lett. 10, 945–949 Shakespeare, W. et al. (2000) Structure-based design of an osteoclastselective, nonpeptide src homology 2 inhibitor with in vivo antiresorptive activity. PNAS 97, 9373–9378
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Hennequin, L.F. et al. (2005) The discovery of AZD0530: a novel, oral, highly selective and dual-specific inhibitor of the Src and Abl family kinases. 96th Annual Mtg. of Amer. Assoc. Cancer Res. (A2537) Herynk, M.H. et al. (2005) c-Src inhibition with AZD0530 reduces estrogen mediated growth and invasion in breast cancer cells expressing the K303R ERa mutant. 96th Annual Mtg. of Amer. Assoc. Cancer Res. (A264) Hiscox, S. et al. (2005) Adhesion-independent focal adhesion kinase activation involves Src and promotes cell adhesion and motility in tamoxifen-resistant MCF-7 cells and is inhibited by the Src/Abl kinase inhibitor AZD0530. 96th Annual Mtg. of Amer. Assoc. Cancer Res. (A266) Logie, A. et al. (2005) Pharmacokinetics, tissue distribution and anti-tumor activity of the Src/Abl kinase inhibitor AZD0530 in a rat xenograft model. 96th Annual Mtg. of Amer. Assoc. Cancer Res. (A5989) Mullender, M.G. et al. (2005) Inhibition of osteoclastic bone resorption by the novel, potent, and selective c-Src/Abl kinase inhibitor, AZD0530. 96th Annual Mtg. of Amer. Assoc. Cancer Res. (A2923) Eastell, R. et al. (2005) The effect of AZD0530, a highly selective, orally available Src/Abl kinase inhibitor, on biomarkers of bone resorption in healthy males. 2005 ASCO Annual Meeting (A3041) Lockton, J.A. et al. (2005) Phase I ascending single and multiple dose studies to assess the safety, tolerability and pharmacokinetics of AZD0530, a highly selective, dual-specific Src-Abl inhibitor. 2005 ASCO Annual Meeting (A3125) Shah, N.P. et al. (2004) Overriding Imatinib resistance with a novel ABL kinase inhibitor. Science 305, 399–401 Evans, T.R. et al. (2005) Phase I dose-escalation study of the src and multikinase inhibitor BMS-354825 in patients with GIST and other solid tumors. 2005 ASCO Annual Meeting (A3034) Neckers, L. et al. (2002) Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol. Med. 8 (Suppl.), S55–S61 Ramanathan, R.K. et al. (2005) Phase I pharmacokinetic– pharmacodynamic study of 17-(allylamino)-17-demethoxygeldanamycin (17AAG, NSC 330507), a novel inhibitor of heat shock protein 90, in patients with refractory advance cancers. Clin. Cancer Res. 11, 3385–3391 Goetz, M.P. et al. (2005) Phase I trial of 17-allylamino-17demethoxygeldanamycin in patients with advanced cancer. J. Clin Oncol. 23, 1078–1087 Parsons, S.J. (2001) Src. In Encyclopedic Reference of Cancer (Schwab, M., ed.), pp. 841–843, Springer Sawyer, T. et al. (2001) Src inhibitors: genomics to therapeutics. Expert Opin. Invest. Drugs 10, 1327–1344
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Drug Discovery Today: Therapeutic Strategies Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Cancer
The Src pathway as a therapeutic strategy Allison P. Belsches-Jablonski1,2,z, Michelle L. Demory2,z, J. Thomas Parsons2, Sarah J. Parsons2,* 1 2
Biology Program, Lynchburg College, 1501 Lakeside Drive, Lynchburg, VA 24501-3199, USA Department of Microbiology and Cancer Center, Box 800734, University of Virginia Health, System, Charlottesville, VA 22908-0734, USA
The protein and activity levels of the non-receptor tyrosine kinase Src are elevated in many cancers. In addition, Src has been shown to be involved in the regulation of important cellular processes including migration, survival and proliferation. Given the participation of Src in many diverse cell functions and the data implicating Src in tumor formation, there has been significant effort to develop Src inhibitors. Here, we review the cell culture, animal and clinical studies that document the use and specificity of Src inhibitors. Introduction Src is a 60 kDa non-receptor, membrane-associated TYROSINE (see Glossary). It is the cellular homologue of v-Src, a protein encoded by Rous Sarcoma virus, a retrovirus that causes solid tumors in chickens. Src is expressed in many cells and tissues, most notably in brain, platelets and osteoclasts [1]. The major defect in Src / knockout mice occurs in osteoclasts [2]. Therefore, many studies regarding Src have been performed in tissues (and cell lines derived from them) that express high levels of Src. The structure of Src is conserved among the Src family of kinases (Src, Lck, Hck, Fyn, Blk, Lyn, Fgr, Yes and Yrk). Src contains a MYRISTOYLATION (see Glossary) site at the N-terminus, followed by an Unique region, SH3 (see Glossary) domain, SH2 (see Glossary) domain, kinase domain and a short, C-terminal autoinhibitory domain (Fig. 1). The kinase domain is inactive when tyrosine KINASE
*Corresponding author: S.J. Parsons ([email protected]) z These authors have contributed equally to this work. 1740-6773/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2005.11.005
Section Editors: Tona Gilmer – GlaxoSmithKline, NC, USA Mike Luzzio – Pfizer Global Research and Development, USA 527, which resides within the C-terminal autoinhibitory domain, is phosphorylated by the Src negative regulator, C-terminal src kinase (Csk), and binds the SH2 domain. Dephosphorylation of tyrosine 527 or binding of cellular proteins, such as focal adhesion kinase (FAK), to the SH2 and/or SH3 domains results in a conformational change, allowing trans-phosphorylation on tyrosine 416 and kinase activity [3,4] (Fig. 1).
Significance of Src in targeted therapies Elevated protein levels and catalytic activity of Src have been detected in many cancers including breast, colon, lung, pancreatic, skin and head and neck. Src itself is only weakly oncogenic; therefore, it is hypothesized that it works in association with other proteins to regulate and/or promote multiple cellular processes [5]. Src has been shown to associate with tyrosine kinase receptors, cytokine receptors, steroid receptors, integrin receptors and G-protein coupled receptors. By associating with these molecules, Src is involved in the downstream pathways that, in combination, modulate a variety of cellular processes including growth, survival, adhesion and migration [5,2,6]. One example of Src involvement in cellular processes is its interactions with the epidermal growth factor receptor (EGFR). Src has been shown to phosphorylate tyrosine 845 www.drugdiscoverytoday.com
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Glossary Endocytosis: a process by which plasma membrane proteins are internalized into vesicles, which migrate into the cytoplasm of the cell. There are several types of endocytosis including, clathrin-mediated (discussed in this paper) and caveolin-mediated (not discussed). Several proteins regulate the clathrin mediated endocytic pathway, including, but not limited to, clathrin and dynamin. IC50: the molar concentration of receptor ligand in a competition experiment between two ligands at which one half of the agonist is bound to the receptor. Myristoylation: a protein modification that occurs through a cotranslational process. A 14-carbon saturated fatty acid, myristate, is covalently attached through an amide bond to an N-terminal glycine residue. The myristoylation of Src allows for its association with the plasma membrane. SH2: Src homology domain 2. This domain specifically binds to phosphotyrosine residues and their flanking sequences. In the case of Src, the SH2 domain participates in intramolecular interactions that maintain the protein in an inactive conformation. SH3: Src homology domain 3. This domain is involved in protein– protein interactions. Most simply, it associates with proline rich regions with the consensus sequence PxxP. Tyrosine kinase: an enzyme that transfers a phosphate group from ATP to a target tyrosine of a protein substrate. Ubiquitination: the process of transferring ubiquitin to a protein. This covalent modification has several functions, including degradation and sub-cellular localization of the modified protein.
of the EGFR, which is required for mitogenic and cellular survival signals emanating from the receptor [5]. Src phosphorylation can also modulate the activities of Akt, PLCg, PKCd and PP2A downstream of EGFR [7,8]. The endocytic components in the cytoplasm, clathrin and dynamin, are also targets of Src, which when phosphorylated, facilitate receptor internalization and continued signaling. By contrast,
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phosphorylation of Cbl by Src enhances UBIQUITINATION (see Glossary) and downregulation of EGFR [5] (Fig. 2). Through its involvement in cellular migration, Src might also play a role in metastasis of primary tumors. Indeed, Srctransformed cells exhibit decreased cell–extracellular matrix (ECM) interactions and loss of actin organization. Molecular mechanisms regulating these processes are dependent upon the interaction of Src with FAK. Binding of integrin receptors to ECM promotes integrin clustering and the phosphorylation and activation of FAK. Subsequently, FAK acts as a scaffold to recruit and maintain additional proteins in focal adhesions [8]. One of the recruited proteins is Src, which subsequently phosphorylates p130Cas, paxillin, vinculin, tensin, ezrin and cortactin [8] (Fig. 3).
An array of inhibitors The literature points to Src as a prime target for cancer therapy for two major reasons: (1) the key roles it plays in mediating cellular mitogenic, survival and migration processes; and (2) its increased protein levels and activation state in a broad spectrum of human tumors. Recently, numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. Src inhibitor studies can be divided into three general categories: in vitro studies only; preclinical studies in animal models; and clinical trials. Within these studies, three different types of inhibitors have been tested: ATP competitive inhibitors; molecules that disrupt protein–protein interaction with Src; and small molecule inhibitors that result in destabilization of Src. Tables 1 and 2 present a summary of recent studies describing Src inhibitors, grouped by their mechanism of inhibition.
Figure 1. Structure of Src. Src is a membrane-associated nonreceptor tyrosine kinase involved in several cell signaling pathways involved in cancer. It is composed of several domains, and can assume an active or an inactive configuration based on phosphorylation of a C-terminal tyrosine. Reproduced, with permission, from Ref. [41].
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Figure 2. Src phosphorylation and EGFR signaling. Src can phosphorylate the EGFR itself, downstream targets of EGFR signaling and/or mediators of EGFR ENDOCYTOSIS and downregulation.
In vitro studies 4-Anilinoquinazolines In 2001, Tian et al. [9] investigated several members of a class of protein kinase inhibitors, the 4-anilinoquinazolines (see Fig. 4 for backbone structure of Src inhibitors). Src kinase activity was tested in vitro using spectrophotometry, and one
such compound with a bulky 4-position group [4-(40 -phenoxyanilino-6,7-dimethoxyquinazoline, compound 4] exhibits specific inhibition of Src at 15 nM potency. Based on computer modeling of Src and the inhibitor, the authors suggested that this inhibitor might be a dual site inhibitor, with the inhibitor preventing binding of ATP and the peptide
Figure 3. (a) Src mechanism at focal adhesions. Src phosphorylation propagates adhesion and migration signals received by integrin receptors at focal adhesions. (b) The role of Hsp90. The chaperone protein Hsp90 aids newly synthesized proteins, including Src, in their maturation, stability and appropriate subcellular localization.
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Table 1. Src inhibitor compounds with clinical or preclinical data Inhibitor compound
Pros
Cons
Developments
Who is working on this compound
Refs
AP22408
Promising novel Src SH2amediated therapy for osteoporosis and bonerelated diseases, metastasis
No further advances since 2000
Binding studies of compound with Src or Lck SH2; shows decrease of bone resorption in bone culture assays and preclinical rat model
Ariad Pharmaceuticals http://www.ariad.com/
[28]
AZD0530
Promising antitumor results in preclinical rodent models of different cancers
Not yet tested in clinical trials of cancer patients
Clinical trials (Phase I) determined safety, tolerability, pharmacokinetics, biomarkers of bone resorption in healthy volunteers. Preclinical mouse and rat xenograft models of prostate cancer, pancreatic cancer tumors reduced in presence of AZD0530
AstraZeneca-US http://www.astrazeneca-us.com
[35,34,15,32, 29,33,30,31]
AZM475271
Effective treatment for pancreatic cancer
Requires gemcitabine co-treatment for greatest effects
Preclinical mouse models of human prostate or pancreatic cancer. Cell-based assays of cell death, migration and proliferation
AstraZeneca-UK http://www.astrazeneca.co.uk
[15,16]
BMS-354825
ABLb-specific inhibitor; very effective antitumor activity in mouse models of imatinib-resistant CMLc; low toxicity in clinical trial
No objective GISTd tumor responses by CTe in small Phase I trial (14 patients)
Clinical trial (Phase I) in patients with GIST; determined MTDf and toxicity; no objective tumor responses by CT. Preclinical studies of mouse B-cell or CML tumor growth show prolonged survival
Bristol Myers Squibb http://www.bms.com
[37,36]
CGP77675
Effectively reduces bone
Reduces tyrosine phosphorylation of Src substrates, but not Src itself
Preclinical study of bone resorption in rats shows decrease in bone resorption and loss after treatment
Novartis http://www.novartis.com/
[26,27]
resorption in rats 17-AAG
Targeted to peripheral blood mononuclear cells; one trial showed induction of HSP70g, an indicator that HSP90h is affected
No objective tumor responses in 66 patients over 2 trials
Clinical trials (Phase I) of patients with advanced cancer to determine MTD, toxicity, bioavailability, changes in Hsp90 levels; no tumor responses
Kosan Biosciences http://www.kosan.com
[39,40,38]
PP2
Allows gemcitabineresistant pancreatic cancer to become treatment sensitive
Works best in combination with other chemotherapeutic agents (gemcitabine, NI-5.12)
Preclinical studies in mouse xenografts of human pancreatic adenocarcinoma cells show decrease in tumor growth and inhibition of metastasis. PP2 pretreatment of mice prevents acute lung injury mediated by LPSi airway challenge
Pfizer http://www.pfizer.com
[24,22,25,21]
SKI-606
Dual inhibitor of Src and ABL; limited toxicity in animal models
Less obvious effects in cell culture than other compounds already used in clinic
Preclinical mouse xenografts of human colon cancer and CML tumors in nude mice show growth inhibition or complete regression
Wyeth Research http://www.wyeth.com/
[18,19,17]
SU6656
Potent inhibitor of Src family kinases in PDGFjmediated signalling
Similar effects as PP2 and tyrphostin A1; difficult to determine JAKk vs. Src specificity
SU6656 pretreatment of mice prevents acute lung injury mediated by LPS airway challenge
SUGEN/Pfizer http://www.pfizer.com
[21,10,20]
a
Src homology domain 2. Abelson murine leukemia viral oncogene. Chronic myelogenous leukaemia. d Gastrointestinal stromal tumor. e Computed tomography. f Maximum tolerated dose. g Heat shock protein 70 kDa. h Heat shock protein 90 kDa. i Lipopolysaccharide. j Platelet-derived growth factor. k Janus kinase. b c
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Table 2. Src inhibitors with test-tube or cell-based data only Inhibitor compound
Pros
Cons
Developments
Who is working on this compound
Refs
4-Anilinoquinazolines
Dual specificity for ATP binding site and peptide substrate binding site
Very preliminary work, no cell-based system
Test-tube assays of kinase activity; computer modeling of binding shows compound competitive with both ATP and peptide
GlaxoSmithKline http://www.gsk.com/index.htm
[9]
PD173952
Shows significant effects on downstream targets of Src pathway
Signaling in normal epithelial cells not necessarily indicative of malignant or transformed cells
Cell culture system (NHKa cells), compound inhibits cell proliferation and migration
Parke-Davis/Pfizer http://www.pfizer.com
[10]
PD173955
Strong antimitotic activity in cancer cell lines; competition not readily reversible
Inhibitor more effective for Yes than Src
Treatment of multiple cancer cell lines shows decrease in cellular Src and Yes kinase activity and block at G2/M phase of cell cycle
Parke-Davis/Pfizer http://www.pfizer.com
[11]
Terphenylquinone
Highly specific, IC50 = 3.9 mmol
Preliminary in vitro work, no cell-based system
Inhibitors from Stilbella sp. show significant inhibition of Src by test-tube kinase assays
Boehringer Ingelheim Pharma http://www.boehringer-ingelheim.com
[12]
UCS15A
Novel mechanism; shows significant effects on downstream targets of Src pathway
Not specific for Src, but for many SH3b-mediated protein–protein interactions
In vitro studies of compound disruption of SH3-mediated protein–protein interactions via proline-rich domains
Pharmaceutical Research Laboratories; Kyowa Hakko Kygyo Co., Ltd., Japan http://www.kyowa.co.jp
[13,14]
a b
Normal human keratinocytes. Src homology domain 3.
substrate. Other anilinoquinazolines such as AZM475271 and AZD0530 have been tested in preclinical and clinical models (see below).
PD173952 The pyrido-pyrimidine inhibitor PD173952 was examined for effects on Src activity in normal human keratinocytes
(NHK) [10]. In this system, PD173952 (as well as SU6656 and PP1) inhibits EGF-stimulated EGFR-Y1148 phosphorylation (with no decrease in EGFR kinase activity), ERK phosphorylation, cell proliferation, colony formation and migration. However, apoptosis is not seen in treated cells. Reduction of phosphorylation of EGFR-Y845 was evidence that Src kinase activity itself is inhibited. We suggest that
Figure 4. Heterocyclic templates used to design Src tyrosine kinase inhibitors. As reviewed in Sawyer et al. [42], below are the chemical structures of various starting compounds used in synthesis of Src inhibitors. Reproduced, with permission, from Ref. [42].
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inhibition of Src kinase activity affects erbB-family-driven growth and migration.
PD173955 This pyrido-pyrimidine is an ATP-competitor and tyrosine kinase inhibitor selective for Src and Yes, a Src family member. PD173955 effectively inhibits Src and Yes kinase activities in test-tube kinase assays using enolase as a synthetic substrate (IC50 = 22 nM; see Glossary), and in a study of eight breast cancer cell lines, it was found to block cell cycle progression, with the cells arresting at G2/M [11].
Terphenylquinone Identified in a natural product extract, terphenylquinone compound 4 (a benzoquinone) was isolated from the fungus Stilbella sp. strain 1586 and it exhibited inhibition of Src kinase activity (IC50 = 3.9 mM) [12]. Src kinase activity was measured by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA).
UCS15A Originally identified as an antibiotic produced by Streptomyces sp., UCS15A has been studied as a completely novel small-molecule inhibitor of Src [13]. It is not specific for Src and does not inhibit Src kinase activity, yet studies by Oneyama et al. [14] in HCT-116 colorectal carcinoma cells suggest that UCS15A disrupts SH3-mediated protein interactions by directly interacting with proline-rich (P-xx-P) domains in selective proteins. This mechanism is borne out by studies in HCT116 cells in which UCS15A prevents Src association with and phosphorylation of Sam68 and cortactin, substrates of Src. Further studies in a mouse calvaria organ culture system have shown that inhibition of Src by USC15A results in a dose-dependent inhibition of bone resorption, with no osteoclast toxicity [13].
Preclinical studies AZM475271 AZM475271 is a novel anilinoquinazoline, ATP-competitive inhibitor of Src. In a promising study by Yang et al. [15], mouse xenografts of LNCap prostate carcinoma cells engineered to express gastrin-releasing peptide (GRP) that were grown in cell culture or soft agar in the absence of androgen. AZM475271 shows significant inhibition of colony formation, suggesting that the compound might have therapeutic benefit for patients with androgen-independent prostate cancer. In a more complete study of AZM475271, Yezhelyev et al. [16] studied the response of nude mice with orthotopic L3.6pl human pancreatic cancer tumors to oral treatment with this compound alone and in conjunction with gemcitabine. With AZM475271 alone, primary pancreatic tumor volume 318
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is reduced by 40%, and together with gemcitabine, this was reduced by 90%, with no metastasis to lymph nodes. Cellbased studies noted that AZM475271 treatment (alone or with gemcitabine) reduces Src, Lck and Yes kinase activity (with IC50 = 0.01, 0.03 and 0.08 mM, respectively), reduces L3.6pl cell proliferation and cell migration and increases the rate of apoptosis.
SKI-606 SKI-606 (also known as compound 77) is a small-molecule ATP-competitive inhibitor, classified as a 4-anilino-3-quinolincarbonitrile, and is a potent dual inhibitor of Src and BCRABL kinase activity. Thaimattam et al. [17] have performed three-dimensional quantitative, structure–activity relationship (3D-QSAR) analyses and docking analyses of SKI-606 with Src and BCR-ABL kinases and have shown that SKI606 docks in the active site of Src and Abl. A cell culture model studied by Golas et al. [18] using the human CML cell lines, K562 and KU812, shows that SKI-606 inhibits autophosphorylation of Src family kinases Lyn and Hck, blocks phosphorylation of BCR-ABL and ablates phosphorylation of STAT5. Using test-tube Src kinase assays, an IC50 = 1.2 nM was measured [18]. In the colon carcinoma cell line HT-29, SKI-606 decreases phosphorylation of FAK and inhibits HT-29 colony formation in soft agar [19]. This group also studied preclinical in vivo mouse models of HT-29 and K562 xenografts. After oral administration of SKI-606 to nude mice, xenograft tumors of both origins either fully regress or fail to form. No toxicity is seen, even after prolonged administration [18,19].
SU6656 As a small molecule, indolinone-based inhibitor of Src family kinases (Src, Fyn, Yes, Lyn), SU6656 has been studied in a variety of cell types. Blake et al. [20] have used this inhibitor to probe signaling pathways of the PDGF receptor because SU6656 does not inhibit this receptor but does inhibit Src (IC50 = 0.28 mM), a mediator of PDGF signaling. From their findings they conclude that in NIH3T3 cells, SU6656 inhibits PDGF and Src-driven mitogenesis and also inhibits PDGFdriven c-Myc induction. Downstream Src substrates also exhibit decreased or ablated tyrosine phosphorylation after treatment with SU6656. Severgnini et al. [21] examined the role of Src in a lung injury model in mice, which involved a lethal lipopolysaccharide (LPS) challenge in the airway. In mice pretreated with SU6656, lung homogenates were examined by western blotting for phosphorylated Src; SU6656 sharply decreases Src phosphorylation to basal levels. Pretreatment with SU6656 (or PP2) decreases lung injury (neutrophil infiltration, hemorrhage, endothelial and epithelial damage, respiratory distress and cytokine production) after LPS treatment. Mortality from LPS challenge is also prevented.
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PP2 PP1 and PP2 were identified in 1995 as novel, pyrazolopyrimidine compounds that are selective inhibitors of Src family kinases [22]. Later studies suggested that PP1 was not as selective as first believed because it also inhibits the PDGFb receptor kinase (IC50 for Src = 0.7 mM; IC50 for PDGFb = 3 mM) [23]. Boyd et al. [24] examined the effects of PP2 in human colon cancer cells (HT-29; SW480). PP2 causes a dose-dependent decrease in urokinase-type plasminogen activator receptor (u-PAR) gene expression, u-PAR protein expression, cell invasiveness and Src kinase activity. Combined with NI-5.12 (u-PAR antagonist), PP2 treatment leads to a synergistic inhibition of invasiveness of cells. We noted that u-PAR expression can however be controlled by factors other than Src. Duxbury et al. [25] tested a panel of pancreatic adenocarcinoma cell lines for Src expression, Src phosphorylation and kinase activity after treatment with PP2 alone or in combination with gemcitabine. They reported that a decrease in Src Y419 phosphorylation is associated with a decrease in gemcitabine resistance, suggesting that Src is an important player in acquisition of gemcitabine resistance. In addition, PP2 augments apoptosis, decreases tumor growth and inhibits metastasis in nude mice xenografts with PNAC1 pancreatic adenocarcinoma cells.
CGP77675 Missbach et al. [26,27] examined several members of a class of di-phenyl-pyrrolo-pyrimidines for test-tube inhibition of Src. CGP77675 was the most promising of these compounds, inhibiting Src autophosphorylation and phosphorylation of FAK and paxillin (IC50 = 0.2–5.0 mM) in IC8.1 cells. In vivo studies of CGP77675 in ovariectomized rats show prevention of osteoclast-driven bone loss, bone resorption and bone architectural changes.
AP22408 Phosphotyrosine mimic AP22408 binds the Src and/or Lck SH2 domain, as shown by molecular modeling, X-ray crystallography and test-tube binding assays [28]. The compound was designed to be bone-specific and was shown through hydroxyapatite chromatography to specifically associate with bone. AP22408 inhibits rabbit osteoclast bone resorption (IC50 = 1.6 mM) and demonstrates bone antiresorptive activity in a rat model. We suggest that AP22408 will prove useful in treatment of bone cancer metastasis, osteoporosis and other bone diseases.
Clinical studies AZD0530 AZD0530 is a highly selective, dual-specificity small molecule inhibitor of Src and BCR-ABL. It is another anilinoquinazoline that has recently been studied primarily in cell culture
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models and preclinical animal models, but at least two Phase I clinical trials in healthy volunteers have been published as abstracts. In tissue culture, it shows promise as an inhibitor in breast cancer cell lines and in models of osteoclastic bone resorption, so it is a natural therapeutic candidate for clinical trials in metastatic cancers involving bone. AZD0530 inhibits migration of MDA-MB-231 breast carcinoma cells and decreases tyrosine phosphorylation of Src, FAK and paxillin in wild-type MCF7 breast carcinoma cells and MCF7 cells expressing an estrogen receptor-a mutant [29]. AZD0530 also blocks MCF7 cell proliferation and invasion stimulated by estrogen [30]. In wild-type and tamoxifenresistant MCF7 cells, AZD0530 reduces motility, FAK Y861 phosphorylation, integrin expression and cell attachment [31]. Preclinical animal models have shown that AZD0530 completely prevents growth of Src-expressing 3T3 xenografts in nude rats, and significantly increases survival rate in a rat orthotopic model of human pancreatic cancer (BxPC-3 cells) [29]. In a separate rat tumor model with Src-expressing 3T3 xenografts, AZD0530 inhibits tumor growth in a dose-related fashion, with >90% of tumor growth inhibition at 6 and 10 mg/kg [32]. Tissue distribution and pharmacokinetics were also determined in this study. In a preclinical model of bone dynamics by Mullender et al. [33], AZD0530 reduced rabbit osteoclastic bone resorption area, specifically the number and average area of resorption pits in a pit-formation assay. The authors concluded that AZD0530 can show therapeutic benefit in metastatic bone disease. In a Phase I clinical study designed to examine effects of AZD0530 on bone resorption in healthy male volunteers, Eastell et al. [34] reported inhibition of osteoclast-mediated bone resorption at a 1000 mg dose, as assayed by markers of serum crosslinked C telopeptide of type I collagen (sCTX) and urinary crosslinked N telopeptide (NTX). These authors also noted the therapeutic potential of AZD0530 for metastatic bone disease and osteoporosis. In a Phase I clinical trial to determine safety, tolerability and pharmacokinetics of AZD0530 in healthy male volunteers, Lockton et al. [35] reported that a once-daily oral dosing of 250 mg is well tolerated with mild adverse effects. The plasma concentrations of AZD0530 remain above the IC50 for Src kinase in these patients.
BMS-354825 BMS-354825 (a methyl-pyrimidine carboxamide) is an orally active, ATP-competitive, potent inhibitor of several kinase molecules including Src family members, Kit, PDGFR and BCR-ABL. Much of the preclinical work using BMS-354825 has been done in mouse models of imatinib-resistant CML, examining the signaling of BCR-ABL rather than Src. (Imatinib is a specific BCR-ABL inhibitor used as front-line therapy for CML.) In preclinical studies by Shah et al. [36], SCID mice www.drugdiscoverytoday.com
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were injected with mouse B-cells (Ba/F3) expressing BCR-ABL (either wild-type or an imatinib-resistant mutant). The authors report a reduction of tumor burden and prolonged survival with BMS-354825, regardless of the BCR-ABL isoform expressed. In addition, BMS-354825 inhibits growth of bone progenitor cells from CML patients but does not affect bone cell growth in healthy volunteers. A Phase I dose-escalation study of BMS-354825 in treatment-resistant gastrointestinal stromal tumors (GIST) was recently published as an abstract at the American Society of Clinical Oncology (ASCO; http://www.asco.org). Evans et al. [37] reported that BMS-354825 can be safely administered at doses of up to 70 mg BID on a 5-days on, 2-days off, weekly schedule. No objective tumor responses are seen by CT analysis, although the abstract suggests that the clinical benefits noted in a subset of patients are encouraging.
impact on human disease is disappointing. Among the challenges to investigators is defining how inhibition of Src activity impacts normal cell function as well as tumor cell homeostasis. For this, standard assays for the Src-dependent signaling pathways need to be evaluated. Fortunately, the characterization of two such pathways, EGF receptor signaling and integrin signaling, provide ample opportunity to carefully assess the efficacy of Src inhibitors both in cells in culture and in model systems. Application of these assays to well characterized preclinical animal models and human tumors where the relevant pathways are functional might speed the understanding of how Src inhibitors work. It is clear, however, that the careful characterization of compound activity and specificity is essential if new Src inhibitors are to be successful drugs in the clinic.
17-AAG/geldanamycin
Acknowledgements
17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an analog of geldanamycin and specifically binds to the cytosolic chaperone protein HSP90. HSP90 binds to Src, HER2/ neu, and other signaling molecules, and has been shown to aid in their maturation, stability and appropriate subcellular localization [38]. Presumably, 17-AAG disrupts this interaction between its target and HSP90, resulting in increased rates of degradation and inhibition of signaling activity (reviewed in Ref. [38]). Ramanathan et al. [39] reported a Phase I study of 17-AAG in 45 patients with refractory advanced cancers of many different origins. Doses of 17-AAG between 10 and 295 mg/ m2 have been found to be safe and of limited toxicity, given on a weekly basis. However, no objective tumor responses are observed. A similar Phase I trial published by Goetz et al. [40] determined that the maximum tolerated dose of 17-AAG in 21 patients with advanced cancer is 308 mg/m2, with no tumor response.
Conclusions Src and Src family kinases represent an attractive target for inhibitor development because of their roles in the regulation of cellular processes and the link to cancer. As noted above, several inhibitors have been identified and are in different stages of development, some showing promise in early clinical studies. Researchers and clinicians face a daunting task when deciding on potentially therapeutic compounds to move to clinical trials. Currently, compounds active against enzymes such as Src are identified using classic test-tube based assays, subsequently tested in cell culture systems, and if efficacious, the studies are moved into animal models of tumorigenesis. Finally, human trials are conducted. Given the thorough, time-consuming research and planning, the correlation between test-tube inhibitory activity, inhibitory activity in cell culture and antitumor activity in animals and 320
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We gratefully acknowledge the support of the following granting agencies: A.P.B-J. is supported by Lynchburg College and NIH-NCI grants CA104237 and CA71449, awarded to S.J.P. M.L.D. is a recipient of a Dissertation Research Award from the Susan G. Komen Breast Cancer Foundation, #DISS0402970, awarded to S.J.P. J.T.P. and S.J.P. acknowledge support from the NIH-NCI, CA40042 and CA29243 to J.T.P. and CA104237 and CA71449 to S.J.P.
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