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The Development of Novel Small Molecules as
Protein Tyrosine Kinase Inhibitors for the Treatment
of Cancer
Julia X Su
Review Article
Fourth Year Undergraduate Student in Pharmaceutical Chemistry Specialist, Deparment of Pharmaceutical Science, University of Toronto, Toronto, Canada
Abstract
Protein Tyrosine Kinases (PTKs) are important regulators of signalling, growth and proliferation of cells. In cancer cells, overexpression or gain-of-function mutations causes PTKs to become constitutively active, causing uncontrolled growth and proliferation.
Studies of the common PTKs mutations can lead to potential treatment of cancer through developing novel small molecules which
selectively target the mutated PTKs. This review summarizes the diseases associated with four different mutated PTKs, Bcr-Abl, Anaplastic Lymphoma Kinase (ALK), Fms-Like Tyrosine Kinase 3 (FLTK3) and Fibroblast Growth Factor Receptor 3 (FGFR3). This review will
also discuss the small molecules developed to specifically inhibit each of these mutated PTKs. Being on different stages of the drug
development process, the small molecule inhibitors have so far been shown to be effective at PTK inhibition, demonstrating their potential as therapeutic anti-cancer agents. Though a large numbers of tests and clinical trials are still needed before these molecules can
be proven to be safe and efficacious in humans, continuous studies on small molecule inhibitors of PTKs will lead to new understanding
of cellular pathways and more effective therapies for the treatment of cancer. .
Protein Tyrosine Kinase
Protein Tyrosine Kinases (PTKs) play important roles
in signalling, growth and proliferation of cells. Upon ligand
binding, PTKs catalyze the transfer of the phosphate group of
ATP to tyrosine residues of their protein substrates. PTKs consist of two subclasses, the Receptor Tyrosine Kinases (RTKs),
a type of cell surface receptor that has intrinsic kinase activity
and the non-receptor tyrosine kinases, which can be either cytoplasmic or nuclear proteins. Currently, there are 90 known
PTK genes in the human genome, out of which, 58 encoding
transmembrane RTK genes, and 32 encoding non-receptor
PTKs [1].
Both subclasses of PTKs are involved in critical growth
and proliferative pathways in the cell. In tumours with mutated
and constitutively active PTKs, the growth and proliferation of
cancer cells become highly dependent on the mutated PTKs
and their associated signalling pathways [1]. The growth and
proliferation of cells can thus become uncontrolled.
Because of PTKs’ involvement in cell proliferation in various cancers, they have been identified as excellent therapeutic
targets in the rational drug discovery process. Based on the
crystal structure of the PTK, various small chemical molecules are designed using computational tools to selectively
inhibit the targeted PTK [2]. Currently, in the pharmaceutical
industry, larger efforts are devoted to designing these small
molecules and testing their efficacy in inhibiting PTKs for the
treatment of cancer.
This review summarizes the mutations and diseases associated with four different PTKs, their mechanism of actions,
and the inhibitory efficacy of several small molecules devel-
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Journal of Undergraduate Life Sciences
oped specifically for mutated PTKs.
Bcr-Abl
One of the most familiar examples of PTK inhibition for
cancer treatment is the targeting of Bcr-Abl, the mutant PTK
resulted from a chromosomal translocation. The Philadelphia
chromosome (Ph-chromosome), produced by the fusion of
portions of chromosome 9 and chromosome 22, is the genetic
cause of 95% of chronic myeloid leukemia (CML) and 20%
of acute lymphoblastic leukemia (ALL) [3]. The resultant fusion protein, Bcr-Abl PTK becomes constitutively active and
transfers the phosphate group of ATP to various substrate proteins, which in turn activates a whole cascade of downstream
pathways, resulting in the activation or improper stabilization
of some well-known oncoproteins, such as MYC and JAK2 [4].
In 1996, a small molecule STI571 (imatinib) was discovered
and developed by Novartis (Basel, Switzerland) to selectively
inhibit Bcr-Abl [5]. Imatinib was shown to bind to the ATP
binding site on the Abl protein and stabilize the fusion protein
in its inactive conformation, thereby inhibiting its kinase activity and preventing the activation of downstream pathways
[5]. In clinical studies, imatinib has substantial tumour reduction activity in CML and in Ph-chromosome positive ALL [6].
Imatinib was later introduced to the market under the name
Gleevec, as a frontline therapy for CML.
However, since the introduction of Gleevec to the market,
its secondary resistance in patients is commonly encountered
and has attracted enormous research interests. Clinical resistance is primarily caused by point mutations in the kinase
The Development of Novel Small Molecules as Protein Tyrosine Kinase Inhibitors for the Treatment of Cancer
domain of Bcr-Abl [7]. Since imatinib can only bind to the
ATP binding site of Abl when the activation loop is closed, it
is believed that the majority of the amino acid substitutions
impairs the ability of Abl to adopt its specific closed conformation and prevents imatinib from binding [8]. As a result, a
derivative of imatinib that has less rigid binding requirements
is needed to overcome the resistance. Another small molecule,
BMS-354825 (dasatinib) was discovered in 2004, which is able
to bind to Bcr-Abl in both open and closed conformations,
thus demonstrating its ability to target most imatinib-resistant
Bcr-Abl mutations [9].
Recently, another derivative of imatinib, nilotinib was
brought to clinical trials with a more favourable safety profile
and reduced side effects when administered at a similar dose
[10]. Nilotinib caused less myelosuppression and fewer nonhematologic adverse effects than imatinib. In a clinical trial,
86 patients who were intolerant to imatinib were administered
nilotinib, and only 2 out of these 86 patients developed grade
3 or 4 adverse events in the nilotinib treatment (Table 1) [10].
This reduction in side effects can be explained by nilotinib’s
higher selectivity for Bcr-Abl. Nilotinib provides an alternative
treatment for diseases that have become resistant or intolerant
to imatinib.
Table 1. Comparison of nonhematologic adverse events observed
in imatinib-intolerant patients treated with nilotinib.
Grade 3/4 adverse
events
Rash/skin
Liver toxicity
Fluid retention
Gastrointestinal
intolerance
Musculoskeletal
Other
Total
Patients with toxicity on
imatinib, no.
25
10
17
15
Patients with toxicity on
nilotinib, no.
0
1
0
1
9
10
86
0
0
2
86 patients who were intolerant to imatinib were administered
nilotinib. [10].
ALK (Anaplastic Lymphoma Kinase)
After the success of Gleevec, the efforts in developing
mechanism-based therapy for cancer have brought forth many
more small molecules to inhibit other mutated PTKs. One
example is the inhibition of mutated anaplastic lymphoma
kinase (ALK). Mutated ALK is the cause of cancers like
anaplastic large-cell lymphoma (ALCL) and neuroblastoma
[13]. In ALCL, 50-60% of the cases are associated with the
fusion of the nucleophosmin (NPM) gene on chromosome 5
to a portion of the ALK gene on chromosome 2, generating
a chimeric NPM-ALK protein that is constitutively active,
and leading to tumour formation [14]. In neuroblastoma, the
ALK positive tumours do not show the same chromosomal
translocation, but acquire point mutations in the ALK gene.
These non-synonymous sequence variations in conserved
positions of the tyrosine kinase domain, such as F1174L and
R1275Q, are associated with constitutive phosphorylation of
ALKs and downstream signalling proteins, such as STAT3 and
AKT, which are regulators that can induce protein synthesis
pathways and promote oncogenesis [15].
Galkin et. al developed a cell proliferation assay using
a NPM-ALK transformed murine cell line and were able to
identify a small molecule, TAE684, which selectively blocked
ALK activity and signal transduction by occupying the ALK’s
ATP binding site [16]. The inhibition was shown to be accompanied by a rapid and sustained reduction in ALK autophosphorylation, dose-dependent reduction of phosphorylation of
downstream proteins ERK and AKT, and down-regulation of
CD30 expression, a marker of ALCL [14]. With the administration of TAE684 in the cell culture, the proliferation and
survival of NPM-ALK transformed cancer cells decreased
dramatically (Figure 1) [16]. In addition, TAE684 was shown
efficacious in tumours involving both a translocation and
point mutations of the ALK gene in a mouse model [16].
Currently, TAE684 is still under investigation for optimization
and testing. There are currently no clinical trial data regarding
the use of TAE684 in humans. It will be intriguing to know if
TAE684 demonstrates safety and the same level of efficacy in
humans as it has in cell cultures and animal models.
FLT3 (Fms-Like Tyrosine Kinase 3)
Fms-like tyrosine kinase (FLT3), a member of the receptor PTKs, plays an important role in the proliferation,
differentiation and survival of hematopoietic stem cells and
progenitor cells [17]. Thirty percent of acute myeloid leukemia
(AML) patients show FLT3 positive tumours [18]. The mutations involve either an internal tandem duplication of amino
acids in the juxtamembrane (JM) domain of the receptor, or a
point mutation in the activating loop which changes the conformation of this domain and causes the receptor to adopt an
active conformation [18]. Both types of FLT3 mutations result
Journal of Undergraduate Life Sciences
Volume 4 • No. 1 • Spring 2010
Another advantage of nilotinib over imatinib comes from
its differential susceptibility to drug efflux pumps, which are
highly expressed in hematopoietic stem cells [9]. These drug
efflux pumps actively transport substrates such as anti-cancer
drug molecules out of the cell, resulting in a decreased concentration of drugs at the target sites, and therefore a decreased
drug efficacy. Imatinib is a substrate of P-glycoprotein, a
multidrug resistance protein, while nilotinib is able to inhibit
these efflux pumps, such as the human multidrug resistance
protein [11]. Therefore, nilotinb can achieve higher therapeutic concentration at target sites and longer duration of action
than imatinib, improving its effectiveness in the human body
[11].
Furthermore, there are benefits to explore combinatorial
therapy for the treatment of CML. The possibility of developing a cocktail of kinase inhibitors with activity against all drug
resistant Bcr-Abl mutations is presently being investigated [9].
Studies have also showed that combining Bcr-Abl targeted
small molecules with other current standard treatments for
CML, which include stem cell transplantation, interferonalpha-containing regimens, cytosine arabinoside and daunorubicin showed additive or synergistic effects [12].
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in FLT3s that are active in the absence of the endogenous FLT3
ligand, a growth factor, leading to tumour formation [17].
Figure 1. TAE684 inhibits cell proliferation of Karpas-299 and
Su-DHL-1 cell lines, which are established human ALCL cell lines
expressing NPM-ALK [16].
In 2002, Weisberg discovered that small molecule PKC412
(N-benzoyl staurosporine) had the ability to directly inhibit
both mutant and wild-type FLT3 [19]. PKC412 is a synthetic
derivative of the naturally occurring alkaloid staurosporine
and was developed originally as an inhibitor for tyrosine kinases such as c-kit and the vascular endothelial growth factor
receptor [19]. In cell culture studies, PKC412 inhibits the autophosphorylation of mutant FLT3 receptors and reduces proliferation of FLT3 positive tumour cells by inducing apoptosis
and cell cycle arrest [19]. PKC412 also proves to be efficacious
in an animal model of leukemia, using mice transplanted with
marrow cells infected by FLT3 mutant allele retrovirus [19].
In clinical studies, PK412 was also shown to be effective and
well tolerated by cancer patients with FLT3 mutations, and no
significant side effects were found when it was administered
at its therapeutic level [18]. However, the serum level of free
PKC412 was generally low due to a large extent of plasma
protein binding, leading to decreased concentration of free
active drug at the target site, which has a negative impact on
its efficacy [18]. Because only the free unbound drug in the
blood will enter cells, it may be useful to alter the structure of
PKC412 in a way that decreases its binding affinity to albumin
in the blood, and increase the drug efficacy.
FGFR3 (Fibroblast Growth Factor Receptor 3)
In an oncogenic form of fibroblast growth factor receptor
3 (FGFR3), the immunoglobulin heavy chain (IgH) locus on
chromosome 14 can recombine with the locus on chromosome 4, giving rise to the aberrant expression and dysregulation of FGFR3 [20]. This reciprocal chromosomal translocation occurs in 15% of patients with multiple myeloma (MM),
a B cell neoplasm characterized by clonal expansion of plasma
cells in the bone marrow [21]. MM patients with this specific
translocation show a significantly worse prognosis, with minimal benefit from receiving a stem cell transplant [21]. A novel
treatment approach is therefore necessary for these patients,
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Journal of Undergraduate Life Sciences
and the development of a small molecule to inhibit mutant
FGFR3 would be highly beneficial.
CHIR258, a novel benzimidazole-quinolinone is a small
molecule that inhibits several classes of PTKs, including
FGFR3, platelet-derived growth factor receptor (PDGFR) and
C-kit [21]. In cell culture studies, CHIR258 has been shown
to inhibit FGFR3 autophosphorylation and therefore cell proliferation in human myeloma cell lines expressing wild type
or activated mutant FGFR3 [21]. CHIR258 administration
also resulted in a reduction of the phosphorylation of a downstream signalling molecule, FRS2, which directly interacted
with FGFR3 [22]. Trudel et al. demonstrated the therapeutic
efficacy of CHIR258 in a xenograft mouse model of FGFR3
MM [21]. In an orthotopic MM model in mice, Xin et al. demonstrated that CHIR258’s efficacy exceeded the proteosome
inhibitor Velcade, the current standard therapy approved for
treatment of MM in humans, providing strong support for
starting clinical trials of CHIR258 in FGFR3 positive MM
patients [22].
Because CHIR258 is known to inhibit other PTKs, such as
the PDGFR, it is likely that the off-target inhibition of PDGFR
contributes to the efficacy of CHIR258 in the orthotopic model
of MM [22]. More studies need to be conducted for a detailed
understanding of the contribution of PDGFR to MM and the
molecular interaction of CHIR258 with PDGFR.
Conclusion
Cancer is becoming more prevalent in today’s society.
According to the Canadian Cancer Society, almost 40% of
Canadian women and 45% of Canadian men will develop
cancer during their lifetimes [23]. Standard chemotherapy for
cancer is associated with toxicity and may be ineffective for
some patients. Highly efficacious and targeted small molecules
are promising alternative treatments.
The success of the oncoprotein-directed compound
Gleevec has led to a breakthrough in the treatment of cancer.
Gleevec demonstrates the power of rational drug discovery,
presenting PTKs as suitable molecular targets. Small molecules
can be developed to selectively inhibit the mutant PTKs, turn
off the downstream signalling pathways and induce apoptosis
or growth arrest of cancer cells. Though proteins downstream
of a mutant PTK receptor in proliferative or survival pathways
can also serve as therapeutic targets, directly targeting the
activated kinase itself is often the better choice, because many
signal transduction pathways have a high degree of redundancy [18].
In this review, four different receptor tyrosine kinases and
their small molecule inhibitors are investigated and characterized (Table 2). The four PTKs summarized are associated with
different genetic mutations that lead to various cancers.
The Development of Novel Small Molecules as Protein Tyrosine Kinase Inhibitors for the Treatment of Cancer
Table 2. Summary of the Protein Tyrosine kinases reviewed in this paper, the causes of their defects and the small molecule inhibitors targeting each
Protein Tyrosine Kinase
Disease Associated
Genetic Mutation
Small Molecule
Inhibitor
Bcr-Abl
chronic myeloid leukemia (CML)
Translocation between chromosome 9
and 22
Imatinib
(Gleevec);
acute lymphoblastic leukemia
(ALL)
Dasatinib;
Nilotinib
NPM-ALK
Nucleophosmin- Anaplastic
Lymphoma Kinase
ALK
anaplastic large-cell lymphomas
(ALCL)
Translocation between chromosome 2
and 5
neuroblastoma
Point mutation in Tyrosine kinase
domain
acute myeloid leukemia (AML)
internal tandem duplication of amino
acids in the juxtamembrane (JM)
domain
Anaplastic Lymphoma Kinase
FLT3
Fms-Like Tyrosine Kinase 3
TAE684
PKC412
or
a point mutation in the activating loop
FGFR3
multiple myeloma (MM)
Fibroblast Growth Factor
Receptor 3
Translocation between chromosome 4
and 14
CHIR258
The small molecules developed against each of the four PTKs are in different stages of the drug development process. They have been shown to be effective at PTK
inhibition in several assays, bringing hope to patients waiting for suitable treatments. However, some of these small molecules are under preclinical investigation, and
more tests and clinical trials need to be conducted before they are proven safe and efficacious in humans. There are various challenges that need to be overcome, such
as undesirable absorption profile, low drug level at the therapeutic site due to the presence of efflux proteins, and secondary resistance that arises from PTK mutations.
The treatment and prevention of cancer requires an enormous effort from the scientific community and health care professionals. Continuing studies on small
molecule inhibitors of PTKs will lead to new understandings of critical signalling pathways and certainly more breakthrough therapies for the treatment of cancer.
.References
1. Blume-Jense, P. and T. Hunter, Oncogenic kinase signalling. Nature, 2001. 411: p.
355–365.
3. Kurzrock, R, et al., The molecular genetics of Philadelphia chromosome positive
leukemias. N Engl J Med, 1988. 319: p. 990-998.
4. Cardama, A, and J. Cortes, Molecular biology of bcr-abl1-positive chronic myeloid
leukemia. Blood, 2009. 113: p. 1619-1630.
5. Mauro, M.J., et al., ST1572, a tyrosine kinase inhibitor for the treatment of chronic
meylogenous leukemia: validating the promise of molecularly targeted therapy.
Cancer Chemother Pharmacol, 2001. 48(suppl 1): p. S77-78.
6. Bunchdunger, E., et al., Inhibition of the Abl protein-tyrosine kinase in vitro and in
vivo by a 2-phenylaminopyrimidine derivative. Cancer Res, 1996. 56: p. 100-104.
7. Druker, B.J., et al., Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in
the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with
the Philadelphia chromosome. N Engl J Med, 2001. 344(14): p. 1038-1042.
9. Talpaz, M., et al., Dasatinib in imatinib resistant philadelphia chromosome positive
leukemias. N Engl J Med, 2006. 354(24): p. 2531-2541.
10. Kantarijian, H.M., et al., Nilotinib (formerly AMN107), a highly selective Bcr-Abl TK
inhibitor is effective in patients with philadelphia chromosome positive chronic
myelogenous leukemia in chronic phase following imatinib resistance and intolerance. Blood, 2007. 110(10): p. 3540-3546.
11. Tiwari, A.K., et al., Nilotinib (AMN107, Tasigna) reverses multidrug resistance by
inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters.
Biochem Pharmacol, 2009. 78(2): p.153-161.
12. Thiesing, J.T., et al., Efficacy of ST1571 and abl tyrosine kinase inhibitor in conjunction with other antileukemic agents against bcr-abl positive cells. Blood, 2000. 96
(9): p. 3195-3199.
13. Benharroch, D., et al., ALK positive lymphoma: single disease with broad morphology. Blood, 1998. 91(6): p. 2076-2084.
14. Morris, S.W., et al., Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM,
in non-Hodgkin’s lymphoma. Science, 1994. 263: p. 1281-1284.
15.George, E.R., et al., Activating mutations in ALK provide a therapeutic target in
Journal of Undergraduate Life Sciences
Volume 4 • No. 1 • Spring 2010
2.Ghose, A.K., et al., Knowledge based prediction of ligand binding modes and rational
inhibitor design for kinase drug discovery. Journal of Medicinal Chemistry, 2008.
51(17): p. 5149-5170.
8. Shah, N.P., et al.,Overriding imatinib resistance with a novel abl kinase inhibitor.
Science, 2004. 305(16): p. 399-401.
5
neuroblastoma. Nature, 2008. 455(16): p. 975-978.
16. Galkin, A.V., et al., Identification of NVP-TAE684, a potent, selective, and efficacious
inhibitor of NPM-ALK. PNAS, 2007. 104(1): p. 270-275.
17. Naoe, T., et al.,FLT3 tyrosine kinase as a target molecule for selective antileukemia
therapy. Cancer Chemother Pharmacol, 2001. 48(suppl 1): p. S27-S30.
18. Stone, R.M., et al., Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412.
Blood, 2005. 105: p. 54-60.
19. Weisberg, E., et al., Inhibition of mutant FLT3 receptors in leukemia cells by the
small molecule tyrosine kinase inhibitor PKC412. Cancer Cell, 2002. 1: p. 433-443.
20. Chesi, M., et al., Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma
is associated with increased expression and activating mutations of fibroblast
growth factor receptor 3. Nature Genetics, 1997. 16: p. 260-264.
21. Trudel, S., et al., CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the
potential treatment of t(4;14) multiple myeloma. Blood, 2005. 105: p. 2941-2948.
22. Xin, X., et al., CHIR-258 is efficacious in a newly developed fibroblast growth factor
receptor 3-expressing orthotopic multiple myeloma model in mice. Clinical Cancer
Res, 2006. 12(16): p. 4908-4915.
23. Canadian Cancer Society/National Cancer Institute of Canada. Canadian Cancer
Statistics 2008. 2008.
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Journal of Undergraduate Life Sciences