Key words
Kinome - Protein kinases - Kinase oncogenic mutations - Kinase identification - Kinase
inhibitors - Kinase targets in oncology - gene editing - kinome knockout screen
Introduction
Protein kinases are an important part of almost every aspect of cell functioning
[1]. Ever since protein phosphorylation
was discovered by Ed Krebs and Ed Fisher around 1950s, protein kinases got
recognition as prominent regulators of cellular signalling [2]. Initially, the focus of researchers was
metabolic pathways, mainly emphasizing on glycogen metabolism and insulin
signalling. An extensive work in the field of biochemistry led to the origin of
protein kinase, which includes working on insulin action, eventually leading to the
mitogen-activated protein kinase identification [3]
[4]. A complete set of protein
kinases, which encode for them in the genome of an organism is called the kinome,
especially in the fields of molecular biology, biochemistry and cell signalling.
Another breakthrough for understanding protein kinome came from establishing their
variation with earlier identified oncogenes, for example; Rous-sarcoma virus
proto-oncogene (Src) encoded viral gene of cellular protein kinases [5]
[6].
These protein kinases are involved in almost all cellular functions such as
transcription, translation, cell division, other metabolism and even in apoptosis.
Reversibility is maintained between phosphorylation and dephosphorylation via
balanced action of protein kinases and phosphoprotein phosphatases during the
protein phosphorylation [1]
[7]. The protein kinases mediate the
phosphorylation process i. e. catalyse phosphate group transfer from a high
energy source such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP)
to proteins or even lipids as depicted in [fig.
1]. Generally, a serine, threonine or tyrosine residue containing a
hydroxyl group acts as the phosphate group receiver in proteins [8].
Fig. 1 Protein phosphorylation facilitated by protein kinases.
In disease pathophysiology, the importance of protein kinases was understood because
mutations and alterations in kinases can cause altered cellular functioning, which
can lead to various diseases [9]. According to
Manning et al., the human kinome contains 518 protein kinases and about 106 pseudo
protein kinases [1], among which, 218 protein
kinases are such whose alterations can cause human diseases. Since the Manning
study, the human genome's count has advanced, and as of now, 483 human
proteins have been identified by Uniprot as possessing 496 typical kinase domains
(https://www.uniprot.org/docs/pkinfam) [10]. For this reason, protein kinases are
undoubtedly promising therapeutic targets for various diseases including some viral
infections [1].
In this work, we have discussed the importance of kinome in context of cancer
pathogenesis and how mutations of the kinome contribute to the same. An overview has
been presented about the protein kinases as therapeutics, including novel kinase
targets in oncology, their identification, causes of kinase gene mutations during
cancer, various techniques for detection of protein kinase activities, different
types of protein kinase inhibitors and various FDA approved drugs.
Active and Pseudo kinases
Active and Pseudo kinases
Protein kinase domains comprise of two main lobes. First is a small N-terminal lobe
made up of an α-helix, also known as C-helix and five β-sheet
strands that are prominent for coordinating ATP binding. Second is a large
C-terminal lobe, which is composed of 6 α-helices for protein-substrate
binding and phosphorylation catalysis [11]
[12]. The activation and
catalytic loops that combine to generate the ATP and substrate binding sites are
found in the active site, which are situated between these two lobes [10]
[13].
Protein kinase activity regulation takes place through multiple post-translational
modifications (i. e. phosphorylation), autoinhibition, regulatory partner
binding, including both activator and inhibitor proteins along with expression
changes [14]
[15]. Most of the protein kinases utilize the above-mentioned mechanism
for stabilizing and promoting the active conformation and enhancing protein kinase
domain capabilities such as supporting ATP and substrate binding [11].
The human genome contains many non-functional, non-enzymatic members of kinase genes.
According to the data of sequence analysis, it was found that about 10% of
human kinase domains are catalytically inactive but have common core kinase scaffold
as that of catalytically active protein kinases and lacks the presence of key motifs
necessary for effective catalytic activity [10]. For instance, the absence of one of the amino acids like Lys30,
Asp125, and Asp143 in the catalytic domain of the protein kinases. These inactive
pseudokinases are divided into 3 groups [16].
In the first group, the Pseudokinomes are potential modulators of their
catalytically active kinomes [17]. Second are
those kinases, which have high similarity to the eukaryotic protein kinase (ePK)
domain’s canonical structure [18]. In
the third group, members are dissimilar to their active domains. The
three-dimensional structures of pseudokinases and protein kinases are very much
similar. The pseudoactive site (ATP-binding site) is hidden in between the two lobes
i. e., the N-terminal lobe, which is dominated by β-sheets and
connected to the C-terminal lobe by the hinge region [19]. Pseudokinomes show almost zero levels of
enzymatic activity but in few cases, these bind in normal body concentration of
nucleotides and start working as a molecular signalling switch, which occurs in
cells through druggable ligand-induced transitions. Sometimes, they indirectly take
part in enzymatic action by allosteric modification, competition in substrate
binding or change in the signalling pathway. Two well-known examples are Human
epidermal growth factor receptor 3 (HER3) and Erb-B2 Receptor Tyrosine Kinase
3 (ERBB3), which are prominent HER2 signalling partners in cancerous
cells. These activate and maintain the activity of Phosphoinositide 3-kinases
(PI3K) and also take part in Kinase Suppressor of Ras, KSR1 and KSR2 in
the Epidermal growth factor receptor-Ras-Mitogen-activated protein (EGFR-Ras-MAP)
kinase pathway via allosteric interaction, with respect to its active kinase, which
is RAF (Rapidly Accelerated Fibrosarcoma) relative.
Identification of novel kinase targets in oncology
Identification of novel kinase targets in oncology
The human genome contains more than 500 different kinases [20] which comprise about 2% of the
entire genome. Most protein kinases promote cell survival, proliferation and
migration, therefore when constitutively overexpressed, they can be associated with
oncogenesis [21]. Deregulated kinases are
often found in oncogenic cells and can be central in their survival and
proliferation [22]. Hundreds of kinases play
intricate and overlapping roles in cell transformation, tumour initiation, tumour
survival and proliferation [21]. Therefore, to
identify novel protein kinase targets in oncology, it becomes necessary to catalogue
kinases in the human body so that any abnormalities leading to cancer and other
disorders can be identified and treated.
Identifying and cataloguing protein kinases
Protein kinases enzymatically phosphorylate proteins to
“activate” them. Up to 30% of all human proteins may be
modified by kinase activity. Since kinases play a role in several processes such
as gene expression, proliferation, differentiation, metabolism, membrane
transport, and apoptosis, it is not surprising that dysregulation of their
activity may lead to different diseases and disorders including cancer [23]. Cancer-associated phenotypes as
classified by Gross et al. [24], their
role in oncogenesis and protein kinase examples are given below in [table 1].
Table 1 Tumorigenic phenotypes.
|
Cancer associated phenotype
|
Role in oncogenesis
|
Protein kinase examples
|
|
Immuneoevasion
|
Tumours escaping immunosurveillance and
immunosuppression.
|
MAP4K1 (Mitogen Activated Protein Kinase)
|
|
Cell cycle progression
|
Proceed through cell cycles despite the damage.
|
B-RAF (B-Rapidly Accelerated Fibrosarcoma)
|
|
DNA damage response
|
Not arresting cell cycle or recruitment of DNA repair
mechanisms despite significant unrepairable damage.
|
ATM (Ataxia-Telangiectasia Mutated), ATR
(Ataxia-Telangiectasia & Rad3)
|
|
Angiogenesis
|
Increase blood supply and therefore nutrients in the tumor
microenvironment.
|
VEGF (Vascular Endothelial Growth Factor)
|
|
Metabolism
|
Increased nutrient metabolism.
|
mTor (Target of Rapamycin), PI3K
(Phosphoinositide-3-Kinase)/ATK,
|
|
Anti-apoptosis
|
Bypassing apoptosis leading to uncontrolled growth.
|
RIPK3 (Receptor Interacting Serine/Tyrosine Kinase
3)
|
|
Metastasis & invasion
|
Metastasis and cell invasion
|
RTKs (Receptor Tyrosine Kinases)
|
Therefore, it becomes clear that the first step in developing novel protein
kinase targets is the identification of a particular protein kinase, which is
associated with a specific cancer phenotype. This can be done by identifying the
enzyme or receptor, which is abnormally expressed and then associating it with a
kinase, which activates it.
One recent emerging method of associating protein kinases with their
corresponding substrates is using an established process called Biomolecular
Fluorescence Complementation (BiFC), which has been elucidated in [fig. 2], where a fluorescent protein is
divided into two parts; one attached with the protein kinase under study and
another with the cancer phenotype proteins under study. In performing BiFC
assay, the BiFC labelled protein kinase and cancer phenotype proteins can be
reacted together and on conjugation, the associated fluorescent marker can be
identified. This method has been used recently to study the interaction of
regulatory 14-3-3 proteins with Cyclin B1 in tumour promotion sensitive mouse
epithelial JB6P+ cells [25].
Fig. 2 Kinase-Substrate identification using Bimolecular
fluorescence complementation (BiFC).
Bioinformatics can also be used to statistically study common mutations across
cancer cells compared to normal cells. Paired tumour normal exome sequencing has
revealed millions of somatic mutations across many thousands of patients.
However, only a fraction of them have any biological significance and a majority
of those are inclined towards cancer development [26]. One possibility might also be to
subject different variations of the same protein kinase to mutagenic screens to
model and predict abnormal kinases, and their associated genotype [21]. With increasing computing power, large
scale complex data analysis is becoming easier and hence is now widely used in
research. One of the method of employing computational analysis is using reputed
cancer genomic repositories such as The Cancer Genomic Atlas (TCGA) and putting
it through a clinical kinase index (CKI) or kinases, whose mRNA expression seems
to be associated with the prognosis of cancer [27].
Protein kinase structure prediction
Kinases are a class of enzymes and being so, their sensitivity and functionality
largely depend on their structure. Therefore, to develop novel selective agents
against protein kinases involved in cancer, it becomes necessary to identify and
study their structure in detail. Advancements in X-Ray crystallography, NMR
spectroscopy, cryo-electron microscopy and computational protein modelling have
made it possible to study protein structures in greater detail.
All protein kinases contain an ATP binding site, where the ATP binds and
“powers” the transfer of the phosphate group onto the target
protein to activate it. Until recently, it was thought that these ATP binding
residues were conserved among kinases and therefore it was not possible to
selectively target them [21]. However
detailed crystallographic studies have revealed some inter-class differences,
which can be exploited to develop more specific inhibitors [28]. Blocking substrate binding sites such
as exposed hydrophobic groove in Cyclin-Dependent Kinases (CDKs) reduce
recruitment of cell cycle substrates [29].
Other non-active site regions such as blocking the polo box domains of Polo Like
Kinase (PLK1) have been shown to decrease its kinase activity [30]. Normal cells rely on a full complement
of cell cycle checkpoint kinases, whereas most cancer cells have faulty G1 DNA
damage checkpoint and thus depend on G2 or S phase checkpoints [29]. Using alteration of kinase-mediated
cytological pathways like this, it may be possible to downregulate overexpressed
kinases, while upregulating those, which are functional and can partially
restore normal cellular function.
Kinase gene mutations in cancer
Kinase gene mutations in cancer
Analysing the role of protein kinases and thus kinome behind phosphorylation of a
large section of proteins, it’s quite easy to understand that dysregulation
of the same would lead to many diseases and disorders including cancer. However,
about 24% of human kinases including pseudo kinases are still under study
and only 8% have been the target of any therapeutics, so a lot more needs to
be done with regards to identifying the role of different kinases in cancer and the
mutations which may result because of the same [27]. Mutations are random changes in the genome in both normal and
cancerous cells. However, genomic mutations can be divided into three broad
categories based on the extent of the mutation; base substitution, frameshift, and
chromosomal. In base substitutions, one nucleotide is erroneously replaced with
another, which may or may not change the associated protein functionality. One such
mutated kinase gene is BRAF (encoding for B-Raf protein), which is regularly
mutated at Val-600 and leads to colorectal, melanoma, thyroid or non-small cell lung
cancers [22]. RAS point mutations are also
responsible for about 10–20% of thyroid cancers [23].
In case of frameshift defects, it is possible for genes to carry tens of thousands
of
aberrant insertions or deletions in tandem repeats, while most occur in intronic
regions, few can happen in the exon regions also, leading to frameshift mutations,
altering several codons. It is likely that some of these mutations may have a role
in oncogenesis [31]. In gene or chromosomal
level mutations, entire genes or portions of genes become translocated on one
another, causing extensive damage. One of the most well-known examples of this is
the Philadelphia Chromosome, which involves the translocation and fusion of
Breakpoint Cluster Region (BCR) and Abelson murine leukemia viral oncogene
homolog 1 (ABL1) genes to form a fusion BCR-ABL1 gene, which is
present in 95% of people with chronic myelogenous leukemia and 25%
with acute lymphoblastic leukemia [18]. The
BCR-ABL1 fusion gene produces a hybrid tyrosine kinase signalling
protein, which is always activated leading to uncontrolled cell division by
undermining many normal cell signalling pathways [32]. The same fusion gene is also responsible for imatinib resistance,
which is a tyrosine kinase inhibiting anti-cancer drug, leading to poor prognosis
[30]. Rearrangement and fusion of RET gene
has also been associated with several RET dependent cancers such as
10–20% of papillary thyroid carcinomas [23]
[33].
Several kinase genes have been categorized as proto oncogenes and oncogenes.
Genomic study of protein kinases involves accurate identification of genes
responsible for protein kinase, their sequencing and finally a comparison of the
healthy kinomes with corresponding ones from cancer cells. Thereafter several
statistical analytical tools can be applied to the mutations to reveal any
reoccurring pattern. Two more terms, which must be discussed for the analysis of
kinome mutations are driver and passenger mutations. Driver mutations are those
mutations, which are significant for cancer progression because they offer an
advantage in the clonal expansion [34]. For
the individual cell, it is beneficial but for the organism, it leads to tumour
formation and progression. Passenger mutations are mutations, which can be a result
of driver mutations, or accumulated over the cell’s life and are less
significant than driver mutations because they do not directly contribute to
oncogenesis [35]. However, their study may
shed light on patterns of mutations leading to oncogenesis. This phenomenon is shown
in [fig. 3], where a random mutation on a
kinome leads to the formation of a driver mutation, which gives a clonal advantage
and may further lead to passenger mutations. The result is a protein kinome enzyme,
which is insensitive to its repressor, and therefore catalyses excess of
pro-proliferative proteins. One prominent example of kinase drivers is receptor
tyrosine kinases because of the activating point mutations, which leads to
oncogenesis [36]. In a report, resequencing
518 kinases across seven lung cancer cell lines and 26 primary lung neoplasms across
141 kinomes, 188 somatic mutations were found, three-fourths of which were probably
passenger mutations [36]. In a more
comprehensive study, 518 protein kinase genes amounting to 274 MB of genetic data
across a total of 210 different cancers including breast, lung, colorectal, gastric,
testis, renal, skin, brain, leukaemia, etc. amount to 1000 somatic mutations were
analysed, where 158 were classified as driver mutations and the rest as passenger
mutations [37]. Perhaps the most comprehensive
tumour sequencing study preformed till date was by Wood and colleagues, where out
of
a total of 18,191 exon genes in eleven breast and colorectal tumours, 280 were found
to be of candidate cancer genes [38]. These
were then studied in a cohort of 96 patients, where it was found that most of these
genes were mutated in less than 5% of the tumours. An extreme exception to
this would be in case of BRAF kinase mutations in melanomas, where about
60% of the mutations were present in the catalytic core [36]. Specific protein kinase cancer drivers are
rare; however, few hotspots do exist and are vital in our understanding of
proto-oncogenes [36]. It may be an attractive
approach to identify cancer driver mutations but recent studies on cancer genomics
have found less domination of high frequency driver mutations than study of
individual cancers seem to imply [39].
Fig. 3 Kinome mutations leading to cancer phenotypes.
Techniques to detect activities of Protein Kinases
Techniques to detect activities of Protein Kinases
Direct or indirect quantification of the catalysed products i. e.
phosphorylated substrates, ATP or GTP can be detected by using enzymatic-activity
and kinase functional based assays [40].
According to a worldwide survey, the functional assays formats are considered better
as compared to cellular and binding based assays [41]. Radiometric, luminescence based, mobility shift based, and
fluorescence-based assays are included in functional assays and some of them have
been elaborated in the following section.
Fluorescence resonance energy transfer (FRET) based assay
FRET is fluorescence based microscopic technique. It involves the radiation less
transfer of energy from a donor, which can be a dye or a chromophore molecule
(eg. coumarin) to an acceptor chromophore (eg. fluorescein) [42]. FRET is frequently used in many
biomedical researches and in drug delivery. The advantages of this technique
include its simple applications and homogenous format, which means, the donor
and acceptor must be in closer proximity. The energy transfer in FRET is
distance dependent. This assay gives a quantitative analysis of kinase reaction
by calculating the ratio between the emission of donor and acceptor. In this
method, the user must avoid the materials, which have autofluorescence property
to avoid any variation in the signal intensity. One of the important challenges
of this assay is designing of specific substrate for binding with a particular
kinase. This assay consists of limited number of substrates.
Time-resolved fluorescence (TRF) based assay
TRF is based on the use of fluorophores, which have long decay time. In this, the
fluorescence output is observed as a function of time after excitation of the
complex [43]. The lanthanide ions like
samarium, europium and terbium are frequently used in this assay, due to their
longer emission lifetimes. The use of lanthanide ions reduces the background
noise which in turn enhances the sensitivity of detection.
The commercially available TRF based assay is PerkinElmer’s DELFIA1 kit
[44]. This includes the
fluorophore-conjugated substrate binding, separation and detection. In this
assay, the biotinylated substrate has been used for the reaction, then the
mixture is transferred onto a streptavidin coated plate. After multiple
washings, the europium labelled antibody is used to detect the phosphorylated
complex. Now for the dissociation of lanthanide ions, an enhancement solution is
added and the dissociated lanthanide ions forms a fluorescent chelating complex
with the enhancement solution [42]. One of
the biggest disadvantages of this assay is that, it is time consuming due to its
multistep process like transferring, washing, developing and detecting. In
addition to this, the selection and development of antibody is another
challenging task.
Time-resolved fluorescence resonance energy transfer (TR-FRET) based
assay
TR-FRET is a combined assay of FRET and TRF but it only detects the
phosphorylated substrate. In this, the signal is produced through FRET between
donor and acceptor compound in close proximity. This assay contains an acceptor
fluorophore tagged with peptide substrate for the detection and an
anti-phospho-peptide antibody tagged with a donor fluorophore is used [45]. The commercially available TR-FRET
assay kits are PerkinElmer’s Lance1 and Life Technology’s
LanthaScreenTM
[46]. The
PerkinElmer’s Lance1 assay uses lanthanoids (eg. europium) as a
chelating agent, which binds with the anti-phosphorylated antibodies and uses
streptavidin–allophycocyanin as an acceptor. In the
LanthaScreenTM assay, the peptide substrate is tagged with
fluorescein (acceptor), which reacts with a donor (terbium chelating agent). The
product of this reaction is terbium-labelled anti-phosphopeptide antibody which
is used for the detection. Due to this, there is no requirement of additional
acceptor fluorophore. This makes it more cost-effective, highly sensitive,
robust and can be performed in 384 and 1536 well plates. The major challenge of
the assay is to develop a specific detection antibody.
Fluorescence lifetime technology (FLT) based assay
FLT is widely used as an alternative of traditional fluorescence-based methods.
It is an automated assay, free from antibodies and free from the interference
due to fluorescent molecules. Developments in recent years have pointed the
target class suitability, assay authorization and assay reagent suitability
problems using FLT. The market available assay is AlmacFLEXYTETM
protein kinase assay. In this assay, a fluorescent tagged peptide substrate is
phosphorylated by kinase reaction [47].
Then a small molecule chelating agent (SMC) or Lifetime Modulator (LiM) is used
to bind with the phosphate group, which results in decrease in the fluorescence
lifetime. This method is only limited to some tyrosine kinases and
serine/ threonine kinases due to the availability of a limited number of
generic peptide substance.
Luminescence-based assay
These assays are used to calculate the depletion of ATP employing luciferase,
which in the presence of ATP converts luciferin to oxyluciferin and give rise to
the emission of light. Luminescence had been extensively employed in numerous
kinase assays developed by various companies like PerkinElmer and Promega [48]. The commercially available luminescent
kinase assay Promega’s Kinase-GloTM records the depletion of
ATP or the formation of ADP. The other available luminescence assay is
PerkinElmer’s ATPLiteTM, which is based on the similar
approach of Promega’s Kinase-GloTM
[49]. These assays usually show low
sensitivity towards the low ATP concentration.
Enzyme-linked immunosorbent (ELISA) based assay
ELISA was extensively used for the detection of phosphorylated substrates prior
to the use of latest fluorescence-based technologies. In this assay, the
substrate is picked by the matrix and washed several times before the detection
so that the compound fluorescence could not interfere with the measurement.
After washing, a specific anti-phosphorylated antibody is used for its
detection. In this, highly fluorescent dye labelled antibodies are used for more
sensitive measurements. But somehow, the High-throughput screening (HTS)
applicability of ELISA is restricted due to separation and continuous washing
steps [49]. In addition to this, the major
challenge in the application is to design specific antibodies that can bind to
serine / threonine kinase. Kinase assay kits based on the phenomena of
ELISA, dedicated for few specific kinases are available in the market for use.
These are provided via various companies like Abcam, Life Technology, Ray
Biotech, and Cell Biolabs, etc.
Radiometric filtration binding based assay
These are one of the most common and effective kinase profiling assays. This
method directly detects the original products without doing any modification in
substrates or enzymes coupling. This assay working involves incubation of test
compounds along with kinase, reactants, appropriate co-factors and
radio-isotopically labelled ATP like 33P-g-ATP or
32P-g-ATP. The radioisotopically labelled catalytic product obtained
from the incubated reaction mixture is spotted on p-81 phosphocellulose filter
papers. After that, continuous washing is done to remove an excess of
radioactive ATP. The inclusion of radio-isotopically labelled phosphate along
with the kinase substrate is evaluated in order to detect the transfer activity
of kinase phosphorylation, which directly depends on the extent of substrate
phosphorylated [50]. The biggest advantage
of this assay is that it is applicable to all the protein kinases. This assay
does not require any modification in the substrate and its detection is also
free from background interference (fluorescence), but its major disadvantage is
that it can’t be applied on a large-scale HTS. The commercially
available radioisotope filtration binding assays are Eurofins’s Kinase
ProfilerTM and Reaction Biology Corporation (RBC)’s
HotSpotSM, which are used for assessing a large amount of kinase.
The RBC assay provides largest kinase panel among the commercially available
assays. It has total of 578 kinases, which have 366 wild type, 175 mutant, 20
atypical and 17 lipid kinases [51].
Most of the detection techniques for protein kinases with their advantages and
disadvantages have been summarized in [table
2]. It was observed that no single technique is fully acceptable for
drug discovery against kinases. The radioisotope filter binding assay remains
the much accepted amongst the others, due to its various advantages and
applicability to all the protein kinases in an HTS format.
Table 2 Some common kinase profiling
technologies
|
S.No
|
Techniques
|
Available assays
|
Advantage
|
Disadvantage
|
|
1
|
Fluorescence resonance energy transfer (FRET)
|
-
AlphaScreenTM
-
AlphaLISA1®
|
-
Highly sensitive in detection
-
Applied to all fluorescent derived molecule
-
Effectively used for Biological macromolecules
|
|
|
2
|
Time-resolved fluorescence assay (TRF)
|
▪ DELFIA1
|
▪ No background disturbance from sample
|
|
|
3
|
Time-resolved fluorescence resonance energy transfer
(TR-FRET)
|
▪ LanthaScreenTM
|
|
|
|
4
|
Fluorescence lifetime technology (FLT)
|
|
-
Homogenous
-
Antibody free assay
-
HTS compatible
|
|
|
5
|
Luminescence-based assay
|
Kinase-GloTM
|
-
Homogenous set up
-
HTS compatible
-
Less interference
|
|
|
6
|
Radiometric filtration binding assay
|
-
KinaseProfilerTM
-
HotSpotSM
|
-
No background interference
-
Applicable to all protein kinases
-
No modification in substrate is required
|
|
Protein Kinase Inhibitors
Protein Kinase Inhibitors
Enzymatic protein kinase inhibitors are special class of inhibitors that hinder the
phosphorylation of a protein. These inhibitors are employed to block the action of
protein kinases generally on serine, tyrosine, and threonine amino acids of the
protein, as these are the most readily phosphorylating targets in protein kinases.
Generally, most of the kinases function on serine and threonine, whereas for
tyrosine, tyrosine kinase is employed. Some kinases can act with dual specificity
and can phosphorylate any protein based on the three above-mentioned amino acids of
the protein. Since these protein kinases can mutate or alter the cellular
functioning and can cause various diseases including cancer, hence these protein
kinase inhibitors can be utilized as a promising candidate for treating diseases
including cancer, which might have been caused because of the abnormal protein
kinases. Due to this, quite substantial work has been carried out to develop a
therapeutic tool for inhibiting protein kinases and further examining cellular
functioning [9]. Nowadays, numerous examples
of protein kinase inhibitors are available having high selectivity and apt
pharmaceutical applicability properties, which can bring about a significant change
in the concerned field [52]. Imatinib is an
excellent example of such inhibitors, which is employed for inhibition of
BCR-ABL1 in case of acute lymphoblastic leukemia and chronic myelogenous
leukemia (CML) with the Philadelphia chromosomes [53]. Some other examples are vemurafenib for mutant tumor, which is
caused because of the proto-oncogene BRAF
[54]; crizotinib and alternative anaplastic lymphoma kinase (ALK)
inhibitors for cancer caused via ALK fusions [55]; erlotinib and gefitinib for mutated tumor caused by EGFR and
lapatinib employed for HER2/ ERBB2 amplified tumor [56]. Focusing on the target inhibiting
specificity of the protein kinase inhibitors, the multiple targeted kinase
inhibitors are the most prominent inhibitors of all, which are discussed in the
following section.
Multiple Targeted Kinase Inhibitors
As mentioned earlier, there are about 518 protein kinases, which have pivotal
roles in regulating various cellular signalling pathways, having different
cellular metabolisms such as cell apoptosis, cell division, angiogenesis, and
cell separation, etc. Several kinases like BCR-ABL, mutant EGFR, BRAF and
tyrosine protein kinases are proclaimed to be oncogenic and their
hindrance/ inhibition is beneficial for anticancer treatment [57]. Continuous efforts are being made to
convert the most efficient kinase inhibitors to work on multiple kinases by the
collegial action [58]. This multi-targeted
inhibition using a single reagent, also known as polypharmacology, offers
considerable therapeutic benefits like satisfactory patient consent,
interruption of drug-drug cooperation, simple and minimal combined off-target
effects of protein kinases [59]
[60]
[61]
[62]. Sorafenib was the
first multiple targeted inhibitor drug, which was approved in 2005. Sorafenib
can be utilized for the treatment of non-resectable hepatocellular and final
stages of renal cell carcinomas [63]
[64]
[65]. Other than sorafenib, many other FDA approved multi-kinase
targeting inhibiting drugs are nilotinib, imatinib, pralsetinib, and dasatinib,
etc., which are mentioned in [table 3]
[66]
[67]. These are validated to be inhibitors of various kinases such as
p38, ABL, B-RAF, and c-KIT kinases, etc. [68]
[69]
[70]
[71]
[72]
[73]
[74].
Table 3 FDA Approved Multi-Kinase Targeting
Inhibitors.
|
Drug
|
Target Protein Kinase
|
Targeted Cancer
|
Developing Company
|
|
Imatinib
|
ABL1–2, KIT, PDGFR
|
MML, GIST. CML, CEL, Ph+ B-ALL
|
Novartis
|
|
Dasatinib
|
SRC, ABL1–2, KIT, PDGFR
|
CML
|
Bristol Myers
|
|
Sorafenib
|
KIT, BRAF, VEGFR2, FLT3, PDGFR
|
RCC
|
Onyx and Bayer Pharmaceuticals
|
|
Lapatinib
|
ERBB2, EGFR
|
Breast Cancer
|
Glaxo SmithKline
|
|
Gefitinib
|
EGFR
|
NSCLC
|
AstraZeneca
|
|
Temsirolimus
|
mTOR
|
RCC
|
Wyeth
|
|
Erlotinib
|
EGFFR
|
Pancreatic Cancer, NSCLC
|
Roche, OSI, Genentech Inc
|
|
Nilotinib
|
KIT, ABL1–2, PDGFR
|
CML
|
Novartis
|
|
Everolimus
|
mTOR
|
RCC
|
Novartis
|
|
Pralsetinib
|
RET
|
NSCLC, Thyroid Cancer
|
Genentech and Blueprint Medicines
|
|
Selpercatinib
|
RET
|
NSCLC, Thyroid Cancer
|
Eli Lilly and Company
|
|
Ripretinib
|
PDGFRA, KIT
|
GIST
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Deciphera Pharmaceuticals
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FDA Approved Drugs as Targets
FDA Approved Drugs as Targets
Kinases play a vital role in cell survival, proliferation, transformation and tumour
initiation. A key challenge is to understand their role in oncology process and then
to develop drugs against them [75]. On the
other hand, it is hard to understand the functionalities of all the kinases, due to
which these are categorized in different groups on the basis of their roles in
oncology. Many intensive efforts have been made for the detection of novel targets
in the human genome. Many clinical trials have been done successfully for drugs
discovery to mark the infection. The advancement in kinase inhibitors had started
in
the mid-1970s. From the past few years, the protein kinase attracts a lot of
attention in targeted drug discovery in cancer [76]. Currently, Structure-based medication discovery is made easier by
more than 5,000 protein kinase structures that are publicly available. Additionally,
the pharmaceutical industry uses a greater number of proprietary structures in the
drug discovery process. Around 180 kinases showed the potential as primary targets
of oral kinase inhibitors, which are under clinical trial. One can find a
comprehensive list of these agents, which is updated periodically, at www.icoa.
fr/pkidb/[77]
[78]
[79].
Since there are vast number of drugs in clinical Phase I-III, related to protein
kinase inhibition, therefore, it is too difficult to cover all of them in this
review. However, this article tends to review some of recent FDA approved drugs that
bear high clinical importance for kinase inhibition, and these have been summarized
in [fig. 4] along with their targets [21]
[80].
Fig.4 FDA approved kinome therapeutics with their approval year and
major kinase targets.
The first successful protein kinase Rho-associated Protein Kinase (ROCK) inhibitor
Fasudil was approved for treatment in 1995 in China and Japan. After 4 years (in
1999), Rapamycin, which is an inhibitor of mammalian target of rapamycin complex 1
(mMTORC1) complex was approved by the FDA [81]. Rapamycin was the first kinase inhibitor, which got FDA approval [82].
One of the biggest breakthroughs was the discovery of CGP57148Bin 2001, which was
later named as Imatinib [83]
[84]
[85].
It showed potential binding to the inactive conformation of ABL1 kinase for the
treatment of chronic myeloid leukaemia (CML). Imatinib targets the BCR-Abl
gene, the BCR part of the gene is found on chromosome 22 and the Abl gene is
located at chromosome 9 [86], which showed the
clinical improvement in the leukaemia patients [87]. This discovery leads to the development of other drugs for kinase
inhibition like nilotinib, which was used against the imatinib resistant CML [88]. After this, sunitinib, which has a
broad-spectrum activity against the FMS-like tyrosine kinase 3 (FLT3), a
receptor tyrosine kinase (KIT), fibroblast growth factor receptors
(FGFR), Platelet-derived growth factor receptors (PDGFR) and
vascular endothelial growth factor receptors (VEGFR), got approved by FDA for
the treatment of renal cell carcinoma (RCC) and second line therapy of GIST
(imatinib-resistant gastrointestinal stromal tumour) [89]. Similarly, a drug sorafenib which
effectively binds with the inactive conformation of VEGFR kinase, was approved for
the cure of RCC and hepatocellular carcinoma [90]. In 2009, a 2-amino pyrimidine derivative pazopanib was approved for
the advanced stage of RCC. After 2009, an increase was observed in the approval of
kinase inhibitor drugs. From 2011 to 2013, total of 14 kinase inhibitor drugs were
approved by the FDA for various types of cancers. Since 1999, around 70 drugs got
approval from FDA till Dec 2021 and still many more drugs are under clinical trial
for many new kinase inhibitors [25]
[91]
[92].
Outlook and Future Prospects
Outlook and Future Prospects
Protein kinases, encoded by genes collectively called the kinome of the organism,
mainly function to phosphorylate proteins and thus activate them. Since this form
of
protein activation is so common, this highlights the omnipresence and importance of
protein kinases and by extension of kinomes in the physiological and biochemical
functioning of the body. Mutations in the kinome may lead to alterations in the
protein kinase, resulting in their deregulation, which leads to several diseases
including cancers. To throw some light on this significant issue, we have briefly
discussed the mechanism of kinase action, various methods for their detection, and
their roles in direct phosphorylation and indirect cell signalling. Use of
bioinformatics for comparison of mutated kinomes in cancer patients against healthy
kinomes to identify patterns, mutation hotspots and detailed structural study of
protein kinases to develop more specific targeted therapies is very essential. We
have also discussed the potential, importance, and types of kinome silencing using
protein kinase inhibitors to check deregulated protein kinases and a selection of
FDA approved drugs, to address this dysregulation. Detailed genomic studies to
identify common mutation hotspots aided by computational methods to predict possible
mutations resultant in dysregulated protein kinases would help in diagnostics.
