Protein kinases: Role of their dysregulation in carcinogenesis, identification and inhibition

Abstract

Protein kinases belong to the phosphor-transferases superfamily of enzymes, which “activate” enzymes via phosphorylation. The kinome of an organism is the total set of genes in the genome, which encode for all the protein kinases. Certain mutations in the kinome have been linked to dysregulation of protein kinases, which in turn can lead to several diseases and disorders including cancer. In this review, we have briefly discussed the role of protein kinases in various biochemical processes by categorizing cancer associated phenotypes and giving their protein kinase examples. Various techniques have also been discussed, which are being used to analyze the structure of protein kinases, and associate their roles in the oncogenesis. We have also discussed protein kinase inhibitors and United States Federal Drug Administration (USFDA) approved drugs, which target protein kinases and can serve as a counter to protein kinase dysregulation and mitigate the effects of oncogenesis. Overall, this review briefs about the importance of protein kinases, their roles in oncogenesis on dysregulation and how their inhibition via various drugs can be used to mitigate their effects.

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

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

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

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

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 LanthaScreen^TM [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 LanthaScreen^TM 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 AlmacFLEXYTE^TM 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-Glo^TM records the depletion of ATP or the formation of ADP. The other available luminescence assay is PerkinElmer’s ATPLite^TM, which is based on the similar approach of Promega’s Kinase-Glo^TM [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 Profiler^TM and Reaction Biology Corporation (RBC)’s HotSpot^SM, 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	Modified molecular system can change the fluorescence properties. Tagging of fluorescent probes can change the character of substrate.
2	Time-resolved fluorescence assay (TRF)	▪ DELFIA1	▪ No background disturbance from sample	Use of limited lanthanide dye. More expensive
3	Time-resolved fluorescence resonance energy transfer (TR-FRET)	▪ LanthaScreenTM	Does not depend on radioactive material for detection High sensitivity HTS Friendly	Modification of substrate for fluorophore labeling. Development of specific antibody
4	Fluorescence lifetime technology (FLT)	FLEXYTETM ATPLiteTM	Homogenous Antibody free assay HTS compatible	Limited to few kinases Requires specifically designed peptide substrate
5	Luminescence-based assay	Kinase-GloTM	Homogenous set up HTS compatible Less interference	Low sensitivity in case of low ATP Requires anti screening against coupling enzyme
6	Radiometric filtration binding assay	KinaseProfilerTM HotSpotSM	No background interference Applicable to all protein kinases No modification in substrate is required	Additional washing step required Radio isotopic materials safety issues.

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	Deciphera Pharmaceuticals

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

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.

Conflict of interest

Authors declare no conflict of interest regarding the present work.

Acknowledgments

MK is thankful for the research and development grant by University of Delhi, Delhi, India. NS, PK, and AS would like to acknowledge their fellowships as UGC-CSIR fellowship no. F.16–6(DEC 2016)/2017(NET) and CSIR fellowship no. 09/045(1646)/2019-EMR-1 and Ref.No.: Sch/139/Non-Net/Chem./Ph.D/2018–19/625 fellowship from Dept. of Chemistry, Delhi University respectively.

^* These authors contributed equally: Niloy Sarkar, Amit Singh, Pankaj Kumar

• References

• 1 Manning G, Whyte D.B, Martinez R. et al. The protein kinase compliment of the human genome. Science 298: 1912-1934 2002; DOI: 10.1126/science.1075762.
• 2 Fischer EH, Krebs E.G. Conversion of phosphorylase b to phosphorylase a in muscle extracts. Journal of Biological Chemistry 216: 121-132 1955; No DOI found
• 3 Boulton T.G, Gregory JS, Cobb MH. Purification and properties of extracellular regulated kinase 1, an insulin-stimulated microtubule-associated protein 2 kinase. Biochemistry 30: 278-286 1991; DOI: 10.1021/bi00215a038.
• 4 Rossomando AJ, Payne DM, Weber MJ. et al. Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. Proceedings of the National Academy of Sciences 86: 6940-6943 1989; DOI: 10.1073/pnas.86.18.6940.
• 5 Hunter T, Sefton BM. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proceedings of the National Academy of Sciences 77: 1311-1315 1980; DOI: 10.1073/pnas.77.3.1311.
• 6 Parker RC, Varmus HE, Bishop JM. Cellular homologue (c-src) of the transforming gene of Rous sarcoma virus: isolation, mapping, and transcriptional analysis of c-src and flanking regions. Proceedings of the National Academy of Sciences 78: 5842-5846 1981; DOI: 10.1073/pnas.78.9.5842.
• 7 Alonso A, Sasin J, Bottini N, Friedberg I. et al. Protein tyrosine phosphatases in the human genome. Cell 117: 699-711 2004; DOI: 10.1016/j.cell.2004.05.018.
• 8 Duong-Ly KC, Peterson JR. The human kinome and kinase inhibition. Current protocols in pharmacology 60: 2-9 2013; DOI: 10.1002/0471141755.ph0209s60.
• 9 Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9: 28-39 2009; DOI: 10.1038/nrc2559.
• 10 Modi V, Dunbrack RL.. A Structurally Validated Sequence Alignment of All 497 Typical Human Protein Kinase Domains. bioRxiv 776740 2019; DOI: 10.1101/776740.
• 11 Taylor SS, Kornev AP. Protein kinases: evolution of dynamic regulatory proteins. Trends in biochemical sciences 36: 65-77 2011; DOI: 10.1016/j.tibs.2010.09.006.
• 12 Ten Eyck LF, Taylor SS, Kornev AP. Conserved spatial patterns across the protein kinase family. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1784: 238-243 2008; DOI: 10.1016/j.bbapap.2007.11.002.
• 13 Modi V, Dunbrack RL.. Kincore: a web resource for structural classification of protein kinases and their inhibitors. Nucleic Acids Research 50: D654-D664 2022; DOI: 10.1093/nar/gkab920.
• 14 Johnson LN, Noble ME, Owen DJ. Active and inactive protein kinases: structural basis for regulation. Cell 85: 149-158 1996; DOI: 10.1016/S0092-8674(00)81092-2.
• 15 Bayliss R, Fry A, Haq T. et al. On the molecular mechanisms of mitotic kinase activation. Open Biology 2: 2046-2441 2012; DOI: 10.1098/rsob.120136.
• 16 Eyers PA, Murphy JM. Dawn of the dead: protein pseudokinases signal new adventures in cell biology. Biochemical Society Transactions 41: 969-974 2013; DOI: 10.1042/BST20130115.
• 17 Chen M, Cheng A, Candotti F. et al. Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains. Molecular and Cellular Biology 20: 947-956 2000; DOI: 10.1128/MCB.20.3.947-956.2000.
• 18 Morrison DK. KSR: a MAPK scaffold of the Raspathway?. Journal of cell science 114: 1609-1612 2001; No DOI found
• 19 Tomoni A, Lees J, Santana AG. et al. Pseudokinases: From allosteric regulation of catalytic domains and the formation of macromolecular assemblies to emerging drug targets. Catalysts. Sep 19 9: 778 2019; DOI: 10.3390/catal9090778.
• 20 Walter C, Marada A, Suhm T. et al. Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery. Nature communications. 13 12: 1-2 2021; DOI: 10.1038/s41467-021-24426-9.
• 21 Bhullar KS, Lagaron NO, McGowan E. et al. Kinase-targeted cancer therapies: progress, challenges and future directions. Molecular Cancer 17: 48 DOI: 10.1186/s12943-018-0804-2.
• 22 Cicenas J, Zalyte E, Bairoch A. et al. Kinases and cancer. Cancers 10: 63 2018; DOI: 10.3390/cancers10030063.
• 23 Hofmann MC, Kunnimalaiyaan M, Wang JR. et al. Molecular mechanisms of resistance to kinase inhibitors and redifferentiation in thyroid cancers. Endocrine-Related Cancer. 1 29: R173-R190 2022; DOI: 10.1530/ERC-22-0129.
• 24 Gross S, Rahal R, Stransky N. et al. Targeting cancer with kinase inhibitors. The Journal of clinical investigation 125: 1780-1789 2015; DOI: 10.1172/JCI76094.
• 25 Fan M, Ahmed KM, Coleman MC. et al. Nuclear Factor-KB and Manganese Superoxide Dismutase Mediate Adaptive Radioresistance in Low-Dose Irradiated Mouse Skin Epithelial Cells. Cancer Research 67: 3220-3228 2007; DOI: 10.1158/0008-5472.
• 26 Kumar RD, Bose R. Analysis of somatic mutations across the kinome reveals loss-of-function mutations in multiple cancer types. Scientific Reports 7: 6418 2017; DOI: 10.1038/s41598-017-06366-x.
• 27 Essegian D, Khurana R, Stathias V. et al. The clinical kinase index: a method to prioritize understudied kinases as drug targets for the treatment of cancer. Cell Reports Medicine. 20 1: 100128 2020; DOI: 10.1016/j.xcrm.2020.100128.
• 28 Roskoski R. Classification of small molecule protein kinase inhibitors based upon the structures of their drug-enzyme complexes. Pharmacological Research 103: 26-48 2016; DOI: 10.1016/j.phrs.2015.10.021.
• 29 Lapenna S, Giordano A. Cell cycle kinases as therapeutic targets for cancer. Nature Reviews Drug Discovery 8: 547-566 2009; DOI: 10.1038/nrd2907.
• 30 Chandran RK, Geetha N, Sakthivel KM. et al. Genomic amplification of BCR-ABL1 fusion gene and its impact on the disease progression mechanism in patients with chronic myelogenous leukemia. Gene 686: 85-91 2019; DOI: 10.1016/j.gene.2018.11.005.
• 31 Futreal PA, Coin L, Marshall M. et al. A census of human cancer genes. Nature Reviews Cancer 4: 177-183 2004; DOI: 10.1038/nrc1299.
• 32 Wapner J, Weinburg RA. The Philadelphia Chromosome: A Genetic Mystery, A Lethal Cancer, and the improbable Invention of a Life-Saving Treatment. The Experiment, New York, (. 2014 ISBN 9781615191970
• 33 Subbiah V, Yang D, Velcheti V. et al. State-of-the-art strategies for targeting RET-dependent cancers. Journal of Clinical Oncology. 4 38: 1209 2020; DOI: 10.1200/JCO.19.02551.
• 34 Chatterjee A, Rodger EJ, Eccles MR. Epigenetic drivers of tumorigenesis and cancer metastasis. Seminars in Cancer Biology 51: 149-159 2018; DOI: 10.1016/j.semcancer.2017.08.004.
• 35 Salvadores M, Mass-Ponte D, Supek F. Passenger mutations accurately classify human tumors. PLoS Computational Biology 15 2019; DOI: 10.1371/journal.pcbi.1006953.
• 36 Torkamani A, Verkhivker G, Schork NJ. Cancer driver mutations in protein kinase genes. Cancer Letters 281: 117-127 2009; DOI: 10.1016/j.canlet.2008.11.008.
• 37 Greenman C, Stephens P, Smith R. et al. Patterns of somatic mutation in human cancer genomes. Nature 446: 153-158 2007; DOI: 10.1038/nature05610.
• 38 Wood LD, Parsons DW, Jones S. et al. The genomic landscapes of human breast and colorectal cancer. Science 318: 1108-1113 2007; DOI: 10.1126/science.1145720.
• 39 Mendiratta G, Ke E, Aziz M. et al. Cancer gene mutation frequencies for the US population. Nature communications. 13 12: 1-1 2021; DOI: 10.1038/s41467-021-26213-y.
• 40 Goldstein DM, Gray NS, Zarrinkar PP. High-throughput kinase profiling as a platform for drug discovery. Nature reviews Drug discovery 7: 391-397 2008; DOI: 10.1038/nrd2541.
• 41 Comley J. Outsourced kinase profiling services-adding value to in-house kinase programmes. Drug Discov World Fall 2013: 26-45 2013; No DOI found
• 42 Wang Y, Ma H. Protein kinase profiling assays: a technology review. Drug Discovery Today: Technologies 18: 1-8 2015; DOI: 10.1016/j.ddtec.2015.10.007.
• 43 Peruski AH, Johnson LH, Peruski LF. Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays. Journal of immunological methods 263: 35-41 2002; DOI: 10.1016/S0022-1759(02)00030-3.
• 44 Lu X, Bergelson S. Development of a sensitive potency assay to measure the anti-proliferation effect of an anti-HER2 anx tibody. Journal of immunological methods 415: 80-85 2014; DOI: 10.1016/j.jim.2014.10.007.
• 45 Gaborit N, Larbouret C, Vallaghe J. et al. Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) to analyze the Disruption of EGFR/HER2 Dimers A new method to evaluate the efficiency of targeted therapy using monoclonal antibodies. Journal of Biological Chemistry 286: 11337-11345 2011; DOI: 10.1074/jbc.M111.223503.
• 46 Ergin E, Dogan A, Parmaksiz M. et al. Time-resolved fluorescence resonance energy transfer [TR-FRET] assays for biochemical processes. Current pharmaceutical biotechnology 17: 1222-1230 2016; DOI: 10.2174/1389201017666160809164527.
• 47 Cotton G, Bowen W. Fluorescence Lifetime Assays: New Technology Enables Primary and Secondary Screening of Broad Panels. Genetic Engineering & Biotechnology News 33: 28-29 2013; DOI: 10.1089/gen.33.10.12.
• 48 Hagan AK, Zuchner T. Lanthanide-based time-resolved luminescence immunoassays. Analytical and bioanalytical chemistry 400: 2847-2864 2011; DOI: 10.1007/s00216-011-5047-7.
• 49 Roda A, Mirasoli M, Michelini E. et al. Progress in chemical luminescence-based biosensors: a critical review. Biosensors and Bioelectronics 76: 164-179 2016; DOI: 10.1016/j.bios.2015.06.017.
• 50 Ma H., Deacon S, Horiuchi K. The challenge of selecting protein kinase assays for lead discovery optimization. Expert opinion on drug discovery 3: 607-621 2008; DOI: 10.1517/17460441.3.6.607.
• 51 Inglese J, Johnson RL, Simeonov A. et al. High-throughput screening assays for the identification of chemical probes. Nature chemical biology 3: 466-479 2007; DOI: 10.1038/nchembio.2007.17.
• 52 Gross S, Rahal R, Stransky N. et al. Targeting cancer with kinase inhibitors. The Journal of clinical investigation 125: 1780-1789 2015; DOI: 10.1172/JCI76094.
• 53 Kantarjian H, Sawyers C, Hochhaus A. et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. The New England Journal of Medicine 346: 645-652 2002; DOI: 10.1056/NEJMoa011573.
• 54 Chapman PB, Hauschild A, Robert C. et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. The New England Journal of Medicine 364: 2507-2516 2011; DOI: 10.1056/NEJMoa1103782.
• 55 Shaw AT, Kim DW, Mehra R. et al. Ceritinib in ALK-rearranged non–small-cell lung cancer. The New England Journal of Medicine 370: 1189-1197 2014; DOI: 10.1056/NEJMoa1311107.
• 56 Arteaga CL, Engelman JA. ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics. Cancer cell 25: 282-303 2014; DOI: 10.1016/j.ccr.2014.02.025.
• 57 Traxler P. Tyrosine kinases as targets in cancer therapy–successes and failures. Expert opinion on therapeutic target 7: 215-234 2003; DOI: 10.1517/14728222.7.2.215.
• 58 Knight ZA, Lin H, Shokat KM. Targeting the cancer kinome through polypharmacology. Nature Reviews Cancer 10: 130-137 2010; DOI: 10.1038/nrc2787.
• 59 Hoeflich KP, Merchant M, Orr C. et al. Intermittent administration of MEK inhibitor GDC-0973 plus PI3K inhibitor GDC-0941 triggers robust apoptosis and tumor growth inhibition. Cancer research 72: 210-219 2012; DOI: 10.1158/0008-5472.
• 60 Zhang X, Zhang J, Tong L. et al. The discovery of colchicine-SAHA hybrids as a new class of antitumor agents. Bioorganic & medicinal chemistry 21: 3240-3244 2013; DOI: 10.1016/j.bmc.2013.03.049.
• 61 Guerrant W, Patil V, Canzoneri JC. et al. Dual targeting of histone deacetylase and topoisomerase II with novel bifunctional inhibitors. Journal of Medicinal Chemistry 55: 1465-1477 2012; DOI: 10.1021/jm200799p.
• 62 Renshaw J, Taylor KR, Bishop R. et al. Dual blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) pathways synergistically inhibits rhabdomyosarcoma cell growth in vitro and in vivo. Clinical Cancer Research 19: 5940-5951 2013; DOI: 10.1158/1078-0432.
• 63 Dietrich J, Hulme C, Hurley LH. The design, synthesis, and evaluation of 8 hybrid DFG-out allosteric kinase inhibitors: A structural analysis of the binding interactions of Gleevec^®, Nexavar^®, and BIRB-796. Bioorganic & medicinal chemistry 18: 5738-5748 2010; DOI: 10.1016/j.bmc.2010.05.063.
• 64 Liu L, Cao Y, Chen C. et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer research 66: 11851-11858 2006; DOI: 10.1158/0008-5472.
• 65 Escudier B. Randomized phase III trial of the multi-kinase inhibitor sorafenib (BAY 43-9006) in patients with advanced renal cell carcinoma (RCC). European Journal of Cancer Supplements 3. 226 No DOI found
• 66 Kuster B. (Ed) Kinase inhibitors: methods and protocols. Humana Press ; 795. 1-44 2012. DOI: 10.1007/978-1-61779-337-0
• 67 Cicenas J, Račienė A. Anti-Cancer Drugs Targeting Protein Kinases Approved by FDA in 2020. Cancers 13: 947 2021; DOI: 10.3390/cancers13050947.
• 68 Trudel S, Li ZH, Wei E. et al. CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t (4; 14) multiple myeloma. Blood 105: 2941-2948 2005; DOI: 10.1182/blood-2004-10-3913.
• 69 OʼHare T, Shakespeare WC, Zhu X. et al. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer cell 16: 401-412 2005; DOI: 10.1016/j.ccr.2009.09.028.
• 70 Gajiwala KS, Wu JC, Christensen J.. et al. KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients. Proceedings of the National Academy of Sciences 106: 1542-1547 2009; DOI: 10.1073/pnas.0812413100036.
• 71 Simard JR, Getlik M, Grütter C. et al. Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors. Journal of the American Chemical Society 131: 13286-13296 DOI: 10.1021/ja902010p.
• 72 Hu M, Subbiah V, Wirth LJ. et al. 1913O Results from the registrational phase I/II ARROW trial of pralsetinib (BLU-667) in patients (pts) with advanced RET mutation-positive medullary thyroid cancer (RET+ MTC). Annals of Oncology 31: S1084 2020; DOI: 10.1016/j.annonc.2020.08.1401.
• 73 Wirth LJ, Sherman E, Robinson B. et al. Efficacy of selpercatinib in RET-altered thyroid cancers. New England Journal of Medicine 383: 825-835 2020; DOI: 10.1056/NEJMoa2005651.
• 74 Blay JY, Serrano C, Heinrich MC. et al. Ripretinib in patients with advanced gastrointestinal stromal tumours (INVICTUS): a double-blind, randomised, placebo-controlled, phase 3 trial. The Lancet Oncology 21: 923-934 2020; DOI: 10.1016/S1470-2045(20)30168-6.
• 75 Wilson LJ, Linley A, Hammond DE. et al. New perspectives, opportunities, and challenges in exploring the human protein kinome. Cancer research 78: 15-29 2018; DOI: 10.1158/0008-5472.
• 76 Collett MS, Erikson RL. Protein kinase activity associated with the avian sarcoma virus src gene product. Proceedings of the National Academy of Sciences 75: 2021-2024 1978; DOI: 10.1073/pnas.75.4.2021.
• 77 Hu Y, Furtmann N, Bajorath J. Current compound coverage of the kinome: miniperspective. Journal of medicinal chemistry 58: 30-40 2014; DOI: 10.1021/jm5008159.
• 78 Santos R, Ursu O, Gaulton A. et al. A comprehensive map of molecular drug targets. Nature reviews Drug discovery 16: 19-34 2017; DOI: 10.1038/nrd.2016.230.
• 79 Viossat Y, Noble R. A theoretical analysis of tumour containment. Nature ecology & evolution 5: 826-835 2021; DOI: 10.1038/s41559-021-01428-w.
• 80 Roskoski R. 2021; Properties of FDA-approved small molecule protein kinase inhibitors: a 2021 update. Pharmacological research 105463
• 81 Gaubitz C, Oliveira TM, Prouteau M. et al. Molecular basis of the rapamycin insensitivity of target of rapamycin complex 2. Molecular cell 58: 977-988 2015; DOI: 10.1016/j.molcel.2015.04.031.
• 82 Tym JE, Mitsopoulos C, Coker EA. et al. canSAR updated cancer research and drug discovery knowledgebase. Nucleic acids research 42: D938-D943 2015; DOI: 10.1093/nar/gkv1030.
• 83 Hopkin MD, Baxendale IR, Ley SV. A flow-based synthesis of Imatinib: the API of Gleevec. Chemical communications 46: 2450-2452 2010; DOI: 10.1039/C001550D.
• 84 Cohen P, Cross D, Jänne PA. Kinase drug discovery 20 years after imatinib: Progress and future directions. Nature reviews drug discovery 20: 551-569 2021; DOI: 10.1038/s41573-021-00195-4.
• 85 Attwood MM, Fabbro D, Sokolov AV. et al. Trends in kinase drug discovery: Targets, indications and inhibitor design. Nature Reviews Drug Discovery 20: 839-861 2021; DOI: 10.1038/s41573-021-00252-y.
• 86 Schultz KR, Bowman WP, Aledo A. et al. Improved early event-free survival with imatinib in Philadelphia chromosome–positive acute lymphoblastic leukemia: A children's oncology group study. Journal of Clinical Oncology 27: 5175-5181 2009; DOI: 10.1200/JCO.2008.21.2514.
• 87 Lozzio CB, Lozzio BB. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45: 321-334 1975; No DOI found
• 88 Kantarjian H, Giles F, Wunderle L. et al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome–positive ALL. The New England Journal of Medicine 354: 2542-2551 2006; DOI: 10.1056/NEJMoa055104.
• 89 Motzer RJ, Hoosen S, Bello CL. et al. Sunitinib malate for the treatment of solid tumours: a review of current clinical data. Expert opinion on investigational drugs 15: 553-561 2006; DOI: 10.1517/13543784.15.5.553.
• 90 Sternberg CN, Szczylik C, Lee E. et al. A randomized, double-blind phase III study of pazopanib in treatment-naive and cytokine-pretreated patients with advanced renal cell carcinoma (RCC). Journal of Clinical Oncology 27: 5021-5021 2009; DOI: 10.1200/jco.2009.27.15s.5021.
• 91 Roskoski R. Properties of FDA-approved small molecule protein kinase inhibitors: A 2022 update. Pharmacological research 106037 2021; DOI: 10.1016/j.phrs.2021.106037.
• 92 Lee PY, Yeoh Y, Low TY. A recent update on small-molecule kinase inhibitors for targeted cancer therapy and their therapeutic insights from mass spectrometry-based proteomic analysis. The FEBS Journal 2022; DOI: 10.1111/febs.16442.

Publication History

Received: 28 April 2022

Accepted: 07 November 2022

Article published online:
23 February 2023