**2. Kinase structure and function**

Protein kinase function was first observed in 1954 by Burnett and Kennedy who discovered an enzyme that phosphorylates casein [1]. Protein kinases are a group of protein enzymes that phosphorylate protein and other targets in the cell in order to modulate these target proteins' function. Protein kinases are often target proteins themselves. Protein kinases catalyze the transfer of a phosphate group from adenosine triphosphate (ATP) to amino acid sidechains that bear a hydroxyl group on target proteins, namely serine, threonine, and tyrosine. The phosphorylation of one of these amino acid sidechains can drastically change the local physicochemical environment as the relatively small polar unionized hydroxyl group is transformed into the relatively large polar ionized phosphate group. This change in the local environment can initiate a conformational change in the newly phosphorylated target protein, in an attempt to find a new low energy conformation, which may modulate the function of the target protein. The phosphorylation event may serve to activate or inhibit target protein enzyme function, or simply modify the target proteins' surface to modulate protein/protein interactions. For example, Src homology 2 (SH2) domain containing proteins recognize certain phospho-tyrosine sequences within protein targets which enable them to perform their adaptor or scaffolding function [2].

**Figure 1.** The EGFR kinase domain with color coded structural elements. Cyan; N-terminal lobe. Red; C-terminal lobe. Blue; P-loop. Magenta; hinge. Green; activation loop. Orange; catalytic loop.

Protein kinases are classified into groups based on sequence similarity [3]. The AGC group is named for protein kinases A, G and C. The CaM group is named for calcium/calmodulindependent kinases. The CK1 group is named for casein kinase 1. The CMGC group is named for cyclin dependent, mitogen-activated, glycogen synthase, and CDK-like kinases. The STE group is named for sterile phenotype kinase. The TK group is named for tyrosine kinases, and the TKL group is named for tyrosine kinase-like kinases. The groups are further broken down to individual protein kinase families and these families may be further broken down to subfamilies. For example, within the TK group is the family called epidermal growth factor receptor (EGFR) tyrosine kinase. The EGFR family includes members EGFR (Her1/ErbB1), Her2/ErbB2, Her3/ErbB3 and Her4/ErbB4. These family members have a high degree of sequence similarity and function.

Unfortunately, resistance to kinase inhibitors can develop through up-regulation of the target

In the most ideal sense of targeted therapy for cancer, kinase inhibitors have the potential to fulfill the goal of personalized cancer therapy. Studies have shown kinase inhibitors targeting specific kinases that drive a proportion of patients' tumors can provide better outcomes for those patients. Combine this outcome with the possibility of mutant selective kinase inhibitors which may leave tissue that expresses wild-type kinase largely unaffected and provide a

Protein kinase function was first observed in 1954 by Burnett and Kennedy who discovered an enzyme that phosphorylates casein [1]. Protein kinases are a group of protein enzymes that phosphorylate protein and other targets in the cell in order to modulate these target proteins' function. Protein kinases are often target proteins themselves. Protein kinases catalyze the transfer of a phosphate group from adenosine triphosphate (ATP) to amino acid sidechains that bear a hydroxyl group on target proteins, namely serine, threonine, and tyrosine. The phosphorylation of one of these amino acid sidechains can drastically change the local physicochemical environment as the relatively small polar unionized hydroxyl group is transformed into the relatively large polar ionized phosphate group. This change in the local environment can initiate a conformational change in the newly phosphorylated target protein, in an attempt to find a new low energy conformation, which may modulate the function of the target protein. The phosphorylation event may serve to activate or inhibit target protein enzyme function, or simply modify the target proteins' surface to modulate protein/protein interactions. For example, Src homology 2 (SH2) domain containing proteins recognize certain phospho-tyrosine sequences within protein targets which enable them to perform their

**Figure 1.** The EGFR kinase domain with color coded structural elements. Cyan; N-terminal lobe. Red; C-terminal lobe.

Blue; P-loop. Magenta; hinge. Green; activation loop. Orange; catalytic loop.

kinase, or mutation of the target kinase resulting in decreased drug binding.

potentially better safety profile.

14 Updates on Cancer Treatment

**2. Kinase structure and function**

adaptor or scaffolding function [2].

All protein kinases have a somewhat homologous kinase domain that adopts a common tertiary structure [4]. Figure 1 represents the epidermal growth factor receptor (EGFR) kinase domain and will serve to identify the various secondary structural elements of kinase domain structure and function. Starting from the N-terminus, the basic structural elements of a kinase domain include an N-terminal lobe, composed mostly of beta-sheet structure, and a C-terminal lobe, composed mostly of alpha-helix structure. The two lobes are connected by a hinge region. Of particular interest are the conserved mobile elements of the kinase domain which contribute to kinase function.

**Figure 2.** EGFR kinase domain (ribbon) in the active conformation bound to an ATP analog (stick). The active confor‐ mation is characterized by alignment of key amino acid sidechains that form a hydrophobic spine (surface representa‐ tion).

Conformational mobility is essential for kinase function. A number of flexible loop regions are important for the ability to interconvert between active and inactive conformations. These loop regions include the activation loop (A-loop), catalytic loop, nucleotide binding loop (P-loop), and the hinge region. The P-loop is a long loop that joins two anti-parallel beta-strands that are a part of the N-terminal lobe beta-sheet structure of the kinase domain. This loop typically contains a number of glycine residues that are able to access a large range of phi and psi angles and enable considerable conformational flexibility. In the inactive conformation the P-loop may be largely disordered. In the active kinase conformation the P-loop may resemble more of a classic beta-turn and is able to interact with the triphosphate group of bound ATP. The Aloop includes one or more tyrosine residues that when phosphorylated induce the conversion from the inactive to the active kinase conformation. The A-loop is long, containing around 27 amino acid residues that may fold into a number of isolated single turn helices. The A-loop has a conserved DFG sequence, where in the non-phosphorylated inactive kinase conforma‐ tion the phenylalanine sidechain is oriented toward the triphosphate binding area of the ATP binding site, the so called DFG-out conformation. In the phosphorylated active kinase conformation, the DFG phenylalanine sidechain is oriented toward the interior of the protein, the so-called DFG-in conformation [5-7]. While the DFG orientation related to active and inactive kinase conformations is clear in some cases it is not clear in others, and is not as general in differentiating active and inactive conformations as first thought. It has subsequently been found through surface comparison of x-ray derived crystal structures of both active and inactive forms of specific kinases, that the assembly of a hydrophobic spine through sidechain interaction of discontiguous amino acids within the N-lobe, C-lobe and activation loop regions offers a better structural definition of kinase activation for all kinase families [8]. Figure 2 demonstrates the hydrophobic spine assembly of a representative kinase. Both active and inactive kinase conformations are able to bind ATP. However, only the active kinase confor‐ mation can perform the kinase function on substrates.

Protein kinases are important to many biological processes, as is evidenced by the more than 500 kinases encoded in the human genome. Kinases are involved in processes ranging from modulation of intracellular trafficking of proteins, to assembly of complicated multiprotein complexes, to gene expression. Kinase modulation of gene expression through signal trans‐ duction is of particular interest for cancer therapy.

The underlying cause of cancer is genomic instability. The emergence of a tumor cell typically requires multiple mutations in the genome that both activate oncogenes and inactivate tumor suppressor genes. These mutations synergize to transform a cell to a state of uncontrolled growth, proliferation, and enhanced survival. Mutations and/or epigenetic alterations that increase expression of, or produce constitutive active forms of some kinase proteins are known to drive the proliferation and/or survival of some tumors [9]. The types of kinases that are often mutated in tumor cells, although not exclusively, are found in signal transduction pathways. These pathways are kinase cascades that transmit extracellular signals to the nucleus and ultimately affect gene expression patterns. When a kinase in such a pathway is mutated where it is constitutively active, the extracellular signal is no longer needed and the pathway is always active and not regulated. Alternatively, epigenetic alterations or an increase in copy number of a protein kinase gene may cause overexpression of the kinase to increase activation of a signaling pathway. For example, a significant portion of non-small cell lung cancers (NSCLC) over express or have EGFR mutations. This causes over active signaling through one or more of the Ras/Raf/ERK, PI3K/AKT/mTOR, and STAT pathways that lead to increased growth, proliferation, motility and survival of the tumor cells.

The KIT receptor tyrosine kinase, or stem cell factor receptor, is a good example for represent‐ ing the different types of mutations that occur in kinases that contribute to the development of cancer. KIT possesses an extracellular domain, a juxtamembrane domain, and a kinase domain. Point mutations have been observed to occur in all domains of KIT [10]. The most common mutations occur in the juxtamembrane domain followed by the extracellular domain, and finally the kinase domain. Within the kinase domain mutations occur within the ATP binding site and the activation loop. Multiple muatations may occur within a tumor cell. All of these mutations produce constitutive active KIT. For example, juxtamembrane mutations result in the disruption of the autoinhibited form of the kinase, which results in a similar conformation to the phosphorylated activated kinase [11] Additionally, a KIT isoform with a GNNK insert in the extracellular domain has been identified that, once phosphorylated remains so for longer than the isoform that does not contain this insert, increases survival of myeloma cells [12, 13]. Overexpression of wildtype KIT may also drive tumor development. Overexpression may be due to an increase in gene copy number and/or hypomethylation of the gene promoter region.

The link between the development of some tumors, and aberrant modulation of certain protein kinases-either wildtype of mutant- has been established. Some of these aberrations, such as the BCR-Abl protein, represent driver mutations that have lead to the successful translation of experimental protein kinase inhibitors into the clinic for therapeutic treatment of cancer patients.
