Kinases as drug targets

1 Introduction

1.1 Kinases as drug targets 1.1.1 The human kinome

All living organisms rely on manifold biological processes that are simultaneously or subsequently active. These processes are mainly carried out by proteins, which react to external and internal stimuli, catalyze cellular reactions and regulate development, growth, division, and death of a cell.

One important group of proteins involved in these processes is protein kinase family. They are organized in signaling cascades and responsible for the transmission of external stimuli from the cell membrane anchored receptor tyrosine kinases to substrate kinases eventually resulting in gene transcription, cell division, cell growth or apoptosis (as exemplarily shown for EGFR, Figure 1a). This signal transmission is characterized by a tight interplay of phosphorylation and dephosphorylation events. Kinases catalyze the transfer of the γ-phosphate group of adenosine triphosphate (ATP) to a substrate, which activates this protein. The phosphate group can again be removed by phosphatases resulting in a reversible control mechanism (Figure 1b). This tightly controlled process allows the cell to alter enzyme activity and react to external and internal changes^{1, 2}. Its discovery by Edmond Fischer and Edwin Krebs was awarded with the Nobel Prize in Physiology or Medicine.

Figure 1: The human kinome. a) Schematic of EGFR-signaling cascade. b) Schematic representation of protein phosphorylation by kinases and dephosphorylation by phosphatases. c) Phylogenetic tree of human kinases (Courtesy of Cell Signaling Technologies).

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To date, 518 protein kinases have been identified in man, also referred to as the human kinome. It represents almost 2% of the known genome and, therefore, is one of the largest gene families encoded by eukaryotes. Protein kinases can be classified according to the sequence of their catalytic center, the kinase domains. This is often represented as phylogenetic tree (Figure 1c). The major groups are tyrosine kinases (TK, divided into receptor TK and non-receptor TK), ‘tyrosine kinase like’

kinases (TKL), sterile 20 kinases (STE), AGC family containing PKA, PKG and PKC, calcium/calmodulin dependent kinases (CAMK), casein kinases 1 (CK1) and a group comprised of cyclin dependent kinases, MAP kinases, glycogen synthase kinase and casein kinase 2 (CMGC). Kinases that cannot be grouped into these major groups are attributed to the so-called ‘other’ group. The kinome is completed by atypical kinases that do not share sequence similarity with the kinases in the major groups but have shown protein kinase activity^3-6. Besides tyrosine, most kinases phosphorylate a serine or threonine⁴. Roughly 10% of all kinases are non-catalytic (pseudo-kinases). They can still bind ATP and may execute important regulatory functions like scaffolding or allosteric regulation of kinases^7-9. Apart from protein kinases, phosphatidylinositol kinases (PI) play central roles in signaling pathways. They phosphorylate lipids in the cell membrane, which then again recruit protein kinases.

1.1.2 Structural insights into the kinase domain

A kinase has to be structurally flexible allowing simultaneous binding of both protein substrate and ATP to exert its catalytic function. During the phosphate group transfer, the kinase domain changes its conformation, which has been investigated in crystallization studies by several groups (Figure 2)^10-12.

The protein kinase domain consists of two structurally and functionally different lobes, the N- and C- lobe. Both lobes are connected via a hinge region, which forms a cleft - the active site (Figure 2a, b). Here, ATP or other nucleotides can be bound, hydrolyzed and released again.

The motif between the first two β-strands of the N-lobe is called glycine-rich loop (Gly-rich loop). It can fold over ATP and positions the phosphate group for catalysis (see top left of Figure 2b). The AxKmotif in the β3strand pairs the ATPphosphates to the αChelix. The Nterminus of the αC -helix is connected with the activation loop, therefore the positioning of the αC --helix is a crucial step for the activation of a kinase and its catalytic activity¹³. The C-lobe mainly consists of helices and acts as anchoring surface for protein or peptide substrates. Its beta-subdomain comprises motifs necessary for the catalytic transfer of the phosphate group from ATP to the substrate. This includes the magnesium-binding loop (at the beginning of the activation loop) containing the DFG-motif, a conserved sequence of aspartate (D), phenylalanine (F) and glycine (G). The aspartate (D) interacts with all three ATP phosphates by polar interactions or through coordinating atoms of magnesium.

The phenylalanine (F) contacts the αC-helix and the conserved HRD-motif (histidine, arginine, aspartate) of the catalytic loop and thereby alters the position of the DFG-motif. The glycine (G) acts as ‘bipositional switch’ between inactive and active conformation and leads to proper positioning of the aspartate¹¹. The activation loop is interrupted by the β9-strand, forming an antiparallel β-sheet with the β6-strand in the catalytic loop. Within inactive kinases, this formation is often disordered and therefore considered important for proper magnesium-binding loop configuration. Most protein kinases are activated by phosphorylation of a residue in the activation loop (P-loop), the most variable and diverse part of a kinase, leading to rearrangement of the loop and an increase in enzymatic activity.

3 Figure 2: Structure of the kinase domain. a) Ribbon view on the catalytic center of the kinase highlighting the relevant residues and structures (from ¹⁰). b) Schematic of kinase domain with ATP in the active conformation (from ¹¹). c) Conformational changes in active or inactive conformation as well as the SRC/CDK like inactive DFG-in, helix αC out conformation (from ⁹).

In the active, so-called ‘DFG-in’ conformation, the D coordinates with magnesium and the F points in the direction of the αC-helix. In this position, the αC-helix can then interact with the ß3-strand.

When D and F switch positions, the bulky phenyl ring of the F prevents binding of the nucleotide and induces conformational changes in the activation loop. This is referred to as ‘DFG-out’ or the inactive conformation. Besides the ‘DFG-out’ inactive conformation, another inactive conformation exists. Here, the αC-helix is turned away from the lysine, with the DFG-motif still pointing inwards.

The activation loop then rearranges in a short helix. This inactive state has been discovered for SRC and CDKs and has been found for other kinases as well (Figure 2c)^{7, 9, 14}.

1.1.3 Kinases in disease

Miss-regulation of protein kinases has been found to be involved in various diseases like cancer, immunological, neurological, metabolic and infectious disorders because of their key function in cellular signaling^{15, 16}. Pioneering studies by Collet and Erikson, who found that the rous sarcoma virus transforming factor was a protein kinase¹⁷, as well as the discovery of tumor-promoting phorbol esters as activators of protein kinase C by Castagna and colleagues¹⁸ implicated an important role of kinase activity in tumor biology. The role of kinases in the development of cancer is also reflected in the hallmarks of cancer. They are directly involved in sustained proliferative signaling, evasion of growth suppression, induction of angiogenesis as well as invasion and metastasis^{19, 20}. Looking at the distribution of drugs across various protein families, protein kinases have become the second major target class after G-protein coupled receptors²¹.

Kinase inhibition directly interferes with the signaling cascade and, thus, can lead to a real physiological response. Targeting kinase deregulation in cancer can be divided into three groups depending on their involvement in cellular pathways. In the first group, the kinases have undergone genetic mutation or translocation and are therefore unaffected by normal cellular regulatory

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mechanisms. Often, they are constitutively active, which makes them indispensable for the survival and proliferation of a cell. This so-called oncogene addiction makes the cancer susceptible for appropriate kinase inhibitors^{22, 23}. Inhibition of the mutated kinase has direct impact on tumor survival. Well-known examples for this are BCR-ABL in chronic myeloid leukemia (CML)²⁴, EGFR and/or ALK mutations in lung cancer^{25, 26}, BRAF(V600E) in malignant melanoma²⁷, or PIK3CA in various cancers²⁸. These mutations can be easily identified by DNA sequencing²⁹ and represent addressable targets as inhibition prevents oncogenic signaling^{22, 23}.

The next group consists of kinases, which are essential for cell survival and/or proliferation. Usually, these kinases are downstream of the oncogenic kinases mentioned before. Examples here are MEK1/2 (MAP2K1, MAP2K2), mTOR, RPS6K, CDKs, Aurora kinases or PLK. Inhibition of these kinases is synthetically lethal to the tumor in combination with a cancer-driving mutation³⁰.

The last group of kinases is relevant for tumor formation and interaction of cancer cells with the human host. Examples are VEGFR, FGFR or NTRK2. They can promote vessel growth towards the tumor³¹ and are required for metastasis development³². Targeting the two latter groups interferes with healthy cell signaling and has to be carefully evaluated in the specific disease background³³.

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