As outlined above, kinases have emerged as interesting targets to tackle diseases dependent on the function of these proteins. In light of personalized medicine, specific targeting of kinases has moved in the focus of research supporting conventional chemotherapy34. The arsenal of molecular targeting techniques comprises monoclonal antibodies, small molecule inhibitors, peptide mimetics or gene therapy with antisense oligonucleotides35. Antibodies interact with cancer-specific proteins expressed on the cell surface, interfere with ligand-receptor interactions and activate the immune system. Targets of antibodies currently approved include HER2, VEGFR, TNFα and antigens (CD20, CD25, CD33, CD52). Whereas antibodies are large biomolecules (around 150 kDa) and are administered intravenously, small molecule inhibitors are small synthetic chemicals (around 500 Da) and can be taken orally. The latter can not only target proteins on the cell surface but also interact with kinases inside the cell and further downstream of signaling cascades by interrupting signal transduction36. The following work will focus on small molecule kinase inhibitors, which will therefore be described in more detail.
1.2.1 Small molecule kinase inhibitors in clinical trials
The first medicinal chemistry efforts in the development of small molecule inhibitors were based on lead compounds like the natural molecule Staurosporine and synthetic tyrphostins37, 38. Fasudil, targeting Rho-kinase, was the first inhibitor approved in Japan in 1995 for treatment of cerebral vasospasm39. Four years later, the allosteric inhibitor Rapamycin, was approved for immunosuppression after organ transplants40. It targets the protein kinase mTOR (mammalian target of Rapamycin), which was discovered in 199341 and is a component of the PI3K/mTOR pathway resulting in protein translation.
In 2001, Imatinib (Gleevec, STI-571) was approved for inhibition of BCR-ABL positive chronic myeloid leukemia (CML). It was the first rational, target-based kinase inhibitor and has been very successful since then42. Besides BCR-ABL, also KIT and PDGFR are inhibited by the drug43. This polypharmacology of Imatinib led to application in gastro-intestinal stromal tumors (GIST), hyper-eosinophilic syndrome (HES) and other indications44. CML and GIST have been fatal diagnoses before the use of Imatinib, but application of the drug turned them into manageable diseases. This success encouraged pharmaceutical companies to invest more into the design of protein and lipid kinase inhibitors in light of targeted therapies45, 46. Around 50-70% of today’s cancer drug discovery efforts concentrate on protein kinase inhibitors47. The timeline in Figure 3 showing the FDA approval of small molecule kinase inhibitors is representative for the clinical success of these molecules48. In 2015, another three small molecule inhibitors received FDA approval, namely Alectinib, Osimertinib and Cobimetinib49. Currently, 37 inhibitors are approved worldwide;
Imatinib, Nilotinib, Dasatinib, Bosutinib and Ponatinib are indicated for CML, but only Ponatinib can target the T315I Imatinib resistant gatekeeper mutation50. Lapatinib and Palbociclib are used for the treatment of HER2 positive breast cancer, Ibrutinib and Idelalisib for various types of blood cancers. Gefitinib, Erlotinib, Icotinib, Afatinib and Osimertinib are applied in non-small cell lung cancer (NSCLC) with activating EGFR mutations, whereas ALK-translocations in NSCLC are treated with Ceritinib, Crizotinib or Alectinib. Sorafenib, Sunitinib, Everolimus, Temsirolimus, Axitinib and Pazopanib are used in renal cell cancer. Sunitinib and Imatinib as well as Regorafenib can also be applied in GIST.
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Figure 3: Approved small molecule inhibitors. Timeline for FDA approval of small molecule inhibitors (adapted from48). The increasing number of approved molecules in recent years indicates clinical success of these molecules.
Cabozantinib, Lenvatinib and Vandetanib treat medullary thyroid carcinoma. BRAF(V600E) mutated metastatic melanoma can be managed with either Vemurafenib or Dabrafenib alone or in combination with the MEK-inhibitors Trametinib or Cobimetinib. Besides oncology, kinase inhibitors can also be applied in other diseases; Tofacitinib is approved for rheumatoid arthritis, Ruxolitinib for myeloid fibrosis, Rapamycin as immunosuppressive in organ transplants, Fasudil for cerebral vasospasm and Nintedanib for idiopathic pulmonary fibrosis. Masitinib is a KIT inhibitor with orphan drug status in Europe for pancreatic cancer in human and mast-cell tumors in dogs48,
49. Additionally, more than 300 small molecules are tested in clinical trials for various indications nowadays, ranging from oncology to transplantation or infectious diseases (www.clinicaltrials.gov)47, 49, 51.
1.2.2 Kinase binding of small molecule inhibitors
With a few exceptions - MEK-inhibitors and analogues of Rapamycin (rapalogs) - most of the inhibitors described above target the structurally conserved ATP-binding pocket. They interact with amino acids in the hinge region, simulating the hydrogen bond, which is formed by the adenine ring upon ATP binding52, 53. Small molecule kinase inhibitors can be categorized akin to their mode of binding to their kinase target (Figure 4a, reviewed in48, 49, 54). The following classification scheme is based on Roskoski55. Kinase inhibitors binding to the active conformation of a kinase are so-called type 1 inhibitors. In the active conformation, the D of the DFG-motif points into the ATP binding pocket (‘DFG-in’). Selectivity is achieved by variation of shape and size of the inhibitor and interactions with the gatekeeper residue at the entrance of the ATP-binding pocket as well as non-conserved residues at the solvent exposed sites (Erlotinib, Figure 4b)56. A subtype are the type 1.5 inhibitors, the kinase is also fixed in a ‘DFG-in’ position but the αC-helix is pointing outwards (C-helix-out). This is the case for BRAF bound by Vemurafenib and has also been found in other kinases (Vemurafenib, Figure 4b)7, 14. Inhibitors locking the kinase in its inactive state, with the DFG-motif pointing outwards, are termed type 2 inhibitors. The ‘DFG-out’ conformation exposes a hydrophobic pocket next to the ATP-binding site, which can be targeted by these type 2 molecules (Sorafenib, Figure 4b). Furthermore, C-helix, activation and P-loop are also more flexible in the inactive conformation, which therefore can vary between kinases. This is why type 2 inhibitors seem to be more selective for a specific kinase than the type 1 inhibitors binding the more conserved active conformation7, 57.
Other molecules bind the kinase non-ATP competitively on an allosteric site. Inhibitors in clinical trials of the type 3 binding mode are mainly MEK1/2 inhibitors. These drugs exploit a unique binding pocket adjacent to the ATP-binding site, only present in these proteins. Inhibitor binding leads to
7 conformational changes and blocks the kinase in its inactive state (i.e. TAK-733, Figure 4b)58. Inhibitors binding to the substrate-binding site or other motifs along the kinase are called type 4 inhibitors (MK-2206, in Figure 4b) or type 5 inhibitors if exploiting two different binding sites along the kinase.
Figure 4: Kinase inhibitor binding modes. a) Overview of possible small molecule binding types to kinases (modified from). b) Example kinase inhibitors for different binding modes. Chemical moieties targeting the adenine, hydrophobic, allosteric or MEK-selective pocket are highlighted. Arrows indicate direct kinase-drug interactions. Erlotinib is an example for type 1 EGFR binding; Vemurafenib inhibits BRAF in DGF-in helix αC out conformation. Afatinib irreversibly interacts with Cys797 of EGFR after Michael-addition with the double bound. Sorafenib binds to VEGFR in type 2 binding mode whereas TAK-733 exploits the MEK selective pocket for binding. MK-2206 is an example for an allosteric inhibitor targeting the pleckstrin homolog domain (modified after48).
Some inhibitors (e.g. Afatinib, Ibrutinib, Osimertinib) can bind to their target covalently (sometimes referred to as type 6 inhibitors). They bind to a lysine or cysteine, either in the ATP binding pocket or in close proximity to it (Afatinib, Figure 4b). Most irreversible inhibitors in clinical evaluation today are targeting a cysteine in the active center of EGFR and BTK59. They feature a Michael acceptor site in their structure. Ibrutinib, Afatinib and Osimertinib are approved by the FDA.
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1.2.3 Evaluating selectivity and affinity of small molecules
As the ATP-binding pocket is structurally conserved, these ‘ATP-mimetics’ potentially interact with more than one kinase. This promiscuity might lead to toxicity60 or can be used for repositioning of a drug in another disease setting, pioneered by the use of Imatinib for GIST (see above). The hitherto known target landscape of small molecule kinase inhibitors revealed that only about 80 protein and lipid kinases can be successfully targeted61. These inhibitors are mainly indicated for oncology and there are several inhibitors against one target in one indication54.
The selectivity and efficacy of drugs is often influenced by their binding affinity towards a target.
Common measures for ligand-receptor interactions include the half maximal inhibitory concentration (IC50), the half maximal effective concentration (EC50), the inhibition constant (Ki) and the equilibrium dissociation constant (Kd). The IC50 value describes the concentration that leads to 50% inhibition and is dependent on factors like substrate concentration, target accessibility, duration of incubation, or cell permeability62. EC50 describes the drug concentration needed to achieve 50% of the maximum effect. For inhibitors, this value is halfway between the baseline and maximum of a measured effect. Cheng and Prusoff introduced the inhibition constant (Ki), which describes the inhibitor concentration at 50% inhibition63. This constant is an absolute value for any inhibitor-protein combination, whereas the IC50 can vary between experiments. Ki can be calculated as shown in equation (1). [S] refers to the concentration of substrate and the Michaelis-Menten constant Km is the substrate concentration (for kinase inhibitors ATP) at which velocity of the
For competitive inhibitors, Ki equals Kd of the kinase-inhibitor complex. However, affinity is not the only parameter for assessing effectiveness. Another important parameter is residence time, the time how long a receptor-ligand complex exists. It is affected by the association (kon) and dissociation (koff) rates. Especially the koff rate influences this time. In closed systems (used in the laboratory to investigate drug-target interactions), affinity is determined in equilibrium. Here, kon
can often be neglected and thus, Kd correlates strongly with koff (with Kd=kon/koff). In open systems like the human body, the pharmacodynamics of a drug (efficacy and duration of efficacy) are subject to drug distribution, drug absorption and metabolism in the body (pharmacokinetics). Here, the actual residence time of a drug-protein complex is an important factor to be considered. Moreover, Kd and residence time also influence drug selectivity and thus off-target toxicity. Higher affinity of a drug towards its intended target and longer residence time at this target result in better safety characteristics as less drug can be used, whereas long residence time at an off-target might lead to toxicity64, 65. Upon irreversible binding, residence time is very high. In this binding mode, inhibitor potency is dependent on reversible binding first (Ki) and then on the efficiency of the covalent bond formation (Kinact, rate of inactivation). The IC50 of irreversible inhibitors decreases over time, because maximal inhibition might only be reached after longer incubation time66. The targeted proteins need to be expressed de novo in order to perform their function again. Hence, also lower amounts of drug are sufficient to achieve a pharmacological effect67.
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