Cancer Biology and the Principles of Targeted Cancer Drug Discovery 13

researchers knocked out the various CDKs showed that CDK2, CDK4, and CDK6 are dispensable for the cell cycle of most normal cell
types, while CDK1 is not. 92 As CDK1 appears to be essential for the division of normal cells, inhibiting CDK1 in cancer shows a much
narrower therapeutic window with the same type of toxicities as traditional chemotherapy. In contrast, CDK4/CDK6 inhibitors may show
a better therapeutic window as certain cancers may be addicted to CDK4/CDK6 activity and therefore will respond to these agents even
though most normal cells may not require them for proliferation. Therefore, some of the early pan-CDK inhibitors have been
fi
discontinued.91,93 At present, P zer (palbociclib), Lilly (abemaciclib), and Novartis (LEE011) are developing selective CDK4/CDK6 inhibitors
that appear to be less toxic and have shown strong progression-free survival (PFS) in ER-positive breast
cancer.91,94

5.01.5.1.5 Approved kinase inhibitors

Since the approval of fasudil in 1995, sirolimus in 1999, and imatinib in 2001, the number of approved kinase inhibitors has increased to
36 with many others still in preclinical development (Table 1). These inhibitors are mainly for oncological indications and are directed
against only a handful of protein and lipid kinases, leaving 70% of the kinome untapped ( Table 1). Despite these successes in kinase drug
discovery, the development of kinase inhibitors with outstanding selectivity, the identification and validation of driver kinase(s) in
diseases, and the emerging problem of resistance to the inhibition of key target kinases remain major challenges. Kinase inhibitors with
outstanding selectivity are likely to become important not only for minimizing side effects and allowing chronic treatment of non-life-
threatening diseases.76,95
Most of the approved kinase drugs are active against more than one type of cancer. Only few of them have been used for the
treatment of non-oncological indications (tofacitinib for rheumatoid arthritis, nintedanib for idiopathic pulmonary fibrosis, everolimus
for organ rejection of heart and kidney transplants, and fasudil for cerebral vasospasm; Table 1). On the other hand, there are multiple
kinase drugs for one single indication. For example, imatinib, nilotinib, dasatinib, bosutinib, and ponatinib have all been approved for
CML (chronic myeloid leukemia), while sorafenib, sunitinib, everolimus, temsirolimus, axitinib, and pazopanib are indicated for various
stages of renal cell cancer. Ceritinib and crizotinib are indicated for non-small cell lung cancer (NSCLC) with ALK translocations, while
gefitinib, erlotinib, afatinib, and osimertinib are indicated for NSCLC with activated EGFR. Finally, vemurafenib alone or the combination
of dabrafenib with trametinib is indicated for metastatic melanoma with BRAF V600 mutations (Table 1).

5.01.5.1.6 Kinase inhibitor mode of action

With a few exceptions, such as the rapalogs (sirolimus, everolimus, and temsirolimus), trametinib, or cobimetinib, all of the
smallmolecular-weight kinase inhibitors are directed toward the ATP binding site (see Table 1). The conservation of the ATP binding in
the human kinome causes these “ATP mimics” to often cross-react with many other different kinases resulting in compounds with
promiscuous profiles, for example, dasatinib96 and sunitinib.97
Kinase inhibitors show different modes of binding to kinases. In principle, one can divide kinase inhibitors into those that bind
covalently or reversibly to the kinase.83,95 ATP-competitive inhibitors bind to the hinge region of the kinase 79,81,95,98; allosteric kinase
inhibitors are non-ATP-competitive and do not have physical contact to the hinge. As they exploit binding sites and regulatory
mechanisms that are unique to a particular kinase, they, therefore, show the highest degree of selectivity. 79,81,95,98 Similarly, agents
targeting the extracellular domains of RTKs act outside the kinase domain. These include peptide mimetics, “peptoids” or antibodies, 99 or
small molecules like SSR128129E (Sanofi), which targets the extracellular domains of the fibroblast growth factor receptor (FGFR). 100
Targeting the allosteric sites of protein kinases may also lead to the identification of activators rather than inhibitors, which could be
useful for therapeutic intervention or as pharmacological tools. There are a few protein kinases that require activation rather than
inhibition to fulfill their therapeutic task as in the case of 5 0-adenosine monophosphate-activated protein kinase (AMPK) and the insulin
receptor for which activators have been identified. 101 Protein kinase C (PKC) activation by exogenous compounds acting at the
diaclyglycerol binding site can have tumor-promoting or tumor-suppressive effects. 102 Another example of kinase activators includes a
mimetic of the brain-derived neurotrophic factor that activates tropomyosin receptor kinase B or neurotrophin receptor kinase 2. 103 In
some cases, kinase inhibitors can lead to unintended paradoxical activation either directly or via modulation of feedback loops. The most
striking example is the paradoxical effect of BRAF inhibitors, which can activate the MAPK pathway in certain genetic backgrounds
resulting in the promotion of hyperproliferative toxicities. 104 There is currently no general strategy for the identification of allosteric
kinase inhibitors or activators as most of them have been discovered serendipitously by diverse approaches ranging from phenotypic
screenings to highly sophisticated structure-based drug design.

5.01.5.1.7 Kinase mutations and resistance

Deregulation of protein kinase activity by gain of function mutations is essential to maintain the oncogenic state, and in many instances,
these mutations can be linked to specific cancers. 84 Inhibitors are being and have been designed to specifically target kinase alleles with
fi
gain of functions.74,105 Despite these successes, it should be emphasized that patients who bene t from these kinase inhibitors often
relapse after an initial response (see section “Challenges of Targeted Cancer Therapy”). Multiple mechanisms of resistance have been
and are being elucidated to improve the efficacy of these types of targeted therapies. Drug resistance to kinase inhibitors can occur
 fi
either by compensatory mechanism or by mutations reducing the af nity of the kinase to its inhibitors. 76,106 Approaches to overcome
resistances include the development of inhibitors that can tolerate mutations, that bind covalently to their target, or that bind to
allosteric sites. Another option is targeting another protein in the same pathway or targeting alternate, activated pathways in
combination with the first inhibitor used.96,107
14 Cancer Biology and the Principles of Targeted Cancer Drug Discovery

Table 1 Approved kinase inhibitors as of Dec. 2015

Year Generic name (compound code, Kinase target Disease Company (year, type)
trade names)

1995 Fasudil (HA-1077) [5181] ROCK1/2 Cerebral vasospam, PAH Asahi Kasei (1995, type 1)
1999 Sirolimus (Rapamune) [6031] mTOR Kidney transplants Pfizer, Wyeth (1999, type 3)
2001 Imatinib (STI571, Glivec, Gleevec) ABL, PDGFR, KIT CML, PhGIST þB-ALL, CMML, Novartis (2001, type 2)
[5687] HES,
2003 Gefitinib (ZD1839, Iressa) [4941] EGFR NSCLC AZ (2003, type 1)
2004 Erlotinib (OSI-774, Tarceva) [4920] EGFR NSCLC, pancreatic cancer Roche, OSI (2004, type 1)
2005 Sorafenib (BAY 43–9006, Nexavar) VEGFR2, PDGFR, KIT, RCC, HCC Bayer, Onyx (2005, type 2)
[5711] FLT3, BRAF
2006 Sunitinib (SU11248, Sutent) [5713] VEGFR, KIT, PDGFR, RCC, imatinib-resistant GIST Pfizer (2006, type 1)
RET, CSF1R, FLT3
2007 Lapatinib (GW2016, Tykerb) [5692] EGFR, ERBB2 BC GSK (2007, type 1.5)
2007 Dasatinib (BMS-354825, Sprycel) ABL, PDGFR, KIT, SRC CML BMS (2007, type 1)
[5678]
2007 Nilotinib (AMN107, Tasigna) [5697] ABL, PDGFR, KIT CML Novartis (2007, type 2)
2009 Everolimus (Rad001, Certican, Zortress, mTOR RCC, SEGA, transplantation Novartis (2009, type 3)
Afinitor, Votubia) [5889]
2009 Temsirolimus (CCI-779, Torisel) [5892] mTOR RCC Pfizer, Wyeth (2009, type 3)
2011 Crizotinib (PF-02341066, Xalkori) MET and ALK NSCLC with ALK translocations Pfizer (2011, type 1)
[4903]
2011 Vandetanib (ZD6474, Caprelsa) RET, VEGFR1-2, FGFR, MTC AZ (2011, type 1)
[5717] EGFR
2011 Ruxolitinib (INC424, Jakafi) [5688] JAK2 IMF with JAK2V617F mutations Novartis, Incyte (2011, type 1)
2011 Vemurafenib (PLX4032, RG7204, BRAF Metastatic melanoma with Roche, Plexxikon (2011, type 2)
Zelboraf) [5893] BRAFV600E mutations
2011 Axitinib (AG013736, Inlyta) [5659] VEGFR, KIT, PDGFR, RCC Pfizer (2012, type 1)
RET, CSF1R, FLT3
2012 Regorafenib (BAY 73-4506, Stivarga) VEGFR2, Tie2 CRC, GIST Bayer (2012, type 2)
[5891]
2012 Pazopanib (GW-786034, Votrient) VEGFR, PDGFR, KIT RCC GSK (2012, type 1)
[5698]
2012 Tofacitinib (CP-690550, Xeljanz, JAK3 RA Pfizer (2012, type 1)
tasocitinib) [5677]
2012 Cabozantinib (XL184, BMS-907351, VEGFR2, PDGFR, KIT, MTC Exelexis (2012, type 1)
Cometriq) [5887] FLT3
2012 Ponatinib (AP24534, Iclusig) [5890] ABL Imatinib-resistant CML with Ariad (2012, type 1)
T315I mutations
2012 Bosutinib (SKI-606, Bosulif) [5710] ABL CML resistant/intolerant to Pfizer (2012, type 1)
therapy
2013 Dabrafenib (Tafinlar) [6494] BRAF Metastatic melanoma with GSK (2013, type 2)
BRAFV600E mutations
2013 Trametinib (Mekinist) [6495] MEK Metastatic melanoma with GSK (2013, type 3)
BRAFV600E mutations
2013 Afatinib (Gilotrif, Tomtovok, Tovok) EGFR NSCLC with EGFR-activating BI (2013, covalent)
[5667] mutations
2013 Ibrutinib (PCI-32765, Imbruvica) BTK MCL, CLL Janssen, Pharmacyclic (2013,
[6912] covalent)
2014 Ceritinib (LDK378, Zykadia) [7397] ALK NSCLC with ALK translocations Novartis (2014, type 1)
2014 Idelalisib (CAL101, GS1101, Zydelig) PI3Kdelta CLL, FL, and SLL Gilead, Calistoga, ICOS (2014,
[6741] type 1)
2014 Nintedanib (BIBF 1120, Vargatef, VEGFR, PDGFR, FGFR Idiopathic pulmonary fibrosis BI (2014, type 1)
Intedanib) [5936]
 2014 Alectinib (AF802, Aliens) [7739] ALK ALK-rearranged NSCLC (brain Roche, Chugai (2014, type 1)
mets) appr.
in Japan
2015 Palbociclib (PD-0332991, Ibrance) CDK4/6 Advanced (metastatic) BC Pfizer (2015, type 1)
[7380]
2015 Lenvatinib (E7080, Lenvima) [7426] VEGFRs multikinase Thyroid cancer (DTC); kidney Eisai Co (2015, type 1)
cancer
2015 Cobimetinib (GDC-0973, XL-518, MEK Melanoma in combination Roche, Exelexis (2015, type 3)
Cotellic) with vemurafenib
Cancer Biology and the Principles of Targeted Cancer Drug Discovery 15

Table 1 Approved kinase inhibitors as of Dec. 2015dcont'd

Generic name (compound code, trade
names)
Year Kinase target Disease Company (year, type)
2012 Radotinib (Supect) BCR-ABL, PDGFR CML Ilyang, Daewoong Pharmaceutical
(2015, type 2). Approved in South
Korea only
2015 Osimertinib (mereletinib, AZD9291; EGFR (T790M) NSCLC with EGFR-activating AZ (2015, covalent)
Tagrisso) mutations or T790M
CML, chronic myeloid leukemia; PhþB-ALL, Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia;NSCLC, non-small cell lung cancer; RCC, renal clear
cell carcinoma;CMML, chronic monomyelocytic leukemia;HCC, hepatocellular carcinoma;HES, hypereosinophilic syndrome; GIST, gastrointestinal stromal tumor;
BC, breast cancer; SEGA, subependymal giant cell astrocytoma; MTC, medullary thyroid cancer; IMF, interstitial myelofibrosis; CRC, colorectal cancer; RA, rheumatoid
arthritis; MCL, mantle cell lymphoma; CLL, chronic lymphocytic leukemia; FL, follicular lymphoma; SLL, small lymphocytic leukemia; PAH, pulmonary arterial
hypertension; Rock, Rho-associated protein kinase; RET RTK, rearranged during transfection RTK; CSF1R, colony-stimulating factor 1 receptor; JAK, Janus kinase;
VEGFR, vascular endothelial growth factor receptor; mTOR, mammalian target of rapamycin; SRC, proto-oncogene tyrosine-protein kinase Src; BTK, Bruton’s tyrosine
kinase; ABL, Abelson kinase; BCR, breakpoint cluster region; PDGFR, platelet-derived growth factor receptor; FLT3, fetal liver kinase 3; ALK, anaplastic lymphoma
receptor tyrosine kinase; MET, MET receptor¼hepatocyte growth factor receptor (HGFR); MEK, MAP2K, mitogen-activated protein kinase kinase; FGFR, fibroblast
growth factor receptor.
The 36 kinase inhibitors approved to date are shown with generic name, compound code, trade name, primary indications, year of approval, company, and mode of
binding. GSK, Glaxo Wellcome; AZ, AstraZeneca; BI, Boehringer Ingelheim. The chemical structures and biochemical profiles of the 36 approved kinase inhibitors can
be viewed in the IUPHAR database (http://www.guidetopharmacology.org/GRAC/LigandListForward?type¼Approved&database¼all).

5.01.5.1.8 Outlook
The future of protein kinase-targeted therapeutics in cancer appears promising, despite the fact that several protein kinase inhibitors
that have entered human clinical trials are not very specific and did not achieve the anticipated results. This situation may be improved
by the upcoming second generation of kinase inhibitors with a better selectivity that will be applied to a genetically better defined
patient population. The development of kinase inhibitors for non-life-threatening indications where chronic regimens are being used will
require a priori a better target selectivity to minimize side effects. Identification of highly selective kinase inhibitors and activators should
lead to an expansion of the chemical and biological kinase space as well as to an improved understanding of their therapeutic limitations
and potentials.
Reversible, noncovalent kinase inhibitors can be classified as type 1, type 2, and type 3 reversible kinase inhibitors. 98 Type 1 inhibitors
fulfill all the criteria of an ATP mimetic by binding to the hinge and targeting an active conformation of the kinase (often referred to as
DFG-in). The type 1.5 inhibitor is a subtype of the type 1 inhibitor that binds to an inactive kinase conformation (DFG-in conformation,
typical of an active kinase, but the C-helix is pushed out). Type 2 inhibitors bind to the inactive, so-called DFG-out conformation,
maintaining contacts to the hinge region and usually displaying an ATP-competitive behavior similar to type 1 inhibitors. The transition of
the active DFG-in to the inactive DFG-out conformation exposes an additional hydrophobic pocket adjacent to the ATP site that is used by
type 2 inhibitors locking the kinase in the inactive conformation. Type 3 inhibitors are non-ATP-competitive (allosteric) kinase inhibitors
that have no physical contact to the hinge. As they exploit binding sites and regulatory mechanisms that are unique to a particular
kinase, they, therefore, show the highest degree of selectivity. Type 3 inhibitors can bind either to the kinase domain (close to or
removed from the ATP site) or to sites that are located outside of the kinase domain (rapamycins).

5.01.5.2 Targets in Induction of Apoptosis

Programmed cell death, apoptosis, is a tightly regulated suicide program that plays a critical role in maintaining the survival/ death
balance. It is part of the machinery that eliminates cells with disturbed function. Impaired apoptosis can lead to the development of
cancers or autoimmunity; increased apoptosis can induce degenerative disease. 108 DNA-damaged cells that do not undergo apoptosis can
form cancerous lesions. Therefore, cancer cells frequently exhibit apoptotic defects resulting not only in oncogenicity but also in
resistance to cytotoxic cancer treatments that induce apoptosis as a mode of action. 109 Affected cells survive beyond their intended life
span and are protected from oxidative stress and hypoxiadboth common features of solid tumors.
Apoptosis is initiated by different pathways. The so-called extrinsic (death receptor-dependent) or the intrinsic (mitochondrial)
pathway is the most relevant in the cancer context. The intrinsic and extrinsic pathways activate upstream caspases, caspase 9 and
caspase 8, respectively, and converge further downstream in the caspase activation cascade. 110 Caspases are intracellular proteases that
cleave key cellular components and activate degrading enzymes (Fig. 3).108,109,111
Activation of the extrinsic pathway is triggered by cytokine ligands binding to death receptors of the tumor necrosis factor (TNF)
family. An intracellular binding site recruits adaptor proteins like procaspases to form a death-inducing signaling complex that triggers
caspase activation,112 which initiates apoptosis. Tumor necrosis factor a (TNFa), Fas ligand (FASL), and tumor necrosis factorrelated
apoptosis-inducing ligand (TRAIL) are the most prominent death receptor ligands.