25 Years of Small Molecular Weight Kinase Inhibitors: Potentials and Limitations

Abstract

Deregulation of protein and lipid kinases of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. Protein kinase and lipid kinases represent, therefore, an important target for the pharmaceutical industry. In fact, approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases. To date 27 kinase inhibitors that have been approved, which, with few exceptions, are mainly for oncological indications and are directed against only a handful of protein and lipid kinases, leaving 70% of the kinome untapped. Despite of these successes the major challenges 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 resistance to the inhibition of key target kinases remain major challenges. This review will provide an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors and novel avenues for the generation of second generation kinase inhibitors to treat cancers.
The American Society for Pharmacology and Experimental Therapeutics.

Full text

1521-0111/87/5/766–775$25.00 http://dx.doi.org/10.1124/mol.114.095489
MOLECULAR PHARMACOLOGY Mol Pharmacol 87:766–775, May 2015
Copyright ª2015 by The American Society for Pharmacology and Experimental Therapeutics
MINIREVIEW
25 Years of Small Molecular Weight Kinase Inhibitors: Potentials
and Limitations
Doriano Fabbro
PIQUR Therapeutics AG, Basel, Switzerland
Received August 20, 2014; accepted December 30, 2014
ABSTRACT
Deregulation of protein and lipid kinase activities leads to a variety
of pathologies, ranging from cancer inflammatory diseases, dia-
betes, infectious diseases, and cardiovascular disorders. Protein
kinases and lipid kinases represent, therefore, an important target
for the pharmaceutical industry. In fact, approximately one-third of
all protein targets under investigation in the pharmaceutical in-
dustry are protein or lipid kinases. To date, 30 kinase inhibitors
have been approved, which, with few exceptions, are mainly for
oncological indications and directed against only a handful of
protein and lipid kinases, leaving 70% of the kinome untapped.
Despite these successes in kinase drug discovery, the develop-
ment of kinase inhibitors with outstanding selectivity, 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. This minireview provides an insight into protein
and lipid kinase drug discovery with respect to achievements,
binding modes of inhibitors, and novel avenues for the generation
of second-generation kinase inhibitors to treat cancers.
Introduction
Protein and lipid kinases represent, after GTP protein–
coupled receptors and proteases, one of the most important
target classes for treating human disorders. A set of divergent
kinases play essential roles in eukaryotic signaling. These
include various protein kinases like BRAF, ABL, ALK, as
well as kinases that are able to phosphorylate phosphatidyl-
inositol (PI) like PI3Ks and PI4Ks (Engelman, 2009; Courtney
et al., 2010). Many human malignancies are associated with
deregulated activation of protein or lipid kinases due to
mutations, chromosomal rearrangements, and/or gene am-
plification (Hanahan and Weinberg, 2000; Hunter, 2000;
Blume-Jensen and Hunter, 2001; Cohen, 2002; Hahn and
Weinberg, 2002; Weinstein, 2002; Bardelli et al., 2003; Levitzki,
2003; Vieth et al., 2004, 2005; Luo et al., 2009; Fedorov et al.,
2010; Fabbro et al., 2011).
The human kinome contains typical, atypical protein
kinases, as well as pseudo-kinases (Manning et al., 2002;
http://kinase.com/kinbase). The latter makes up 10% of human
protein kinases and are either inactive or only weakly active
due to the lack of at least one of three motifs in the catalytic
domain that are essential for catalysis (Boudeau et al., 2006;
Kannan and Taylor, 2008). Despite lacking the ability to
phosphorylate substrates, pseudo-kinases are still very impor-
tant in regulating diverse cellular processes, as indicated by the
regulation of LKB1 by STRAD and the activation of JAK2 by
V617F on its JH2 pseudo-kinase domain (Boudeau et al., 2006;
Kannan and Taylor, 2008).
Protein kinases have a bilobal architecture, with one N-terminal
lobe with mainly b-sheets and one C-terminal domain with
ahelices. The two lobes are connected by the hinge region,
which lines the ATP-binding site, which is targeted by the
majority of small molecular weight kinase inhibitors (Cohen,
2002; Levitzki, 2003; Vieth et al., 2004, 2005; Cowan-Jacob,
2006; Fabbro et al., 2011). In past decades, 29 small-molecule
kinase inhibitors have been approved for clinical use, mainly
for oncological indications. Despite these successes, three major
challenges in kinase drug discovery remain:
1. 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, but also to better understand the on- and off-
target pharmacology of kinase inhibitors (Noble et al.,
2004; Fabbro et al., 2011; Cowan-Jacob et al., 2014).
2. Identification and validation of the driver kinase(s) in
human malignancies and disorders (Weinstein, 2002;
Luo et al., 2009) by genome-wide screening for kinase
amplifications/translocations/mutations in conjunction
dx.doi.org/10.1124/mol.114.095489.
ABBREVIATIONS: CML, chronic myeloid leukemia; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; myr,
myristate; PI, phosphatidyl-inositol; PX-866, 1E,4S,4aR,5R,6aS,9aR)-5-(acetyloxy)-1-[(di-2-propen-1-ylamino)methylene]-4,4a,5,6,6a,8,9,9a-octa-
hydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione; SSR128129E, benzoic acid, 2-amino-
5-[(1-methoxy-2-methyl-3-indolizinyl)carbonyl]-, sodium salt.
766
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with proteomic technologies. These studies have re-
vealed how the mutational status of kinases may be
associated with various cancer conditions (Hunter,
2000; Blume-Jensen and Hunter, 2001; Cohen, 2002;
Bardelli et al., 2003; Sawyers, 2004; Vieth et al., 2004;
Takano et al., 2005; Ventura and Nebreda, 2006; Wolf-
Yadlin et al., 2006; Ali and Ali, 2007; Engelman et al.,
2007; Greenman et al., 2007; Thomas et al., 2007).
3. Emerging resistance to the inhibition of key target
kinases. Multiple mechanisms of resistance have been
and are being elucidated to improve the efficacy of these
types of targeted therapies.
In this review, we will update the current status of kinase
drug discovery and discuss strategies of how to override these
various types of resistances, including compounds capable of
circumventing the target related drug resistance (Lombardo
et al., 2004; Weisberg et al., 2005; Adrian et al., 2006; Quintas-
Cardama et al., 2007; Engelman et al., 2008b; Zhang et al.,
2010).
Current Status of Protein and Lipid Kinase Drug
Discovery
Protein Kinase Domain. Thanks to the many available
structures of kinase domains, we have a reasonable under-
standing of the various structural elements that are required for
the phosphorylation reaction (Cowan-Jacob et al., 2014). Protein
kinase domains consist of a small, mostly b-structured N-lobe
connected by a short hinge fragment to a larger a-helical
C-lobe. ATP binds in the cleft between the N- and C-terminal
lobes of the kinase domain where the adenine group of ATP is
sandwiched between hydrophobic residues and makes contact
via hydrogen bonds to the hinge (Nolen et al., 2004; Taylor and
Kornev, 2011; Fabbro et al., 2011). Just N-terminal of the
hinge, deep in the ATP pocket, is an important residue called
the “gatekeeper”, which is often mutated in kinase alleles
resistant to inhibitors. The gatekeeper controls the access to
the hydrophobic “back pocket”of the kinase (Nolen et al., 2004;
Kornev et al., 2006; Cowan-Jacob et al., 2009; Taylor and
Kornev, 2011; Moebitz and Fabbro, 2012). An outstanding
structural element of the ATP-binding sites is the activation
loop (also called the activation segment or A-loop), with its
N-terminal Asp-Phe-Gly (DFG) motif, whose orientation is piv-
otal for the access of the protein substrate sites (Nolen et al.,
2004; Cowan-Jacob, 2006). Other elements required for catalysis
are the C-helix, which contains the Glu that forms a salt bridge
with the active site Lys (the Ala-X-Lys motif in the b3-strand),
thereby anchoring and orienting the ATP, P-loop (a Gly-rich loop
also known as the G-loop), which contributes to coordination of
the phosphates of ATP, catalytic loop (the Y/HRD motif in the
b6/b7-strand), where Asp functions as a base acceptor for the
proton transfer, and finally, the abovementioned DFG motif,
where Asp binds the Mg
21
ions that coordinate the b-and
g-phosphates of ATP in the ATP-binding cleft (Hanks and
Hunter, 1995; Nolen et al., 2004; Cowan-Jacob, 2006; Taylor
and Kornev, 2011).
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 30, with
many others still in the preclinical state (Table 1). More than
130 kinase inhibitors are reported to be in phase 1–3 clinical
trials (http://www.clinicaltrials.gov/) (Vieth et al., 2005). It is
beyond the scope of this review to discuss all of the protein
kinase inhibitors that are in preclinical or clinical development.
Although there are many more kinase inhibitors in development,
it should be emphasized that all of the mentioned approved and
advanced kinase inhibitors do not cover more than 10–15% of
the whole kinome (Fedorov et al., 2010).
With a few exceptions, like the rapalogs (everolimus and
temsirolimus) and trametinib, all of the small molecular
weight kinase inhibitors are directed toward the ATP-binding
site. The conservation of ATP binding in the human kinome
causes these ATP mimics to often crossreact with many other
different kinases, resulting in compounds with promiscuous
profiles like, for example, dasatinib (Lombardo et al., 2004) or
sunitinib (Motzer et al., 2006; Faivre et al., 2007). The relative
restrictions imposed by the ATP pharmacophore make it
increasingly difficult to operate in a free intellectual property
space and has increased the interest in identifying inhibitors
that do not compete with ATP (Fabbro et al., 2011; Moebitz and
Fabbro, 2012; Cowan-Jacob et al., 2014).
Most of the approved kinase drugs are active against more
than one type of cancer. Only a few of them have been used for
the treatment of nononcological indications, namely, tofacitinib
for rheumatoid arthritis, nitedanib for idiopathic pulmonary
fibrosis, everolimus for organ rejection of the heart and kidney,
and fasudil for cerebral vasospasm (Table 1).
Also, most of the approved kinase drugs are active against
more than one type of cancer. 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 chronic myeloid leukemia (CML), while
sorafenib, sunitinib, everolimus, temsirolimus, axitinib, or
pazopanib are indicated for various stages of renal cell cancer.
Ceritinib and crizotinib are indicated for non–small-cell lung
cancer with ALK translocations, while gefitinib, erlotinib, and
afatinib are indicated for non–small-cell lung cancer with an
activated epidermal growth factor receptor (EGFR). Vandetanib
and cabozantinib are used for the treatment of medullary thyroid
carcinoma, while imatinib, sunitinib, and regorafenib are in-
dicated for gastrointestinal tumors. Finally, vemurafenib alone
or the combination of dabrafenib with trametinib is indicated
for metastatic melanoma with BRAFV600 mutations (Table 1).
The actual landscape of kinase inhibitor drugs developed
over the last two decades shows that only a handful of protein
kinases (about 10% of the kinome) have been successfully targeted
with inhibitors, which mainly target oncological indications. It
should be emphasized that selective protein kinase inhibitors
will not only be important for the treatment of diseases but also
as useful reagents to better understand the on- and off-target
kinase biology (Robert et al., 2005; Force et al., 2007; Knapp
et al., 2013).
Binding Modes of Kinase Inhibitors
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 (Zhang et al., 2009; Cowan-
Jacob et al., 2014).
Covalent Inhibitors
Inhibitors that bind covalently may have reversible or ir-
reversible binding modes, depending on the reactivity of the
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war-head (Cowan-Jacob, 2006; Zhang et al., 2009; Liu et al.,
2013). Many inhibitors bind covalently to the ATP site to form
a bond to a Cys residue in or around the active site, preventing
the binding of ATP to the protein kinase (Zhang et al., 2009;
Liu et al., 2013). The most prominent example is the recently
approved BTK inhibitor ibrutinib and the EGFR inhibi-
tor afatinib, which targets the Cys in the bottom part of the
ATP-binding site [receptor protein tyrosine kinase (RTK)]
(Zhou et al., 2009, 2011; Wiestner, 2013; Akinleye et al., 2014).
More covalent inhibitors targeting these cysteines are being
developed to target the most resistant form of the EGFR RTK
that emerges upon treatment with noncovalent EGFR in-
hibitors (Zhang et al., 2009; Zhou et al., 2011; Liu et al., 2013).
An early discovered type of covalent binder is exemplified by
Wortmannin (or its derivative PX-866 [1E,4S,4aR,5R,6aS,
9aR)-5-(acetyloxy)-1-[(di-2-propen-1-ylamino)methylene]-4,4a,
5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,
6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione]),
TABLE 1
Approved kinase inhibitors as of October 2014
The 30 kinase inhibitors approved to date are shown with generic name, compound code, trade name, primary indications, year of approval, company, and mode of binding. The
chemical structures and biochemical profiles of the 30 approved kinase inhibitors can be viewed in the IUPHAR database (http://www.guidetopharmacology.org/GRAC/
LigandListForward?type=Approved&database=all). The approved kinase inhbitors include fasudil (HA-1077), sirolimus, imatinib (Glivec), gefitinib (Iressa), erlotinib
(Tarceva), lapatinib (Tykerb), sorafenib (Nexavar), sunitinib (Sutent), dasatinib (Sprycel), nilotinib (Tasigna), torisel (Temsirolimus), everolimus (Rad001) as Afinitor, Zortress,
Certican, and Votubia, crizotinib (Xalcori), vandetanib (Caprelsa), ruxolitinib (Jakafi), vemurafenib (Zelboraf), axitinib (Inlyta), regorafenib (Stivarga), pazopanib (Votrient),
tofacitnib (Xeljanz), cabozantinib (Cometriq), ponatinib (Iclusig), bosutinib (Bosulif), dabrafenib (Tafinlar), trametinib (Mekinist), afatinib (Gilotrif), ibrutinib (Imbruvica),
ceritinib (Zykadia), idelalisib (Zydelig), and nintedanib (Vargatef). All compounds are commercially available.
Generic Name (Compound Code, Trade Names) Kinase Target Disease Company (Year, Type)
Fasudil (HA-1077) ROCK Cerebral vasospasm, PAH Asahi Kasei (1995,
type 1)
Sirolimus (Rapamune) mTOR Kidney transplants Pfizer, Wyeth (1999,
type 3)
Imatinib (STI571, Glivec, Gleevec) ABL, PDGFR, KIT CML, Ph+ B-ALL, CMML,
HES, GIST
Novartis (2001, type 2)
Gefitinib (ZD1839, Iressa) EGFR NSCLC AZ (2003, type 1)
Erlotinib (OSI-774,Tarceva) EGFR NSCLC, pancreatic cancer Roche, OSI (2004, type 1)
Sorafenib (BAY 43-9006, Nexavar) VEGFR2, PDGFR, KIT, FLT3,
BRAF
RCC, HCC Bayer, Onyx (2005, type 2)
Sunitinib (SU11248, Sutent) VEGFR, KIT, PDGFR, RET,
CSF1R, FLT3
RCC, imatinib-resistant GIST Pfizer (2006, type 1)
Lapatinib (GW2016, Tykerb) EGFR, ERBB2 BC GSK (2007, type 1.5)
Dasatinib (BM-354825,Sprycel) ABL, PDGFR, KIT, SRC CML BMS (2007, type 1)
Nilotinib (AMN107,Tasigna) ABL, PDGFR, KIT CML Novartis (2007, type 2)
Everolimus (Rad001, Certican, Zortress,
Afinitor, Votubia)
mTOR RCC, SEGA, transplantation Novartis (2009, type 3)
Temsirolimus (CCI-779, Torisel) mTOR RCC Pfizer, Wyeth (2009,
type 3)
Crizotinib (PF-02341066, Xalcori) MET and ALK NSCLC with ALK translocations Pfizer (2011, type 1)
Vandetanib (ZD6474, Caprelsa) RET, VEGFR1, VEGFR2, FGFR,
EGFR
MTC AZ (2011, type 1)
Ruxolitinib (INC424, Jakafi) JAK2 IMF with JAK2V617F mutations Novartis, Incyte (2011,
type 1)
Vemurafenib (PLX4032, RG7204,
Zelboraf)
BRAF Metastatic melanoma with
BRAFV600E mutations
Roche, Plexxikon (2011,
type 2)
Axitinib (AG013736, Inlyta) VEGFR, KIT, PDGFR, RET,
CSF1R, FLT3
RCC Pfizer (2012, type 1)
Regorafenib (BAY 73-4506, Stivarga) VEGFR2, Tie2 CRC, GIST Bayer (2012, type 2)
Pazopanib (GW-786034, Votrient) VEGFR, PDGFR, KIT RCC GSK (2012, type 1)
Tofacitinib (CP-690550, Xeljanz,
tasocitinib)
JAK3 RA Pfizer (2012, type 1)
Cabozantinib (XL184, BMS907351,
Cometriq)
VEGFR2, PDGFR, KIT, FLT3 MTC Exelexis (2012, type 1)
Ponatinib (AP24534, Iclusig) ABL Imatinib-resistant CML with
T315I mutations
Ariad (2012, type 1)
Bosutinib (SKI-606, Bosulif) ABL CML resistant/intolerant to therapy Pfizer (2012, type 1)
Dabrafenib (Tafinlar) BRAF Metastatic melanoma with
BRAFV600E mutations
GSK (2013, type 2)
Trametinib (Mekinist) MEK Metastatic melanoma with
BRAFV600E mutations
GSK (2013, type 3)
Afatnib (Gilotrif, Tomtovok, Tovok) EGFR NSCLC with EGFR-activating
mutations
BI (2013, covalent)
Ibrutinib (PCI-32765, Imbruvica) BTK MCL, CLL Janssen, Pharmacyclic
(2013, covalent)
Ceritinib (LDK378, Zykadia) ALK NSCLC with ALK translocations Novartis (2014, type 1)
Idelalisib (CAL101, GS1101, Zydelig) PI3Kdelta CLL, FL and SLL Gilead, Calistoga, ICOS
(2014, type 1)
Nintedanib (BIBF 1120, Vargatef,
intedanib)
VEGFR, PDGFR, FGFR Idiopathic pulmonary fibrosis BI (2014, type 1)
AZ, Astra-Zeneca; BC, breast cancer; BI, Boehringer-Ingelheim; BMS, Bristol-Myers Squibb; CLL, chronic lymphocytic leukemia; CMML, chronic monomyelocytic
leukemia; CRC, colorectal cancer; FL, follicular lymphoma; HCC, hepatocellular carcinoma; HES, hyper-eosinophilic syndrome; IMF, interstitial myelofibrosis; GIST,
gastrointestinal stromal tumor; GSK, Glaxo-Wellcome; MCL, mantle cell lymphoma; MTC, medullary thyroid cancer; PAH, Pulmonary Arterial Hpertension; Ph+ B-ALL,
Philadelphia chromosome positive B-cell acute lymphoblastic leukemia; RA, rheumatoid arthritis; RCC, renal clear cell carcinoma; SEGA, subependymal giant cell
astrocytoma; SLL, small lymphocytic leukemia.
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which binds to the active site Lys in the ATP-binding site of the
PI3K (Wymann et al., 1996; Ihle et al., 2004). Various covalent
inhibitors targeting different cysteines in various kinases have
been reported (Liu et al., 2013; Kwiatkowski et al., 2014).
Reversible (Noncovalent) Inhibitors
Noncovalent kinase inhibitors can be further classified into
those which either bind or do not bind to the hinge region of
the kinase, leading to the classification of type 1–3 reversible
kinase inhibitors (Liu and Gray, 2006; Zhang et al., 2009).
These have been briefly summarized below.
Type 1 and 1.5 Inhibitors. These inhibitors fulfill all of
the criteria of ATP mimetics by binding to the hinge and
targeting an active conformation of the kinase (often referred
to as DFG-in). In the active kinase, the A-loop adopts an open
conformation typical for the ATP-bound state of the kinase
where the Asp in the DFG motif coordinates the phosphates of
ATP, while Phe stabilizes helix C and A-loop for catalysis
(Nolen et al., 2004; Cowan-Jacob, 2006; Liu and Gray, 2006;
Zhang et al., 2009; Cowan-Jacob et al., 2014). Many of the type
1 inhibitors use variation in the size, shape, and polarity of
the gatekeeper residue to gain selectivity and knowledge about
the various inactive conformations of different kinases, which
allows one to combine these features (Liu and Gray, 2006;
Cowan-Jacob et al., 2014). The bias toward kinase inhibitors
that target the active conformation of the kinase stems from
the early days of kinase drug discovery, where an ATP mimetic
design, together with the use of enzymatic assays displaying
the highest level of activity, was used. Classic examples for this
type of approved kinase inhibitor class are gefitinib, erlotinib,
dasatinib, and sunitinib (Table 1).
The type 1.5 inhibitor is a subtype of the type 1 inhibitor
that binds to an inactive kinase conformation (Nolen et al.,
2004; Cowan-Jacob, 2006; Liu and Gray, 2006; Zhang et al.,
2009; Cowan-Jacob et al., 2014). Laptinib in the EGFR-RTK
adopts a DFG-in conformation, which is typical of an active
kinase, but helix C is pushed out by lapatinib, effectively
disrupting the ion pairing between the active site Lys and the
Glu from helix C (Wood et al., 2004). This type of binding to
the DFG-in inactive conformation, which is referred to as type
1.5 inhibition, has also been observed in other kinases (Cowan-
Jacob et al., 2014).
Although the structures of protein kinases in the active
ATP-bound state are very similar, specificity can be gained
through particular featuresintheATP-bindingsitesof
kinases. More selective type 1 inhibitors are those that, like
the type 1.5 inhibitors, use additional sites close to the ATP
binding, like the adjacent hydrophobic pockets whose entry is
regulated by the gatekeeper (Zuccotto et al., 2010). Other type
1 inhibitors interact with two different sites on the kinase, such
as the ATP site and the peptide-binding site like the bivalent/
bitopic inhibitors (Hill et al., 2012), macrocycles (Tao et al.,
2007), or some of the covalent inhibitors (Liu et al., 2013).
Type 2 Inhibitors. Type 2 kinase inhibitors bind to the
inactive, so-called “DFG-out conformation”, maintaining con-
tacts to the hinge region and usually displaying an ATP-
competitive behavior similar to type 1 inhibitors (Nolen et al.,
2004; Cowan-Jacob, 2006; Liu and Gray, 2006; Zhang et al.,
2009). The transition of the active DFG-in to the inactive DFG-
out conformation exposes an additional hydrophobic pocket
adjacent to the ATP site, which is used by type 2 inhibitors,
locking the kinase in the inactive conformation (Nolen et al.,
2004; Cowan-Jacob, 2006; Liu and Gray, 2006; Zhang et al.,
2009; Cowan-Jacob et al., 2014). Approved kinase inhibitors
binding to or stabilizing the DFG-out conformations are, for
example, imatinib, nilotinib, or sorafenib (Table 1).
In addition to DFG-out, combinations of different confor-
mational states of helix C, the A-loop, and/or P-loop can gen-
erate various inactive conformations of the kinase domain
(Cowan-Jacob, 2006; Cowan-Jacob et al., 2014). Each individ-
ual kinase has a preferred inactive conformation, dependingon
its phosphorylation state and regulatory mechanisms involving
structures outside the kinase domain (Cowan-Jacob et al.,
2014).
Type 3 Inhibitors. Type 3 kinase inhibitors are non-ATP
competitive (allosteric) kinase inhibitors that have no phys-
ical contact with 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 (Nolen
et al., 2004; Cowan-Jacob, 2006; Liu and Gray, 2006; Zhang
et al., 2009; Cowan-Jacob et al., 2014). The noncatalytic role of
kinases involving unique nonconserved interactions and which
increase the target space on the kinome have only recently
been appreciated (Rauch et al., 2011; Cowan-Jacob et al., 2014).
Type 3 inhibitors can either bind to the kinase domain (close to
or removed from the ATP site) or to sites that are located
outside of the kinase domain. These type 3 inhibitors include
very diverse compounds, ranging from the MEK inhibitors to
rapamycin derivatives.
The allosteric inhibitor of MEK binds to a pocket adjacent
to the ATP-binding site, which is referred to as the “allosteric
back-pocket”(Ohren et al., 2004). These type 3 allosteric
inhibitors can either bind in the presence or absence of ATP.
Those that bind in the presence of ATP (DFG-in conformation,
such as MEK inhibitors) can be referred to as “allosteric back-
pocket DFG-in inhibitors”.Alternatively,“allosteric back-pocket
DFG-out inhibitors”can bind to the allosteric back-pocket in the
absence of ATP (DFG-out conformation) and include allosteric
inhibitors of insulin-like growth factor 1 receptor (IGF-1R)
(Heinrich et al., 2010), FAK (Tomita et al., 2013), or p38 (Over
et al., 2013). In the case of IGF-1R, the inhibitor binds in the
DFG-out pocket and extends over the catalytic loop rather than
pushing it out from underneath like the FAK inhibitor, requiring
additional conformational changes (Tomita et al., 2013). The
allosteric AKT inhibitors are a special case of the allosteric back-
pocket DFG-out inhibitors, as they only bind to this site when
the pleckstrin homology domain is present (Barnett et al., 2005;
Lindsley et al., 2005). Thus, the identification of this allosteric
inhibitor that binds at the interface between the kinase domain
and pleckstrin homology domain was only possible using the full-
length protein for AKT (Barnett et al., 2005; Lindsley et al.,
2005). While lack of competition with ATP has, in some cases,
proven to be a useful way to identify type 3 inhibitors, it should
be pointed out that the allosteric back-pocket DFG-out inhib-
itors will score as ATP competitive.
Type 3 inhibitors that are further away from the ATP site
are the myristate (myr) pocket binders located in the bottom
of the C-lobe of ABL (Adrian et al., 2006; Zhang et al., 2009;
Fabbro et al., 2010), CHK1 inhibitors that occupy, in part,
the substrate-binding site (Converso et al., 2009), and JNK1
inhibitors that occupy the mitogen-activated protein kinase
insert region and A-loop (Comess et al., 2011), to only cite
a few. A more comprehensive review on type 3 inhibitors has
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been recently assembled by Cowan-Jacob et al. (Cowan-Jacob
et al., 2014).
Rapamycins appear to be further removed from the kinase
domain, as they specifically target the mTOR kinase in the
context of the Raptor-containing mTORC1 complex (Wang
and Sun, 2009; Yang et al., 2013). Similarly, agents targeting
the extracellular domains of RTKs act outside the kinase
domain (Christopoulos et al., 2014). The extracellular do-
mains of RTKs can be targeted by peptide mimetics, peptoids,
or antibodies (Fleishman et al., 2002; Udugamasooriya et al.,
2008; Cazorla et al., 2010; Jura et al., 2011). The monoclonal
antibodies trastuzumab (Herceptin) and pertuzumab (2C4,
Perjeta) act at different domains, with trastuzumab binding
to domain IV and pertuzumab binding to subdomain II of the
extracellular segments of the HER2/neu receptor, respectively
(Cho et al., 2003; Hynes and Lane, 2005; Hsieh and Moasser,
2007). On the other hand, small molecules like SSR128129E
[benzoic acid, 2-amino-5-[(1-methoxy-2-methyl-3-indolizinyl)
carbonyl]-, sodium salt], which targets the extracellular D2D3
domains of the fibroblast growth factor receptor (FGFR), mod-
ulate signaling of the FGFR RTKs (Bono et al., 2013; Herbert
et al., 2013). Examples of approved type 3 inhibitors are
trametinib and the rapamycins.
Activators, Paradoxical Activation, and Priming.
Targeting the allosteric sites on protein kinases may also
lead to the identification of activators rather than inhibitors,
which could be useful for therapeutic intervention or as phar-
macological tools. In particular, compounds targeting the PIF
pocket (the hydrophobic motif present in the N-terminal lobe
of AGC kinases) of either PDK1 or PKCzcan either act as
activators (Hindie et al., 2009) or substrate-selective inhibitors
(Lopez-Garcia et al., 2011; Sadowsky et al., 2011; Busschots
et al., 2012). Similarly, the myr-pocket binders of ABL can be
converted into activators if they are designed not to bend helix I
of the ABL kinase domain (Jahnke et al., 2010; Yang et al.,
2011).
On the other hand, there are a few protein kinases that
require activation rather than inhibition to fulfill their
therapeutic task, like in the case of AMP-activated protein
kinase and the insulin receptor for which activators have been
identified (Li et al., 2001; Pender et al., 2002; Sanders et al.,
2007; Lee et al., 2011; Salt and Palmer, 2012). PKC activation
by exogenous compounds acting at the diaclyglycerol-binding
site can have tumor-promoting or tumor-suppressive effects
(Martiny-Baron and Fabbro, 2007). These include phorbol
esters, bryostatin, and other compounds acting as diaclyglycerol
mimetics (Martiny-Baron and Fabbro, 2007). Another example
of kinase activators includes a mimetic of the brain-derived
neurotrophic factor that activates TrkB (NTRK-2) (Massa et al.,
2010).
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
activation of RAF inhibitors, which can activate the mitogen-
activated protein kinase pathway in certain genetic backgrounds
(Hall-Jackson et al., 1999). This phenomenon is linked to a
complex regulation of RAF due to crossactivation of the wild-
type RAF isoforms, which is just beginning to be understood,
almost a decade after the first so-called RAF inhibitor sorafenib
was approved (Hall-Jackson et al., 1999; Hatzivassiliou et al.,
2010; Poulikakos et al., 2010; Holderfield et al., 2013).
Thus, BRAF inhibitors can activate MEK and extracellular
signal-regulated kinase (ERK) signaling in normal cells, result-
ing not only in the absence of hypoproliferative toxicities but
actual promotion of hyperproliferative toxicities (Su et al., 2012).
Another phenomenon is priming, which can lead to ac-
tivation via kinase inhibitors, which has been observed for
several kinases like AKT, MEK, and JAK (Okuzumi et al.,
2009; Andraos et al., 2012; Hatzivassiliou et al., 2013). Prim-
ing describes the up-regulation of the phosphorylated form of
the targeted kinase upon inhibition, which can lead to an
activation of the pathway once the inhibitor is removed. Prim-
ing may also depend on the mode of action of the inhibitor.
Inhibitors binding to active AKT do cause priming, whereas
allosteric inhibitors targeting the inactive conformation of AKT
do not (Lin et al., 2012).
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.
Resistance to Kinase Inhibitors (in Oncology)
Introduction to Drug Resistance. Most tumor cells
express only a few of the 150 so-called “driver genes”, which,
when mutated, promote tumorigenesis by affecting a dozen
signaling pathways regulating cell fate, cell survival, and
genome maintenance (Vogelstein et al., 2013). Insights into
particular mutations and their consequences for signaling
have already led to the development of more effective drugs
(Siegel et al., 2012). The finding that deregulation of protein
kinase activity by the gain of function mutations is essential
to maintain the oncogenic state is also reflected by the fact that
30 kinase inhibitors have been approved for various oncological
indications (Janne et al., 2009; Luo et al., 2009; Sharma and
Settleman, 2010; Yarden and Pines, 2012) (Table 1). In many
instances, these mutations can be linked to specific cancers
(Greenman et al., 2007; Thomas et al., 2007). Although many
activating mutations in various kinases have been found in
a variety of cancers, it will take another effort to unambigu-
ously identify the dependence of tumor growth on a particular
kinase or its pathway(s). The major difficulty is the intrinsic
heterogeneity of the late-stage forms of cancer that harbor
multiple mutations, chromosomal aberrations, and display
genomic instability. Demonstrating the cancer dependence of
the kinase target will not only lead to the identification of
inhibitors of the kinase, but may also accelerate the proof
of concept in clinical trials, allowing a better selection of pa-
tients most likely to respond to targeted therapy.
Kinase inhibitors are being and have been designed to spe-
cifically target kinase alleles with a gain of functions (Blume-
Jensen and Hunter, 2001; Fabbro and García-Echeverría,
2002a). Despite these successes, it should be emphasized that
patients most likely to benefit from these kinase inhibitors
often relapse after an initial response. Thus, the emergence of
drug resistance is not limited to conventional chemotherapeu-
tic drugs but extends to drugs with targeted modes of action
(Engelman and Settleman, 2008a). The mechanisms of multi-
drug resistance to chemotherapeutic drugs have been studied
and are not limited to reduced drug accumulation, but also
involve changes in the level of target proteins, mutations that
diminish drug binding, trapping of drugs in acidic vesicles,
enhanced metabolism of drugs by cytochrome P450 mixed
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function oxidases, increased tolerance of cellular DNA damage,
and diminished apoptotic signaling (Gottesman, 2002; Szakacs
et al., 2006; Hall et al., 2009). Apart from the usual mechanisms
of drug inactivation in cancer (Szakacs et al., 2006; Hall et al.,
2009) as well as the findings that quiescent tumor stem cells are
refractory to kinase inhibitors (Graham et al., 2002), there are
additional target-related mechanisms for resistance that are not
based on mutations of the target kinase.
Resistance to Kinase Inhibitors. Drug resistance to tar-
geted agents like kinase inhibitors can either occur by compen-
satory mechanism or by reducing the affinity of the kinase to its
inhibitors (Szakacs et al., 2006; Fabbro et al., 2011).
The most commonly found point mutation leading to
resistance concomitant with relapses affects the gatekeeper
residue, whose size and shape regulates the properties of the
hydrophobic pocket located at the back of the ATP-binding
site. These mutations include the Thr gatekeeper of BCR-
ABL1 (T315I) (Gorre et al., 2001; Sawyers, 2004; Fabbro et al.,
2005), Kit (T670I) (Heinrich et al., 2003; Fletcher and Rubin,
2007), PDGFRa(T674I) (Cools et al., 2003), PDGFRb(T681I)
(Daub et al., 2004), SRC T341M (Bishop, 2004), as well as
other types of gatekeepers like L1196M in ALK (Katayama
et al., 2012), G697R in FLT3 (Cools et al., 2004), and V561M in
FGFR1 (Blencke et al., 2004). This is either due to a steric
clash between the inhibitor and mutated gatekeeper (Blencke
et al., 2004; Fabbro et al., 2005; Fabbro et al., 2011) or by
significantly increasing the affinity for ATP and thereby
reducing the affinity for the kinase inhibitors (Kobayashi et al.,
2005; Pao et al., 2005). Mutations in the gatekeeper as well as
other kinase domain mutations confer resistance to a wide spec-
trum of kinase inhibitors without affecting the kinase activity
and may explain a fraction of cases of acquired resistance.
There are additional mechanisms to circumvent kinase
inhibition (Sawyers, 2004; Rubin and Duensing, 2006, Fabbro
et al., 2011; Serra et al., 2011; Logue and Morrison, 2012;
Trusolino and Berlotti, 2012; Workman et al., 2013). Com-
pensatory changes in the signaling pathways of treated
cancer cells that can bypass drug-mediated kinase inhibition
and so restore downstream signaling in the presence of drug
include
1. Amplification of the target, like in the case of BCR-
ABL1 in CML (le Coutre et al., 2000) or dimerization of
aberrantly spliced BRAF (V-600E) (Poulikakos et al.,
2011); and
2. Up-regulation of RTKs following inhibition of PI3K
(Serra et al., 2011; Rodon et al., 2013) or up-regulation
of alternative kinase pathways through the receptors
for hepatocyte growth factor (MET), IGF-1R, or AXL in
the acquisition of resistance to EGFR kinase inhibition
(Engelman et al., 2007; Turke et al., 2010; Logue and
Morrison, 2012). Activation downstream of RTKs, like
the RAS-RAF-ERK and/or PI3K/AKT pathways, by
several mechanisms can override the effects of any RTK
inhibitor. Examples include activation of the PI3K/AKT
pathway by activating point mutations in PI3K sub-
units, loss-of-function/deletions of the PTEN phospha-
tase in EGFR-mutated lung cancer, mutations in RAS
isoforms downstream of oncogenic RTKs, MEK activation
by COT1 bypassing inhibited BRAF-V600E, and activa-
tion or the EGFR-mediating resistance to vemurafenib in
BRAF-V600E (She et al., 2003; Johannessen et al., 2010;
Prahallad et al., 2012). Signaling redundancies, inter-
connections through pathway crosstalk, and negative
feedback loops have also been identified as contrib-
utors to drug resistance or weaken the efficacy of kinase
inhibitors (Janne et al., 2009; Rodrik-Outmezguine et al.,
2011; Chandarlapaty, 2012). Negative feedback loops,
crossinhibition, crossactivation, and pathway conver-
gence connect signaling pathways through activated
components and are crucial for the maintenance of
normal cell functions as well as the dynamic and
adaptive responses to extracellular signals (Mendoza
et al., 2011; Logue and Morrison, 2012). Even if a driver
kinase is suppressed by the inhibitor, the tumor cells
can exploit its interconnected signaling pathways to
evolve drug resistance (Trusolino and Berlotti, 2012).
The Ras-ERK and PI3K-mTOR signaling provide major
mechanisms for the regulation of cell survival, differen-
tiation, proliferation, metabolism, and motility in re-
sponse to extracellular ligands and are activated in over
80% of all tumors (Engelman, 2009; Wong et al., 2010).
Selective inhibition of mTORC1 leads to disruption of
a negative feedback loop, which enhances the activity of
PI3K and its effector AKT, counteracting the antiproli-
ferative effects of mTOR inhibition (Chandarlapaty,
2012). Combined inhibition of the mTOR kinase and
RTKs therefore seems a promising approach to abolish
AKT signaling and prevent resistance formation (Rodrik-
Outmezguine et al., 2011). Alternatively, inhibition of
PI3K/mTOR signaling resulted in the activation of the
Jak/STAT5 pathway, suggesting a combination strategy
targeting both the PI3K/mTOR and JAK/STAT5 signal-
ing (Britschgi et al., 2012). Negative feedback regulation
hasalsobeenobservedintheRAS-RAF-ERKcascade
(Mendoza et al., 2011). The binding of vemurafenib to
wild-type BRAF in cells can cause BRAF/CRAF hetero-
dimerization and paradoxical activation of ERK1/2,
which can result in the development of kerato-acanthomas
in patients (Chapman et al., 2011; Poulikakos et al.,
2011; Su et al., 2012; Holderfield et al., 2014). This is
further exacerbated in the case where oncogenic RAS
and BRAF mutants are present in the same cells that
support RAS-dependent dimerization and paradoxical
ERK activation.
3. Factors regulating the bioavailability and intracellular
concentration of inhibitors like poor intestinal absorption,
tight binding to blood plasma proteins, overexpression of
the multidrug resistance genes, and/or increased metab-
olism of the drug by liver cytochrome P450 proteins have
also been linked to primary resistance (Mahon et al.,
2003; Apperley, 2007).
All of these mechanisms demonstrate the plasticity of
cancer cells and the many ways by which a tumor can evade
targeted therapies. The only way to approach these problems is
to use a rational combination of drugs, which is a mainstay in
cancer therapy.
Breaking the Resistance to Kinase Inhibitors. To cir-
cumvent target-dependent drug resistance, second-generation
kinase inhibitors have been developed. Inhibitors that
bind covalently to the ATP-binding site of EGFR have been
developed for the emerging resistance to gefitinib and erlotinib
(Kwak et al., 2005; Heymach et al., 2006; Felip et al., 2007;
Kinase Inhibitors 771
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Zhou et al., 2011). Several of these covalent inhibitors are in
late-stage clinical trials (Zhang et al., 2009; Liu et al., 2013).
Alternatively, these types of covalent inhibitors, as in the case
of ibrutinib, have been designed upfront to bind covalently to
Cys481 of BTK and recently approved for B-cell malignancies
(Byrd et al., 2013; Wiestner, 2013; Akinleye et al., 2014). Although
ibrutinib has shown impressive clinical results, patients that have
disease progression revealed a C481S mutation in their BTK,
which abrogates the covalent binding to ibrutinib (Furman et al.,
2014). Additional approaches have attempted to develop non-
covalent inhibitors that can tolerate the amino acid exchange at
the gatekeeper position. Several ATP-site directed inhibitors
that are active against the T315I ABL1 gatekeeper mutant
have been designed, but thus far only ponatinib has been
approved (O’Hare et al., 2009; Hoy, 2014). To target the gate-
keeper mutation, these types of compounds display a rather
low selectivity that can have deleterious side effects, which may
lead to a temporary pull off the market over safety issues (Force
et al., 2007; Cheng and Force, 2010; Dalzell, 2013).
A further approach is to target the kinase outside the ATP-
binding sites with the goal of combining the ATP–site directed
inhibitors (type 1 and 2) with type 3 (Fabbro et al., 2011;
Cowan-Jacob et al., 2014) or to combine different kinase
inhibitors targeting kinases in the same pathway, like in the
case of vemurafenib, where the emerging resistance is not due to
mutations in BRAF (V600E) but rather in the downstream
MEK1 (Wagle et al., 2011; Medina et al., 2013). Unfortunately,
only a very limited number of non-ATP competitive kinase
inhibitors have thus far been identified, which could also be
used to address the resistance caused by mutations in the ATP
binding site (Fabbro et al, 2011; Cowan-Jacob et al., 2014)
A remote binding site on the kinase domain is addressed
by the GNF-2 compound, which was found by a phenotypic
screen shown to target the myr pocket–binding site of ABL
(Adrian et al., 2006; Zhang et al., 2009; Fabbro et al., 2010).
Exploration of the combined efficacy between the myr pocket
and ATP-binding sites significantly increased the survival of
mice in bone-marrow transplantation CML models relative
to treatment with either agent alone (Zhang et al., 2010). In
addition, the improved potency of a second-generation myr
pocket binder against wt-ABL and T315-ABL also translated
into a high level of degree of synergy in BaF3 cells trans-
formed with BcrABL-T315I when combined ATP–site di-
rected inhibitors like nilotinib or dasatinib, as it has been
noted in previous studies (Fabbro et al., 2002b; Zhang et al.,
2010). Surprisingly, NMR and small-angle X-ray scattering
analysis revealed an open state of the ABL when bound to
ATP–site directed inhibitors, like imatinib, leading to the
detachment of the SH3-SH2 domains from the kinase domain
and the formation of an open inactive state, which is inhibited
in the ATP site, which can be reverted by the addition of the
myr pocket binder (Skora et al., 2013). Whether these data
explain the synergy between the myr pocket binder and ATP-
directed inhibitors that appears to overcome the T315I-ABL–
mediated drug resistance remains to be seen. The findings on
the actions of the two classes of inhibitors on a single target
kinase may help to devise new strategies for drug development.
Conclusions and Perspectives
Several kinase inhibitors have become successful drugs. A
large number of kinase inhibitors are in clinical development
mainly for oncology indications, which is a testimony for the
greater tolerability in this indication with respect to potential
side effects. One of the issues that is perceived as a major
challenge in kinase drug discovery is the selectivity for the
target kinases, which defines the on- and off-target pharma-
cology (Cohen, 2002; Fabbro and García-Echeverría, 2002a,b;
Vieth et al., 2004, 2005; Cowan-Jacob, 2006; Liu and Gray,
2006; Zhang et al., 2009). While development of selective
kinase inhibitors is likely to be extremely important from the
standpoint of minimizing drug side effects, inhibitors with
exquisite selectivity are a must for chronic administration in
non–life-threatening diseases like many immunologic dysfunc-
tions (Cohen, 2002; Fabbro and García-Echeverría, 2002a;
Fabbro et al., 2002b; Vieth et al., 2004, 2005; Liu and Gray,
2006; Zhang et al., 2009). While the lack of selectivity of kinase
inhibitors can be advantageous in cancer treatment, it can also
lead to side effects (Cheng and Force, 2010). On the other hand,
low selectivity complicates mechanism of action studies as well
as biomarker discovery. The information linking adverse side
effects of protein and lipid kinase inhibitors with a particular
protein kinase selectivity profile is only now beginning to
emerge (Force et al., 2007; Olaharski et al., 2009). Moreover, we
are still trying to understand the side effects that are generated
by inhibiting multiple kinases, including undesired kinases that
may generate, at least in part, the adverse side effects. Recent
data, for example, suggest that simultaneous inhibition of ATM,
ATR, DNA-PK, Aurora, and/or other kinases involved in mitosis
may be genotoxic (Force et al., 2007; Olaharski et al., 2009).
The development of kinase inhibitors for non–life-threatening
indications where chronic regimens are being used will require
a better target selectivity profile to minimize side effects.
Therefore, only few attempts have been made to position kinase
inhibitors in chronic diseases, such as inflammation and
immune disorders. Most prominent is the p38 kinase, which
has been targeted for treatment of nononcology indications
like rheumatoid arthritis.
Addressing the noncatalytic functions of kinases will result
in novel types of kinase inhibitors with improved selectivity.
The design and synthesis of non-ATP competitive (allosteric)
kinase inhibitors with high selectivity will not only lead to the
expansion of the kinase target space with less studied mem-
bers of the family like the atypical and the pseudo-kinases.
Having highly selective kinase inhibitors (ideally against
most of the members of the whole kinome) at hand will propel
the pharmacology as well as understanding of their roles in
signaling and regulation. Thanks to the available kinase
inhibitors, we have learned a lot about drug-protein inter-
actions and cancer cell signaling, in some cases only after
their use in the clinic. We only now begin to appreciate the
complex rules determining the mechanism of action and spec-
ificity of these types of inhibitors. In addition to its clinical
use, the various new drugs and drug candidates will be
instrumental and indispensable reagents for many biologic
disciplines to help decipher kinase regulation, interrogate
signaling pathways, perturb cellular networks, and study
complex cell biologic processes.
One of the major challenges in future kinase drug discovery
is to better understand the cancer dependence of the target
kinase and anticipate the emerging resistance to kinase
inhibitor treatment. With a few exceptions, the development
of kinase inhibitor resistance in cancer has lowered the en-
thusiasm about the initially observed outstanding clinical
772 Fabbro
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effects. Although, in some cases, we have seen long-lasting
clinical responses with these kinase inhibitors, we have
inevitably and not unexpectedly seen relapses under treat-
ment. Understanding resistance mechanisms has not only
lead to a deeper understanding of cancer cell biology, but also
instructed ways to overcome resistance using second-generation
kinase inhibitors with a better selectivity and appropriate
potency, which are being applied to a genetically better defined
patient population that is most likely to respond. In addition,
clever combinations and sequential treatment regimens are
being tested. It should be noted that clinical responses have
also been noted with many other kinase inhibitors without
a clear rationale, offering encouragement that single agents
can be active in genetically complex neoplasms, although it is
formally possible that the single-agent activity may be due to
its fortuitous ability to inhibit multiple kinases. The recent
introduction of the concept of individualized cancer therapy
along with the development of selective drugs targeting specific
alteration in tumors has provided hope for the development of
more effective treatment strategies.
In summary, the available complement of clinically used
kinase inhibitors do not cover more than 10–15% of the whole
kinome. Ongoing efforts using genome-wide screening in con-
junction with the use of genetic organisms will unravel new
disease associations and pave the way for the discovery of
many more new protein kinase targets in the coming years.
Finally, there are many selective protein kinase inhibitors
that cannot be used as drugs for reasons of toxicity or solubility
but which are still extremely useful as research reagents (Robert
et al., 2005; Force et al., 2007). Extending the complement of
very selective (allosteric) protein kinase inhibitors in conjunction
with a system biology approach will advance our understanding
of kinase biology in cellular networking and diseases.
Acknowledgments
The author thanks Adam Pawson and Elena Faccenda from the
IUPHAR database in helping to compile Table 1. This mini-review
could only be written with the valuable help of many publications.
Due to space restrictions, not all of the relevant publications could be
cited.
Authorship Contributions:
Wrote or contributed to the writing of the manuscript: Fabbro.
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Address correspondence to: Doriano Fabbro, Ph.D., PIQUR Therapeutics
AG, Hochbergerstrasse 60C, CH-4057 Basel, Switzerland. E-mail: doriano.
fabbro@piqur.com
Kinase Inhibitors 775
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