One of the fundamental medical advances of the last decades is targeted chemotherapy.
Conventional chemotherapy acts indiscriminately on rapidly dividing cells in tumors but also in normal tissues throughout the whole body. This lack of specificity causes undesirable effects, which considerably limit the use of chemotherapy, and forces to limit the doses administered. Targeted therapies directed against TKRs or signaling proteins expressed in cancer cells have been proposed. There are several approaches to targeted therapy, including monoclonal antibodies and tyrosine kinase inhibitors (TKIs) (Dassonville et al., 2007).
Targeted therapies present high specificity of action and are less toxic. They are also useful when combined with conventional chemotherapy or radiotherapy to achieve additive or even synergistic action of treatment.
The first monoclonal antibodies were developed by Milstein and Kohler (Kohler & Milstein, 1975) and since then its production and application have grown exponentially. Examples of humanized monoclonal antibodies that are currently used in the clinic are Rituximab and Trastuzumab, which inhibit CD20 and HER2 (Harries et al., 2002; Mendelsohn & Baselga, 2000; Shawver et al., 2002). However, monoclonal antibodies did not achieve a complete blockade when targeted against TKRs (Krystal et al., 1996). With the development of TKIs, a new possibility of inhibition of these receptors arose.
Tyrosine kinases are enzymes that phosphorylate tyrosine residues of an effector protein by transferring a phosphate group from ATP (Lehninger et al., 2008). TKRs induce signaling pathways that are deregulated in cancer and play a central role in tumor progression.
Therefore, TKRs have become good therapeutic targets and have prompted the development of TKIs. TKIs are molecules of low molecular mass, which competitively bind at the ATP binding site of the tyrosine kinase catalytic domain (Hartmann et al., 2009). The pharmaceutical industry has developed many TKIs that can block intracellular signaling triggered by oncogenic proteins. Several of these inhibitors are commercially available, such as imatinib (Glivec®), erlotinib (Tarceva®), lapatinib (Tyverb®), sunitinib (Sutent®), sorafenib (Nexavar®) and dasatinib (Sprycel®). However, their use is limited, and resistance or lack of a therapeutic response raised the question of how to improve the response to treatment.
These problems can be overcome by identifying new molecular targets.
Small-molecule inhibitors have been classified according to their binding site and their mechanism of action in three main groups (Table 3). The type I comprises those molecules that bind to the active conformation of a kinase while those grouped as type II prefer inactive conformation. The type III group includes non-ATP competitors or allosteric inhibitors which bind next to the ATP-binding pocket. For a review of the subject see (Roskoski, 2016). Below we present features of some of the TKIs used in my work.
Table 3. Classification of small molecule protein kinase inhibitors. Adapted from (Roskoski, 2016)
Properties Type I Type II Type III
ATP-competitive Yes Yes No
Conformation Active Inactive Allosteric
DFG In Out Variable
Activation segment Out Variable Variable
Drug-kinase Bosutinib-Src, Dasatinib-Abl/Lyn, Ruxolitinib-Src, Tofacitinib-JAK
Bosutinib-Abl Imatinib-Abl/c-Kit, Nilotinib-Abl,
Sunitinib-c-Kit/VEGFR
TAK-733-MEK1
The first group of TKIs includes those molecules that bind to the ATP binding site and recognize the active “DFG-in” conformation of a kinase. Inhibitors of this group mimic the adenine ATP, thus competing with the ATP (Gotink & Verheul, 2010). However, the ATP binding site is very similar from one kinase to another (Y. Liu & Gray, 2006). These types of inhibitors are therefore less specific than those targeting the inactive conformation.
Nevertheless, they may be useful to target several oncogenes at the same time. Among the TKIs that can be used in the clinic, we can find dasatinib, bosutinib, ruxolitinib, tofacitinib, axitinib, erlotinib, gefitinib, pazopanib, vemurafenib, dabrafenib, and crizotinib.
Dasatinib (Sprycel®)
Crystal structure suggests that Dasatinib binds to the active conformation ((Lombardo et al., 2004)) regardless the conformation of the A-loop. Dasatinib is a second-generation Bcr-Abl inhibitor. This inhibitor has a broad spectrum of action as it also inhibits SFK, PDGFR, c-Kit and other tyrosine kinases. The FDA approved dasatinib for the management of CML resistant or intolerant to imatinib. This drug elicits an excellent response rate, independently of the
mutational status of Bcr-Abl (Muller et al., 2009) at low concentrations (Schiffer, 2007).
Dasatinib can block c-Kit mutants of the A-loop including the activating mutant D816V, which is insensitive to most of the TKIs. Because of this, dasatinib has raised high hopes for targeting c-KitD816V in other diseases such as systemic mastocytosis. Unfortunately, preliminary results are disappointing and showed high toxicity (Pardanani, 2013; Verstovsek et al., 2008). An alternative for patients suffering systemic mastocytosis positive for c-KitD816V is midostaurin, a small-molecule inhibiting various kinase including c-Kit (Gotlib et al., 2016).
The ATP binding pocket is formed by the displacement of the DFG motif located at the base of the A-loop. Once bound to the kinase, the type II drugs prevent conformational changes that activate the kinase. They are more specific because they interact with residues of the hydrophobic pocket, which are less conserved than those of the ATP binding site. These hydrophobic interactions cannot occur when the kinase is activated and phosphorylated.
They, therefore, fix the inactive “DFG-out” conformation of the kinase.
Imatinib
In 1992, the pharmaceutical company Novartis® developed a specific inhibitor against BCR-Abl. Imatinib (also known as imatinib mesylate, Gleevec® or Glivec®, or STI-571) is the drug of reference for the treatment of CML patients. It also blocks the kinase activity of other TKRs such as c-Kit (Heinrich et al., 2000) and PDGFR (Buchdunger et al., 2000). Subsequently, it was successfully used to treat GIST patients who have a significant risk for relapse (Demetri et al., 2002; Joensuu et al., 2011). It can also be used in other cancers such as systemic mastocytosis, small cell lung cancer or glioblastomas (Buchdunger et al., 2002; Krystal, 2004; Manley et al., 2002).
Under KitL stimulation, the JMD of c-Kit no longer interacts with the TKD, releasing access to the catalytic pocket (Mol et al., 2003). The crystal structure of the c-KitWT in complex with imatinib shows that the latter is lodged in the catalytic pocket of c-Kit, in a conformation identical to the one adopted in its inactive kinase conformation. By binding to the ATP and hydrophobic back pocket, imatinib competes with and induces the release of the JMD but blocks the A-loop in its inhibitory conformation (Mol et al., 2004). Thus, imatinib can bind to and block the kinase domain, even in the presence of activating JMD mutations. Because of its mode of action, it appears that imatinib is ideal for JMD, but not optimal for all pathologies presenting a c-Kit activating mutation, for example, those affecting the A-loop. In fact,
mutations affecting the A-loop block the receptor in the active conformation, in which A-loop residues can no longer be used to stabilize the binding of imatinib (Foster et al., 2004) The use of imatinib in c-kit inhibition has been particularly relevant in those cases of GIST which have a c-kit activating mutation in the JMD (Demetri, 2001; Fletcher et al., 2002;
Heinrich et al., 2002; Joensuu et al., 2001; Tamborini et al., 2004). A second mutation (T670I) has been found in GIST patients carrying the JMD c-KitV560G mutation, but did not respond to imatinib (Demetri, 2001; Fletcher et al., 2002; Heinrich et al., 2002; Joensuu et al., 2001;
Tamborini et al., 2004). The c-KitT670I mutation deforms the catalytic (ATP) pocket and breaks the hydrogen bonds between c-Kit and imatinib, therefore, conferring insensitivity to imatinib (Tamborini et al., 2006).
At the molecular level, treatment with imatinib inhibits various intracellular signaling pathways involved in cell proliferation and survival. These include Akt (Burchert et al., 2005;
Marley et al., 2004), STAT3 (Coppo et al., 2006; Paner et al., 2003), and β-catenin (L. Zhou et al., 2003). Imatinib effectively inhibits c-KitWT as well as c-Kit carrying mutations in the TMD and the JMD (Akin et al., 2003; Ma et al., 2002; Zermati et al., 2003). However, inhibition of c-KitD816V is only obtained at drug concentrations that are not safe for humans (Akin et al., 2003; Ma et al., 2002). Imatinib is quite safe and well-tolerated by patients when administered at doses inhibiting the JMD mutations. Despite its clinical success, several cases of gained-resistance point to the acquisition of secondary mutations. Indeed, mutations that alter the inactive conformation, more specifically those located in the A-loop (e.g., c-KitD814V) are expected to be less sensitive to imatinib. That’s the new generation drugs have been developed to target active kinases with less conformational restrictions.
Nilotinib (Tasigna)
Nilotinib inhibits Bcr-Abl activity and is used to treat CML patients after failure or intolerance to imatinib. Nilotinib also inhibits PDGFR and c-Kit. In the case of c-Kit, it targets c-Kit mutants of the JMD and the c-KitV654A but not c-KitT670I. Its activity on the mutants of the kinase domain is not defined. Some studies showed that the mutant D816 is resistant to Nilotinib (Roberts et al., 2007), while others show the opposite (Gleixner et al., 2006; von Bubnoff et al., 2005).
Nilotinib has been shown to stabilize GIST patients who are resistant to imatinib and sunitinib (Gramza et al., 2009). Other trials are ongoing, particularly in GIST and melanoma expressing c-Kit.
Sunitinib (Sutent®)
Sunitinib belongs to the type II group of c-Kit inhibitors, but it does not insert into the back cleft of a kinase (reviewed in (Roskoski, 2016)). In addition to c-Kit, sunitinib also targets VEGFR, PDGFR, Ret, and FLT3. Thanks to its action on VEGFR and PDGFR, sunitinib has a strong anti-angiogenic capacity (Kerbel & Folkman, 2002) being a potent antitumor drug. Sunitinib is used as a second-line for GIST patients who are resistant or intolerant to imatinib (Hopkins et al., 2008). This drug effectively blocks mutants of the JMD, as well as secondary mutations that cause insensitivity to imatinib. For example, the c-Kit mutations V654A and T670I at the ATP binding site can be inhibited by sunitinib (Prenen et al., 2006). Like imatinib, sunitinib does not have any effect on c-KitD816V kinase. Primary mutations found in patients with GISTs most often affect the JMD. However, when under treatment, this group of patients often develops resistance to imatinib due to the acquisition of secondary mutations in the c-Kit KD/A-loop. In these cases, sunitinib is prescribed as a second-line of treatment (Hsueh et al., 2013).
The development of imatinib has revolutionized the oncologic field and smoothened the way for targeted therapies. In addition to Bcr-Abl, imatinib also targets c-Kit. However, not all mutant forms of c-Kit are sensitive to imatinib and resistance or intolerance to treatment is a common phenomenon. Dasatinib and other second-generation drugs have overcome this problem. The spectrum of action on the different kinase mutants being quite variable from one drug to another. To date, many molecules that target c-Kit are being used clinically to improve the management of patients. These drugs cover the spectrum of c-Kit mutations, in a way that the activity of each of these mutants can be prevented.
One major problem with ITKs is their failure to remove all the tumor cells. Therefore, it exists the possibility for the remaining cells to develop resistant mutations or evade other therapies.
Thus, the use of ITKs in the management of patients with cancer may lead, in the long term, to the escape of the tumor due to the development of resistance. This resistance is often a consequence of the acquisition of new mutations or potential changes in the tumor microenvironment. Also, although TKIs are more precise than chemotherapies, they inhibit several kinases at the time and are causing undesirable side effects.