Bcr-Abl tyrosine-kinase inhibitors (TKI) are the first-line therapy for most patients with chronic myelogenous leukemia (CML). More than 90% of CML cases are caused by a chromosomal abnormality that results in the formation of a so-called Philadelphia chromosome. This abnormality was discovered by Peter Nowell in 1960 and is a consequence of fusion between the Abelson (Abl) tyrosine kinase gene at chromosome 9 and the break point cluster (Bcr) gene at chromosome 22, resulting in a chimeric oncogene (Bcr-Abl) and a constitutively active Bcr-Abl tyrosine kinase that has been implicated in the pathogenesis of CML. Compounds have been developed to selectively inhibit the tyrosine kinase.
Before the 2001 U.S. Food and Drug Administration (FDA) approval of imatinib, no drugs were available to alter the natural progression of CML. Only cytotoxic drugs such as busulfan, hydroxyurea or interferon-alpha (rIFN-α) were utilized. Even though the first Bcr-Abl TK inhibitor was named "the magic bullet" to cure cancer by Time magazine, a second generation of Bcr-Abl TKI was subsequently developed to combat the initial resistance that emerged.
New forms of resistance can arise as: missense mutations within the Abl kinase domain, over-expression of Bcr-Abl, increased production of transmembrane plasma proteins, or the constitutive activation of downstream signaling molecules such as Src-family kinases.
Bcr-Abl TKIs are also being investigated as potential disease-modifying treatments for Parkinson’s disease. While initial results have shown modest efficacy, further studies involving highly potent representatives of this drug class are necessary.
History CML has a well defined molecular target and relatively selective therapies aimed at that target, which is not the case for the majority of cancers and chemotherapies today. Bcr-Abl was regarded as highly attractive target for drug intervention since the Bcr-Abl fusion gene encodes a constitutively activated kinase. Drug discovery that specifically targeted the ATP binding site of a single kinase was regarded as quite a challenging task since hundreds of protein kinases were known in the human genome. In the presence of TKI the binding of ATP is blocked, phosphorylation is prevented and Bcr-Abl expressing cells either have a selective growth disadvantage or undergo apoptotic cell death.
Due to increasing resistance and intolerance to imatinib, efforts were made to develop new drugs that could inhibit the Bcr-Abl tyrosine kinase. This led to the discovery of second generation drugs. While drug screening was used to develop imatinib, second generation TKI's were developed with a rational drug design approach due to increased knowledge in structural biology of the Bcr-Abl tyrosine kinase.
First generation
Imatinib (STI571) Imatinib (Gleevec) was discovered in 1992 and is regarded as first generation drug since it is the first Bcr-Abl tyrosine kinase inhibitor to be used in the treatment of CML.
Development
In the development of imatinib, the structure of Bcr-Abl tyrosine kinase played a limited role because it was unknown. A high-throughput screening of chemical libraries at Novartis was performed to identify a starting molecule, which was called "Pyrimidine A". This compound served as a lead compound and was then tested and modified to develop imatinib. With a replacement of the imidazole group with a benzamido group, the compound's specificity increased while its activity as a kinase inhibitor remained the same. Subsequently, introducing a methyl substituent ortho to the pyrimidinyl-amino group enhanced the potency.
Binding
Since then crystallographic studies have revealed that imatinib binds to the kinase domain of Abl only when the domain adopts the inactive or "closed" conformation. This is where the glycine-rich, P-binding phosphate loop (P-loop) folds over the ATP binding site and the activation-loop adopts a conformation in which it occludes the substrate binding site and disrupts the ATP phosphate binding site to block the catalytic activity of the enzyme. The shift of the AspPheGly (DFG) triad at the N-terminal end of the activation loop results in the exposure of a binding pocket which can be utilized by inhibitors. This conformation is referred to as DFGout.
Imatinib binds to Abl domain via six hydrogen bond interactions. This stabilizes the imatinib Bcr-Abl complex and prevents ATP from reaching its binding site. The hydrogen bonds involve the pyridine-N and backbone-NH of Met-318, the aminopyrimidine and side chain hydroxyl of Thr-315, the amide-NH and side chain carboxylate of Glu-286, the carbonyl and backbone-NH of Asp-381, the protonated methylpiperazine with the backbone-carbonyl atoms of Ile-360 and His-361. Additionally, a number of van der Waals interactions contribute to binding. A hydrophobic pocket is formed by amino acid residues Ile-293, Leu-298, Leu-354 and Val-379 around the phenyl ring adjacent to the piperazinyl-methyl group of imatinib. At the time of its discovery, in the absence of structural information, no clear explanation for the impressive selectivity of imatinib could be found.
Although first-generation treatment achieved an extremely high response rate and a low relapse rate in CML patients, some patients do experience resistance or intolerance to imatinib.
Drug resistance Drug resistance is the main drive in continuing research and development of Bcr-Abl TKI. Shortly after the introduction of imatinib, investigators began to describe a number of in vitro derived cell lines with resistance to the drug. This was rapidly followed by the clinical description of imatinib resistant cells in patients, which has resulted in efforts to better understand the biology behind these observations. Assessments of therapeutic response of imatinib in patients with CML are based upon meeting hematologic, cytogenic and molecular milestones. Patients that fail to achieve defined responses at predefined time points are described as primarily resistant to therapy, and those losing previously obtained milestones in disease regression are termed secondarily resistant. Before a conclusion is drawn, it is important to consider that retrospective data has shown a high incidence of imatinib non-compliance in CML patients and this could lead to undesired clinical outcomes.
In general, imatinib resistance can be subdivided into Bcr-Abl dependent and independent mechanisms. Bcr-Abl dependent mechanisms include over expression or amplification of the Bcr-Abl gene and point mutations within the Bcr-Abl kinase domain that interfere with imatinib binding. Bcr-Abl independent mechanisms include factors influencing the concentration of imatinib within the cell, for example by alterations in drug influx and efflux and activation of Bcr-Abl independent pathways, such as members of the Src kinase family. Imatinib resistance can also be produced by other mechanisms that will not be mentioned here as the importance of those mechanisms still remain a question due to lack of clinical data.
Bcr-Abl dependent mechanisms of resistance
Bcr-Abl duplication
The first reports of resistance to imatinib described a development of oncogene amplification. That is, the gene that encodes for the pathogenic Bcr-Abl tyrosine kinase is duplicated in the DNA sequence, leading to higher expression of the pathogen. Increasing the imatinib dose could surmount this kind of resistance, provided that severe or intolerable adverse effects are not produced.
Bcr-Abl mutation
Point mutations can cause amino acid substitutions inside the kinase domain of the Bcr-Abl protein and disrupt the binding site of imatinib on the tyrosine kinase, resulting in a loss of sensitivity to the drug. These mutations normally affect the structure of the Bcr-Abl protein, leading either to interruption of critical contact points between the drug and the Bcr-Abl protein or induction of a conformational change, resulting in a protein that imatinib is unable to bind to.
Mutational frequencies appear to increase as the disease, CML, progresses from chronic phase to the blast phase. The most important mutations are the P-loop mutations and the T315I mutation. Mutations on other sites of the kinase have also been reported, for example on the C-helix, SH2 domain, substrate binding site, activation loop and C-terminal lobe. Some of these mutations have clinical significance, but none as much as P-loop and T315I mutations.
T315I mutation
The T315I is a unique mutation because of its resistance to all approved Bcr-Abl inhibitors, prior to ponatinib. It is caused by a single cytosine to thymine (C -> T) base pair substitution at position 944 of the Abl gene (codon '315' of the Abl protein) sequence resulting in amino acid Threonine being substituted by Isoleucine at that position - thus 'T315I'. This substitution eliminates a critical oxygen atom needed for hydrogen bonding between imatinib and the Abl kinase, and also creates steric hindrance to the binding of most TKIs. When discovered, it was estimated that every 6 out of 9 cases of advanced stage CML with imatinib resistance carried this mutation. T315I produces the highest magnitude of resistance of any mutation both to imatinib and second generations TKIs. Ponatinib (Iclusig) by Ariad was approved in 2013 for use as second-line CML treatment, and is the only licensed TKI which binds to the T315I mutated kinase successfully.
P-loop mutations
The structure of Bcr-Abl contains two flexible loops, the ATP-binding P-loop and the activation loop. These loops have specific arrangements in the inactive conformation of Bcr-Abl that stabilize the basal conformation. Mutations in these loops destabilize arrangement of the loops such that the kinase domain cannot assume the inactive conformation required for imatinib binding. Mutations in the P-loop region are the most common, accounting for 36-48% of all mutations. There are clinical data indicating that Bcr-Abl mutations in the P-loop is 70-100 fold less sensitive to imatinib compared with native Bcr-Abl.
Bcr-Abl Independent mechanisms of resistance Additional mechanisms have been postulated to describe resistance seen in various model systems although none have been clearly identified as a sole source of clinical resistance.
Drug efflux caused by P-glycoproteins
Some investigations in cell lines have shown that imatinib resistance may be partly due to an increase in the expression of the P-glycoprotein efflux pump. By utilizing agents that inhibit P-glycoprotein activity imatinib susceptibility has been restored in some cases.
Drug import by organic cation transporter 1
The entry of imatinib into cells is dependent on an organic cation transporter (OCT1). OCT1 plays a significant role in imatinib resistance by inhibiting its influx and thus decreasing the intracellular bioavailability of imatinib. Patients with low expression, activity or polymorphisms of OCT1 had significantly lower intracellular levels of imatinib. The response of patients with low OCT1 activity was significantly dose-dependent. This data indicates that OCT1 activity is an important determinant in the molecular response to imatinib.
Alternative signaling pathway activation
In a few patient groups, resistance may be caused by the activation of other signaling pathways, particularly the Src family kinases. The Src family kinases have been implicated in Bcr-Abl signaling and mediate imatinib resistance by stabilizing the active conformation of Bcr-Abl, a conformation that does not bind imatinib. Furthermore, increasing evidence suggests that Src family kinases are also involved in Bcr-Abl-independent forms of imatinib resistance.
Solutions