Bcr-Abl tyrosine-kinase inhibitor
Encyclopedia
Bcr-Abl tyrosine-kinase inhibitors (TKI) are the first-line therapy for most patients with chronic myelogenous leukemia
(CML). In more than 90% cases CML is caused by chromosomal abnormality resulting in the formation of a so-called Philadelphia chromosome
. This abnormality was discovered by Janet Rowley
in 1972 and is due to fusion between Abelson (Abl
) tyrosine kinase gene at chromosome 9 and break point cluster (Bcr
) gene at chromosome 22, resulting in the 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 that selectively inhibit this tyrosine kinase. Before the U.S. Food and Drug Administration
(FDA) approval of imatinib in 2001 no drugs were used that changed the natural progression of CML, only cytotoxic drugs such as busulfan
, hydroxyurea
or interferon
-alpha (rIFN-α). 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 has been developed as a cause of resistance
to imatinib emerged. New forms of resistance in patients can arise as; missense mutations within the Abl kinase domain
, over-expression of Bcr-Abl, increased production of transmembrane plasma proteins, or constitutive activating of downstream signaling molecules such as Src-family kinases. Most of the drugs are adenosine triphosphate
(ATP)-competitive inhibitors.
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 they 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 rational drug design
approach due to increased knowledge in structural biology
of the Bcr-Abl tyrosine kinase.
, (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.
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 compounds specificity increased while its activity
as a kinase inhibitor remained the same. Subsequently, introducing a methyl subtituent ortho
to the pyrimidinyl-amino group enhanced the potency
.
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 AspPhe
Gly
triad at the N-terminal end of the activation loop results in the exposure of a binding pocket which can be utilized by inhibitors.
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
-285, 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.
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 understand better 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 chronological time points are described as primarily resistant to therapy, and those losing previously obtained milestones in disease regression are termed secondarily resistant. Before conclusion is drawn, it is important to consider that retrospective
data has showed 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.
. 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.
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.
The T315I is a unique mutation because of its resistance to all approved Bcr-Abl inhibitors. 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 (T)hreonine
being substituted by (I)soleucine
at that position - thus 'T315I'. This substitution eliminates a critical oxygen
molecule needed for hydrogen bond
ing 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.
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.
efflux pump. By utilizing agents that inhibit P-glycoprotein activity imatinib susceptibility has been restored in some cases.
). 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-dependant. This data indicates that OCT1 activity is an important determinant in the molecular response to imatinib.
patients may include strategies such as increasing the dose of imatinib or the use of second-generation drugs. Escalation of imatinib-doses has shown to overcome some cases of primary resistance to imatinib, such as Bcr-Abl duplication, but the response is usually short acting. In the case of resistance or intolerance, it could be helpful to test for Bcr-Abl mutations to direct the choice of second line treatment as the variable options have different function profile against the different mechanisms of resistance. Second-generation drugs offer improved potency
and a greater likelihood of success in resistant patients. There is also a growing interest in testing the hypothesis
that administration of multiple Abl kinase inhibitors in early phase patients could be used to delay or prevent the emergence of drug resistant clones
. The combination of two agents targeting different pathways
involved in CML may significantly improve response rates and potentially increase survival.
s.
is a phenylamino-pyrimidine derivative that is structurally related to imatinib. It was developed based on the structure of the Abl-imatinib complex to address the need associated with imatinib intolerance and resistance. Small changes were made on the imatinib molecule to make it more potent
and selective as a Bcr-Abl inhibitor and these changes resulted in the discovery of nilotinib. Nilotinib is a selective Bcr-Abl kinase inhibitor.
Nilotinib is 10-30 fold more potent than imatinib in inhibiting activity of the Bcr-Abl tyrosine kinase and proliferation
of Bcr-Abl expressing cells. The drug effectively inhibits the auto phosphorylation of Bcr-Abl on Tyr
-177 that is involved in CML pathogenesis. Synergistic
activity of imatinib and nilotinib has been reported following coadministration. This might be a result of the fact that the drugs are taken up in cells by different mechanisms: imatinib influx is dependent on OCT1 but nilotinib is not. Nilotinib is also not a substrate for the efflux transporter P-glycoprotein pump, unlike imatinib. Although the two dimensional
molecular structures of these two drugs might look similar, they are dissimilar in terms of spatial
structure and molecular properties.
-N and the backbone NH of Met-318, the anilino
-NH and the side chain OH of Thr-315, the amido-NH and side chain carboxylate of Glu-286 and the amido carbonyl with the backbone NH of the Asp-381. The [4-(3-pyridinyl)-2-pyrimidinyl] anilino segment of nilotinib has close binding interactions with Met-318, Phe-317 and Thr-315 residues of a region within the ATP binding site. The remaining half of the compound extends beyond the Thr-315 gatekeeper residue to bind within an additional pocket. The 3-methylimidazole and trifluoro-methyl groups of nilotinib make important interactions with the Abl kinase domain. These groups also make the shape of nilotinib very different from that of imatinib. Nilotinib also binds to the kinase through a large number of weak van der Waals interactions.
is a thiazol
ylaminopyrimidine developed as the hydrochloride
salt. It was discovered with a program directed towards immunosuppressive drugs and is 325-fold more potent against cells expressing wild type Bcr-Abl than imatinib. Dasatinib is a multi targeted inhibitor of Bcr-Abl and Src family kinases. It also has inhibitory activity against additional downstream kinases.
Dasatinib has some structural elements in common with nilotinib, in particular the juxtaposition of the aminopyrimidine and the carboxamide
groups. The aminothiazole
segment of dasatinib makes a bi-dentate H-bonding interaction with the backbone CO and NH of Met-318 and the amide-NH makes an H-bond with the side chain oxygen of Thr-315.
's structure is based on a quinoline
scaffold and is structurally related to the AstraZeneca quinazoline
template. Src kinase dependent yeast screening led to characterization of a 4-anilino-3-quinolinecarbonitrile
as an Src inhibitor. Combination of the features of this hit and a related compound, and attachment of solubilizing
groups, led to the discovery of bosutinib. It was suggested to be an Abl kinase inhibitor and when tested as such it turned out to be slightly more potent against Abl than Src (IC50
1,4 nM vs. 3,5 nM). Bosutinib's activity was first described in 2001 and it was disclosed as an Abl kinase inhibitor in 2003. At first it was believed that bosutinib was a selective Src kinase inhibitor but now it is known that its kinase inhibition profile is far less restricted than originally thought. Bosutinib inhibits Src, Abl and a wide range of both tyrosine and serine-threonine kinases.
that promotes efflux of foreign molecules from cells. Bosutinib even inhibits these transporter proteins in higher concentrations.
, Inc. announced on September 10, 2010 that ponatinib
, an orally active Bcr-Abl TKI effective against the T315I mutation had been approved for a phase II clinical trial.
The road to discovery can be linked to AP23464, one of the first of Ariad's ATP competitive dual Src/Abl inhibitors. AP23464 was identified using structure base drug design and focused synthetic libraries of trisubstituted purine
analogs. The substance potently inhibits, on nanomolar scale, Src and Bcr-Abl kinases including many common imatinib resistant Bcr-Abl mutations. AP23464 does not inhibit the T315I mutation, however, whereas AP24534 (ponatinib) does.
amide structure, that was known to have a high affinity to the inactive conformation by forming crucial hydrogen bonds and filling hydrophobic pockets on the kinase. Furthermore it was determined that the cyclopentyl
group on the purine core clashed with a glycine rich P-loop in that confirmation and was thus removed from the molecule. Then with in-vitro testing on inhibitory activity and in-vivo oral absorption assays a more lipophilic, amide bound, cyclopropyl
group on C6 on the purine core was found to display both satisfactory pharmacokinetics and efficacy. Finally modifications on the diarylamide side chain by adding imidazole appendages were inspired by then newly released nilotinib structure. Those modifications resulted in what was called AP24163. During this development cycle, Ariad tested several substances against cells transfected with T315I mutated Bcr-Abl kinase and, surprisingly, found AP24163 demonstrated reasonable inhibitory action on top of potent inhibition of native Bcr-Abl.
Following up on that breakthrough Ariad began further research to increase the efficacy of compound AP24163 against the T315I mutation. Docking of the molecule into the ATP binding site of T315I mutated Bcr-Abl kinase revealed that the expected steric clash with isoleucine was not present due to a lesser sterically demanding vinyl
linkage between the purine core and the diarylamide side chain compared to other TKIs. The first step was to try and find an even less sterically demanding structure. First an acetylene
linkage was tested, that resulted in higher potency but unfavorable pharmacokinetics. Later, a more stable 2-butyne
linkage was selected. To achieve this linkage an imidazol[1,2-a]pyridine core was used as a starting material for a Sonogashira
reaction; but the pharmacokinetics were still poor. When developing AP24163, adding a cyclopropane side chain on C8 in the purine core resulted in favorable pharmacokinetics. Several different side chains were then tested but, interestingly, the best results were obtained with no side chain at all; resulting a substance with satisfactory pharmacokinetics, but now with reduced potency against T315I also. The first step in increasing the potency again was to look at other TKI’s. Imatinib has a terminal methyl piperazine group which has been shown to form a hydrogen bond with the carbonyl oxygen atom of residue Ile-360 in the activation loop of the Abl kinase. The piperazine ring is also a common solubilizing group that could further improve the pharmacokinetic properties of the molecule. Those speculations were confirmed with a two-fold increase in inhibitory action against Bcr-Abl T315I mutated kinase and the silver lining was the plasma protein binding
of the substance (named '19a') appeared to have decreased, allowing for smaller doses with the same potency. Whilst '19a' exhibited good oral pharmacokinetics in both mice and rats, it also retained high partition coefficient
at 6.69. So, in attempts to reduce the molecule's lipophilicity further, substitution of a single carbon atom on the imidazo[1,2-a]pyridine core was made; which resulted in what is now known as the compound ponatinib.
core rests in the adenine pocket of the enzyme. The methylphenyl group occupies a hydrophobic pocket behind I315, the ethynyl
linkage forms favorable van der Waals interactions with the amino acid and the trifluoromethyl group binds to a pocket induced by the inactive conformation kinase. Also in the conformation of the kinase that ponatinb rests in, additional favorable van der Waals interactions between the drug and Tyr-253 and Phe-382. Five hydrogen bonds are generated, with the backbone of Met-318 in the hinge region, with the backbone of Asp-381, with the side chain of Glu-286 and the protonated methylpiperazine with the backbone-carbonyl atoms of Ile-360 and His-361.
With this structure ponatinib has been shown to have a relatively broad kinase specificity profile which can probably be linked to the linearity of the linkage section of the molecule. With this linear structure the drug appears to avoid steric clashes with hydrophobic TK gatekeeper residues. Despite, or even because of this, ponatinib is a potent drug and targets not just most of the known mutations on the Bcr-Abl TK but, most importantly of all, T315I. This mutation is emerging as a common pathway to failure of both first and second line treatments. Unlike other T315I targeting inhibitors in development, ponatinib does not target Aurora kinases, which clearly distinguishes it from them and emphasizes the significance of its discovery.
, bromo
and chloro
substituents. Finally a trifluoromethyl group at position 3 was found to give the best results, with approximately 36-fold improvement over imatinib. The addition of a hydrophobic group now needed to be countered to sustain the solubility of the substance. Closer examination of the crystal structure of imatinib-kinase complex revealed Tyr-236 was in close proximity to the pyridine ring of imatinib, suggesting there was little or no room for a larger group there. With that in mind a more hydrophilic
pyrimidine ring was substituted for the pyridine, which was found to increase solubility while leaving efficacy the same or even slightly greater. Finally to improve the hydrogen bonding of the piperazine ring of imatinib with Ile-360 and His-361, pyrrolidine and azetidine derivatives were introduced. The most promising substance from these final modifications was labeled NS-187.
Bafetinib has its place in TKI therapy as it is effective both against most imatinib resistant mutations (not including T315I) and some dasatinib resistant mutations. Bafetinib also has more affinity for Bcr-Abl than nilotinib (but less than dasatinib) but only targets Bcr-Abl and Src family kinases Lck and Lyn; with unrivalled specificity which suggests the probability of fewer adverse effects. CytRx has bafetinb in phase II clinical trial as a treatment for leukemia as of May 2010.
ring. Though it has to be noted this analysis was done with comparing the crystal structure of Abl and dasatinib, which is the inactive conformation of Abl, the knowledge gathered from the docking and structure analysis led to identification of a compound, referred to as substance 14, with a high affinity to Abl.
-benzene falls nicely into a hydrophobic pocket created by Val 256, Ala 253, Lys 271 and Ala 380. Whilst the similar binding properties to those of dasatinib, suggests the possibility of producing Bcr-Abl TKI’s from thiazole cores is real, the question remains open whether this research will just lead to a dasatinib analog or a novel way to inhibit TKs.
Chronic myelogenous leukemia
Chronic myelogenous leukemia , also known as chronic granulocytic leukemia , is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood...
(CML). In more than 90% cases CML is caused by chromosomal abnormality resulting in the formation of a so-called Philadelphia chromosome
Philadelphia chromosome
Philadelphia chromosome or Philadelphia translocation is a specific chromosomal abnormality that is associated with chronic myelogenous leukemia . It is the result of a reciprocal translocation between chromosome 9 and 22, and is specifically designated t...
. This abnormality was discovered by Janet Rowley
Janet Rowley
Janet Davison Rowley is an American human geneticist and the first scientist to identify a chromosomal translocation as the cause of leukemia and other cancers....
in 1972 and is due to fusion between Abelson (Abl
Abl gene
V-abl Abelson murine leukemia viral oncogene homolog 1 also known as ABL1 is a protein that, in humans, is encoded by the ABL1 gene located on chromosome 9.- Function :...
) tyrosine kinase gene at chromosome 9 and break point cluster (Bcr
BCR gene
The BCR gene is one of the two genes in the bcr-abl complex, which is associated with the Philadelphia chromosome.-Pathology:...
) gene at chromosome 22, resulting in the chimeric oncogene
Oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.An oncogene is a gene found in the chromosomes of tumor cells whose activation is associated with the initial and continuing conversion of normal cells into cancer...
Bcr-Abl and a constitutively active Bcr-Abl tyrosine kinase that has been implicated in the pathogenesis
Pathogenesis
The pathogenesis of a disease is the mechanism by which the disease is caused. The term can also be used to describe the origin and development of the disease and whether it is acute, chronic or recurrent...
of CML. Compounds have been developed that selectively inhibit this tyrosine kinase. Before the U.S. Food and Drug Administration
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...
(FDA) approval of imatinib in 2001 no drugs were used that changed the natural progression of CML, only cytotoxic drugs such as busulfan
Busulfan
Busulfan is a cancer drug, in use since 1959.Busulfan is a cell cycle non-specific alkylating antineoplastic agent, in the class of alkyl sulfonates...
, hydroxyurea
Hydroxyurea
Hydroxycarbamide or hydroxyurea is an antineoplastic drug, first synthesized in 1869, used in myeloproliferative disorders, specifically polycythemia vera and essential thrombocythemia...
or interferon
Interferon
Interferons are proteins made and released by host cells in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.IFNs belong to...
-alpha (rIFN-α). Even though the first Bcr-Abl TK inhibitor was named “the magic bullet” to cure cancer by TIME
Time
Time is a part of the measuring system used to sequence events, to compare the durations of events and the intervals between them, and to quantify rates of change such as the motions of objects....
magazine, a second generation of Bcr-Abl TKI has been developed as a cause of resistance
Drug resistance
Drug resistance is the reduction in effectiveness of a drug such as an antimicrobial or an antineoplastic in curing a disease or condition. When the drug is not intended to kill or inhibit a pathogen, then the term is equivalent to dosage failure or drug tolerance. More commonly, the term is used...
to imatinib emerged. New forms of resistance in patients can arise as; missense mutations within the Abl kinase domain
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
, over-expression of Bcr-Abl, increased production of transmembrane plasma proteins, or constitutive activating of downstream signaling molecules such as Src-family kinases. Most of the drugs are adenosine triphosphate
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
(ATP)-competitive inhibitors.
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 interventionPharmacotherapy
Pharmacotherapy is the treatment of disease through the administration of drugs. As such, it is considered part of the larger category of therapy....
since the Bcr-Abl fusion gene
Fusion gene
A fusion gene is a hybrid gene formed from two previously separate genes. It can occur as the result of a translocation, interstitial deletion, or chromosomal inversion...
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
Human genome
The human genome is the genome of Homo sapiens, which is stored on 23 chromosome pairs plus the small mitochondrial DNA. 22 of the 23 chromosomes are autosomal chromosome pairs, while the remaining pair is sex-determining...
. In the presence of TKI the binding of ATP is blocked, phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
is prevented and Bcr-Abl expressing cells either have a selective growth disadvantage or they 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 rational drug design
Drug design
Drug design, also sometimes referred to as rational drug design or structure-based drug design, is the inventive process of finding new medications based on the knowledge of the biological target...
approach due to increased knowledge in structural biology
Structural biology
Structural biology is a branch of molecular biology, biochemistry, and biophysics concerned with the molecular structure of biological macromolecules, especially proteins and nucleic acids, how they acquire the structures they have, and how alterations in their structures affect their function...
of the Bcr-Abl tyrosine kinase.
Imatinib (STI571)
ImatinibImatinib
Imatinib is a drug used to treat certain types of cancer. It is currently marketed by Novartis as Gleevec or Glivec as its mesylate salt, imatinib mesilate . It is used in treating chronic myelogenous leukemia , gastrointestinal stromal tumors and some other diseases...
, (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 structureChemical structure
A chemical structure includes molecular geometry, electronic structure and crystal structure of molecules. Molecular geometry refers to the spatial arrangement of atoms in a molecule and the chemical bonds that hold the atoms together. Molecular geometry can range from the very simple, such as...
of Bcr-Abl tyrosine kinase played a limited role because it was unknown. A high-throughput screening
High-throughput screening
High-throughput screening is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, High-Throughput Screening allows a...
of chemical libraries at Novartis
Novartis
Novartis International AG is a multinational pharmaceutical company based in Basel, Switzerland, ranking number three in sales among the world-wide industry...
was performed to identify a starting molecule, which was called pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
A. This compound served as a lead compound
Lead compound
A lead compound in drug discovery is a chemical compound that has pharmacological or biological activity and whose chemical structure is used as a starting point for chemical modifications in order to improve potency, selectivity, or pharmacokinetic parameters.Lead compounds are often found in...
and was then tested and modified to develop imatinib. With a replacement of the imidazole
Imidazole
Imidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...
group with a benzamido group, the compounds specificity increased while its activity
Activity (chemistry)
In chemical thermodynamics, activity is a measure of the “effective concentration” of a species in a mixture, meaning that the species' chemical potential depends on the activity of a real solution in the same way that it would depend on concentration for an ideal solution.By convention, activity...
as a kinase inhibitor remained the same. Subsequently, introducing a methyl subtituent ortho
Arene substitution patterns
Arene substitution patterns are part of organic chemistry IUPAC nomenclature and pinpoint the position of substituents other than hydrogen in relation to each other on an aromatic hydrocarbon.- Ortho, meta, and para substitution :...
to the pyrimidinyl-amino group enhanced the potency
Potency
Potency may refer to:* Potency , a measure of the activity of a drug in a biological system* Virility* Potency is a measure of the differentiation potential of stem cells...
.
Binding
Since then crystallographic studiesX-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
have revealed that imatinib binds to the kinase domain
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
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
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
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 AspPhe
Phenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
Gly
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
triad at the N-terminal end of the activation loop results in the exposure of a binding pocket which can be utilized by inhibitors.
Imatinib binds to Abl domain via six hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
interactions. This stabilizes the imatinib Bcr-Abl complex and prevents ATP from reaching its binding site. The hydrogen bonds involve the pyridine
Pyridine
Pyridine is a basic heterocyclic organic compound with the chemical formula C5H5N. It is structurally related to benzene, with one C-H group replaced by a nitrogen atom...
-N and backbone
Backbone chain
In polymer science, the backbone chain or main chain of a polymer is the series of covalently bonded atoms that together create the continuous chain of the molecule....
-NH of Met
Methionine
Methionine is an α-amino acid with the chemical formula HO2CCHCH2CH2SCH3. This essential amino acid is classified as nonpolar. This amino-acid is coded by the codon AUG, also known as the initiation codon, since it indicates mRNA's coding region where translation into protein...
-318, the aminopyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
and side chain hydroxyl
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...
of Thr
Threonine
Threonine is an α-amino acid with the chemical formula HO2CCHCHCH3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar...
-315, the amide-NH and side chain carboxylate of Glu
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
-285, the carbonyl and backbone-NH of Asp
Aspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
-381, the protonated methylpiperazine
Piperazine
Piperazine is an organic compound that consists of a six-membered ring containing two opposing nitrogen atoms. Piperazine exists as small alkaline deliquescent crystals with a saline taste....
with the backbone-carbonyl atoms of Ile
Isoleucine
Isoleucine is an α-amino acid with the chemical formula HO2CCHCHCH2CH3. It is an essential amino acid, which means that humans cannot synthesize it, so it must be ingested. Its codons are AUU, AUC and AUA....
-360 and His
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
-361. Additionally, a number of van der Waals
Van der Waals force
In physical chemistry, the van der Waals force , named after Dutch scientist Johannes Diderik van der Waals, is the sum of the attractive or repulsive forces between molecules other than those due to covalent bonds or to the electrostatic interaction of ions with one another or with neutral...
interactions contribute to binding. A hydrophobic pocket is formed by amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
residues Ile-293, Leu
Leucine
Leucine is a branched-chain α-amino acid with the chemical formula HO2CCHCH2CH2. Leucine is classified as a hydrophobic amino acid due to its aliphatic isobutyl side chain. It is encoded by six codons and is a major component of the subunits in ferritin, astacin and other 'buffer' proteins...
-298, Leu-354 and Val
Valine
Valine is an α-amino acid with the chemical formula HO2CCHCH2. L-Valine is one of 20 proteinogenic amino acids. Its codons are GUU, GUC, GUA, and GUG. This essential amino acid is classified as nonpolar...
-379 around the phenyl ring adjacent to the piperazinyl
Piperazine
Piperazine is an organic compound that consists of a six-membered ring containing two opposing nitrogen atoms. Piperazine exists as small alkaline deliquescent crystals with a saline taste....
-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
Drug intolerance
Drug intolerance or drug sensitivity is a lower threshold to the normal pharmacologic action of a drug. It is not to be confused with drug allergy. Drug intolerance is uncommon and idiopathic, thus extremely difficult to predict except in persons with a prior history or a family history of...
to imatinib.
Drug resistance
Drug resistanceDrug resistance
Drug resistance is the reduction in effectiveness of a drug such as an antimicrobial or an antineoplastic in curing a disease or condition. When the drug is not intended to kill or inhibit a pathogen, then the term is equivalent to dosage failure or drug tolerance. More commonly, the term is used...
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
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
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 understand better 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 chronological time points are described as primarily resistant to therapy, and those losing previously obtained milestones in disease regression are termed secondarily resistant. Before conclusion is drawn, it is important to consider that retrospective
Retrospective
Retrospective generally means to take a look back at events that already have taken place. For example, the term is used in medicine, describing a look back at a patient's medical history or lifestyle.-Music:...
data has showed a high incidence of imatinib non-compliance
Compliance (medicine)
In medicine, compliance describes the degree to which a patient correctly follows medical advice...
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 duplication
The first reports of resistance to imatinib described a development of oncogene amplificationGene duplication
Gene duplication is any duplication of a region of DNA that contains a gene; it may occur as an error in homologous recombination, a retrotransposition event, or duplication of an entire chromosome.The second copy of the gene is often free from selective pressure — that is, mutations of it have no...
. That is, the gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
that encodes for the pathogenic Bcr-Abl tyrosine kinase is duplicated in the DNA sequence
DNA sequence
The sequence or primary structure of a nucleic acid is the composition of atoms that make up the nucleic acid and the chemical bonds that bond those atoms. Because nucleic acids, such as DNA and RNA, are unbranched polymers, this specification is equivalent to specifying the sequence of...
, 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
CML
- Computing :* Cache Meta Language, a language for configuring web server caching* Chemical Markup Language, a representation of chemistry using XML* Column Managed Lengths, a representation of data in columns...
, progresses from chronic phase
Chronic phase chronic myelogenous leukemia
Chronic phase chronic myelogenous leukemia is a phase of chronic myelogenous leukemia in which 5% or fewer of the cells in the blood and bone marrow are blast cells . This phase may last from several months to several years, and there may be no symptoms of leukemia.- External links :* entry in the...
to the blast phase
Blastic phase chronic myelogenous leukemia
Blastic phase chronic myelogenous leukemia is a phase of chronic myelogenous leukemia in which more than 30% of the cells in the blood or bone marrow are blast cells immature blood cells). When tiredness, fever, and an enlarged spleen occur during the blastic phase, it is called blast crisis.-...
. 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
Helix
A helix is a type of smooth space curve, i.e. a curve in three-dimensional space. It has the property that the tangent line at any point makes a constant angle with a fixed line called the axis. Examples of helixes are coil springs and the handrails of spiral staircases. A "filled-in" helix – for...
, SH2 domain
SH2 domain
The SH2 domain is a structurally conserved protein domain contained within the Src oncoprotein and in many other intracellular signal-transducing proteins...
, 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. It is caused by a single cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
to thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
(C -> T) base pair
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
substitution at position 944 of the Abl gene (codon '315' of the Abl protein) sequence resulting in amino acid (T)hreonine
Threonine
Threonine is an α-amino acid with the chemical formula HO2CCHCHCH3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar...
being substituted by (I)soleucine
Isoleucine
Isoleucine is an α-amino acid with the chemical formula HO2CCHCHCH2CH3. It is an essential amino acid, which means that humans cannot synthesize it, so it must be ingested. Its codons are AUU, AUC and AUA....
at that position - thus 'T315I'. This substitution eliminates a critical oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
molecule needed for hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
ing between imatinib and the Abl kinase, and also creates steric hindrance
Steric effects
Steric effects arise from the fact that each atom within a molecule occupies a certain amount of space. If atoms are brought too close together, there is an associated cost in energy due to overlapping electron clouds , and this may affect the molecule's preferred shape and reactivity.-Steric...
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.
-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-glycoproteinP-glycoprotein
P-glycoprotein 1 also known as multidrug resistance protein 1 or ATP-binding cassette sub-family B member 1 or cluster of differentiation 243 is a glycoprotein that in humans is encoded by the ABCB1 gene...
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 (OCT1SLC22A1
Solute carrier family 22 member 1 is a protein that in humans is encoded by the SLC22A1 gene.-Further reading:...
). 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-dependant. 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
The treatment options for imatinib resistant or intolerant CMLCML
- Computing :* Cache Meta Language, a language for configuring web server caching* Chemical Markup Language, a representation of chemistry using XML* Column Managed Lengths, a representation of data in columns...
patients may include strategies such as increasing the dose of imatinib or the use of second-generation drugs. Escalation of imatinib-doses has shown to overcome some cases of primary resistance to imatinib, such as Bcr-Abl duplication, but the response is usually short acting. In the case of resistance or intolerance, it could be helpful to test for Bcr-Abl mutations to direct the choice of second line treatment as the variable options have different function profile against the different mechanisms of resistance. Second-generation drugs offer improved potency
Potency (pharmacology)
In the field of pharmacology, potency is a measure of drug activity expressed in terms of the amount required to produce an effect of given intensity. A highly potent drug evokes a larger response at low concentrations, while a drug of lower potency evokes a small response at low concentrations...
and a greater likelihood of success in resistant patients. There is also a growing interest in testing the hypothesis
Hypothesis
A hypothesis is a proposed explanation for a phenomenon. The term derives from the Greek, ὑποτιθέναι – hypotithenai meaning "to put under" or "to suppose". For a hypothesis to be put forward as a scientific hypothesis, the scientific method requires that one can test it...
that administration of multiple Abl kinase inhibitors in early phase patients could be used to delay or prevent the emergence of drug resistant clones
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
. The combination of two agents targeting different pathways
Metabolic pathway
In biochemistry, metabolic pathways are series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by a series of chemical reactions. Enzymes catalyze these reactions, and often require dietary minerals, vitamins, and other cofactors in order to function...
involved in CML may significantly improve response rates and potentially increase survival.
Second generation drugs
Second generation drugs are intended to have decreased resistance and intolerance than imatinib. Second generation drugs that are currently marketed are nilotinib and dasatinib and more drugs are being tested in clinical trialClinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...
s.
Nilotinib (AMN107)
Development
NilotinibNilotinib
Nilotinib , in the form of the hydrochloride monohydrate salt, is a tyrosine kinase inhibitor approved for the treatment of chronic myelogenous leukemia.-Uses:...
is a phenylamino-pyrimidine derivative that is structurally related to imatinib. It was developed based on the structure of the Abl-imatinib complex to address the need associated with imatinib intolerance and resistance. Small changes were made on the imatinib molecule to make it more potent
Potency (pharmacology)
In the field of pharmacology, potency is a measure of drug activity expressed in terms of the amount required to produce an effect of given intensity. A highly potent drug evokes a larger response at low concentrations, while a drug of lower potency evokes a small response at low concentrations...
and selective as a Bcr-Abl inhibitor and these changes resulted in the discovery of nilotinib. Nilotinib is a selective Bcr-Abl kinase inhibitor.
Nilotinib is 10-30 fold more potent than imatinib in inhibiting activity of the Bcr-Abl tyrosine kinase and proliferation
Cell growth
The term cell growth is used in the contexts of cell development and cell division . When used in the context of cell division, it refers to growth of cell populations, where one cell grows and divides to produce two "daughter cells"...
of Bcr-Abl expressing cells. The drug effectively inhibits the auto phosphorylation of Bcr-Abl on Tyr
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
-177 that is involved in CML pathogenesis. Synergistic
Synergy
Synergy may be defined as two or more things functioning together to produce a result not independently obtainable.The term synergy comes from the Greek word from , , meaning "working together".-Definitions and usages:...
activity of imatinib and nilotinib has been reported following coadministration. This might be a result of the fact that the drugs are taken up in cells by different mechanisms: imatinib influx is dependent on OCT1 but nilotinib is not. Nilotinib is also not a substrate for the efflux transporter P-glycoprotein pump, unlike imatinib. Although the two dimensional
Two-dimensional space
- Details :Bi-dimensional space is a geometric model of the planar projection of the physical universe in which we live.The two dimensions are commonly called length and width .Both directions lies in the same plane....
molecular structures of these two drugs might look similar, they are dissimilar in terms of spatial
Three-dimensional space
Three-dimensional space is a geometric 3-parameters model of the physical universe in which we live. These three dimensions are commonly called length, width, and depth , although any three directions can be chosen, provided that they do not lie in the same plane.In physics and mathematics, a...
structure and molecular properties.
Binding
Nilotinib binds to the inactive conformation of the Abl kinase domain, largely through lipophilic interactions and thus blocks its catalytic activity. Nilotinib binds to the kinase domain by making four hydrogen bond interactions involving the pyridylPyridine
Pyridine is a basic heterocyclic organic compound with the chemical formula C5H5N. It is structurally related to benzene, with one C-H group replaced by a nitrogen atom...
-N and the backbone NH of Met-318, the anilino
Aniline
Aniline, phenylamine or aminobenzene is an organic compound with the formula C6H5NH2. Consisting of a phenyl group attached to an amino group, aniline is the prototypical aromatic amine. Being a precursor to many industrial chemicals, its main use is in the manufacture of precursors to polyurethane...
-NH and the side chain OH of Thr-315, the amido-NH and side chain carboxylate of Glu-286 and the amido carbonyl with the backbone NH of the Asp-381. The [4-(3-pyridinyl)-2-pyrimidinyl] anilino segment of nilotinib has close binding interactions with Met-318, Phe-317 and Thr-315 residues of a region within the ATP binding site. The remaining half of the compound extends beyond the Thr-315 gatekeeper residue to bind within an additional pocket. The 3-methylimidazole and trifluoro-methyl groups of nilotinib make important interactions with the Abl kinase domain. These groups also make the shape of nilotinib very different from that of imatinib. Nilotinib also binds to the kinase through a large number of weak van der Waals interactions.
Resistance
Nilotinib has shown effect against most mutations (32/33) that are associated with imatinib resistance but the T315I mutant remains resistant to nilotinib. Its ineffectiveness against the T315I mutant seems to be a consequence of the loss of an H-bond interaction between threonine-O and aniline-NH on nilotinib and a steric clash between the isoleucine-methyl group and 2-methylphenyl phenyl group of nilotinib. On the other hand, resistance to nilotinib is associated with a limited spectrum of Bcr-Abl kinase mutations that mostly affect the P-loop and T315I. However all mutations except T315I were effectively suppressed by increasing nilotinib concentration. Although nilotinib is more potent than imatinib it is possible that its specific mode of binding to Abl may make other sites vulnerable to new kinds of drug resistance.Dasatinib (BMS-345825)
Development
DasatinibDasatinib
Dasatinib, previously known as BMS-354825, is a cancer drug produced by Bristol-Myers Squibb and sold under the trade name Sprycel. Dasatinib is an oral multi- BCR/ABL and Src family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukemia after imatinib treatment...
is a thiazol
Thiazole
Thiazole, or 1,3-thiazole, is a heterocyclic compound that contains both sulfur and nitrogen; the term 'thiazole' also refers to a large family of derivatives. Thiazole itself is a pale yellow liquid with a pyridine-like odor and the molecular formula C3H3NS...
ylaminopyrimidine developed as the hydrochloride
Hydrochloride
In chemistry, hydrochlorides are salts resulting, or regarded as resulting, from the reaction of hydrochloric acid with an organic base . This is also known as muriate, derived from hydrochloric acid's other name: muriatic acid....
salt. It was discovered with a program directed towards immunosuppressive drugs and is 325-fold more potent against cells expressing wild type Bcr-Abl than imatinib. Dasatinib is a multi targeted inhibitor of Bcr-Abl and Src family kinases. It also has inhibitory activity against additional downstream kinases.
Binding
Dasatinib binds to Abl with less stringent conformational requirements than imatinib so it exhibits increased potency but reduced selectivity compared to imatinib. Dasatinib exclusively binds the active conformation of Abl kinase, contrary to most TKIs. Compounds that target the active conformation have been identified but the binding site in all the hundreds of human protein kinases is very similar. Therefore, there is a considerably greater scope for dissimilarities between the inactive conformations so the efforts to discover highly selective kinase inhibitors are being directed towards molecules that bind to the inactive conformation.Dasatinib has some structural elements in common with nilotinib, in particular the juxtaposition of the aminopyrimidine and the carboxamide
Carboxamide
In organic chemistry carboxamides are functional groups with the general structure R-CO-NH2 with R as an organic substituent.Two amino acids, asparagine and glutamine, have a carboxamide group in them....
groups. The aminothiazole
Aminothiazole
2-Aminothiazole is a heterocyclic amine with odor similar to pyridine, soluble in water, alcohol and ether. It is a beginning point for synthesis of many compounds including sulfur drugs, biocides, fungicides, dyes and chemical reaction accelerators. 2-Aminothiazole can be used as a thyroid...
segment of dasatinib makes a bi-dentate H-bonding interaction with the backbone CO and NH of Met-318 and the amide-NH makes an H-bond with the side chain oxygen of Thr-315.
Resistance
Since dasatinib is an inhibitor of Src family kinases, it can overcome resistance due to Src family kinase activation. Because it does not bind to Bcr-Abl with the same stringent conformational requirements as imatinib, it can inhibit all Bcr-Abl kinase domain mutants except for T315I. Dasatinib is also not a substrate of multidrug P-glycoprotein efflux pumps like imatinib. Because of this dasatinib may be active in some patients after failure with both imatinib and nilotinib. Although dasatinib is much more potent than imatinib it is possible, like with nilotinib, that its specific mode of binding to Abl may lead to new vulnerable sites that could confer new kinds of drug resistance. Mutations have been found on Phe317 so that is a potential vulnerable site for this drug.Drugleads
While the first and second generations TKIs are delivering promising results, new mutations of Bcr-Abl TK are still being discovered and some old go unconquered. Those mutations, new and old, are the drive for pharmaceutical companies to continue the search for novel ways to treat patients with CML. In 2010 there were quite a few drugs in development who are either refining current treatments or promising great improvements in efficacy.Development
BosutinibBosutinib
Bosutinib is a tyrosine kinase inhibitor undergoing research for use in the treatment of cancer.It is being developed by Pfizer.-External links:* http://www.eurekalert.org/pub_releases/2007-12/uotm-epi120707.php...
's structure is based on a quinoline
Quinoline
Quinoline is a heterocyclic aromatic organic compound. It has the formula C9H7N and is a colourless hygroscopic liquid with a strong odour. Aged samples, if exposed to light, become yellow and later brown...
scaffold and is structurally related to the AstraZeneca quinazoline
Quinazoline
Quinazoline is a compound made up of two fused six-membered simple aromatic rings, a benzene ring and a pyrimidine ring. Its chemical formula is C8H6N2. Quinazoline is yellow and crystalline...
template. Src kinase dependent yeast screening led to characterization of a 4-anilino-3-quinolinecarbonitrile
Nitrile
A nitrile is any organic compound that has a -C≡N functional group. The prefix cyano- is used interchangeably with the term nitrile in industrial literature. Nitriles are found in many useful compounds, one example being super glue .Inorganic compounds containing the -C≡N group are not called...
as an Src inhibitor. Combination of the features of this hit and a related compound, and attachment of solubilizing
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
groups, led to the discovery of bosutinib. It was suggested to be an Abl kinase inhibitor and when tested as such it turned out to be slightly more potent against Abl than Src (IC50
IC50
The half maximal inhibitory concentration is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance is needed to inhibit a given biological process by half...
1,4 nM vs. 3,5 nM). Bosutinib's activity was first described in 2001 and it was disclosed as an Abl kinase inhibitor in 2003. At first it was believed that bosutinib was a selective Src kinase inhibitor but now it is known that its kinase inhibition profile is far less restricted than originally thought. Bosutinib inhibits Src, Abl and a wide range of both tyrosine and serine-threonine kinases.
Resistance
Bosutinib inhibited cells expressing a variety of mutations, some of which led to imatinib resistance, but the T315 mutation was completely resistant to bosutinib. In contrast to imatinib, nilotinib and dasatinib, bosutinib is not an efficient substrate for multidrug resistance (MDR) transportersP-glycoprotein
P-glycoprotein 1 also known as multidrug resistance protein 1 or ATP-binding cassette sub-family B member 1 or cluster of differentiation 243 is a glycoprotein that in humans is encoded by the ABCB1 gene...
that promotes efflux of foreign molecules from cells. Bosutinib even inhibits these transporter proteins in higher concentrations.
Ponatinib (AP24534)
ARIAD PharmaceuticalsARIAD Pharmaceuticals
ARIAD Pharmaceuticals, Inc. is a small pharmaceutical company working on cancer drugs. The company is located in Cambridge, Massachusetts. It was established in 1991 by Larry Bock, David Blech and Dr. Harvery Berger...
, Inc. announced on September 10, 2010 that ponatinib
Ponatinib
Ponatinib is an experimental oral drug candidate for the treatment of chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia . It is a multi-targeted tyrosine-kinase inhibitor. Some forms of CML, those that have the T315I mutation, are resistant to current...
, an orally active Bcr-Abl TKI effective against the T315I mutation had been approved for a phase II clinical trial.
The road to discovery can be linked to AP23464, one of the first of Ariad's ATP competitive dual Src/Abl inhibitors. AP23464 was identified using structure base drug design and focused synthetic libraries of trisubstituted purine
Purine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
analogs. The substance potently inhibits, on nanomolar scale, Src and Bcr-Abl kinases including many common imatinib resistant Bcr-Abl mutations. AP23464 does not inhibit the T315I mutation, however, whereas AP24534 (ponatinib) does.
Development
Ariad used the highly potent druglead, AP23464 to further investigate inhibitory possibilities of purine cored templates for dual Src/Abl inhibitors. First, searching for substances effective on the inactive conformation of Abl, the side chain bound to the nitrogen on the purine core was replaced with a diarylAryl
In the context of organic molecules, aryl refers to any functional group or substituent derived from an aromatic ring, be it phenyl, naphthyl, thienyl, indolyl, etc....
amide structure, that was known to have a high affinity to the inactive conformation by forming crucial hydrogen bonds and filling hydrophobic pockets on the kinase. Furthermore it was determined that the cyclopentyl
Cyclopentane
Cyclopentane is a highly flammable alicyclic hydrocarbon with chemical formula 510 and CAS number 287-92-3, consisting of a ring of five carbon atoms each bonded with two hydrogen atoms above and below the plane. It occurs as a colorless liquid with a petrol-like odor. Its melting point is −94 °C...
group on the purine core clashed with a glycine rich P-loop in that confirmation and was thus removed from the molecule. Then with in-vitro testing on inhibitory activity and in-vivo oral absorption assays a more lipophilic, amide bound, cyclopropyl
Cyclopropane
Cyclopropane is a cycloalkane molecule with the molecular formula C3H6, consisting of three carbon atoms linked to each other to form a ring, with each carbon atom bearing two hydrogen atoms...
group on C6 on the purine core was found to display both satisfactory pharmacokinetics and efficacy. Finally modifications on the diarylamide side chain by adding imidazole appendages were inspired by then newly released nilotinib structure. Those modifications resulted in what was called AP24163. During this development cycle, Ariad tested several substances against cells transfected with T315I mutated Bcr-Abl kinase and, surprisingly, found AP24163 demonstrated reasonable inhibitory action on top of potent inhibition of native Bcr-Abl.
Following up on that breakthrough Ariad began further research to increase the efficacy of compound AP24163 against the T315I mutation. Docking of the molecule into the ATP binding site of T315I mutated Bcr-Abl kinase revealed that the expected steric clash with isoleucine was not present due to a lesser sterically demanding vinyl
Vinyl
A vinyl compound is any organic compound that contains a vinyl group ,which are derivatives of ethene, CH2=CH2, with one hydrogen atom replaced with some other group...
linkage between the purine core and the diarylamide side chain compared to other TKIs. The first step was to try and find an even less sterically demanding structure. First an acetylene
Acetylene
Acetylene is the chemical compound with the formula C2H2. It is a hydrocarbon and the simplest alkyne. This colorless gas is widely used as a fuel and a chemical building block. It is unstable in pure form and thus is usually handled as a solution.As an alkyne, acetylene is unsaturated because...
linkage was tested, that resulted in higher potency but unfavorable pharmacokinetics. Later, a more stable 2-butyne
Dimethylacetylene
Dimethylacetylene is an alkyne with chemical formula CH3C≡CCH3. Produced artificially, it is a colorless, volatile, pungent liquid at standard temperature and pressure....
linkage was selected. To achieve this linkage an imidazol[1,2-a]pyridine core was used as a starting material for a Sonogashira
Sonogashira coupling
In organic chemistry, a Sonogashira coupling is a coupling reaction of terminal alkynes with aryl or vinyl halides. This reaction was first reported by Kenkichi Sonogashira and Nobue Hagihara in 1975.-Catalyst:...
reaction; but the pharmacokinetics were still poor. When developing AP24163, adding a cyclopropane side chain on C8 in the purine core resulted in favorable pharmacokinetics. Several different side chains were then tested but, interestingly, the best results were obtained with no side chain at all; resulting a substance with satisfactory pharmacokinetics, but now with reduced potency against T315I also. The first step in increasing the potency again was to look at other TKI’s. Imatinib has a terminal methyl piperazine group which has been shown to form a hydrogen bond with the carbonyl oxygen atom of residue Ile-360 in the activation loop of the Abl kinase. The piperazine ring is also a common solubilizing group that could further improve the pharmacokinetic properties of the molecule. Those speculations were confirmed with a two-fold increase in inhibitory action against Bcr-Abl T315I mutated kinase and the silver lining was the plasma protein binding
Plasma protein binding
A drug's efficiency may be affected by the degree to which it binds to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Common blood proteins that drugs bind to are human serum albumin, lipoprotein, glycoprotein, α, β‚ and γ...
of the substance (named '19a') appeared to have decreased, allowing for smaller doses with the same potency. Whilst '19a' exhibited good oral pharmacokinetics in both mice and rats, it also retained high partition coefficient
Partition coefficient
In chemistry and the pharmaceutical sciences, a partition- or distribution coefficient is the ratio of concentrations of a compound in the two phases of a mixture of two immiscible solvents at equilibrium. The terms "gas/liquid partition coefficient" and "air/water partition coefficient" are...
at 6.69. So, in attempts to reduce the molecule's lipophilicity further, substitution of a single carbon atom on the imidazo[1,2-a]pyridine core was made; which resulted in what is now known as the compound ponatinib.
Binding
X-ray crystallographic analysis of ponatinib and T315I Bcr-Abl mutated kinase display that the imidazo[1,2b]pyridazinePyridazine
Pyridazine is a heteroaromatic organic compound with the molecular formula C4H4N2, sometimes called 1,2-diazine. It contains a six-membered ring with two adjacent nitrogen atoms. It is a colorless liquid with a boiling point of 208 °C....
core rests in the adenine pocket of the enzyme. The methylphenyl group occupies a hydrophobic pocket behind I315, the ethynyl
Ethynyl radical
The ethynyl radical, with chemical formula C2H, is an abundant interstellar polyatomic molecule that does not occur naturally on Earth. It was first observed by electron spin resonance isolated in a solid argon matrix at liquid helium temperatures in 1963 by Cochran and coworkers at the Johns...
linkage forms favorable van der Waals interactions with the amino acid and the trifluoromethyl group binds to a pocket induced by the inactive conformation kinase. Also in the conformation of the kinase that ponatinb rests in, additional favorable van der Waals interactions between the drug and Tyr-253 and Phe-382. Five hydrogen bonds are generated, with the backbone of Met-318 in the hinge region, with the backbone of Asp-381, with the side chain of Glu-286 and the protonated methylpiperazine with the backbone-carbonyl atoms of Ile-360 and His-361.
With this structure ponatinib has been shown to have a relatively broad kinase specificity profile which can probably be linked to the linearity of the linkage section of the molecule. With this linear structure the drug appears to avoid steric clashes with hydrophobic TK gatekeeper residues. Despite, or even because of this, ponatinib is a potent drug and targets not just most of the known mutations on the Bcr-Abl TK but, most importantly of all, T315I. This mutation is emerging as a common pathway to failure of both first and second line treatments. Unlike other T315I targeting inhibitors in development, ponatinib does not target Aurora kinases, which clearly distinguishes it from them and emphasizes the significance of its discovery.
Bafetinib (INNO-406)
With the emerging resistance to imatinib treatment after its launch alternative treatment was highly sought after. Bafetinib was the offspring of an attempt to create a more potent drug than imatinib, with efficacy against various point mutations in the Bcr-Abl kinase, with fewer adverse effects and with narrower kinase spectra, namely just Lyn and Bcr-Abl.Development
In the search for a substance that fit the criteria mentioned, the crystal structure of imatinib bound to Abl was examined. This revealed a hydrophobic pocket around the phenyl ring adjacent to the piperazinylmethyl group of imatinib. Attempts to utilize this pocket to increase efficacy led to the addition of various hydrophobic groups including single fluoroFluorine
Fluorine is the chemical element with atomic number 9, represented by the symbol F. It is the lightest element of the halogen column of the periodic table and has a single stable isotope, fluorine-19. At standard pressure and temperature, fluorine is a pale yellow gas composed of diatomic...
, bromo
Bromine
Bromine ") is a chemical element with the symbol Br, an atomic number of 35, and an atomic mass of 79.904. It is in the halogen element group. The element was isolated independently by two chemists, Carl Jacob Löwig and Antoine Jerome Balard, in 1825–1826...
and chloro
Chlorine
Chlorine is the chemical element with atomic number 17 and symbol Cl. It is the second lightest halogen, found in the periodic table in group 17. The element forms diatomic molecules under standard conditions, called dichlorine...
substituents. Finally a trifluoromethyl group at position 3 was found to give the best results, with approximately 36-fold improvement over imatinib. The addition of a hydrophobic group now needed to be countered to sustain the solubility of the substance. Closer examination of the crystal structure of imatinib-kinase complex revealed Tyr-236 was in close proximity to the pyridine ring of imatinib, suggesting there was little or no room for a larger group there. With that in mind a more hydrophilic
Hydrophile
A hydrophile, from the Greek "water" and φιλια "love," is a molecule or other molecular entity that is attracted to, and tends to be dissolved by water. A hydrophilic molecule or portion of a molecule is one that has a tendency to interact with or be dissolved by, water and other polar substances...
pyrimidine ring was substituted for the pyridine, which was found to increase solubility while leaving efficacy the same or even slightly greater. Finally to improve the hydrogen bonding of the piperazine ring of imatinib with Ile-360 and His-361, pyrrolidine and azetidine derivatives were introduced. The most promising substance from these final modifications was labeled NS-187.
Binding
Due to the structural similarities of imatinib and bafetinib, their binding to Bcr-Abl is also quite similar. The only notable difference come from the hydrophobic interaction between the trifluoromethyl group and the hydrophobic pocket created by Ile-293, Leu-298, Leu-354, and Val-379. This group can also be linked to bafetinib's specificity for Lyn, as the binding site there is almost identical to that on Bcr-Abl.Bafetinib has its place in TKI therapy as it is effective both against most imatinib resistant mutations (not including T315I) and some dasatinib resistant mutations. Bafetinib also has more affinity for Bcr-Abl than nilotinib (but less than dasatinib) but only targets Bcr-Abl and Src family kinases Lck and Lyn; with unrivalled specificity which suggests the probability of fewer adverse effects. CytRx has bafetinb in phase II clinical trial as a treatment for leukemia as of May 2010.
1,3,4 thiadiazole derivatives
Some interest has been with thiazol and thiadiazole derivatives and their ability to inhibit Bcr-Abl TKs.Development
One Italian research group discovered through digital screening that commercially available thiadiazole derivatives displayed moderate inhibitory action on both Abl and Src kinases. Using a 1,3,4 thiadiazol core and trying different groups or molecules on the benzene rings, several different substances with inhibitory properties were produced. The flexibility of the core allowed a number of conformations of the substances to bind to the ATP site of the Abl kinase, though all of them bound to the kinase’s active form. Further study of the binding showed that the position of the sulfur that binds to the toluene structure played an important role in regard to Abl binding and also that only one of the nitrogen's one thiadiazole formed a hydrogen bond. Furthermore, computer analysis of the structure showed the amide connected benzene-ketone could be substituted for a more favorable thiopheneThiophene
Thiophene is a heterocyclic compound with the formula C4H4S. Consisting of a flat five-membered ring, it is aromatic as indicated by its extensive substitution reactions. Related to thiophene are benzothiophene and dibenzothiophene, containing the thiophene ring fused with one and two benzene...
ring. Though it has to be noted this analysis was done with comparing the crystal structure of Abl and dasatinib, which is the inactive conformation of Abl, the knowledge gathered from the docking and structure analysis led to identification of a compound, referred to as substance 14, with a high affinity to Abl.
Binding
The binding of substance 14 is partly similar to dasatinib, the aminothiazole segment of substance 14 makes a bi-dentate H-bonding interaction with the backbone CO and NH of Met-318 while the methoxyMethoxy
In chemistry , methoxy refers to the functional group consisting of a methyl group bound to oxygen. This alkoxy group has the formula O–CH3.The word is used in organic nomenclature usually to describe an ether...
-benzene falls nicely into a hydrophobic pocket created by Val 256, Ala 253, Lys 271 and Ala 380. Whilst the similar binding properties to those of dasatinib, suggests the possibility of producing Bcr-Abl TKI’s from thiazole cores is real, the question remains open whether this research will just lead to a dasatinib analog or a novel way to inhibit TKs.
Summary
| Drug | Structure | H-bonds | H-bonding amino acids | Binding confirmation | Discovery | Status on November 4, 2010 |
|---|---|---|---|---|---|---|
| Imatinib Imatinib Imatinib is a drug used to treat certain types of cancer. It is currently marketed by Novartis as Gleevec or Glivec as its mesylate salt, imatinib mesilate . It is used in treating chronic myelogenous leukemia , gastrointestinal stromal tumors and some other diseases... (STI571) |
6 | Met-318, Thr-315, Glu-286, Asp-381, Ile-380, His-361 | Inactive | Drug screening | Marketed as first line therapy | |
| Nilotinib Nilotinib Nilotinib , in the form of the hydrochloride monohydrate salt, is a tyrosine kinase inhibitor approved for the treatment of chronic myelogenous leukemia.-Uses:... (AMN107) |
4 | Met-318, Thr-315, Glu-286, Asp-381 | Inactive | Rational drug design | Marketed as second line therapy | |
| Dasatinib Dasatinib Dasatinib, previously known as BMS-354825, is a cancer drug produced by Bristol-Myers Squibb and sold under the trade name Sprycel. Dasatinib is an oral multi- BCR/ABL and Src family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukemia after imatinib treatment... (BMS-345825) |
3 | Met-318, Thr-315 | Active | Rational drug design | Marketed as second line therapy | |
| Bosutinib Bosutinib Bosutinib is a tyrosine kinase inhibitor undergoing research for use in the treatment of cancer.It is being developed by Pfizer.-External links:* http://www.eurekalert.org/pub_releases/2007-12/uotm-epi120707.php... (SKI-606) |
- | - | Inactive | Rational drug design | Phase III clinical trials | |
| Ponatinib Ponatinib Ponatinib is an experimental oral drug candidate for the treatment of chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia . It is a multi-targeted tyrosine-kinase inhibitor. Some forms of CML, those that have the T315I mutation, are resistant to current... (AP-24534) |
5 | Met-318, Asp-381, Glu-286, His-381, Ile-380 | Inactive | Rational drug design | Phase II clinical trials | |
| Bafetinib (INNO-406) | 6 | Met-318, Thr-315, Glu-286, Asp-381, His-361, Ile-360 | Inactive | Rational drug design | Phase II clinical trials |
Current status
In the near future, there is hope for better treatment results in TKI-resistant CML, based on the availability of second generation TKI and other drugleads. Imatinib still remains the standard frontline TKI, but more potent Bcr-Abl TKIs are available. Two of these drugs, nilotinib and dasatinib, are approved for the treatment of imatinib resistant or intolerant CML. The first line data for these compounds are encouraging and suggest that one or both of them may replace imatinib as a frontline standard TKI in the future.See also
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