Bcr-Abl tyrosine-kinase inhibitor

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. 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 The treatment options for imatinib resistant or intolerant CML 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. == 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, dasatinib, bosutinib and ponatinib (third generation). Nilotinib (AMN107) Development Nilotinib is a phenylamino-pyrimidine derivative that is structurally related to imatinib. (PDB 2GQG) of Abl kinase domain (blue) in complex with dasatinib (red). Binding Dasatinib binds to Abl with less stringent conformational requirements than imatinib so it exhibits increased potency but reduced selectivity compared to imatinib. 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. 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. 1,3,4 thiadiazole derivatives - Substance 14 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. It has had a phase 1 clinical trial for Leukemias (Ph+ CML With T315I Mutation). It is in a phase 1 clinical trial of combination therapy for metastatic breast cancer. Asciminib (ABL001) is an inhibitor of the Abelson kinase targeting the myristoyl pocket to allosterically inhibit the enzyme. As of August 2020, it had completed a phase III study in CML (ASCEMBL) showing superior efficacy to bosutinib. ==Summary==

==Current status - re Ph+ CML==

Imatinib remains a standard frontline TKI. Nilotinib and dasatinib are also approved by the FDA as frontline drugs, in June and October 2010, respectively. Four of these drugs, nilotinib, dasatinib, bosutinib and ponatinib are approved for the treatment of imatinib-resistant or intolerant CML. The first-line data for these compounds are encouraging and suggest that some or all of them may replace imatinib as a frontline standard TKI in the future. ==References==