Imatinib Binding Research Articles

Abstract C158: Predicting TKI responses in PDGFRA-mutant gastrointestinal stromal tumor

Abstract Gastrointestinal stromal tumor (GIST) is the most common sarcoma and frequently harbor oncogenic mutations in the receptor tyrosine kinases KIT or platelet derived growth factor receptor alpha (PDGFRA). Imatinib, a type II tyrosine kinase inhibitor (TKI), can successfully treat many KIT-mutant GIST. Yet, it is generally regarded as less effective against PDGFRA-mutant GIST because patients with the most common mutation, exon 18 D842V, are clinically resistant to imatinib and other GIST type II TKIs. However, there are many other PDGFRA mutations reported in GIST whose imatinib sensitivity is unknown. We curated mutation data from a large cohort of PDGFRA-mutant GIST patients, and over 80% had exon 18 mutations. Many mutations clustered around residues 840-849 and included non-D842V point mutations or complex indels. We previously biochemically profiled some of these frequently observed mutations and showed that some were imatinib sensitive, and that sensitivity was dependent on the properties of the 842-codon residue. As modeling each clinically reported mutation is not feasible, we hypothesized that imatinib sensitivity of PDGFRA exon 18 mutations could be predicted based on the 842 residue. To test our hypothesis, we created individual Ba/F3 cell lines that expressed individual PDGFRA mutant kinases, with one cell line each for every amino acid substitution at the 842 residue. Cells underwent transformation limiting dilution assays and imatinib sensitivity was determined via immunoblotting for phosphorylated and total PDGFRA. We observed that 13 out of the 20 amino acids conferred imatinib sensitivity when in the 842-codon position. 7 out of 8 hydrophobic residues conferred imatinib resistance (IC50>150nM) and all amino acids from other classes (polar, negative charge, positive charge, special case) conferred sensitivity (IC50<150nM). Alanine conferred imatinib sensitivity, despite being hydrophobic. Previous studies describe that imatinib resistance seen in D842V patients is due to PDGFRA structural changes. Therefore, we are currently performing molecular simulations and drug docking studies with our mutant kinases to understand how changes at the 842 position effect kinase structure and imatinib binding. Our predictions will identify patients that would benefit from imatinib therapy, while identifying those that require avapritinib. Avapritinib was recently FDA-approved for first-line treatment of all PDGFRA exon 18 mutation GIST, although it was initially created to target D842V. In Europe, avapritinib is only approved for D842V patients. Identifying non-D842V patients that either require avapritinib or could benefit from imatinib is crucial, as current guidelines may suggest no treatment at all. Also, eligible patients treated with imatinib first have additional treatment options if they progress, but this avenue is not available for those progressing on avapritinib. Using our work to support this paradigm shifting approach to optimize TKI treatment sequencing for PDGFRA-mutant GIST will transform patient care and clinical outcomes. Citation Format: Homma M Khosroyani, Ajia Town, Johanna Falkenhorst, Sebastian Bauer, Michael Heinrich. Predicting TKI responses in PDGFRA-mutant gastrointestinal stromal tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C158.

A Paradigm Shift in Experimental Biochemistry: A Priori Estimation of Physical Parameters of Biomolecules

This paper proposes a new paradigm for the biophysical concept of measuring the affinity of molecular complexes, based on a matrix representation of biological interactions and subsequent numerical analysis of the stability of this matrix. Our numerical criterion of stability (lg(cond([Formula: see text]))) correlates well with experimental values such as [Formula: see text] and IC[Formula: see text] as well as with experimental data of aggregation kinetics in studies of amyloid peptides. The main goal of this work is to reduce the cost of biochemical experiments by obtaining preliminary information on the interaction of chemical compounds. The paper also presents our numerical calculations in comparison with a large amount of experimental data on the examples of binding of small chemical molecules gefitinib, erlotinib, imatinib, naquatinib, and CO-1686 with proteins, protein–peptide interactions of the Bcl-2 protein family, antibody–antigen CD20–rituximab, and aggregation of amyloid peptides. The description of the software package that implements the presented algorithm is given on the website: https://binomlabs.com/ .

Evaluating Imatinib's Affinities and Specificities for Tyrosine Kinases Using Molecular Dynamics Simulations

Computational chemistry lets us model intermolecular interactions in ways assays cannot. My project focuses on the multi-kinase interactions of the cancer drug, imatinib. Most cancer drugs target one kinase, but some affect multiple kinases. Imatinib treats chronic myeloid leukemia by targeting ABL kinase. Proteomics data reveals it can interact with other kinases, such as KIT to treat gastrointestinal stromal tumors, but the mechanisms are unknown. Imatinib has different affinities for similar kinases, such as a 3000x difference between ABL and SRC, despite sharing 50% structural homology. Here, I investigate the conformational differences between free and imatinib-bound ABL, KIT, and SRC using Molecular Dynamics simulations to understand the key imatinib-kinase interactions. The alignment analysis shows the docked conformations are similar to co-crystal structures in the Protein Data Bank. Root-mean-square-deviation and fluctuation (RMSD and RMSF) analysis show that all simulations converge at 45 ns, with some regions exhibiting differential flexibility. Hydrogen bond analysis across 100 ns simulations show that ABL has one main H-bond, KIT has three main H-bonds, and SRC has no main H-bonds. All the drug-kinase complexes feature at least 15 key salt bridge interactions relevant for structural stability. The dihedral distributions reveal that most residues adopt a single conformation, but some can adopt multiple, increasing the protein flexibility. The entropy results quantify the protein disorder, revealing KIT and SRC favors the apoprotein while ABL favors the complex. This signifies that broad protein similarity does not govern imatinib binding, instead, it is explained by smaller structural details.

Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket.

It was recently reported (Xie et al., 2022) that the Abelson tyrosine kinase (Abl) ATP-site inhibitor imatinib also binds to Abl's myristoyl binding pocket, which is the target of allosteric Abl inhibitors. This was based on a crystal structure of a truncated Abl kinase domain construct in complex with imatinib bound to the allosteric site as well as further isothermal titration calorimetry (ITC), NMR, and kinase activity data. Although imatinib's affinity for the allosteric site is significantly weaker (10 M) than for the ATP site (10 nM), imatinib binding to the allosteric site may disassemble the regulatory core of Abl, thereby stimulating kinase activity, in particular for Abl mutants with reduced imatinib ATP-site affinity. It was argued that the previously observed imatinib-induced opening of the Abl regulatory core (Skora et al., 2013; Sonti et al., 2018) may be caused by the binding of imatinib to the allosteric site and not to the ATP site. We show here that this is not the case but that indeed imatinib binding to the ATP site induces the opening of the regulatory core at nanomolar concentrations. This agrees with findings that other type-II ATP-site inhibitors (nilotinib, ponatinib) disassemble the regulatory core despite demonstrated negligible binding to the allosteric site.

Analysis to determine the effect of mutations on binding to small chemical molecules.

In this paper, the authors present and describe, in detail, an original software-implemented numerical methodology used to determine the effect of mutations on binding to small chemical molecules, on the example of gefitinib, AMPPNP, CO-1686, ASP8273, erlotinib binding with EGFR protein, and imatinib binding with PPARgamma. Furthermore, the developed numerical approach makes it possible to determine the stability of a molecular complex, which consists of a protein and a small chemical molecule. The description of the software package that implements the presented algorithm is given in the website: https://binomlabs.com/.

Imatinib can act as an Allosteric Activator of Abl Kinase

Imatinib is an ATP-competitive inhibitor of Bcr-Abl kinase and the first drug approved for chronic myelogenous leukemia (CML) treatment. Here we show that imatinib binds to a secondary, allosteric site located in the myristoyl pocket of Abl to function as an activator of the kinase activity. Abl transitions between an assembled, inhibited state and an extended, activated state. The equilibrium is regulated by the conformation of the αΙ helix, which is located nearby the allosteric pocket. Imatinib binding to the allosteric pocket elicits an αΙ helix conformation that is not compatible with the assembled state, thereby promoting the extended state and stimulating the kinase activity. Although in wild-type Abl the catalytic pocket has a much higher affinity for imatinib than the allosteric pocket does, the two binding affinities are comparable in Abl variants carrying imatinib-resistant mutations in the catalytic site. A previously isolated imatinib-resistant mutation in patients appears to be mediating its function by increasing the affinity of imatinib for the allosteric pocket, providing a hitherto unknown mechanism of drug resistance. Our results highlight the benefit of combining imatinib with allosteric inhibitors to maximize their inhibitory effect on Bcr-Abl.

High Frequency of Point Mutations of BCR/ABL Gene in CML Patients Resistant to Imatinib Therapy - Report of a Novel Mutation.

Imatinib is a drug that competitively targets the adenosine 5′-thriphosphate (ATP)-binding site of the kinase domain of ABL with high specificity and has been stablished as the new standard care in CML. However, resistance to imatinib develops in all phases of the disease, mainly in accelerated phase (AP) and blast crisis (BC). Mutations in the kinase domain of BCR/ABL are the most frequent mechanisms associated to resistance in vivo.Aims: detect mutations in the kinase domain of BCR/ABL in CML patients with primary or secondary resistance to Imatinib.Patients and Methods: we evaluated five patients in blast crisis followed in our institution with primary (1) or secondary resistance (4) to Imatinib. After RNA extraction from peripheral blood samples, amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR. For the first PCR, primers PCR1 were used and for the second PCR, PCR2 primers; PCR 1 - BCRF (sense - 5′ TGA CCA ACT CGT GTG TGA AAC TC) and ABLKinaseR (anti-sense - 5′ TCC ACT TCG TCT GAG ATA CTG GAT T); PCR 2 ABLKinaseF (sense - 5′ CGC AAC AAG CCC ACT GTC T) and ABLKinaseR (anti-sense - 5′ TCC ACT TCG TCT GAG ATA CTG GAT T). PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank).Results: We found mutations in four of five CML patients in blast crisis with secondary resistance to Imatinib (E255K, E279K, F359V). E279K mutation was found in a patient in myeloid blast crisis which become resistant to imatinib after 18 months of therapy. He died of progressive disease(PD). This mutation was not previously described and additional studies are necessary to categorize it. Recently H-bonds were reported between Arg 386, located in A-loop and Asp276/Glu279 located in β3-αC-loop which stabilizes the inactive conformation of the A-loop, necessary for imatinib binding. E255K mutation was found in two patients with lymphoid BC. This mutation is located in P-loop and is related to a poor prognosis. These two patients died with PD. F359V mutation is found outside P-loop and related to a longer survival. In fact, the patient with this mutation was in second CP when mutation was detected and had a good response to dose increase. Later, she was submitted to non-myeloablative BMT and is still alive. Mutations were not identified in one patient, a case with primary resistance, corroborating literature data. In conclusion, BCR/ABL kinase domain mutations are frequent in advanced phases of the disease and have prognostic implications. We described a novel mutation which may have clinical relevance. Early detection of BCR/ABL mutations may help in clinical decisions.

A molecularly imprinted electrochemical sensor based on highly selective and an ultra-trace assay of anti-cancer drug axitinib in its dosage form and biological samples

In this study, a novel, fast, selective, and sensitive molecularly imprinted polymer (MIP)-based electrochemical sensor was developed to determine axitinib (AXI) at low concentrations in pharmaceutical dosage forms and human serum. The newly developed MIP-based sensor (MIP@o-PD/GCE) was designed through electropolymerization of functional monomer o-phenylenediamine (o-PD) in the presence of a template molecule AXI, on a glassy carbon electrode (GCE) using cyclic voltammetry. Differential pulse voltammetry and electrochemical impedance spectroscopy (EIS) techniques were employed for removal and rebinding processes, optimization of conditions, as well as for performance evaluation of MIP@o-PD/GCE using [Fe(CN)6]3−/4– as the redox probe. Under the optimum experimental conditions, MIP@o-PD/GCE shows a linear response toward AXI in a range of 1 × 10−13 M − 1 × 10−12 M. The limit of the detection value of MIP@o-PD/GCE was found as 0.027 pM while the limit of the quantification was obtained as 0.089 pM, respectively. To demonstrate the applicability and validity of the developed sensor, it was successfully applied to tablet dosage form and human serum sample. The selectivity of the sensor was qualified by comparing the binding of AXI, erlotinib, dasatinib, nilotinib, and imatinib, which are similarly structured and in the same group of anticancer drugs. MIP@o-PD/GCE sensor showed a significant selectivity toward AXI. The non-imprinted polymer (NIP) based GCE was prepared and used to control the analytical performance of the MIP-based electrochemical sensor.

Spectroscopic studies of simultaneous binding of cyclophosphamide and imatinib mesylate to human holo-transferrin

The interactions of proteins with drugs are very important from a pharmacological point of view. Holo-transferrin is a blood-plasma glycoprotein whose main function is iron-binding and the transport of other ligands. Additionally, the protein is only transferrin-form recognized by TfR1 and TfR2 receptors at the surface of rapidly proliferating malignant cells. Imatinib mesylate is a tyrosine-kinase inhibitor mainly used in the treatment of blood cancers, frequently in multidrug therapy with cyclophosphamide. In this study the effect of cyclophosphamide on the interaction of imatinib mesylate with human holo-transferrin has been investigated. Using spectroscopic techniques such as fluorescence, circular dichroism, ultraviolet-visible and electrophoretic light scattering additive parameters, system stability and the effect of the ligands on the protein conformation at varying pH values have been defined. Calculated quenching constants are in the order of 2 × 104 M−1 and the type of interaction depends on the reaction medium. Under physiological conditions binding constant is 1.329 × 106 M−1 whereas in an environment similar to that of cancer cells the constant is significantly lower, Ka = 6.060 × 104 M−1. N values are approximate to 1 in all cases. Moreover, some changes are observed in the α-helical structure of the protein after interaction with the drugs and the presence of cyclophosphamide slightly stabilizes the protein secondary structure. All collected data proves the effect of cyclophosphamide on the interaction between imatinib mesylate and human holo-transferrin. It is of great clinical interest due to anticancer, multidrug therapies including both imatinib mesylate and cyclophosphamide.

An SPR investigation into the therapeutic drug monitoring of the anticancer drug imatinib with selective aptamers operating in human plasma.

The anticancer drug imatinib is often involved in therapeutic drug monitoring (TDM) studies aimed at improving the treatment of several forms of leukemia and gastrointestinal stromal tumors (GIST). To further implement the TDM of imatinib in clinical practice, we developed a detection assay by using an ssDNA aptamer, which demonstrated excellent selectivity and was not affected by interference from the components of human plasma samples. The efficient binding of imatinib to the aptamer was demonstrated by means of surface plasmon resonance (SPR) analysis, which allowed the development of a quantitative assay in the concentration range between 400 and 6000 ng mL-1 (0.7-10 μM), where a lower limit of quantification (LLOQ) of 400 ng mL-1 was achieved. The precision of the assay was found to be within 12.0%, whereas the accuracy was in a range between 97.1 and 101.5%. The sample preparation procedure displayed a recovery in the range of 48.8-52.8%. Solid validation data were collected according to the regulatory guidelines and the method was compared with standard analytical techniques, leading to the development of a feasible aptasensor for the TDM of patients administered with imatinib.

Review on Pathophysiological and Pharmacotherapeutic approach on Chronic Myeloid Leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell malignant clonal disorder resulting in elevation of erythroid cells and platelets in peripheral blood and clear myeloid hyperplasia in the bone marrow. Philadelphia (Ph) chromosome detection forms the major diagnosis in CML. It displays the translocation t(9; 22) that generates the oncogene bcr/abl. Constitutive tyrosine kinase activity is been exhibited by the oncoprotein (BCR/ABL) which supports growth and continued existence in cells of CML. Incidence remains almost constant worldwide and it is the most commonest in India majorly in adults. Clinically, Chronic Phase (CP), Accelerated Phase (AP), and Blast Phase (BP) are the three phases of CML. The drugs used in the treatment of CML are known as Tyrosine Kinase Inhibitors (TKI'S). The primary drug which directly target BCR-ABL tyrosine kinase activity was Imatinib. Imatinib resistance is the major drawback of this drug due to mutations. Imatinib binding is prevented by BCR-ABL kinase domain mutations, BCR-ABL gene amplification or over-expression, clonal progression, and reduced bioavailability of imatinib or cell exposure. Stem cell transplantation is the only curative approach till date. In patients who cannot undergo transplantation, the BCR-ABL tyrosine kinase inhibitor STI571 (Gleevec, Imatinib), immune therapy or interferon-alpha and other cytoprotective drugs are used. Currently available data shows that Imatinib is a better compound compared to second line drugs in producing complete molecular and cytogenetic responses. The resistance against STI571 has been reported which has to be tested with mutational analysis for the treatment for using an accurate regimen. Current researchers are consequently challenging to inhibit or work against STI571 resistance by the development of new molecules or combination of molecules contributing for further improvement in the treatment of CML and for better understanding of resistant mechanisms. These strategies, whether will guide to curative treatment in CML in the future remains unclear.

Imatinib modulates pro-inflammatory microenvironment with angiostatic effects in experimental lung carcinogenesis.

Lung cancer has second highest rate of incidence and mortality around the world. Smoking cigarettes is the main stream cause of lung carcinogenesis along with other factors such as spontaneous mutations, inactivation of tumor suppressor genes. The present study was aimed to identify the mechanistic role of Imatinib in the chemoprevention of experimental lung carcinogenesis in rat model. Gross morphological observations for tumor formation, histological examinations, RT-PCR, Western blotting, fluorescence spectroscopy and molecular docking studies were performed to elucidate the chemopreventive effects of Imatinib and support our hypothesis by various experiments. It is evident that immuno-compromised microenvironment inside solid tumors is responsible for tumor progression and drug resistance. Therefore, it is inevitable to modulate the pro-inflammatory signaling inside solid tumors to restrict neoangiogenesis. In the present study, we observed that Imatinib could downregulate the inflammatory signaling and also attributed angiostatic effects. Moreover, Imatinib also altered the biophysical properties of BAL cells such as plasma membrane potential, fluidity and microviscosity to restrict their infiltration and thereby accumulation to mount immuno-compromised environment inside the solid tumors during angiogenesis. Our molecular docking studies suggest that immunomodulatory and angiostatic properties of Imatinib could be either independent of each other or just a case of synergistic pleiotropy. Imatinib was observed to activate the intrinsic or mitochondrial pathway of apoptosis to achieve desired effects in cancer cell killings. Interestingly, binding of Imatinib inside the catalytic domain of PARP-1 also suggests that it has caspase-independent properties in promoting cancer cell deaths.

Structural Basis for the Regulation of PPARγ Activity by Imatinib.

Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2′ and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects.

A Study on Imatinib Binding to c-Src Tyrosine Kinase According to Sequential Conformational Change

A Study on Imatinib Binding to c-Src Tyrosine Kinase According to Sequential Conformational Change

Novel mutations in the kinase domain of BCR-ABL gene causing imatinib resistance in chronic myeloid leukemia patients

Mutations in the drug binding region of BCR-ABL lead to imatinib resistance during the management of chronic myeloid leukemia (CML). In our study, 62 Philadelphia positive (Ph+) CML patients showing conspicuous expression of BCR-ABL gene were treated with imatinib. At the end of 3 months, 21/62 (33.87%) patients did not obtain complete hematological response (CHR) and also showed no significant decrease in BCR-ABL gene expression. In all the imatinib-resistant patients BCR-ABL gene was PCR amplified and sequenced. The sequence analysis showed four novel missense mutations p.(Leu301Ile), p.(Tyr320His), p.(Glu373Asp), p.(Asp381Asn) and six already reported mutations p.(Val256Gly), p.(Thr315Ile), p.(Gly250Glu), p.(Tyr253His), p.(Phe317Leu), p.(Met351Thr) which contributed in the formation of inactive enzyme and also two novel frameshift mutations p.(Glu281*) and p.(Tyr393*), which resulted in truncated protein formation. Further, the structural analysis revealed all these mutations affected P-loop, gatekeeper, catalytic and activation loop domain regions of the enzyme causing poor imatinib binding in the ATP region. The primary intention of the study was to find out the mutations in the BCR-ABL gene causing imatinib resistance. This study highlights the need for BCR-ABL gene sequence analysis to detect the mutations in CML patients in order to properly guide the therapy.

Molecular modelling evaluation of exon 18 His845_Asn848delinsPro PDGFR\u03b1 mutation in a metastatic GIST patient responding to imatinib

Platelet-Derived Growth Factor Receptor Alpha (PDGFRA) mutations occur in approximately 5–7% of gastrointestinal stromal tumours (GIST). Over half of all PDGFRA mutations are represented by the substitution at position 842 in the A-loop of an aspartic acid (D) with a valine (V), recognized as D842V, conferring primary resistance to imatinib in vitro and in clinical observations due to the conformation of the kinase domain, which negatively affects imatinib binding. The lack of interaction between imatinib and the D842V PDGFRA mutated model has been established and widely confirmed in vivo. However, for the other PDGFRA mutations, the correlation between pre-clinical and clinical data is still unclear. An in silico evaluation of the p.His845_Asn848delinsPro mutation involving exon 18 of PDGFRA in a metastatic GIST patient responding to first-line imatinib has been provided. Docking analyses were performed, and the ligand-receptor interactions were evaluated with the jCE algorithm for structural alignment. The docking simulation and structural superimposition analysis show that PDGFRA p.His845_Asn848delinsPro stabilizes the imatinib binding site with the residues that are conserved in KIT. The in vivo evidence that PDGFRA p.His845_Asn848delinsPro is sensitive to imatinib was confirmed by the molecular modelling, which may represent a reliable tool for the prediction of clinical outcomes and treatment selection in GIST, especially for rare mutations.

BCR-ABL Kinase Domain Mutations Exist in Few Imatinib Treated Chronic Myeloid Leukemia (CML) Patients Exhibiting Stable Cytogenetic and Hematologic Responses

Introduction: Complete hematologic response (CHR) and complete cytogenetic response (CCyR) is observed in almost 80-90% freshly diagnosed chronic phase chronic myeloid leukemia patients treated with Imatinib, a tyrosine kinase inhibitor (TKI). The responses are usually persistent; however the residual disease may be detected molecularly by amplifying BCR-ABL transcript. Furthermore, the leukemogenic potential of the residual BCR-ABL positive cells is reflected when disease reoccurs after discontinuation of therapy. There are evidences of occurrence of mutations in patients who have achieved CCyR, which indicates their role in disease persistence and drug resistance.Patients and Methods: We performed BCR-ABL kinase domain (KD) mutation screening in 150 Imatinib treated CML patients (either resistant or sensitive to therapy) including 84 (56%) individuals manifesting CHR and and 67 (44.7%) with CCyR. For this purpose, nested reverse transcription PCR followed by direct sequence analysis was implemented as previously described. Statistical analysis was performed using SPSS version 19 (IBM, Chicago, IL, USA).Results and Conclusion: Thirty five of 150 patients exhibited mutations in BCR-ABL KD, out of which 11 (31.4%) had achieved CHR and 08 (22.9%) attained CCyR. Single rather than multiple mutated sites in ABL-KD was observed in most of mutant patients achieving CHR (27.3%) or CCyR (27.3%) (Figure 1). Eleven nucleotide substitutions were observed in more than one patient, including Imatinib sensitive individuals with CHR and CCyR (Table 1). Those include C-Helix, SH2 contact, Imatinib binding site (IBS), and A-loop mutations within ABL kinase domain. Overall survival among chronic myeloid leukemia patients achieving different levels of hematologic and cytogenetic response irrespective of mutation status show a favorable survival outcome in those attaining CHR and CCyR (Figure 2). Presence of mutations in cytogenetically stable patients of CML with good hematologic outcomes indicates a likely suboptimal response or TKI resistance during the course of treatment. It is believed by some researchers that mutations do not exist previously, but emerge after Imatinib exposure. Whereas, some believe that TKI's do not induce mutations, rather they select them. Many studies reported a rise in BCR-ABL transcript levels during follow up studies in patients who co-exhibited mutated ABL-KD and CCyR. This suggests that emergence of mutations is a disease evolution process independent of TKI therapy. Studies report that monitoring of nucleotide substitutions in optimally responding patients plays an important role in well managed therapeutic approach. Our studies reveal occurrence of ABL-KD mutations in patints with stable hematologic and cytogenetic response, therefore we suggest for regular mutation screening in CML paients irrespective of their response towards TKIs. DisclosuresNo relevant conflicts of interest to declare.

A Multiscale Simulation Approach to Modeling Drug-Protein Binding Kinetics.

Drug-target binding kinetics has recently emerged as a sometimes critical determinant of in vivo efficacy and toxicity. Its rational optimization to improve potency or reduce side effects of drugs is, however, extremely difficult. Molecular simulations can play a crucial role in identifying features and properties of small ligands and their protein targets affecting the binding kinetics, but significant challenges include the long time scales involved in (un)binding events and the limited accuracy of empirical atomistic force fields (lacking, e.g., changes in electronic polarization). In an effort to overcome these hurdles, we propose a method that combines state-of-the-art enhanced sampling simulations and quantum mechanics/molecular mechanics (QM/MM) calculations at the BLYP/VDZ level to compute association free energy profiles and characterize the binding kinetics in terms of structure and dynamics of the transition state ensemble. We test our combined approach on the binding of the anticancer drug Imatinib to Src kinase, a well-characterized target for cancer therapy with a complex binding mechanism involving significant conformational changes. The results indicate significant changes in polarization along the binding pathways, which affect the predicted binding kinetics. This is likely to be of widespread importance in binding of ligands to protein targets.

Abstract 812: New c-KIT mutations in GISTs: Bad for good

Abstract Gastrointestinal stromal tumors are mesenchymal tumors arising in the stomach and small bowel and more rarely in the rectum, esophagus, peritoneum and retroperitoneum. These tumors are characterized by KIT or PDGFRA mutations. KIT mutations are all in frame and lead to constitutive tyrosine kinase domain activation without ligand binding. Mutations concern four exons (9, 11, 13 and 17) but mainly exon 11. In this work we report a case of a GIST patient with wild-type PDGFRA but bearing two mutations in KIT exon 9, i.e., the insertion mutation Y503_F504insAY and the missense mutation K509N. The patient was treated with Imatinib at standard regimen dose (400 mg/day), and responded well. Accordingly, in silico experiments were performed to disclose the effects exerted by these new mutations on the protein structure, its thermodynamic stability, and the ability to promote/interfere with Imatinib binding. Direct drug binding thermodynamics and kinetics of the mutant KIT were measured using isothermal titration calorimetry. FInally, in vitro kinase assays were performed to monitor the phosphorylation status of this new mutant KIT. The prototypical KIT mutations Δ559 and T670I were also analyzed in parallel for comparison. To our knowledge, only AY 502-503 duplication/insertion and K509I substitution have been previously reported. Citation Format: Erik Laurini, Suzana Aulic, Domenico Marson, Maurizio Fermeglia, Roberta Riboni, Marco Lucioni, Elena Dellera, Mario Alessiani, Vittorio Perfetti, Sabrina Pricl. New c-KIT mutations in GISTs: Bad for good [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 812.

Ensemble-based virtual screening: identification of a potential allosteric inhibitor of Bcr-Abl

Ensemble-based virtual screening using different conformations of a target protein is gaining popularity, as it can leverage information from target flexibility for effective lead identification. In this paper, molecular dynamics simulation followed by RMSD-based clustering was employed to generate and choose distinct conformations of Bcr-Abl. Three representative structures from the most-populated clusters along with the crystal structure conformation (PDBID: 3K5V) were used to perform docking-based virtual screening of 14,400 compounds (in the Maybridge database) in order to identify potential allosteric site binders. Seven compounds found as hits in at least three of the four virtual screenings had higher Glide docking scores than the co-crystallized allosteric inhibitor GNF-2. Detailed computational analyses of the seven hits identified SEW02675 (ΔG bind=-164.92kJ/mol with the wild-type (wt) Bcr-Abl and -167.37kJ/mol with the T334I Bcr-Abl mutant) as a better allosteric site binder with both the wt and the mutant Bcr-Abl protein than the reference allosteric inhibitor GNF-2 (ΔG bind=-103.12 with wt and -142.96kJ/mol with T334I). Moreover, the presence of SEW02675 in the allosteric site enhanced the binding of imatinib (ΔG bind=-367.58 with wt and -294.56kJ/mol with T334I) to the ATP sites of the wt and the mutant Bcr-Abl. However, when GNF-2 was present in the allosteric site, the binding of imatinib (ΔG bind=-351.76 with wt and -273.94kJ/mol with T334I) to the ATP site was weaker. The in silico findings suggest that SEW02675 could be used in combination with imatinib to treat chronic myeloid leukemia, and that it could help to overcome resistance due to T334I Bcr-Abl mutation. Graphical abstract Virtual screening strategy to identify allosteric inhbitors of Bcr-Abl for the treatment of Chronic myeloid leukemia.