Approved Kinase Inhibitors Research Articles

Abstract 5168: Effects of point mutations, recombinant tags, activation status, and identity of target kinases on the mode of action of approved kinase inhibitors.

Abstract Protein kinases regulate a variety of biological signaling pathways affecting cell proliferation, differentiation, migration and apoptosis. In tumor cells the activity of many protein kinases are frequently found to be upregulated linking deregulation of protein kinases causally to the development and progression of many human cancers and other diseases. Therefore, protein kinases have become a prime molecular target for therapeutic intervention, and up to now fourteen small molecule inhibitors have been approved for treatment of various types of cancer. These inhibitors block the activity of the target protein kinases by either blocking the ATP binding site in a direct competitive manner (type1-inhibitors) or by indirectly interfering with ATP/kinase interaction by binding to an inactive state, referred to as DFG-out (type2-inhibitors). Due to the fact that these inhibitor types bind to a region which is highly conserved among the protein kinase superfamily, achieving target selectivity represent a major challenge in the development of protein kinase inhibitors. Allosteric inhibitors, also sometimes referred to as type3-inhibitors, bind to structural sites different to the ATP-binding pocket region, and have, therefore, a significant higher potential to inhibit kinases much more selective than ATP-competitive compounds. The majority of the currently clinically approved small molecule protein kinase inhibitors are commonly classified as ATP-competitive inhibitors, and are able to inhibit different protein kinases with high potency. Interestingly, analysing the effect of the ATP-concentration on the IC50 of Sorafinib against different target kinases, we observed that the inhibitory potency against various target kinases is differently affected by the ATP concentration indicating differences in the exact mode of action of the inhibitor. To answer the question whether this is a specific property of Sorafenib, or also relevant for other inhibitors, we perfomed similar studies with seven additional clinically approved kinase inhibitors (Axitinib, Crizotinib, Erlotinib, Gefitinib, Lapatinib, Pazopanib and Sunitinib) using a panel of up to 16 different target protein kinases. We extended these studies and will in addition also present data investigating the influences of (a) the tags to which the respective recombinant kinase was fused, (b) the activation status of the kinase, and (c) of activating point mutations on the mode of action of the different inhibitors. Citation Format: Daniel Müller, Christian Beisenherz-Huss, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Thomas Weber, Michael H.G. Kubbutat. Effects of point mutations, recombinant tags, activation status, and identity of target kinases on the mode of action of approved kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5168. doi:10.1158/1538-7445.AM2013-5168

Metabolomic Response to Sorafenib Treatment in Human Hepatocellular Carcinoma Cells

Untargeted metabolomics has enabled comprehensive overview on physiological system and provides chances for new discoveries linking cellular pathways to biological mechanism, which also is a good platform for better understanding of drug action. In this study, we employed a metabolomics method to examine how the multikinase inhibitor sorafenib affects metabolic pathways in human hepatocellular carcinoma cell line. The results showed that sorafenib treatment in HepG2 cells caused remarkable changes of several metabolic pathways in a concentration‐dependent manner. At a low concentration of sorafenib, the glycerophospholipid metabolism was the most affected pathway, while at a higher concentration, the purine metabolism was most significantly altered. The significant decrease of GTP level after sorafenib treatment could be associated with the toxicity of this drug. These findings have provided insights into how sorafenib acts on distinct metabolic and biochemical pathways that are associated with its therapeutic efficacy and side effects. Metabolic profiling studies for clinically approved kinase inhibitors can serve as a powerful tool to facilitate the understanding of their mechanism of actions, molecular targets and the interactome.

Activity‐based kinase profiling of approved tyrosine kinase inhibitors

The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors.

84IN - Breakthroughs in Basic Research

ABSTRACT Over the last few years several basic research studies have led to new therapeutic insights into the treatment of patients with advanced non-small cell lung cancer (NSCLC). These include genomic studies of lung cancers which have revealed novel potential therapeutic targets. One such target is fibroblast growth factor receptor (FGFR) 1 which has been found to be amplified in a subset of squamous cell lung cancers. Preclinical studies have further demonstrated that inhibition of FGFR1 kinase activity using FGFR tyrosine kinase inhibitors (TKIs) may be clinically effective in this subset of NSCLC. Clinical trials are now underway using FGFR TKIs in this subset of NSCLC. A second example is the identification of novel genomic rearrangements of the RET oncogene in NSCLC. These rearrangements lead to a fusion gene (KIF5B-RET) which is transforming in vitro and in vivo. Several already approved kinase inhibitors, including vandetinib, sorafenib and sunitinib, also inhibit RET and may be effective as clinical therapies in this subset of NSCLC. In addition, several more potent RET TKIs are under preclinical development and undergoing validation in model systems. A second recent basic science advance is the use of mouse models of lung cancer to mirror ongoing or planned clinical trials. These “co-clinical” trials aim to use the murine models to determine if they can predict the outcome of human trials and/or be used to further understand the success and limitations of the therapeutic approach. A recent example evaluated docetaxel and the MEK inhibitor selumetinib compared to docetaxel alone in a genetically engineered mouse model of Kras mutant NSCLC. In this mouse model the combination led to a greater response rate and progression free survival compared to docetaxel alone. The result was similar in a recent clinical trial of the same therapeutic design in KRAS mutant NSCLC. Interestingly, in the mouse model, those tumors with concurrent Lkb1 loss (occurs in about 30% of KRAS mutant NSCLC in humans), the combination of docetaxel/selumetinib was significantly less effective. These findings open the possibility of evaluating the impact of LKB1 loss in KRAS mutant NSCLC patients treated with selumentib/docetaxel and more broadly underscore the potential utility of animal models in informing the design of human clinical trials. Disclosure P.A. Janne: I have acted as a consultant to Pfizer, Boehringer Ingelheim, Astra-Zeneca, Roche, Genentech and Sanofi

Outcomes of patients (pts) with metastatic renal cell carcinoma (mRCC) treated with pazopanib after progression on other targeted therapies (TT): A single-institution experience.

4615 Background: Pazopanib is an approved multi-tyrosine kinase inhibitor that prolongs progression-free survival (PFS) compared to placebo in treatment-naive and cytokine-refractory mRCC. Outcomes and safety data on its use after TT are limited. Methods: We retrospectively reviewed pts with mRCC who received salvage pazopanib between 11/09-11/11. Kaplan-Meier method was used to estimate survival outcomes. PFS was calculated from start of pazopanib until progressive disease (PD) or death. Univariable and multivariable Cox proportional hazards models were fitted to evaluate associations of PFS with covariables. Results: 114 consecutive pts met inclusion criteria (median age 62.6 years, 66% males, 83% clear cell). All pts had PD after other TT (median # of prior TT 2, range 1-5; median time on prior TT 23.3 mos). 79% of pts had PD on sunitinib, 39% on sorafenib, 19% on temsirolimus, 59% on everolimus, and 23% on bevacizumab. 25% received prior chemotherapy and 16% received prior cytokines in addition to TT. 87% had prior nephrectomy. 11% had favorable-risk, 68% intermediate-risk, and 21% poor-risk per MSKCC criteria. 85 events (PD or death) occurred. Median OS was 17 mos (95% CI: 10.3-NA). Median PFS was 6.4 mos (95% CI: 4.5-9.5). By multivariable analysis, PFS was associated with male gender (HR=0.433, 95%CI: 0.269-0.696; p=0.0006), # of metastatic sites (HR=1.252; 95%CI: 1.04-1.503; p=0.016), hypertension exacerbation (HR=0.378; CI: 0.175-0.813; p=0.0128) and PS 2+ vs.0-1 (HR=2.067; CI: 1.243-3.437; p=0.0052). 58% discontinued pazopanib due to PD, 12% died of PD on treatment, and 11% discontinued pazopanib due to adverse events (AEs), mostly GI complaints or fatigue. There were no treatment related deaths. Common AEs included: fatigue (44%), diarrhea (29%), nausea/vomiting (15%), anorexia (14%), hypertension exacerbation (11%), hypothyroidism (11%), hand-foot skin reaction (9%), and increase LFTs (4%). 86% of AEs were grade 1/2. Conclusions: In this retrospective study, pazopanib demonstrated efficacy in mRCC following PD with other TT. AEs were mild/moderate and manageable.

Abstract 1791: CO-1686, a novel mutant-selective EGFR inhibitor, overcomes T790M mediated resistance in Non-Small Cell Lung Cancer (NSCLC)

Abstract Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond well to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. However, clinical efficacy is limited by the development of resistance. The most common mechanism of resistance is a second site mutation within exon 20 of EGFR (T790M), observed in ∼50% of cases. CO-1686 is an irreversible kinase inhibitor that targets the mutant forms of EGFR by inhibiting the common activating mutations (L858R, delE746-A750) and the gatekeeper mutation (T790M) but not the wild-type receptor. Therefore, CO-1686 has the potential to effectively treat first- and second-line NSCLC patients with EGFR mutations without causing the dose limiting toxicities associated with approved EGFR kinase inhibitors or those in clinical development. Experimental procedures: Using structure-based drug design, CO-1686, a covalent, irreversible small molecule, which selectively inhibits mutant EGFR, was identified. CO-1686 potency was assessed against four common EGFR mutations (L858R, delE746-A750, L858R/T790M and delE746-A750/T790M) using in vitro biochemical and cell-based assays. Antitumor activity of CO-1686 as a single agent was assessed in NSCLC xenograft models harboring EGFR mutations: H1975 (L858R/T790M) and HCC827 (delE746-A750). Pharmacodynamic studies were performed to evaluate effects on cell survival and EGFR signaling. Results: In vitro and in vivo pharmacology studies focused on evaluating CO-1686 potency in four EGFR mutations common in NSCLC patients: L858R, delE746-A750, L858R/T790M and delE746-A750/T790M. CO-1686 was shown to be active against all four EGFR mutants. Effects of CO-1686 on cell proliferation and EGFR signaling were evaluated in HCC827 cells (delE746-A750) and its erlotinib-resistant clone, HCC827-EPR harboring the second site mutation T790M (delE746-A750/T790M). CO-1686 inhibited cell proliferation in both cell lines equally (GI50 values of 12 nM in the parental line and 19 nM in the T790M-positive clone). In mouse xenograft studies, oral dosing of CO-1686 in H1975 double mutant (L858R/T790M) and in HCC827 single mutant (delE746-A750) models caused tumor shrinkage as a single agent in a dose-dependent manner. Different dosing schedules were explored. Conclusions: Our results establish CO-1686 as a potent, mutant-selective EGFR inhibitor with excellent in vivo activity in mice bearing tumors with activating EGFR mutations as well as the resistance mutation T790M. These data suggest that treatment with CO-1686 as a single agent can overcome T790M-mediated drug resistance in NSCLC. Initially, clinical development will focus on NSCLC patients with mutant EGFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1791. doi:1538-7445.AM2012-1791

The Epidermal Growth Factor Receptor-L861Q Mutation Increases Kinase Activity without Leading to Enhanced Sensitivity Toward Epidermal Growth Factor Receptor Kinase Inhibitors

Mutations in the epidermal growth factor receptor (EGFR) kinase domain such as EGFR-L858R and EGFR-G719S have been reported to activate the kinase and also sensitize a subset of patients with non-small cell lung cancer to EGFR kinase inhibitor treatment. Nevertheless, for other common point mutations such as EGFR-L861Q, it is unclear whether and to what extent they sensitize toward gefitinib and erlotinib. Thus far, there is no reliable cellular assay to compare in a ligand-independent manner intrinsic kinase activity and drug sensitivity of the unmutated (wild type) and mutated EGFR kinase domain. To overcome this obstacle, we introduced L858R, G719S, and L861Q into the backbone of EGFRvIII. EGFRvIII has a wild type-kinase domain but is activated in a ligand-independent manner through a deletion in the extracellular domain. Using this tool, we show that the L861Q mutation displays enhanced kinase activity and transforming potential compared with L858R, G719S, and also to the wild type-EGFR kinase domain. Interestingly, L861Q does not increase drug sensitivity toward clinically used EGFR kinase inhibitors in contrast to the L858R and G719S mutation. In addition, we demonstrate that EGFR-L861Q could be effectively inhibited with the irreversible second-generation EGFR inhibitor WZ-4002. Thus, in the common EGFR-L861Q mutation, activation of the kinase domain is uncoupled from a sensitizing effect toward clinically approved kinase inhibitors. Therefore, patients with EGFR-L861Q may not have the same clinical benefit from gefitinib/erlotinib treatment as patients with EGFR-L858R and EGFR-G719S mutations. Treatment with irreversible second-generation kinase inhibitors such as WZ-4002 may be an attractive option in the future for patients with EGFR-L861Q.

Metabolism considerations for kinase inhibitors in cancer treatment

Importance of the field: A concerted effort by the pharmaceutical industry over the last decade has led to the successful clinical development of protein kinase inhibitors as effective targeted therapies for certain cancers.Areas covered in this review: This review details eight small molecule kinase inhibitors that have been approved for the treatment of cancer in either the US or Europe as of March 2010: imatinib, sorafenib, gefitinib, erlotinib, dasatinib, lapatinib, sunitinib and nilotinib. These eight compounds vary from the relatively specific inhibitor lapatinib to the more promiscuous kinase inhibitors dasatinib and sunitinib.What the reader will gain: A brief discussion on the biology of each inhibitor, selectivity over other kinases and toxicity is provided. A more detailed discussion on the metabolism, drug transporters, drug–drug interactions and possible roles of metabolism in compound toxicity is provided for each compound.Take home message: The majority of the currently approved kinase inhibitors is heavily influenced by drug transporters and significantly affected by CYP3A4 inhibitors/inducers. At least three, gefitinib, erlotinib and dasatinib, are metabolized to form reactive metabolites capable of covalently-binding biomolecules.

Biochemical Mechanisms of Resistance to Small-Molecule Protein Kinase Inhibitors

Protein kinases have emerged as one of the most frequently targeted families of proteins in drug discovery. While the development of small-molecule inhibitors that have the potency and selectivity necessary to be effective cancer drugs is still a formidable challenge, there have been several notable successes in this area over the past decade. However, in the course of the clinical use of these inhibitors, it has become apparent that drug resistance is a recurring problem. Because kinase inhibitors act by targeting a specific kinase or set of kinases, there is a strong selective pressure for the development of mutations that hinder drug binding but preserve the catalytic activity of these enzymes. To date, resistance mutations to clinically approved kinase inhibitors have been identified in a number of kinases. This review will highlight recent work that has been performed to understand how mutations in the kinase catalytic domain confer drug resistance. In addition, recent experimental efforts to predict potential sites of clinical drug resistance will be discussed.

Non-ATP competitive protein kinase inhibitors as anti-tumor therapeutics

Alternative approaches for inhibitor development in targeting sites other than the ATP cleft are increasingly being pursued in the search for new therapeutics based on inhibition of protein kinases. While recently approved kinase inhibitor drugs offer benefit in cancer treatment, further advances are required to affect tumor selective cell killing, avoid off-target related toxicities and improve survival rates. Protein–protein interactions involved in kinase regulation and substrate recognition as well as exploiting allosteric pockets, offer the potential for selectivity and avoid decreased efficacy as a result of competition with high intracellular ATP concentrations. We discuss several preliminary examples where regulatory and substrate binding sites present potential druggable interfaces. These include the cell cycle targets which are the cyclin-dependent and polo-like kinases among several others.

Pharmacokinetic Considerations for New Targeted Therapies

Targeted therapies are increasingly being incorporated into standard treatment regimens for cancer. Tyrosine kinase inhibitors represent one class of targeted agents, with eight drugs receiving approval for the treatment of cancer in recent years. Although they hold the promise of being more effective and less toxic to normal cells than conventional cytotoxic chemotherapy, tyrosine kinase inhibitors are associated with different adverse effects, sometimes severe, as well as wide pharmacokinetic variability. Thus, a “therapeutic window” may exist for these agents. Clinical Pharmacology of Tyrosine Kinase Inhibitors The majority of tyrosine kinase inhibitors approved for the treatment of cancer have selective activity in certain populations such as imatinib, dasatinib, and nilotinib in patients with Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemia (CML) with the BcrAbl oncogene, 1, 2 sunitinib in patients with gastrointestinal stromal tumors and activating mutations in the tyrosine kinase c-Kit, 3, 4 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib in patients with non-small cell lung cancer (NSCLC) harboring EGFR mutations. 5 However not all patients in these selected populations respond and emerging drug resistance becomes a clinical problem. Although life-threatening myelosuppression is less frequent with many of the approved kinase inhibitors, they are associated with other adverse effects such as fatigue, nausea, diarrhea, rash and hand-foot syndrome, mucositis, as well as newly recognized ones such as cardiotoxicity and hypothyroidism. 6-10 Since these drugs are administered daily and for some diseases are given for years until disease progression, toxicities can greatly affect patient quality of life and may lead to treatment cessation and relapse. Tyrosine kinase inhibitors registered for the treatment of cancer are administered orally and generally exhibit similar pharmacokinetic characteristics to each other. They are highly bound in human plasma (>90%), they undergo extensive metabolism by cytochrome P-450 3A (CYP3A), and are substrates for and/or inhibitors of transporters in the intestinal epithelial cells and hepatocytes. Variation in drug metabolizing enzymes and transporters contribute to systemic pharmacokinetic variability, but they are also localized in cancer cells and can contribute to intracellular pharmacokinetic variability and drug action. Figure 1 illustrates the concept of how variable expression of solute carriers (involved in drug uptake), ABC transporters (involved in drug efflux), and drug-metabolizing enzymes can affect systemic and intratumoral exposure to orally administered drugs. Similar to most drugs that undergo extensive metabolism, tyrosine kinase inhibitors exhibit wide pharmacokinetic variability. For example, steady-state trough concentrations of gefitinib varied 20-fold in 14 adult cancer

Functions of Paracrine PDGF Signaling in the Proangiogenic Tumor Stroma Revealed by Pharmacological Targeting

BackgroundImportant support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting.Methods and FindingsWe used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment.ConclusionsThis study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.

Molecule of the Month

Molecule of the Month

Molecular Design and Clinical Development of VEGFR Kinase Inhibitors

Vascular angiogenesis has been shown to play a key role in many solid tumors. The vascular endothelial growth factor (VEGF) isoforms and their tyrosine kinase receptors (VEGFRs) have been under intense research for effective anticancer drug candidates. Epidermal growth factor (EGF) and its receptor (EGFR) provide another pathway critical in monitoring angiogenesis. VEGF exerts its effect through binding to tyrosine kinase receptors, mainly VEGFR-1 (Flt-1, the fms-like tyrosine kinase-1) and VEGFR-2 (Flk-1/KDR, fetal liver kinase-1). This paper reviews the progress, mechanism, and binding modes of recently approved kinase inhibitors, such as sunitinib (Sutent), sorafenib (Nexavar) and dasatinib (Sprycel), as well as other inhibitors that are still under clinical development. Recent clinical treatments suggest that most inhibitors of VEGFR (and/or EGFR) exert their therapeutic effect through not only targeting the VEGFR (and/or EGFR) pathway, but also inhibiting other pathways, such as RAF/MEK/ERK pathway. A new pharmacophore model for second generation of type II tyrosine kinase inhibitors and recent advances in the combination of VEGFR tyrosine kinase inhibitors and other chemotherapeutics are also covered.

Cultured ES cells come unstuck

A clinically approved kinase inhibitor can stop human ES cells from clumping, and should make them easier to grow.

Monitoring Imatinib Resistance with a “Polony” Assay: Towards Tailored Therapy of Chronic Myelogenous Leukemia (CML).

Although tyrosine kinase inhibitor therapy with imatinib in CML patients leads to a high rate of long-term disease control, the development of resistance largely due to bcr-abl point mutations has become a therapeutic challenge. Dasatinib, a recently approved kinase inhibitor, and other new agents invariably have a unique pattern of activity against particular drug-resistant forms of BCR-ABL. It will therefore be helpful to determine the specific mutations that mediate resistance in order to employ the optimal alternative agents in relapsed or refractory patients. As such, there is a need for a rapid, sensitive, and quantitative assay to identify and monitor drug resistance mutations. We have adapted a method for genotyping single nucleotide polymorphisms to mutation detection in clinical samples of CML patients. This “polony” technique entails solid phase PCR amplification of single DNA molecules in a polyacrylamide gel (forming a PCR-colony, or “polony”), followed by discrimination of wild type and mutated nucleotides by a fluorescent single nucleotide base extension assay. The technique is versatile and sensitive (detection limit: 1 mutant in 10−4 wild type cells); it allows us to quantify the BCR-ABL to ABL ratio and to haplotype and screen for several mutations in the same cell population. We have used this assay to screen for the most common BCR-ABL mutations in CML to discern the dynamics of specific mutations following treatment with BCR-ABL kinase inhibitors. One imatinib-treated patient sustained a cytogenetic relapse and was switched to dasatinib. This patient was found to have a T315I mutation that caused subsequent dasatinib resistance. The polony assay detected this mutation from a rare presence to the dramatic takeover in concert with the patient's rapid disease progression. In another imatinib-treated patient who sustained a cytogenetic relapse on imatinib the polony assay detected an M244V mutation, modestly resistant to imatinib (IC50 3.1μM). Doubling the imatinib dose the patient regained cytogenetic response, which was documented by the polony assay. A third patient who exhibited primary resistance to imatinib was switched to nilotinib after progression to accelerated phase, but without success. The polony assay identified an E255K mutation, known to be quite resistant to imatinib (IC50 5.2μM), relatively resistant to nilotinib (IC50 200nM) but sensitive to dasatinib (IC50 5.6nM). Dasatinib therapy resulted in a rapid hematologic response, with a drop in WBC from 60,000 to 5000, and a complete clearance of blasts. The polony assay showed a rapid decrease of the E255K mutation from a frequency of almost 100 percent to about 20 percent. These patients and others in our clinical surveys illustrate the importance of having a reliable means of detecting and quantifying the mutational burden of patients in order to make informed treatment decisions. A prospective clinical trial testing the role of mutational monitoring in guiding the choice of novel agents is required to prove the likely clinical utility of the polony assay in the current era of burgeoning target-directed therapy in CML.

A Liquid Chromatography/Mass Spectrometry-Based Method for the Selection of ATP Competitive Kinase Inhibitors

The currently approved kinase inhibitors for therapeutic uses and a number of kinase inhibitors that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. The 5'-fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an ATP-affinity reagent that covalently modifies a conserved lysine present in the nucleotide-binding site of most kinases. The authors have developed a liquid chromatography/mass spectrometry-based method to monitor binding of ATP competitive protein kinase inhibitors using FSBA as a nonselective activity-based probe for protein kinases. Their method provides a general, rapid, and reproducible means to screen and validate selective ATP competitive inhibitors of protein kinases.

Helicobacter pylori eradication with tetracycline, furazolidone and bismuth (TFB), and rate of reinfection one year after successful treatment

In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 104. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as − 5.234 kcal mol− 1 for CBZ-AAG interaction and − 6.237 kcal mol− 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are − 9.553 kcal mol− 1 and − 14.618 cal mol− 1K− 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol− 1 and 7.206 cal mol− 1K− 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.