ATP-competitive Kinase Inhibitors Research Articles

Allosteric regulation of epidermal growth factor (EGF) receptor ligand binding by tyrosine kinase inhibitors

The epidermal growth factor (EGF) receptor is a classical receptor tyrosine kinase with an extracellular ligand-binding domain and an intracellular kinase domain. Mutations in the EGF receptor have been shown to drive uncontrolled cell growth and are associated with a number of different tumors. Two different types of ATP-competitive EGF receptor tyrosine kinase inhibitors have been identified that bind to either the active (type I) or inactive (type II) conformation of the kinase domain. Despite the fact that both types of inhibitors block tyrosine kinase activity, they exhibit differential efficacies in different tumor types. Here, we show that in addition to inhibiting kinase activity, these inhibitors allosterically modulate ligand binding. Our data suggest that the conformations of the EGF receptor extracellular domain and intracellular kinase domain are coupled and that these conformations exist in equilibrium. Allosteric regulators, such as the small-molecule tyrosine kinase inhibitors, as well as mutations in the EGF receptor itself, shift the conformational equilibrium among the active and inactive species, leading to changes in EGF receptor-binding affinity. Our studies also reveal unexpected positive cooperativity between EGF receptor subunits in dimers formed in the presence of type II inhibitors. These findings indicate that there is strong functional coupling between the intracellular and extracellular domains of this receptor. Such coupling may impact the therapeutic synergy between small-molecule tyrosine kinase inhibitors and monoclonal antibodies in vivo.

Fishing wild-type sparing inhibitors of proto-oncogene c-met variants in renal cell carcinoma from a curated tyrosine kinase inhibitor pool using analog-sensitive kinase technology

A variety of missense mutations in proto-oncogene protein tyrosine kinase c-Met have been clinically observed in the patients and families of papillary renal cell carcinoma (pRCC), imparting that the kinase mutations can be exploited as a new and potential therapeutic target for pRCC. Here, a systematic inhibitor response-to-kinase mutation profile for ATP-competitive tyrosine kinase inhibitors (TKIs) against pRCC-related c-Met mutations is created using a rigorous thermodynamic cycle scheme, from which we are able to identify a number of representative inhibitor/mutation pairs with passivation and sensitization. It is revealed that passivation is commonly caused by steric hindrance between the mutated residue and inhibitor ligand, while sensitization usually results from the formation of favorable nonbonded interactions upon the mutation. The type II inhibitor Nintedanib possesses a high selectivity (7.2-fold) for c-MetY1248H variant over wild type. Structural and energetic analysis revealed that the Y1248H mutation is located in kinase's activation loop which can directly contact the extended moiety of type II inhibitor. The titratable variant residue His1248 is protonated with stabilization by its vicinal negatively charged residue Asp1246, which can form a geometrically satisfactory hydrogen bond and a weak cation-π interaction with the inhibitor ligand, thus conferring variant selectivity to the Nintedanib/Y1248H sensitization.

Abstract 3924: Aurora B, a potential new target in non-T790M lung cancer cells with acquired resistance to anti-EGFR therapy, is effectively blocked by the MET/AXL/FGFR inhibitor S49076

Abstract Background: Non-small cell lung cancer (NSCLC) tumors harboring mutations in the EGF receptor (EGFR) ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (TKIs). S49076 is a potent ATP-competitive tyrosine kinase inhibitor of MET, AXL/MER and FGFR1/2/3 currently in a phase I clinical trial in combination with Gefinitib in NSCLC resistant to EGFR inhibitors. Methods: We generated 6 resistant lines by treating EGFR-mutated, TKI sensitive PC9 cells with increasing concentrations of gefitinib (GR1-5) or erlotinib (ER). The six lines conserved the exon 19 deletion but the T790M resistance mutation emerged in two of them (GR1, GR4), which remained sensitive to osimertinib. Six additional cell lines resistant to osimertinib were derived from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. The 12 resistant cell lines were genotyped and characterized for AXL, GAS6, MET, FGFR1 and FGFR2 expression by qRT-PCR, immunohistochemistry and Western blotting. The effects of S49076 and the aurora kinase B (AURKB) inhibitor barasertib were analyzed by MTT, flow cytometry and immunocytochemistry. Western blotting and ELISA of key signal transduction proteins and gene silencing was used to gain insight in the mechanism of action of the drug. Results: Several mechanisms associated with resistance to EGFR TKIs were represented in our panel of resistant cell lines and they frequently co-occurred, including AXL/GAS6 and FGFR1 overexpression, MET activation or emergence and loss of the p.T790M. When using in vitro models, we discovered that the cell lines of our panel were relatively insensitive to single-agent treatment with inhibitors of AXL, MET or FGFR1. In contrast, resistant cells not harboring the p.T790M were sensitive in vitro and also in xenografts to S49076, a drug targeting AXL, MET and FGFR1/2/3. At higher doses (above 300 nM in cultured cells), S49076 blocks AURKB, which could therefore be considered as a further target of the compound. Since partial silencing of AURKB rendered cells resistant to S49076, we investigated the role of this protein. We found that the levels of phospho-histone H3 (pH3), but not Ki67, were increased in all the EGFR TKI resistant cells of our panel, revealing a widespread AURKB activation not related to increased cell proliferation. Treatment with S49076 or barasertib down-regulated pH3 and induced G1/S arrest and polyploidy. In cells harboring the p.T790M, polyploid cells underwent senescence. However, in absence of the resistance mutation, polyploidy was followed by cell death. Conclusions: AURKB is a novel target in non-T790M NSCLC with acquired resistance to first and third line EGFR TKIs. Multitargeted agents such as S49076, which inhibit not only AXL, MET or FGFR1 but also AURKB, might be more effective in this setting than agents targeting the receptor tyrosine kinases alone. Citation Format: Jordi Bertran-Alamillo, Valérie Cattan, Marie Schoumacher, Jordi Codony-Servat, Frederique Cantero, Ruth Roman, Sonia Rodriguez, Ana Gimenez-Capitan, Josep Castellvi, Cristina Teixido, Rafael Rosell, Miguel A. Molina-Vila. Aurora B, a potential new target in non-T790M lung cancer cells with acquired resistance to anti-EGFR therapy, is effectively blocked by the MET/AXL/FGFR inhibitor S49076 [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 3924.

Identification of PKMYT1 inhibitors by screening the GSK published protein kinase inhibitor set I and II

As a member of the Wee-kinase family protein kinase PKMYT1 is involved in G2/M checkpoint regulation of the cell cycle. Recently, a peptide microarray approach led to the identification of a small peptide; EFS247–259 as substrate of PKMYT1, which allowed for subsequent development of an activity assay. The developed activity assay was used to characterize the PKMYT1 catalyzed phosphorylation of EFS247–259. For the first time kinetic parameters for PKMYT1, namely Km, Km, ATP and vmax were determined. The optimized assay was used to screen the published protein kinase inhibitor sets (PKIS I and II), two sets of small molecule ATP-competitive kinase inhibitors reported by GlaxoSmithKline. We identified ten inhibitors, providing different scaffolds. The inhibitors were further characterized by using binding assay, activity and functional assay. In addition, docking studies were carried out in order to rationalize the observed biological activities. The derived results provide the basis for further chemical optimization of PKMYT1 inhibitors and for further analysis of PKMYT1 as target for anti-cancer therapy.

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Rapamycin-insensitive mechanistic target of rapamycin regulates basal and resistance exercise-induced muscle protein synthesis.

We investigated whether rapamycin-insensitive mechanistic target of rapamycin (mTOR) signaling plays a role in regulating resistance exercise-induced muscle protein synthesis. We used a rodent model of resistance exercise and compared the effect of rapamycin, an allosteric mTOR inhibitor, with the effect of AZD8055, an ATP-competitive mTOR kinase inhibitor. The right gastrocnemius muscle of male Sprague-Dawley rats age 11 wk was contracted isometrically via percutaneous electrical stimulation (100 Hz, 5 sets of ten 3-s contractions, 7 s of rest between contractions, 3 min of rest between sets), and the left gastrocnemius muscle served as control. Vehicle, rapamycin, or AZD8055 were intraperitoneally injected 1 h before resistance exercise. Results indicated that both rapamycin and AZD8055 inhibited mTOR complex 1 (mTORC1)/70-kDa ribosomal protein S6 kinase signaling similarly, whereas mTORC1/eukaryotic translation initiation factor 4E-binding protein 1 signaling was greatly inhibited by AZD8055. Moreover, only AZD8055 inhibited the phosphorylation of Akt at Ser473, a downstream target of mTORC2. AZD8055, but not rapamycin, completely inhibited the resistance exercise-induced increase in muscle protein synthesis. We conclude that the resistance exercise-induced increase in muscle protein synthesis is an mTOR signaling-dependent process. Furthermore, both rapamycin-sensitive and -insensitive mTOR signaling regulate this event.-Ogasawara, R., Suginohara, T. Rapamycin-insensitive mechanistic target of rapamycin regulates basal and resistance exercise-induced muscle protein synthesis.

Phase I studies of AZD1208, a proviral integration Moloney virus kinase inhibitor in solid and haematological cancers

BackgroundProviral integration Moloney virus (PIM) kinases (PIM1, 2 and 3) are overexpressed in several tumour types and contribute to oncogenesis. AZD1208 is a potent ATP-competitive PIM kinase inhibitor investigated in patients with recurrent or refractory acute myeloid leukaemia (AML) or advanced solid tumours.MethodsTwo dose-escalation studies were performed to evaluate the safety and tolerability, and to define the maximum tolerated dose (MTD), of AZD1208 in AML and solid tumours. Secondary objectives were to evaluate the pharmacokinetics, pharmacodynamics (PD) and preliminary efficacy of AZD1208.ResultsSixty-seven patients received treatment: 32 in the AML study over a 120–900 mg dose range, and 25 in the solid tumour study over a 120–800 mg dose range. Nearly all patients (98.5%) in both studies experienced adverse events, mostly gastrointestinal (92.5%). Dose-limiting toxicities included rash, fatigue and vomiting. AZD1208 was not tolerated at 900 mg, and the protocol-defined MTD was not confirmed. AZD1208 increased CYP3A4 activity after multiple dosing, resulting in increased drug clearance. There were no clinical responses; PD analysis showed biological activity of AZD1208.ConclusionsDespite the lack of single-agent clinical efficacy with AZD1208, PIM kinase inhibition may hold potential as an anticancer treatment, perhaps in combination with other agents.

The PKC inhibitor BimI functions as a bi‐topic ligand that toggles a conserved allosteric switch to drive potent kinase inhibition

ATP‐competitive small‐molecule kinase inhibitors (SMKIs) are viewed primarily from the lens of tight and specific binding to the conserved catalytic domain. The allosteric effects of ATP‐competitive SMKIs are generally ignored, despite evidence that they can influence the potency and selectivity of kinase inhibition. We have recently showed that the ATP‐competitive PKC inhibitor BimI can displace substrate binding leading to potent inhibition of the kinase (Sommese and Sivaramakrishnan, JBC, 2016). In this study, we examine the substrate‐competitive behavior of a range of SMKIs derived from staurosporine and nucleotide analogs. We find that BimI functions like a bi‐topic ligand that acts on both the orthosteric ATP binding pocket and a remote allosteric site that determines overall kinase conformation. Combining novel FRET‐based sensors and multi‐scale MD simulations we identify a novel, conserved structural mechanism that regulates substrate binding in protein kinases. Bi‐topic ligands such as BimI disrupt a conserved ionic‐lock that drives global reorganization of the kinase domain through long‐range allosteric pathways that connect the N‐ and C‐terminal lobes. Together our studies identify a novel allosteric switch that regulates substrate binding in protein kinases that can be leveraged to develop highly selective kinase inhibitors.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antitumor Properties of RAF709, a Highly Selective and Potent Inhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS or BRAF.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.

Inhibition of mTORC1/C2 signaling improves anti-leukemia efficacy of JAK/STAT blockade in CRLF2 rearranged and/or JAK driven Philadelphia chromosome-like acute B-cell lymphoblastic leukemia.

Patients with cytokine receptor-like factor 2 rearranged (CRLF2-re) subgroup Philadelphia chromosome–like B-cell acute lymphoblastic leukemia (Ph-like B-ALL) have a high relapse rate and poor clinical outcomes. CRFL2-re Ph-like B-ALL is characterized by heightened activation of multiple signaling pathways, including the JAK/STAT and PI3K/AKT/mTOR pathways. We hypothesized that the combined inhibition by JAK2 and mTOR inhibitors would induce an additive antileukemia effect in CRLF2-re Ph-like B-ALL. In this study, we tested the antileukemia efficacy of the type I JAK inhibitor ruxolitinib and type II JAK inhibitor NVP-BBT594 (hereafter abbreviated BBT594) [1] alone and combined with allosteric mTOR inhibitor rapamycin and a second generation ATP-competitive mTOR kinase inhibitor AZD2014. We found that BBT594/AZD2014 combination produced robust anti-leukemic effects in Ph-like cell lines in vitro and in patient-derived xenograft (PDX) cells cultured ex vivo. JAK2/mTOR inhibition arrested the cell cycle and reduced cell survival to a greater extent in Ph-like B-ALL cells with CRLF2-re and JAK2 mutation. Synergistic cell killing was associated with the greater inhibition of JAK2 phosphorylation by BBT594 than by ruxolitinib and the greater inhibition of AKT and 4E-BP1 phosphorylation by AZD2014 than by rapamycin. In vivo, BBT594/AZD2014 co-treatment was most efficacious in reducing spleen size in three Ph-like PDX models, and markedly depleted bone marrow and spleen ALL cells in an ATF7IP-JAK2 fusion PDX. In summary, combined inhibition of JAK/STAT and mTOR pathways by next-generation inhibitors had promising antileukemia efficacy in preclinical models of CRFL2-re Ph-like B-ALL.

PF-114 Mesylate, a Novel Third Generation ATP-Competitive BCR-ABL Tyrosine Kinase Inhibitor: First Safety and Efficacy Data from a Phase I Study in Patients with CML with Failure of Prior TKI Therapy

Background: PF-114 mesylate is a novel third generation oral tyrosine kinase inhibitor (TKI) that blocks native and mutated Bcr-Abl isoforms, including the gatekeeper mutant T315I, which is uniformly resistant to all approved first- and second generation TKIs. At the present time extensive pre-clinical studies of PF-114, including mechanism of action, kinase profiling, in vitro and in vivo efficacy, safety pharmacology, ADME, and toxicology studies in mice, rats and dogs have been completed and supported initiation of phase I clinical trial of PF-114 mesylate in patients with chronic or accelerated phase Ph+ CML who are resistant to at least one of the second generation TKIs or intolerant to previous treatment with TKIs or who have T315I mutation in the BCR-ABL gene (NCT02885766).Methods: The trial represents a classical 3+3 design of dose escalation till the maximum tolerated dose (MTD) followed by the expanded cohorts planned for dose(s) below the MTD. The total expected enrollment is 44 patients. Escalating doses of single-agent PF-114 mesylate were administered orally on a continuous once daily (QD) dosing schedule. Treatment continued until disease progression, unacceptable toxicity, consent withdrawal, or death. The primary objective was to study the dose-limiting toxicities (DLTs) occurring in cycle 1 of treatment and determine the MTD. Secondary objectives included safety, anti-CML activity (based on hematologic, cytogenetic, and molecular assessments), pharmacodynamic and pharmacogenetic properties. Adverse events (AEs) were assessed and graded according to NCI-CTCAE v4.03.Results: At data cutoff, 4-dose cohorts - 50 mg, 100 mg, 200 mg and 400 mg have completed cycle 1 and 500 mg cohort has started cycle 1 of treatment. Overall 18 patients (8 males) had been treated at the following QD doses: 50 mg (n = 1), 200 mg (n = 5), 400 mg (n = 11), 500 mg (n = 1), and the MTD has not yet been reached. A total of 7 patients with T315I mutation were included into the trial thus far. Median age was 50.5 years (range, 32-66 years). Median time from diagnostics to treatment was 11.5 years (range, 16-1 years) All patients had baseline Eastern Cooperative Oncology Group performance status 0-1. Patients were heavily pretreated; 9 (50%) had received ≥ 3 prior TKIs, 6 (33%) had received 2 prior TKIs and 3 (17%) patients with T315I had received 1 prior TKI. In 11 patients, treatment is ongoing at doses 200-500 mg QD, with median duration of exposure of 8 months (range, 2-12 months), and 7 patients discontinued (disease progression [n = 5], AEs [n = 2]). Preliminary data suggest PF-114 pharmacokinetics is dose-proportional. At the end of cycle 1 of study therapy, no drug-related adverse events greater than grade 1 in severity were observed in patients treated at the 50 mg, 100 mg and 200 mg dose levels. At the dose of 400 mg QD, a single DLT case of grade 3 erythematous rash was observed. Most common grade 2/3 AEs on 400 mg QD were dermatologic toxicity (4/11), neutropenia (1/11). No deterioration of the ankle-brachial index (ABI), which is being prospectively measured, or vascular occlusive events were observed so far. The patient with recurrent pleural effusions on previous treatment with dasatinib did not reveal effusions after 11 cycle of therapy; the patient with ischemic stroke on previous treatment with nilotinib did not reveal cardio-vascular events after 9 cycles. To date, no SAEs have been reported. Overall, during the period of 3 cycles, major cytogenetic response (CyR) have been obtained in 4 of 11 patients who completed 3 cycles (2 cases of complete CyR in and 2 cases of partial CyR). Of those patients who revealed cytogenetic response two patients have T315I mutation. Pharmacodynamic and pharmacogenetic assessments are underway.Conclusion: PF-114 mesylate exhibits antitumor activity in a heavily pretreated subgroup of patients with resistant forms of CML including cases with T315I mutation. The evaluation of the safety profile continues. The dose escalation stage of the current phase 1 study continues, while the including of patients into the expanded cohorts with doses below the MTD has already started. DisclosuresChelysheva:Fusion Pharma LLC: Consultancy. Nemchenko:Fusion Pharma LLC: Consultancy. Zaritskey:Janssen: Consultancy; Novartis: Consultancy, Speakers Bureau. Shuvaev:Fusion Pharma LLC: Consultancy. Novikov:Fusion Pharma LLC: Employment. Shulgina:Fusion Pharma LLC: Employment. Chilov:Fusion Pharma LLC: Employment, Patents & Royalties.

2-Alkylsulfanyl-4(5)-aryl-5(4)-heteroarylimidazoles: An Overview on Synthetic Strategies and Biological Activity.

2-Alkylsulfanyl-4(5)-aryl-5(4)-heteroarylimidazoles represent an important class of ATP-competitive protein kinase inhibitors, offering the possibility of multiple interactions with different regions of the target enzyme. The necessity of exploring the effects of diverse chemical decorations around the imidazole core prompted the design of several synthetic routes aimed at achieving both efficiency and flexibility. Additionally, the optimization of established protocols and the extensive use of transition metal-catalyzed cross-coupling reactions have been broadening the spectrum of preparative methodologies within the last decade. This review summarizes the progress in the development of synthetic strategies leading to 2-alkylsulfanyl-4(5)-aryl-5(4)-heteroarylimidazoles and 1-alkyl-2-alkylsulfanyl-4(5)-aryl-5(4)-heteroarylimidazoles and offers a glance at the biological activities of this class of compounds.

Kinase Crystal Miner: A Powerful Approach to Repurposing 3D Hinge Binding Fragments and Its Application to Finding Novel Bruton Tyrosine Kinase Inhibitors

Protein kinases represent an important target class for drug discovery because of their role in signaling pathways involved in disease areas such as oncology and immunology. A key element of many ATP-competitive kinase inhibitors is their hinge-binding motif. Here, we describe Kinase Crystal Miner (KCM)-a new approach developed at Boehringer Ingelheim (BI) that harvests the existing crystallographic information on kinase-inhibitor co-crystal structures from internal and external databases. About 1000 unique three-dimensional kinase inhibitor hinge binding motifs have been extracted from structures covering more than 180 different protein kinases. These hinge binding motifs along with their attachment vectors have been combined in the KCM for the purpose of scaffold hopping, kinase screening deck design, and interactive structure-based design. Prospective scaffold hopping using the KCM identified two potent and selective Bruton tyrosine kinase (BTK) inhibitors with hinge binding fragments novel to BTK.

First-in-human phase I study of oral S49076, a unique MET/AXL/FGFR inhibitor, in advanced solid tumours

Background and objectivesS49076 is a novel ATP-competitive tyrosine kinase inhibitor of MET, AXL and FGFR with a unique selectivity profile. A phase I open-label study was undertaken to establish the tolerability profile and determine the recommended dose (RD) and administration schedule. Materials and methodsPatients with advanced solid tumours received S49076 orally once-daily (qd) or twice-daily (bid) in continuous 21-day cycles at escalating doses guided by a 3 + 3 design and followed by an expansion phase at the RD. Pharmacokinetic (PK) parameters were assessed and pharmacodynamic end-points were evaluated in pre- and post-treatment tumour biopsies. Preliminary anti-tumour activity was evaluated as per the Response Evaluation Criteria In Solid Tumours 1.1 criteria. ResultsA total of 103 patients were treated: 79 in the dose-escalation and 24 in the expansion. Doses from 15 to 900 mg were evaluated. Dose-limiting toxicities were reported in 9 patients and occurred at 30, 760 and 900 mg in the qd arm and at 180, 225 and 285 mg in the bid arm. The RD was defined at 600 mg qd. Adverse events (AEs) occurred with similar frequency in both regimens at an equivalent total daily dose. Overall, 83 patients (81.4%) had drug-related AEs, the majority (93%) of which were grade I–II (National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0) and only 3% led to drug discontinuation. Intratumoural PK analysis at the RD suggested hitting of MET, AXL and FGFR. ConclusionS49076 demonstrated a tolerable safety profile with limited single-agent activity. PK/pharmacodynamic readouts of S49076 are encouraging for further investigation of S49076 in combination therapies. Trial registration numberISRCTN00759419.

Abstract 4186: A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors

Abstract Protein kinases are central to cellular signal transduction and regulation of cellular processes, and are one of the most attractive target classes in modern drug discovery. Multiple kinase inhibitors have already been approved for treatment of various diseases including such severe conditions as cancer. The first and up to today largest group of drugs that effectively inhibits their respective kinase targets belong to the class of ATP competitive compounds. They bind into or near the ATP binding site of the enzymes and inhibit kinase activity by blocking access of ATP to the active site. Although there are numerous examples of highly specific ATP competitive compounds this mode of action is limited by several factors: The ATP binding pocket structures of kinases show a high degree of similarity, which makes finding highly selective compounds challenging. Furthermore, competing with ATP for binding to the same target site, compounds have to be of very high affinity due to the high intracellular ATP concentrations. Therefore, the interest to develop non-ATP competitive inhibitors has risen considerably over the last years. Such inhibitors bind to kinases at sites apart from the ATP binding site, inhibiting their activity e.g. by stabilizing an inactive conformation (like DFG-out state binders), displacing essential cofactors (like cyclins for CDKs) or by blocking activating modifications (like phosphorylation by upstream kinases). We present data of an in-vitro biochemical kinase activity assay setup which is suited to discriminate between ATP-competitive and non-ATP competitive inhibitors. The IC50 of an ATP-competitive inhibitor will increase with increasing ATP concentrations and the IC50 value at a given ATP concentration may be calculated using the equation of Cheng and Prussof: IC50=Ki+(Ki*[ATP]/KM[ATP] (Cheng Y., Prusoff W. H. (1973) Biochem. Pharmacol. 22: 3099-3108). By determining IC50 values for an inhibitor of a specific kinase at different ATP concentrations we examined whether the IC50 value changed according to the Cheng-Prusoff equation, indicating an ATP-competitive mode of action, or if the IC50 values remained unchanged in presence of elevated ATP indicating a non-ATP competitive or mixed type mode of action. In our assay setup we determined the IC50 values at ATP concentrations in a range of 0.1 to 10 fold the ATP KM of the kinase of interest. By comparing the results obtained for the non-ATP competitive MEK1 inhibitor selumetinib and the ATP competitive inhibitor staurosporine we could verify that our assay setup is well suited to discriminate between these different types of kinase inhibitors. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Carolin Heidemann-Dinger, Constance Ketterer, Diane Krämer, Marcel Pathe, Michael H. Kubbutat. A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2017-4186

Identification of a Water-Soluble Indirubin Derivative as Potent Inhibitor of Insulin-like Growth Factor 1 Receptor through Structural Modification of the Parent Natural Molecule.

Indirubins have been identified as potent ATP-competitive protein kinase inhibitors. Structural modifications in the 5- and 3'-position have been extensively investigated, but the impact of substituents in 5'-position is not equally well-studied. Here, we report the synthesis of new indirubin 3'- and 5'-derivatives in the search of water-soluble indirubins by introducing basic centers. Antiproliferative activity of all compounds in tumor cells was evaluated along with kinase inhibition of selected compounds. The results show the 3'-position to tolerate large substituents without compromising activity, whereas bulk and rigid substituents in 5'-position appear unfavorable. Screening molecular targets of water-soluble 3'-oxime ethers revealed 6ha as preferential inhibitor of insulin-like growth factor 1 receptor (IGF-1R) in a panel of 22 protein kinases and in cells. Consistently, 6ha inhibited tumor cell growth in the NCI 60 cell line panel and induced apoptosis. The results indicate that the 5'-position provides limited space for chemical modifications and identify 6ha as a potent water-soluble indirubin-based IGF-1R inhibitor.

AKT Inhibition in Solid Tumors With AKT1 Mutations.

Purpose AKT1 E17K mutations are oncogenic and occur in many cancers at a low prevalence. We performed a multihistology basket study of AZD5363, an ATP-competitive pan-AKT kinase inhibitor, to determine the preliminary activity of AKT inhibition in AKT-mutant cancers. Patients and Methods Fifty-eight patients with advanced solid tumors were treated. The primary end point was safety; secondary end points were progression-free survival (PFS) and response according to Response Evaluation Criteria in Solid Tumors (RECIST). Tumor biopsies and plasma cell-free DNA (cfDNA) were collected in the majority of patients to identify predictive biomarkers of response. Results In patients with AKT1 E17K-mutant tumors (n = 52) and a median of five lines of prior therapy, the median PFS was 5.5 months (95% CI, 2.9 to 6.9 months), 6.6 months (95% CI, 1.5 to 8.3 months), and 4.2 months (95% CI, 2.1 to 12.8 months) in patients with estrogen receptor-positive breast, gynecologic, and other solid tumors, respectively. In an exploratory biomarker analysis, imbalance of the AKT1 E17K-mutant allele, most frequently caused by copy-neutral loss-of-heterozygosity targeting the wild-type allele, was associated with longer PFS (hazard ratio [HR], 0.41; P = .04), as was the presence of coincident PI3K pathway hotspot mutations (HR, 0.21; P = .045). Persistent declines in AKT1 E17K in cfDNA were associated with improved PFS (HR, 0.18; P = .004) and response ( P = .025). Responses were not restricted to patients with detectable AKT1 E17K in pretreatment cfDNA. The most common grade ≥ 3 adverse events were hyperglycemia (24%), diarrhea (17%), and rash (15.5%). Conclusion This study provides the first clinical data that AKT1 E17K is a therapeutic target in human cancer. The genomic context of the AKT1 E17K mutation further conditioned response to AZD5363.

19F-NMR-Based Dual-Site Reporter Assay for the Discovery and Distinction of Catalytic and Allosteric Kinase Inhibitors.

In modern kinase drug discovery, allosteric inhibitors have become a focus of attention due to their potential selectivity, but such compounds are difficult to identify. Here we describe an NMR-based competition assay using 19F-containing reporter molecules, which allows for rapid identification and discrimination between ATP-competitive and allosteric kinase inhibitors. We illustrate the principle of such a dual-site competition assay with the example of catalytic and allosteric ABL1 kinase inhibitors. The assay can also be used to identify and characterize mixed binding modes of well-known drugs, as shown for crizotinib and fingolimod.

Abstract P6-08-04: Preclinical efficacy of dasatinib in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer xenograft model

Abstract Background: Dasatinib is an orally-active ATP-competitive small molecule kinase inhibitor that potently inhibits Abl kinase, Src family kinases and other kinases (Lombardo et al., 2004). Src, one of the key targets of dasatinib is involved in the regulation of cell proliferation, survival and apoptotic ability of cancer cells (Tryfonopoulos et al., 2011; Pusztai et al., 2014). Dasatinib has shown its anti-proliferative and anti-metastatic effectiveness against triple-negative breast cancer (TNBC) in both preclinical and clinical studies (Finn et al., 2011). Several molecular targets including poly ADP ribose polymerase (PARP) are under clinical investigation for the treatment of TNBC. Recently, PARP inhibitors in combination with chemotherapy have shown promising results in this disease in clinical and preclinical studies (Tutt et al., 2010; Kim et al., 2013; De et al., 2014). Here, we hypothesize that dasatinib in combination with PARP inhibitor (ABT888) plus standard cytotoxic agent (carboplatin) will attenuate the growth of both TNBC cell lines and xenograft tumors. Methodology:We have used BT-20 (PIK3CA mutated, H1047R), HCC70 (PTEN null), HCC1937 (BRCA1 mutated, PTEN null), MDA-MB-231 (KRAS/BRAF mutated), MDA-MB-468 (PTEN null) and SUM149PT (BRCA1 mutated, PTEN null) cells for in vitro study. Survival/proliferation, colony formation and apoptosis were examined by using 2D proliferative/growth assay, 3D-ON-TOP assays, and annexinV staining respectively. We next studied the activation status of Src and its downstream signaling. We also have evaluated the effects on tumor growth inhibition of dasatinib/ABT888/carboplatin as a single agent or in combination by using mouse xenograft model. Results: We observed that 1) Dasatinib inhibited Src activation in all tested lines, induced dephosphorylation of ERK1/2 and S6 RP; 2) level of Cyclin D1 was decreased by dasatinib treatment; 3) high anti-proliferative activities were observed following the treatment of dasatinib along with ABT888 plus carboplatin in both 2D proliferation assay and 3D-ON TOP colony formation assay; 4) dasatinib in combination with ABT888 plus carboplatin inducing early stage apoptosis was seen by Annexin V staining in all tested cell lines; 5) dasatinib alone or combined with ABT888 or carboplatin or in triple combination inhibited tumor growth in TNBC xenograft models, the best tumor inhibition result was induced by triple combination (comparing to no treatment control, the mean tumor volume was decreased ~ 87% ). Conclusion: Our in vitro and in vivo studies suggest that dasatinib may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC. Mechanistic studies of xenograft tumor samples are ongoing, the results of which will be presented in the meeting. Citation Format: Sun Y, Lin X, Carlson JH, De P, Dey N, Jepperson T, R & D NCI, Williams C, Leyland-Jones B. Preclinical efficacy of dasatinib in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer xenograft model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-04.

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors.

ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile ∼200 kinases in single analytical runs using as little as 5 μL of kinobeads and 300 μg of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2, and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.