* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Binding Studies of Type I, II, and III Kinase Inhibitors against Bcr
Toxicodynamics wikipedia , lookup
Discovery and development of proton pump inhibitors wikipedia , lookup
Discovery and development of beta-blockers wikipedia , lookup
Discovery and development of cephalosporins wikipedia , lookup
DNA-encoded chemical library wikipedia , lookup
Discovery and development of angiotensin receptor blockers wikipedia , lookup
Discovery and development of dipeptidyl peptidase-4 inhibitors wikipedia , lookup
Discovery and development of direct thrombin inhibitors wikipedia , lookup
Neuropharmacology wikipedia , lookup
Discovery and development of cyclooxygenase 2 inhibitors wikipedia , lookup
Nicotinic agonist wikipedia , lookup
Discovery and development of HIV-protease inhibitors wikipedia , lookup
Drug discovery wikipedia , lookup
Discovery and development of tubulin inhibitors wikipedia , lookup
Discovery and development of non-nucleoside reverse-transcriptase inhibitors wikipedia , lookup
NK1 receptor antagonist wikipedia , lookup
MTOR inhibitors wikipedia , lookup
Discovery and development of neuraminidase inhibitors wikipedia , lookup
Metalloprotease inhibitor wikipedia , lookup
Discovery and development of ACE inhibitors wikipedia , lookup
Discovery and development of direct Xa inhibitors wikipedia , lookup
Discovery and development of antiandrogens wikipedia , lookup
Drug design wikipedia , lookup
Discovery and development of integrase inhibitors wikipedia , lookup
MOLECULAR SENSING AUTHORS Fei Shen Robert R. Lavieri Richard J. Isaacs Nathan Gilbert Stephen Dotson *Scot R. Weinberger POSTER NOTE Binding Studies of Type I, II, and III Kinase Inhibitors against Bcr-Abl Kinase using Back-Scattering Interferometry: A Tale of Allostery [email protected] Molecular Sensing, Inc. Nashville, Tennessee * Corresponding Author Figure 1. A model showing where various inhibitors bind Bcr-Abl kinase is shown. Note the large distance between the ATP binding pocket and site in which GNF-2/ GNF-5 binds. Resistance to imatinib results from the emergence of point mutations within the kinase domain of the Bcr-Abl protein that reduce the binding affinity of imatinib. The most resistant mutations are found in the P-loop of the protein near residues that are in direct contact with the drug. The degree of resistance varies from a few fold for some of the A-loop mutants to complete resistance for the T315I mutation which precludes imatinib from binding. The steady rate of developing resistance to imatinib has suggested that new kinase inhibitors could be of clinical value, if they could override imatinib resistance and bind with higher affinity to Bcr-Abl. INTRODUCTION In addition to playing an essential role in cellular energetics, kinases and their associated signaling pathways are principally responsible for the regulation of intracellular processes. When abnormally expressed or controlled, kinase activity can cause cellular dysregulation and contribute to the onset of several diseases, including cancer. Based on the fundamental understanding of kinase malfunction in cancer biology, the discovery of small organic molecules to alter kinase function has culminated in the development of targeted cancer therapy. However, limited selectivity and the emergence of drug resistance remain fundamental challenges for current modern medicinal chemistry research for the development of kinase inhibitors that are effective in long-term treatments. Most known kinase inhibitors are Type I inhibitors, ATP-competitive compounds such as staurosporine, erlotinib (Tarceva®) and dasatinib (Sprycel®), that bind to the ATP binding site and hydrogen bond with the hinge region of the kinase . Type II inhibitors are compounds which bind partially in the ATP binding site and extend past the gatekeeper and into an adjacent allosteric site that is present only in the inactive kinase conformation. Compared to Type I inhibitors, Type II inhibitors have been shown to possess advantageous pharmacological properties, including improved target specificity 2. As such, many Type II inhibitors currently on the market, such as imatinib (Gleevec®), are very effective anti-cancer drugs. Mutations resistant to classical ATP-competitive (Type I/II) inhibitors are emerging at a rapid pace and often limit the success of newly available targeted cancer therapies. Such mutations often result in a steric hindrance that obstructs inhibitor binding to the hinge region of the ATP pocket. At present, there are more than 50 mutation sites and more than 70 individual mutations conferring different levels of imatinib resistance found within CML patients. More recently, a number of Type III inhibitors that function via allosteric modulation have demonstrated promise towards addressing mutation dependent drug resistance. As such, the need to identify and develop reversible inhibitors that are resistant to such mutations and bind with a high affinity is the focus of many academic and industrial research projects. MOLECULAR SENSING Binding Studies of Type I, II, and III Kinase Inhibitors against Bcr-Abl Kinase using Back-Scattering Interferometry: A Tale of Allostery Figure adapted from: Conn PJ, Christopoulos A, and Lindsley CW “Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders” Nat Rev Drug Discov. 2009 Jan;8(1):41-54 Figure 2. An allosteric modulator compound may exert its action by altering affinity or by altering efficacy. In the case of affinity driven modulation one would expect to see a leftward (or rightward in the case of negative modulation) shift for the binding isotherm of the endogenous ligand. If no change in affinity of the endogenous ligand is detected in the presence of the allosteric modulator, then it is likely an efficacy driven mechanism. MATERIALS AND METHODS Back-Scattering Interferometry The Back-Scattering device is a micro-scale interferometer (see figure 3). The BSI device consists of a HeNe laser, a microchip, and a CCD camera. The microchip receives light from the laser and illuminates the sample containing channel. As light passes into the channel, interference fringe patterns arise. A camera images the fringes as shown in Figure 3. When molecules combine, the resultant complex causes a change in molecular mean polarizability that is measured as a fringe pattern shift. Monitoring the change in fringe phase as a function of ligand concentration allows equilibrium dissociation constant, Kd, measurements to be performed. Setting up the BSI Assay BSI Kd determinations are performed in end-point fashion, with target and ligand pre-incubated to establish equilibrium. Target and ligand concentrations are chosen to initiate pseudo-first order binding conditions, for which the target is typically held at sparing concentration and the ligand in excess to insure against depletion during the binding process. Page 2 Figure 3: Back-Scattering Interferometer schematic. POSTER NOTE Figure 4: BSI sample preparation. Figure 4 illustrates the RI change for a constant concentration of target A (light blue trace, constant RI), the increase in RI as ligand B is increased (red trace) as a control and finally, the binding isotherm RI curve for the AB complex after mixing and equilibration of A and B (dark blue trace). In practice, control B is run simultaneously in a reference channel with complex AB probed in the analytical channel. The difference is plotted as AB-B. A. A single site binding isotherm model is fitted to AB to determine the binding maximum or Bmax. Kd is then established as ½ Bmax. Preparation of Bcr-Abl Kinase Target Solutions and Kinase Inhibitors Wild-type Bcr-Abl Kinase as well as H396P, M351T, Q252H, and T315I mutants were sourced from Millipore (EMD Millipore, Darmstadt, Germany). All Bcr-Abl kinases were expressed via baculovirus in Sf21 insect cells and provided in aliquots of 10 ug of enzyme (27% purity) in 100 uL of 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 270 mM sucrose, 1 mM benzamide, 0.2 mm PMSF, 0.1 mm EGTA, 0.1% 2-mercaptoethanol, 0.03% Brij 35 and kept frozen at -70o C until used. Assay and diluent buffer was 8 mM MOPS, pH 7, 10 mM Mg Acetate, 0.2 mM EDTA, and 1% DMSO. Kinase working solutions were created by diluting the above noted stock solutions to concentrations that approximated 1/50 of the target Kd for each system (concentration range: 0.5 nM – 10 nM). See figure 8 for kinase inhibitor preparation Assay Sample Preparation and BSI Analysis BSI binding affinity determinations were performed under equilibrium based conditions. For these Bcr-Abl kinase – kinase inhibitor assays, enzyme concentration range was varied from 0.5 nM to 10 nM, while kinase concentration ranged from 50 pM – 125 mM. Samples were prepared using 250 uL Eppendorf tubes, and allowed to incubate for four hours at room temperature (typically 22o C). BSI measurements were performed using a dual-channel BSI prototype system (Molecular Sensing, Inc., Nashville, TN, USA). Each sample was measured in triplicate. Difference plots of assay minus control for BSI response at each inhibitor concentration were constructed using GraphPad Prism® (San Diego, CA, USA). Single-site binding model fits were performed to determine the binding maximum (Bmax) and Kd determined as ½ Bmax. Page 3 MOLECULAR SENSING Binding Studies of Type I, II, and III Kinase Inhibitors against Bcr-Abl Kinase using Back-Scattering Interferometry: A Tale of Allostery Figure 5: Preparation of Kinase Inhibitors. Imatinib, disatanib, and nilotinib were purchased from LC Laboratories (Woburn, MA, USA) and were first brought up as 50 uM working stocks in 100% DMSO. Dose response series were created by diluting each working stock with MOPS buffer to establish the appropriate target concentration range for each binding system using a 12-point doubling dilution approach (range: 50 pM – 125 uM). Figure 6: GNF-2 and GNF-5 are fully allosteric inhibitors of BCR-Abl; GNF-5 has improved pharmacokinetic properties compared to GNF-2. Interestingly, combining GNF-5 with ATP-competitive type inhibitors seems to be a viable approach for targeting mutations such as T315I. Note, an ATP-competitive type compound alone is not effective against T315I. DATA ANALYSIS Kinase Inhibitor Binding Curve Analysis The results for measurements of dasatinib, nilotinib, and imatinib binding affinity to wild-type and mutant Bcr-Abl Kinase are illustrated in figure 7. The overall binding affinity for these systems is summarized in Table 1. For each of these systems, resultant assays produced a high degree of concordance between replicate measurements (avg Kd % RSD < 25 %). Table 1 also lists the final concentration of Bcr-Abl Kinase used in each assay. For most of these assay systems, the total protein consumption was quite low [range 72 picomole ( 9 mg) – 0.156 picomole (0.02 mg)]. The binding affinity of Type I, II and III Bcr-Abl kinase inhibitors with wild type and four mutant Bcr-Abl kinases (H396P, M351T, Q252H, and T315I) were measured using the label-free, free solution biosensor Page 4 technology known as Back-Scattering Interferometry (BSI). BSI successfully demonstrated facile determination of equilibrium dissociation constants (Kd) for all systems, with a high degree of concordance with competition assay derived IC50 results. Binding assays were optimized within a couple of days of experimentation, and for the entire study, less than twenty micrograms of total enzyme was consumed. These results indicate that BSI binding studies both class I, II, and III kinase inhibitors can easily be performed, allowing for confirmation of target engagement as well as direct binding assessment of type II and III kinase inhibitors against inactive Bcr-Abl kinase. The latter makes BSI an attractive biophysical technique for the study of second and third generation kinase inhibitors to address the challenges of kinase inhibitor drug resistance. S ignal (pixels ) POSTER NOTE 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 0.01 Wild Type M351T Q252H H396P T3151 0.1 10 100 1000 10000 100000 Log[Dasatinib], nM 7A Signal (pixels) 1 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 0.1 [Bcr-Abl Kinase] (nM) Imatinib-WT 472 +/- 83 5 Imatinib- H396P 1228 +/- 92 Imatinib- T315I 4050 +/- 1001 Nilotinib-WT 12 +/- 1.8 Nilotinib- H396P 32 +/- 6.5 Nilotinib- T315I 761 +/- 100 Dasatinib-WT Wild Type M351T Q252H H396P T3151 1 10 100 1000 0.5 1 +/- 0.24 Dasatinib-H396P 0.7 +/- 0.10 Dasatinib-T315I 87 +/- 6.7 .025 . Table 1: BSI determined binding Kd of desatinib, nilotinib, and imatinib against wild-type and mutant Bcr-Abl kinase. Final Bcr-Abl kinase enzyme concentrations for each assay system are also indicated. See text for further details. 10000 100000 Log[Nilotinib], nM 7B 0.35 Wild Type M351T Q252H H396P T3151 0.30 Signal (pixels) Kd (nM) Assay System 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 100 1000 10000 100000 1000000 Log[Imatinib], nM 7C Figure 7: BSI analysis of Dasatnib (5a), Nilotinib (5b) and Imatinib (5c) binding against Bcr-Abl kinase Wt and H396P, M351T, Q252H, and T315I mutants. All binding systems achieved saturation and appropriate Kd determination easily ensued. See text for further details. Dasatinib (nM) Imatinib (nM) Nilotinib (nM) IC50 KD IC50 KD IC50 KD Wild Type 1.83 1.08 527 472 17.69 13.3 M351T 1.61 0.42 926 1086 7.8 2.7 Q252H 5.6 5.49 733 961 46.7 47.6 T315I 137 86.5 9221 4050 696 761 H3696P 1.95 0.7 1280 1228 42.6 32.6 Bcr-Abl Kinase Table 2: Comparison of BSI determined binding affinity for desatinib, nilotinib, and imatinib for wild-type and mutant Bcr-Abl kinase as compared to determined IC50 values obtained by radio-labeled abl-tide assays. Page 5 MOLECULAR SENSING Binding Studies of Type I, II, and III Kinase Inhibitors against Bcr-Abl Kinase using Back-Scattering Interferometry: A Tale of Allostery Comparison with Known IC50 Data Table 2 compares the obtained binding equilibrium constants for each system with previously reported IC50 data as compiled by O’Hare (8). Figure 8 depicts the correlation between BSI obtained Kd and IC50 values for the studied systems (linear fit R2 = 0.9744). As is clearly demonstrated, BSI binding results highly correlated with previously compiled kinase activity inhibition assays. Figure 8: Correlation of BSI determined Kd vs literature IC50 values. Determined Kd values for each kinase are in high agreement with various kinase inhibitor assays. See text for further details. Negative allosteric modulation of Bcr-Abl by GNF-5 One of BSI’s strengths is that the technique is binding site agnostic, which allows for direct studies of allosteric modulation. The application of BSI to the study of a target, such as Bcr-Abl, with so much structural and functional information available has yielded a powerful, compelling case study that shows both how BSI can be utilized to study allosteric systems of increasing levels of complexity in vitro, and how BSI can provide evidence in support of the mechanism(s) of action of a small molecule. (Reference 1). Data from both a crystal structure and hydrogen-deuterium exchange mass spectrometry support a mechanism in which GNF-5 causes an allosteric perturbation of the amino acids near T315 that should allow for binding of an ATP-competitive kinase inhibitor, even in the presence of an isoleucine residue (Reference 1). This model explains how GNF-5 is able to sensitize the T315I Bcr-Abl mutant to an ATP-competitive kinase inhibitor, but does not fully explain how GNF-5 is able to inhibit the WT Bcr-Abl kinase. The mechanism of action of GNF-5 is particularly interesting from both a biophysical point of view and from a pharmacological point of view. Unlike all other compounds mentioned in this document GNF-5 does not bind anywhere near the ATP-binding site of Bcr-Abl. The crystal structure shown on page 1 suggests that some of structural changes caused by GNF-5 binding to Bcr-Abl lock the kinase into an inactive conformation and this may explain how GNF-5 inhibits the WT kinase (Reference 1 and Figure 1). In fact, GNF-5 binds to a myristate site approximately 30 Angstroms away from the ATP-binding site BSI was utilized to measure Kd values for ATP and GNF-5, both alone and in combination, for both Page 6 WT Bcr-Abl and the T315I mutant construct of Bcr-Abl (Figure 9). Interestingly, in the presence of a saturating amount of GNF-5 the binding curve for ATP on WT Bcr-Abl is substantially right shifted (Figure 9). This suggests that GNF-5’s mechanism of action against the WT enzyme may be to substantially decrease WT Bcr-Abl’s affinity for ATP. In contrast to the WT enzyme, GNF-5 only slightly right shifts the binding curve of the T315I mutant for ATP (Figure 9). The structural and functional data in the literature indicate that GNF-5 sensitizes the T315I mutant to ATP-competitive kinase inhibitors, therefore the addition of an ATP-competitive inhibitor, such as imatinib, to the Bcr-Abl-GNF-5 complex may be able to substantially right shift the ATP binding curve. These BSI experiments are currently in progress. 0.12 0.10 0.10 0.08 0.08 0.06 T315I_Without GNF-5 0.04 T315I_With 50 uM GNF-5 0.02 0.00 -0.02 1 9A Si gnal (pixels) Si gnal (pixels) 0.12 10 L og[ATP] , uM 100 0.06 0.02 0.00 1000 -0.02 0.12 0.10 0.10 0.08 0.08 0.06 Wild Type_Without GNF-5 Wild Type_With 50 uM GNF-5 0.02 0.00 -0.02 0.1 1 10 Log[ATP] , uM 1 10 Log[ATP] , uM 100 1000 9B 0.12 0.04 T315I Wild Type 0.04 100 Si gnal (pixels) Si gnal (pixels) POSTER NOTE Figure 9: A) The Kd of ATP for both WT and T315I BCR-Abl is about 4-5 micromolar. B) The Kd values of GNF-5 for WT and T315I BCR-Abl are about 510 nM and 130 nM respectively. C) Interestingly, in the presence of a saturating amount of GNF-5 the binding curve for ATP is right-shifted substantially. Without GNF-5 present ATP has a Kd of about 3 micromolar, but the presence of GNF-5 shifts the ATP binding curve about 25-fold to the right. This may explain how GNF-5 functions. The compounds mode of action may be to substantially decrease WT BCR-Abl’s affinity for ATP. D) In contrast to the WT BCR-Abl protein’s behavior, the presence of GNF-5 only shifts the binding curve for the T315I mutant about 3-fold to the right. Given that GNF-5 seems to sensitize the T315I to previous generation inhibitors it is likely also necessary to include a compound such as imatinib in this experiment to observe a large rightward shift in the binding isotherm. These experiments are currently in progress. T315I Wild Type 0.04 0.02 0.00 1000 9C 0.06 -0.02 0.01 0.1 1 Log[GNF-5], uM 10 100 9D CONCLUSIONS This study illustrates the utility of BSI in the analysis of type I, type II, and type III inhibitors of wild-type Bcr-Abl kinase as well as four different imatinib resistant mutants. The overall assay development process was quite facile, requiring only a couple of days and consuming minimal amounts of precious target. Assay fidelity was quite high, with substantial agreement of results for three independent replicate analyses for each system. Obtained BSI kinase inhibitor affinities agreed well with previously reported IC50 values and are consistent with theoretical and x-ray crystallographic studies of the same systems, lending credence to the overall approach as a viable means to study potency for kinase inhibitors. GNF-5 was observed to decrease ATP affinity for WT kinase, but only marginally affected T315I. This may account for GNF-5 efficacy against WT and failure to arrest T315I kinase activity. Because BSI evaluates direct target engagement independent of downstream activity, BSI is aptly suited to advance efforts in the discovery of new inhibitors to Bcr-Abl Kinase as well as other valuable kinase targets of medical import. Page 7 MOLECULAR SENSING POSTER NOTE REFERENCES 1. Strebhardt K & Ullrich A (2008) Paul Ehrlich’s Magic Bullet Concept: 100 Years of Progress. Nature Reviews Cancer 8(6):473-480. 2. Backes A, Zech B, Felber B, Klebl B, & Muller G (2008) Small-molecule inhibitors binding to protein kinases. Part I: exceptions from the traditional pharmacophore approach of type I inhibition. Expert Opinion on Drug Discovery 3(12):1409-1425. 3. van der Plas DC, Soekarman D, van Gent AM, Grosveld G, & Hagemeijer A (1991) bcr-abl mRNA lacking abl exon a2 detected by polymerase chain reaction in a chronic myelogeneous leukemia patient. Leukemia 5(6):457-461. 4. O’Dwyer ME & Druker BJ (2000) STI571: an inhibitor of the BCR-ABL tyrosine kinase for the treatment of chronic myelogenous leukaemia. The Lancet Oncology 1:207-211. 5. Daub H, Specht K, & Ulrich A (2004) Strategies to overcome resistance ot targeted protein kinase inhibitors. Nature Reviews Drug Discovery 3(12):1409-1425. 6. Shah NP, et al. (2002) Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2(2):117-125. 7. Corbin AS, et al. (2004) Sensitivity of oncogenic KIT mutants to the kinase inhibitors MLN518 and PD180970. Blood 104(12):3754-3757. 8. O’Hare T, Eide CA, & Deininger MW (2007) Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood 110(7):2242-2249. 9. Redaelli S, et al. (2009) Activity of bosutinib, dasatinib, and nilotinib against 18 imatinib-resistant BCR/ABL mutants. Journal of clinical oncology: Official Journal of the American Society of Clinical Oncology 27(3):469-471. Company Headquarters North America Contacts European Office & Contact Molecular Sensing, Inc. (MSI), is a commercial stage drug discovery tools and contract research services company with headquarters and drug discovery services laboratories in Nashville, Tennessee and an R&D center in Los Gatos, California, along with a European operations center near Frankfurt, Germany. Molecular Sensing, Inc. 111 10th Ave, South Suite 110 Nashville, TN 37203 Technical Support Jake Isaacs +1 615 938-7049 [email protected] ph +1 615-938-7050 fax +1 615-255-0094 Technical Sales Julian Abery +1 919-724-0946 [email protected] Molecular Sensing GmbH Am Frauwald 10 DE 65510 Idstein Germany +49 6126 229050 [email protected] [email protected] www.molsense.com © Copyright 2015 by Molecular Sensing, Inc. All rights reserved. European Operations Wilt Peters +49 171 7604450 [email protected]