Inhibitors to Overcome Secondary Mutations in the Stem Cell Factor

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Article Cite This: J. Med. Chem. 2017, 60, 8801-8815

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Inhibitors to Overcome Secondary Mutations in the Stem Cell Factor Receptor KIT Helena Kaitsiotou,† Marina Keul,† Julia Hardick,† Thomas Mühlenberg,‡,§ Julia Ketzer,‡,§ Christiane Ehrt,† Jasmin Krüll,†,○ Federico Medda,∥,∇ Oliver Koch,† Fabrizio Giordanetto,∥,⊥ Sebastian Bauer,*,‡,§ and Daniel Rauh*,† †

Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 4a, D-44227 Dortmund, Germany Department of Medical Oncology, Sarcoma Centre West German Cancer Centre University Duisburg−Essen, Medical School, Hufelandstraße 55, D-45122 Essen, Germany § Germany and German Cancer Consortium (DKTK), Partner Site University Hospital Essen, D-45147 Essen, Germany ∥ Taros Chemicals GmbH & Co. KG, Emil-Figge-Straße 76a, D-44227 Dortmund, Germany ‡

S Supporting Information *

ABSTRACT: In modern cancer therapy, the use of small organic molecules against receptor tyrosine kinases (RTKs) has been shown to be a valuable strategy. The association of cancer cells with dysregulated signaling pathways linked to RTKs represents a key element in targeted cancer therapies. The tyrosine kinase mast/stem cell growth factor receptor KIT is an example of a clinically relevant RTK. KIT is targeted for cancer therapy in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). However, acquired resistance mutations within the catalytic domain decrease the efficacy of this strategy and are the most common cause of failed therapy. Here, we present the structure-based design and synthesis of novel type II kinase inhibitors to overcome these mutations in KIT. Biochemical and cellular studies revealed promising molecules for the inhibition of mutated KIT.

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INTRODUCTION KIT is a type III receptor tyrosine kinase that is an important signaling protein for the development of melanocytes, erythrocytes, germ cells, mast cells, and interstitial cells of cajal (gastrointestinal pacemaker cells).1−3 Upon binding of its ligand, stem cell factor (SCF), KIT activates downstream signaling pathways that promote cell survival and cell proliferation and inhibit apoptosis.4 Constitutive activation of KIT as an oncogenic driver has first been described in feline sarcomas (“kitten”-KIT) but was later also found in a variety of human cancers including melanomas,5,6 seminomas, acute myeloid leukemias, systemic mastocytosis, sinonasal lymphoma, and gastrointestinal stromal tumors (GIST).7,8 Particularly, GISTs have since then become a paradigm of successful targeted treatment for cancer. GIST represents the most common mesenchymal tumor of the gastrointestinal tract,9−11 and approximately 85% of GISTs harbor oncogenic gain-of-function mutations of KIT or the platelet-derived growth factor receptor (PDGFR).12,13 Activating mutations of KIT represent an early oncogenic event, but KIT also remains the central oncogenic driver in patients with highly advanced GIST disease.7,14,15 Primary, activating mutations of KIT in GIST most commonly occur within the juxtamembrane (exon 11) and the extracellular regions (exon 9) and only very rarely in other exons (8, 13, 17).16−19 Three © 2017 American Chemical Society

inhibitors of KIT have until now been approved for the treatment of GIST. Of note, these three inhibitors had been primarily developed as inhibitors of kinases other than KIT such as BCR-ABL (1 (imatinib) 20 ) and VEGFR (2 (sunitinib),21 and 3 (regorafenib)22) (Figure 1).23−26 The possibility of inhibiting dysregulated tyrosine kinases in kinase mutation-driven cancer has led to the development of tyrosine kinase inhibitor (TKI) therapies. In many cases, TKIs have been shown to be more beneficial than traditional cancer treatments in terms of side effects and the overall clinical outcomes for patients with GIST.17,18 1, a 2-phenylaminopyrimidine derivative, is a rapidly absorbed oral kinase inhibitor that effectively inhibits exon 9 and exon 11 KIT mutants and whose response rates, as well as duration of clinical benefit, correlate with genotype.27,28 Long lasting disease control (median progression-free survival [PFS] exon 11:2.3 years; exon 9:1.6 years)29 paired with a favorable side effect profile have made 1 the standard first-line treatment. Nonetheless, the majority of patients eventually progress and secondary mutations within the ATP-binding pocket (exon 13, V654A; exon 14, T670I) and the activation loop (affecting codons 816−829) represent the major mechanisms of resistance.30,31 Received: June 8, 2017 Published: October 9, 2017 8801

DOI: 10.1021/acs.jmedchem.7b00841 J. Med. Chem. 2017, 60, 8801−8815

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Figure 1. (A) Structural alignment of kinase inhibitors, approved by the FDA for the treatment of GIST, bound to wild-type KIT (pdb 4u0i) (1 (green, pdb 4bkj), 2 (yellow, pdb 3g0e), 3 (blue, modeled), and 4 (red, pdb 4u0i)). Unique substitution patterns and hinge binding elements of the inhibitors highlighted (right). (B) X-ray crystal structure of 4 in complex with wild-type KIT (pdb 4u0i) with modeled secondary mutations (sticks and surfaces). Hydrogen bonds are depicted by blue dotted lines.

hypertension-related side effects. Compared to the substantial benefits obtained with 1 as first-line treatment, the clinical benefits of 2 and 3 are moderate, and the side effect profiles of both inhibitors are much less favorable than those of 1.35−38 Currently, (S)-1-(4-fluorophenyl)-1-(2-(4-(6-(1-methyl-1Hpyrazol-4-yl)pyrrolo[2,1-f ][1,2,4]triazin-4-yl)piperazin-1-yl)pyrimidin-5-yl)ethan-1-amine39 ((BLU-285) (NCT02508532)), a potent inhibitor of PDGFRA-D842 and KIT exon 17 mutations and 1-N′-[2,5-difluoro-4-[2-(1methylpyrazol-4-yl)pyridine-4-yl]oxyphenyl]-1-N-phenylcyclopropane-1,1-dicarboxamide40 ((DCC-2618) (NCT02571036)), a type III inhibitor of KIT and PDGFRA, and PLX9486/PLX339741 (NCT02401815) represent the first wave of novel compounds that were designed especially for KIT to exhibit strong activity against exon 13/14 and 17, which is not only important for GIST but also for KIT-driven leukemias and melanomas.42 4 (ponatinib) is a next-generation, highly potent ATP-competitive inhibitor of BCR-ABL, which was rationally designed to inhibit the notoriously resistant T315I mutation.43,44 Approval was granted for the treatment of

Both 2 and 3 show better activity against KIT mutants with secondary resistance mutations, and approval was granted based on an improved median PFS of 4−5 months over placebo in second-32 and third-line treatment.33 However, response rates for both drugs are low (95% purity was conducted by high-performance liquid chromatography (HPLC). The purity was determined using Agilent 1200 series HPLC systems with UV detection at 210 nm (system: Agilent Eclipse XDB-C18 4.6 mm × 150 mm, 5 μM, 10−100% CH3CN in H2O, with 0.1% TFA, for 15 min at 1.0 mL/min). Reagents and Materials. All supplies for the KIT HTRF assay kit were purchased from CisBio (Bagnols-sur-Cèze, France). Small volume (20 μL fill volume) white round-bottom 384-well plates were obtained from Greiner Bio-One GmbH (Solingen, Germany). Activity-Based Assay for IC50 Determination. IC50 determinations for KIT kinases were measured with the KinEASE-TK assay from Cisbio according to the manufacturer’s instructions. A biotinylated poly-Glu-Tyr substrate peptide was phosphorylated by the specific kinase of interest. After completion of the reaction, an antiphosphotyrosine antibody labeled with europium cryptate and streptavidin labeled with the fluorophore XL665 were added. FRET between europium cryptate and XL665 was measured to quantify the phosphorylation of the substrate peptide. ATP concentrations were set at their respective KM values (30 μM for wild-type KIT, 20 μM for KIT V559D/T670I, 12 μM for KIT D816H, and 50 μM for V559D/ V654A). A substrate concentration of 330 nM was used for KIT wildtype, 450 nM was used for KIT V559D/T670I, 1 μM was used for KIT D816H and 1 μM was used for KIT V559D/V654A, respectively. Kinase and inhibitor were preincubated for 30 min before the reaction was started by addition of ATP and substrate peptide. A PerkinElmer EnVision multimode plate reader was used to measure the fluorescence of the samples at 620 nm (Eu-labeled antibody) and 665 nm (XL665 labeled streptavidin) 50 μs after excitation at 320 nm. The quotient of both intensities for reactions at eight different inhibitor concentrations was then analyzed using the Quattro software suite for IC50 value determination. Each reaction was performed in duplicate, and at least three independent determinations of each IC50 were made. Gist Cell Lines. GIST-T1 and GIST-T1 T670I were cultured in DMEM (Gibco), and GIST430-V654A, GIST-T1-D816E, and GIST48B were cultured in IMDM (Gibco), supplemented with 10−15% FBS (Biochrome) and 1% Pen/Step/Ampho (Gibco). GIST430V654A, GIST-T1-T670I, and GIST-D816E Media were supplemented with 1 100 nM, 200 nM, and 1 μM, respectively. GIST-T1 was established (by Takahiro Taguchi, Kochi University, Japan) from a 8812

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plasma ( f u50%) is extrapolated to the fraction unbound at 100% plasma (f u100%) using the following equation: f u100% = f u50%/(2 − f u50%) Caco-2 Assay. To measure cellular permeability, compounds were applied at a concentration of 10 μM in HBSS to either the apical (A) or basolateral (B) side of a Caco-2 cell monolayer and incubated for 2 h at 37 °C. Compound concentrations on each side of the monolayer were determined by LC-MS/MS and the apparent permeability (Papp) was calculated in the apical to basolateral (A → B) and basolateral to apical (B → A) directions according to the following equation: Papp(A → B) = (ΔCB × VB × 0.001)/(Δt × A × Ct0,A).

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ACKNOWLEDGMENTS

This work was cofunded by the German Federal Ministry for Education and Research (NGFNPlus and e:Med) (grant no. BMBF 01GS08104, 01ZX1303C) and by the Deutsche Forschungsgemeinschaft (DFG). D.R. thanks the German federal state North Rhine Westphalia (NRW) and the European Union (European Regional Development Fund: Investing In Your Future) (EFRE-800400). C.E. is funded by the Kekulé Mobility Fellowship of the Chemical Industry Fund (FCI). O.K. is funded by the German Federal Ministry for Education and Research (BMBF, Medizinische Chemie in Dortmund, grant BMBF 1316053).

ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00841. RMSD profiles for the MD simulations of wild-type KIT, KITT670I, and KITV654A in the presence and absence of ponatinib; RMSF values of the active site of wild-type KIT, KITT670I, and KITV654A; angle between the axis of the α helix C and the unit cell vector x during a 300 ns MD simulation; kinome dendrogram derived from the SelectScreen; Analysis of the ponatinib binding site volume for the MD simulations in the absence of ponatinib; PCA results for the MD simulations in the absence of ponatinib; data of the SelectScreen; in vitro intrinsic clearance CLint in human and murine liver microsomes; relative compound degradation of 7f, 10, ponatinib, imatinib, and regorafenib; pharmacokinetic parameters of 7f, 10, ponatinib, imatinib, and regorafenib; results of the RMSD-based binding site clustering analyses; hydrogen bond occupancies for the MD simulations with ponatinib; free energies of binding as estimated by MM-PB(GB)SA calculations; binding site comparison results; detailed synthetic procedures for the preparation of inhibitors for KIT mutants (PDF) Molecular formula strings (CSV)

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ABBREVIATIONS USED KIT, mast/stem cell growth factor receptor kinase; VEGFR, vascular endothelial growth factor receptor; GIST, gastrointestinal stromal tumors; RTK, receptor tyrosine kinase; SAR, structure−activity relationship: relationship between the chemical structure of a molecule and its biological activity on a target; TKI, tyrosine kinase inhibitor; MD, molecular dynamics; GI50, concentration for 50% of maximal inhibition of cell proliferation; IC50, concentration causing 50% inhibition of enzyme activity; PCA, principle component analysis; MMPB(GB)SA, molecular mechanics Poisson−Boltzmann (Generalized Born) solvent-accessible surface area; R-spine regulatory spine, highly conserved motif, found in every active kinase

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*For D.R.: phone, +49 (0)231-755-7080; fax, +49 (0)231-7557082; E-mail, [email protected]. *For S.B.: phone, +49 (0) 201-723-2112; fax, +49 (0) 201-2015996; E-mail, [email protected]. ORCID

Daniel Rauh: 0000-0002-1970-7642 Present Addresses ⊥

For F.G.: D. E. Shaw Research, 120 West 45th Street, 39th Floor, New York, New York 10036, United States. ∇ For F.M.: CytRx Corporation Drug Discovery Branch, Engesserstraße 4, 79108 Freiburg, Germany. ○ For J.K.: Department of Chemistry and Pharmacy, University Erlangen−Nürnberg, Schuhstraße 19, 91052 Erlangen, Germany. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 8813

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DOI: 10.1021/acs.jmedchem.7b00841 J. Med. Chem. 2017, 60, 8801−8815