Inhibition of Various Receptor Tyrosine Kinase (rtk) Families

Activation of cell surface growth factor receptor tyrosine kinases (RTKs) results in cellular proliferation, differentiation and survival. Humans have 58 RTKs, categorized into twenty subfamilies based on their structural properties and ligand specificity. One of the most well-studied RTKs, the epidermal growth factor receptor (EGFR), is mutated or overexpressed in many human cancers including non-small cell lung cancer and glioblastoma. Several EGFR inhibitors have been approved by the FDA, and numerous additional inhibitors are being pursued as potential cancer therapeutics. Structural studies of the EGFR tyrosine kinase domain suggest that different inhibitors bind selectively to either the active or the inactive conformation of the kinase. Contrary to this supposition, computational studies of inhibitor-stabilized EGFR conformations have suggested that erlotinib can bind equally well to either the active or inactive forms of the EGFR kinase domain. We tested this hypothesis using a mutated EGFR kinase domain that can adopt either the inactive or active kinase conformation in crystals, and determined a structure with erlotinib bound to the inactive conformation – suggesting that this inhibitor can bind either ‘state’. We also determined the crystal structure of this EGFR kinase variant bound to a novel ErbB2 inhibitor (55A) found in screens designed to select inhibitors that bind the active state. When bound to this compound, the EGFR kinase domain adopted only the active conformation, suggesting that erlotinib and 55A select different kinase ‘states’. Collaborative approaches combining biochemical, structural and cell signaling studies suggest new considerations in predicting modes of inhibitor binding in the development of therapeutic agents for cancer. Several activating mutations in the anaplastic lymphoma kinase (ALK) gene have been implicated in neuroblastoma. The small molecule tyrosine kinase inhibitor crizotinib, which inhibits ALK and Met, was recently approved by the FDA for the treatment of non-small cell lung cancer (NSCLC), and is currently in early-phase clinical testing in patients with neuroblastoma (NB). Numerous additional ALK inhibitors are also being pursued as potential therapeutics. However, more and more mutations in the ALK tyrosine kinase domain (TKD) are being found in patients with neuroblastoma. Several of these are associated with primary resistance to currently available ALK inhibitors, and several mutations in oncogenic ALK fusions have been linked to acquired crizotinib resistance. Understanding how different ALK mutations activate its kinase domain, and how they change inhibitor sensitivity, is therefore crucial for further clinical development of ALK-targeted therapeutics. We have studied the biochemical consequences of a wide variety of ALK kinase domain mutations in parallel both with studies of their transforming abilities and computational studies of their structural effects. Our first goal was to use these data to predict the effects of new clinically emerging ALK mutations on ALK’s transforming ability, signaling activity, and drug sensitivity. A second goal was to identify the most potent ALK inhibitor that has inhibitory effects on the widest possible range of ALK mutants using biochemical and cellular assays, with a view to identifying a clinical path forward. Our studies provide valuable mechanistic insight into how ALK is regulated, and also lay important groundwork for guiding more refined targeted therapy in neuroblastoma patients

Mechanistic Insight into RET Kinase Inhibitors Targeting the DFG-out Conformation in RETrearranged Cancer

RET Fusions-Gene können in 1-2% aller Lungen-Adenokarzinom Patienten nachgewiesen werden. Diese genetischen Veränderungen stellen mittels Tyrosin-Kinase Inhibitoren potentiell therapierbare molekulare Zielstrukturen dar (Pao and Hutchinson 2012), jedoch haben klinische Studien im Jahr 2018 für diese Lungenkarzinome bisher noch keine ausreichend erfolgreichen Therapieansätze zeigen können (Drilon et al. 2018). Um systematisch das therapeutische Profil von AD80 und einer Vielzahl weiterer Tyrosinkinase Inhibitoren gegen RET-Fusion getriebene Zellmodelle auswerten zu können, habe ich zunächst Ba/F3 Zellen viral mit KIF5B-RET sowie CCDC6-RET transduziert. Das Wachstum von Ba/F3 Zellen in vitro ist im Normalzustand abhängig von IL-3. Sobald sie mit einem starken Onkogen jedoch transduziert werden, sind sie unabhängig von IL-3 und proliferieren nur noch abhängig von der Aktivität des entsprechenden Onkogens. Durch dieses Modelsystem konnte ich eine große Anzahl verschiedener Inhibitoren gegen RET testen und ihre Potenz untereinander vergleichen. Die selbst etablierten Ba/F3 Zell-Linien zusammen mit der RET-Fusion getriebenen Lungen-Adenokarzinom Zelllinie LC-2/AD bildeten den Ausgangspunkt für mein Projekts. Zusätzlich habe ich ein endogen RET-mutiertes Zell-Model mittels der Genom-Editierungstechnik CRISPR/Cas9 etabliert. Dafür habe ich einen Vektor mit Cas9 mRNA sowie zwei Promotoren für die Expression von spezifischen „single-guided RNAs“ (sgRNA) kloniert und murine Fibroblast Zellen (NIH-3T3) transfiziert. Mittels sgRNAs gegen die jeweiligen spezifischen Introns von RET und KIF5B konnte ich somit in selektionierten NIH-3T3 Zellen KIF5B-RET Translokationen generieren. Indem ich diese neu etablierten Zelllinien zusammen mit einer größeren Anzahl humaner Lungenkrebs Zell-Linien gegen potentielle RET-Inhibitoren getestet habe, konnte ich die klinische Erfahrung in vitro bestätigen, dass die zurzeit gängigen Therapieansätze mit Tyrosinkinase- Inhibitoren wie z.B. Cabozantinib, Alectinib oder Vandetanib wahrscheinlich eine nicht ausreichend hohe therapeutische Potenz besitzen, um eine effektive Wirkung auf RET getriebene Tumore zu entwickeln. Weiterhin deuteten die Daten darauf hin, dass andere Tyrosin-Kinase Inhibitoren, wie z.B. AD80 und Ponatinib, im Vergleich dazu 100 bis 1000-fach potenter sind und spezifisch die RET-Kinase inhibieren. Als nächstes habe ich die Unterschiede in den Zell-Viabilitäts-Assays mit den Veränderungen auf der Protein-Ebene zu verglichen. Die folgenden Western Blot und Phosphoproteom Analysen haben eine entsprechende Reduktion in phospho-RET und den weiteren nachgeschalteten Signal-Molekülen gezeigt. Zusätzlich haben die in vivo Ergebnisse unserer CCDC6-RET PDX-Mausmodelle unsere in vitro Daten mit gutem Tumoransprechen unter Therapie mit AD80 bestätigt. Parallel zu meiner Arbeit für das Projekt haben wir mit anderen Arbeitsgruppen zusammengearbeitet, um ein tieferes Verständnis über die funktionellen Mechanismen hinter der hohen Aktivität von AD80 gegen RET zu erhalten. Mittels computerbasierter Modelle haben wir ableiten können, dass AD80 mit hoher Wahrscheinlichkeit als Typ II Inhibitor die RET-Kinase in der inaktiven „DFG-out“ Konformation bindet, was mit einer erhöhten Kinase- Thermostabilität im Vergleich zu Typ I Inhibitoren als Bindungspartner einhergeht. Dies wiederum ist ein Surrogat-Parameter für eine engere Kinase-Bindung durch die Inhibitoren und könnte eine mögliche Erklärung für die hohe Cytotoxitität in unseren Experimenten sein. Im Folgenden hat sich das Projekt mehr auf die Rolle von Resistenz-Mechanismen in RET mutierten Zelllinien konzentriert. Mittels zielgerichteter Mutagenese (site-directed mutagenesis) habe ich an der sogenannten „Gatekeeper Position“ mutierte Ba/F3 KIFRETV804M und CCDC6-RETV804M etablieren können, um die Wirksamkeit der Inhibitoren dagegen zu testen. Wieder zeigten AD80 und Ponatinib den stärksten inhibitorischen Effekt gegen RET mit nur einer geringen Reduktion der Cytotoxitität in den Zell-Viabilitäts-Assays und RET-Dephosphorylierung im Vergleich zu RETwt. Mittels Sättigungsmutagenese (saturated mutagenesis screening) habe ich versucht, Resistenzmutationen zu finden, die neben der bekannten „Gatekeeper Position“ pV804M zur Resistenz gegen AD80 führen könnten. Es zeigte sich in der Sequenzierung von resistenten Ba/F3CCDC6-RET Zellen die missense Mutation pI788N (c.2363T>A) im Bereich der RET-Kinase Domäne als potentielle sekundäre Resistenzmutation unter Therapie mit AD80. Diesen Resistenz-Effekt durch die neue Mutation konnte ich dann in den folgenden zellulären Modellen mittels Zell-Viabilitäts-Assays und Western Blots bestätigen. Zusätzlich haben Ergebnisse einer sekundär gegen AD80 resistent gewordenen TPC-1 Schilddrüsenkarzinom-Zelllinie sowie RNASequenzierungen von LC-2/AD Zelllinien unter Therapie gegen AD80 ergeben, dass MAPKReaktivierung potentiell eine Rolle als Resistenz-Mechanismus in RET-getriebenen Tumoren besitzen könnte. Um formell die Rolle von MAPK-Reaktivierung in Bezug auf Resistenz-Effekte zu testen, wurden LC-2/AD Zellen von mir lentiviral mit KRASG12V transduziert, was zu einer Überexprimierung von KRAS und zu einer folgenden Resistenz gegen die Behandlung mit AD80 geführt hat. Für Ponatinib gibt es bereits klinische Phase 3 Studien an Patienten mit Chronisch Myeloischer Leukämie, die jedoch trotz gutem initialen Tumor Ansprechen aufgrund von erhöhten Therapie assoziierten Komplikationen beendet werden musste (Lipton et al. 2016). Der RET-Kinase Inhibitor AD80 wäre daher ein geeigneter Kandidat für zukünftige klinische Studien. Bedenkt man den hohen Bedarf an neuen, wirksamen und verträglichen Therapieansätzen im Rahmen der individualisierten Lungenkarzinom-Therapie, bietet unsere Studie eine Vielzahl neuer mechanistischer Einsichten in die aktuelle anti-RET Therapie und trägt zur deren zukünftigen Entwicklung bei

Making NSCLC Crystal Clear:How Kinase Structures Revolutionized Lung Cancer Treatment

The parallel advances of different scientific fields provide a contemporary scenario where collaboration is not a differential, but actually a requirement. In this context, crystallography has had a major contribution on the medical sciences, providing a “face” for targets of diseases that previously were known solely by name or sequence. Worldwide, cancer still leads the number of annual deaths, with 9.6 million associated deaths, with a major contribution from lung cancer and its 1.7 million deaths. Since the relationship between cancer and kinases was unraveled, these proteins have been extensively explored and became associated with drugs that later attained blockbuster status. Crystallographic structures of kinases related to lung cancer and their developed and marketed drugs provided insight on their conformation in the absence or presence of small molecules. Notwithstanding, these structures were also of service once the initially highly successful drugs started to lose their effectiveness in the emergence of mutations. This review focuses on a subclassification of lung cancer, non-small cell lung cancer (NSCLC), and major oncogenic driver mutations in kinases, and how crystallographic structures can be used, not only to provide awareness of the function and inhibition of these mutations, but also how these structures can be used in further computational studies aiming at addressing these novel mutations in the field of personalized medicine

Structural and dynamic determinants for highly selective RET kinase inhibition reveal cryptic druggability.

The structural and dynamic determinants for highly selective RET kinase inhibition are poorly understood. Here we demonstrate by applying an integrated structural, computational and biochemical approach that the druggability landscape of the RET active site is determined by the conformational setting of the ATP-binding (P-) loop and its coordination with the αC helix. Open and intermediate P-loop structures display additional druggable vulnerabilities within the active site that were not exploited by first generation RET inhibitors. We identify a cryptic pocket adjacent to the catalytic lysine formed by K758, L760, E768 and L772, that we name the post-lysine pocket, with higher druggability potential than the adenine-binding site and with important implications in the regulation of phospho-tyrosine kinase activity. Crystal structure and simulation data show that the binding mode of highly-selective RET kinase inhibitors LOXO-292 and BLU-667 is controlled by a synchronous open P-loop and αC-in configuration that allows accessibility to the post-lysine pocket. Molecular dynamics simulation show that these inhibitors efficiently occupy the post-lysine pocket with high stability through the simulation time-scale (300 ns), with both inhibitors forming hydrophobic contacts in the pocket further stabilized by pi-cation interactions with the catalytic K758. Engineered mutants targeting the post-lysine pocket impact on inhibitor binding and sensitivity, as well as RET tyrosine kinase activity. The identification of the post-lysine pocket as a cryptic druggable vulnerability in the RET kinase and its exploitation by second generation RET inhibitors has important implications for future drug design and the development of personalized therapies for patients with RET-driven cancers.We thank the Centro Nacional de Investigaciones Oncológicas (CNIO), which is supported by the Instituto de Salud Carlos III and recognized as a “Severo Ochoa” Centre of Excellence (ref. CEX2019-000891-S, awarded by MCIN/AEI/ 10.13039/501100011033) for core funding and supporting this study. This work was further supported by projects: BFU2017-86710-R funded by MCIN/ AEI /10.13039/501100011033 and ERDF “A way of making Europe”, PID2020-117580RB-I00 funded by MCIN/ AEI /10.13039/501100011033, RYC-2016-1938 funded by MCIN/AEI /10.13039/501100011033 and ESF “Investing in your future”, and a Marie Curie WHRI-ACADEMY International grant (number 608765) to IP-M and a CNIO-Friends predoctoral Carmen Gloria Bonnet Fellowship to MAS.N

Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors

Epithelial-to-mesenchymal transition (EMT) may drive the escape of ALK-rearranged non-small-cell lung cancer (NSCLC) tumors from ALK-tyrosine kinase inhibitors (TKIs). We investigated whether first-generation ALK–TKI therapy-induced EMT promotes cross-resistance to new-generation ALK–TKIs and whether this could be circumvented by the flavonolignan silibinin, an EMT inhibitor. ALK-rearranged NSCLC cells acquiring a bona fide EMT phenotype upon chronic exposure to the first-generation ALK–TKI crizotinib exhibited increased resistance to secondgeneration brigatinib and were fully refractory to third-generation lorlatinib. Such cross-resistance to new-generation ALK–TKIs, which was partially recapitulated upon chronic TGF stimulation, was less pronounced in ALK-rearranged NSCLC cells solely acquiring a partial/hybrid E/M transition state. Silibinin overcame EMT-induced resistance to brigatinib and lorlatinib and restored their efficacy involving the transforming growth factor-beta (TGF )/SMAD signaling pathway. Silibinin deactivated TGF -regulated SMAD2/3 phosphorylation and suppressed the transcriptional activation of genes under the control of SMAD binding elements. Computational modeling studies and kinase binding assays predicted a targeted inhibitory binding of silibinin to the ATP-binding pocket of TGF type-1 receptor 1 (TGFBR1) and TGFBR2 but solely at the two-digit micromolar range. A secretome profiling confirmed the ability of silibinin to normalize the augmented release of TGF into the extracellular fluid of ALK–TKIs-resistant NSCLC cells and reduce constitutive and inducible SMAD2/3 phosphorylation occurring in the presence of ALK–TKIs. In summary, the ab initio plasticity along the EMT spectrum may explain the propensity of ALK-rearranged NSCLC cells to acquire resistance to new-generation ALK–TKIs, a phenomenon that could be abrogated by the silibinin-driven attenuation of the TGF /SMAD signaling axis in mesenchymal ALK-rearranged NSCLC cells.Ministry of Science and Innovation, Spain (MICINN) Spanish GovernmentPlan Nacional de l+D+I PID2019-10455GB-I00 CP20/00003 Spanish GovernmentFundacio Oncolliga Girona (Lliga catalana d'ajuda al malalt de cancer, Girona)Spanish GovernmentCenter for Forestry Research & Experimentation (CIEF)European Commission PI22/00297Grupo Espanol de Cancer de Pulmon (GECP) RTI2019-096724-B-C21La Marato de TV3 foundationHealth Research and Innovation Strategic Plan PROMETEO/2021/059Pla strategic de recerca i innovacio en salut 201906Generalitat de CatalunyaInstituto de Salud Carlos III SLT006/17/11

N1-(3-(Trifluoromethyl)Phenyl) Isophthalamide Derivatives as Promising Inhibitors of Vascular Endothelial Growth Factor Receptor: Pharmacophore-Based Design, Docking, and MM-PBSA/MM-GBSA Binding Energy Estimation

Targeting protein kinases is a common approach for cancer treatment. In this study, a series of novel terephthalic and isophthalic derivatives were constructed as potential type 2 protein kinase inhibitors adapting pharmacophore features of approved anticancer drugs of this class. Inhibitory activity of designed structures was studied in silico against various cancer-related protein kinases and compared with that of known inhibitors. Obtained docking scores, MM-PBSA/MM-GBSA binding energy, and RF-Score-VS affinities suggest that N1-(3-(trifluoromethyl) phenyl) isophthalamide could be considered as promising scaffold for the development of novel protein kinase inhibitors which are able to target the inactive conformation of vascular endothelial growth factor receptor

Importance of Incorporating Protein Flexibility in Molecule Modeling: A Theoretical Study on Type I1/2 NIK Inhibitors

NF-κB inducing kinase (NIK), which is considered as the central component of the non-canonical NF-κB pathway, has been proved to be an important target for the regulation of the immune system. In the past few years, NIK inhibitors with various scaffolds have been successively reported, among which type I1/2 inhibitors that can not only bind in the ATP-binding pocket at the DFG-in state but also extend into an additional back pocket, make up the largest proportion of the NIK inhibitors, and are worthy of more attention. In this study, an integration protocol that combines molecule docking, MD simulations, ensemble docking, MM/GB(PB)SA binding free energy calculations, and decomposition was employed to understand the binding mechanism of 21 tricyclic type I1/2 NIK inhibitors. It is found that the docking accuracy is largely dependent on the selection of docking protocols as well as the crystal structures. The predictions given by the ensemble docking based on multiple receptor conformations (MRCs) and the MM/GB(PB)SA calculations based on MD simulations showed higher linear correlations with the experimental data than those given by conventional rigid receptor docking (RRD) methods (Glide, GOLD, and Autodock Vina), highlighting the importance of incorporating protein flexibility in predicting protein–ligand interactions. Further analysis based on MM/GBSA demonstrates that the hydrophobic interactions play the most essential role in the ligand binding to NIK, and the polar interactions also make an important contribution to the NIK-ligand recognition. A deeper comparison of several pairs of representative derivatives reveals that the hydrophobic interactions are vitally important in the structural optimization of analogs as well. Besides, the H-bond interactions with some key residues and the large desolvation effect in the back pocket devote to the affinity distinction. It is expected that our study could provide valuable insights into the design of novel and potent type I1/2 NIK inhibitors