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1.
J Immunother Cancer ; 11(4)2023 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-37028818

RESUMO

BACKGROUND: Immune responses against tumors are subject to negative feedback regulation. Immune checkpoint inhibitors (ICIs) blocking Programmed cell death protein 1 (PD-1), a receptor expressed on T cells, or its ligand PD-L1 have significantly improved the treatment of cancer, in particular malignant melanoma. Nevertheless, responses and durability are variables, suggesting that additional critical negative feedback mechanisms exist and need to be targeted to improve therapeutic efficacy. METHODS: We used different syngeneic melanoma mouse models and performed PD-1 blockade to identify novel mechanisms of negative immune regulation. Genetic gain-of-function and loss-of-function approaches as well as small molecule inhibitor applications were used for target validation in our melanoma models. We analyzed mouse melanoma tissues from treated and untreated mice by RNA-seq, immunofluorescence and flow cytometry to detect changes in pathway activities and immune cell composition of the tumor microenvironment. We analyzed tissue sections of patients with melanoma by immunohistochemistry as well as publicly available single-cell RNA-seq data and correlated target expression with clinical responses to ICIs. RESULTS: Here, we identified 11-beta-hydroxysteroid dehydrogenase-1 (HSD11B1), an enzyme that converts inert glucocorticoids into active forms in tissues, as negative feedback mechanism in response to T cell immunotherapies. Glucocorticoids are potent suppressors of immune responses. HSD11B1 was expressed in different cellular compartments of melanomas, most notably myeloid cells but also T cells and melanoma cells. Enforced expression of HSD11B1 in mouse melanomas limited the efficacy of PD-1 blockade, whereas small molecule HSD11B1 inhibitors improved responses in a CD8+ T cell-dependent manner. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of interferon-γ by T cells. Interferon pathway activation correlated with sensitivity to PD-1 blockade linked to anti-proliferative effects on melanoma cells. Furthermore, high levels of HSD11B1, predominantly expressed by tumor-associated macrophages, were associated with poor responses to ICI therapy in two independent cohorts of patients with advanced melanomas analyzed by different methods (scRNA-seq, immunohistochemistry). CONCLUSION: As HSD11B1 inhibitors are in the focus of drug development for metabolic diseases, our data suggest a drug repurposing strategy combining HSD11B1 inhibitors with ICIs to improve melanoma immunotherapy. Furthermore, our work also delineated potential caveats emphasizing the need for careful patient stratification.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , Glucocorticoides , Imunoterapia , Melanoma , Animais , Camundongos , Linfócitos T CD8-Positivos , Glucocorticoides/uso terapêutico , Interferon gama/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Reposicionamento de Medicamentos
2.
ChemMedChem ; 17(10): e202100776, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35170857

RESUMO

Akt is a protein kinase that has been implicated in the progression of cancerous tumours. A number of covalent allosteric Akt inhibitors are known, and based on these scaffolds, a small library of novel potential covalent allosteric imidazopyridine-based inhibitors was designed. The envisaged compounds were synthesised, with click chemistry enabling a modular approach to a number of the target compounds. The binding modes, potencies and antiproliferative activities of these synthesised compounds were explored, thereby furthering the structure activity relationship knowledge of this class of Akt inhibitors. Three novel covalent inhibitors were identified, exhibiting moderate activity against Akt1 and various cancer cell lines, potentially paving the way for future covalent allosteric inhibitors with improved properties.


Assuntos
Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt , Regulação Alostérica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-Atividade
3.
Nat Commun ; 12(1): 5297, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489430

RESUMO

The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms' distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties.


Assuntos
Antineoplásicos/farmacologia , Linfócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Antineoplásicos/síntese química , Linhagem Celular , Desenho de Fármacos , Expressão Gênica , Células HEK293 , Humanos , Concentração Inibidora 50 , Linfócitos/citologia , Linfócitos/enzimologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequenas/síntese química , Spodoptera , Relação Estrutura-Atividade
4.
Cell Rep ; 31(4): 107568, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32348765

RESUMO

Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF)-VEGFR2 signaling has shown limited efficacy in lung cancer patients. Here, we demonstrate that inhibition of VEGFR2 in tumor cells, expressed in ∼20% of non-squamous non-small cell lung cancer (NSCLC) patients, leads to a pro-invasive phenotype. Drug-induced inhibition of tumor VEGFR2 interferes with the formation of the EphA2/VEGFR2 heterocomplex, thereby allowing RSK to interact with Serine 897 of EphA2. Inhibition of RSK decreases phosphorylation of Serine 897 EphA2. Selective genetic modeling of Serine 897 of EphA2 or inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, these findings demonstrate that VEGFR2-targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2-targeted anti-angiogenic treatment in lung cancer patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptor EphA2/metabolismo , Serina/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , Metástase Neoplásica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
5.
Angew Chem Int Ed Engl ; 58(52): 18823-18829, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31584233

RESUMO

Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Herein, we present a structure-guided approach for the design of structurally diverse and pharmacologically beneficial covalent-allosteric modifiers, which enabled an investigation of the isoform-specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The isoform-selective covalent-allosteric Akt inhibitors that emerged from this approach showed a conclusive structure-activity relationship and broke ground in the development of selective probes to delineate the isoform-specific functions of Akt kinases.


Assuntos
Regulação Alostérica/fisiologia , Sítio Alostérico/fisiologia , Isoformas de Proteínas/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Humanos , Relação Estrutura-Atividade
6.
Chem Sci ; 10(12): 3573-3585, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30996949

RESUMO

The Ser/Thr kinase Akt (Protein Kinase B/PKB) is a master switch in cellular signal transduction pathways. Its downstream signaling influences cell proliferation, cell growth, and apoptosis, rendering Akt a prominent drug target. The unique activation mechanism of Akt involves a change of the relative orientation of its N-terminal pleckstrin homology (PH) and the kinase domain and makes this kinase suitable for highly specific allosteric modulation. Here we present a unique set of crystal structures of covalent-allosteric interdomain inhibitors in complex with full-length Akt and report the structure-based design, synthesis, biological and pharmacological evaluation of a focused library of these innovative inhibitors.

7.
Cancer Res ; 79(9): 2367-2378, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30858154

RESUMO

Aberrations within the PI3K/AKT signaling axis are frequently observed in numerous cancer types, highlighting the relevance of these pathways in cancer physiology and pathology. However, therapeutic interventions employing AKT inhibitors often suffer from limitations associated with target selectivity, efficacy, or dose-limiting effects. Here we present the first crystal structure of autoinhibited AKT1 in complex with the covalent-allosteric inhibitor borussertib, providing critical insights into the structural basis of AKT1 inhibition by this unique class of compounds. Comprehensive biological and preclinical evaluation of borussertib in cancer-related model systems demonstrated a strong antiproliferative activity in cancer cell lines harboring genetic alterations within the PTEN, PI3K, and RAS signaling pathways. Furthermore, borussertib displayed antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models of mutant KRAS pancreatic and colon cancer. SIGNIFICANCE: Borussertib, a first-in-class covalent-allosteric AKT inhibitor, displays antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models and provides a starting point for further pharmacokinetic/dynamic optimization.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridonas/farmacologia , Pirimidinonas/farmacologia , Animais , Apoptose , Ciclo Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Angew Chem Int Ed Engl ; 57(31): 9970-9975, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29873877

RESUMO

PIK-75 is a phosphoinositide-3-kinase (PI3K) α-isoform-selective inhibitor with high potency. Although published structure-activity relationship data show the importance of the NO2 and the Br substituents in PIK-75, none of the published studies could correctly determine the underlying reason for their importance. In this publication, we report the first X-ray crystal structure of PIK-75 in complex with the kinase GSK-3ß. The structure shows an unusual U-shaped conformation of PIK-75 within the active site of GSK-3ß that is likely stabilized by an atypical intramolecular Br⋅⋅⋅NO2 halogen bond. NMR and MD simulations show that this conformation presumably also exists in solution and leads to a binding-competent preorganization of the PIK-75 molecule, thus explaining its high potency. We therefore suggest that the site-specific incorporation of halogen bonds could be generally used to design conformationally restricted bioactive substances with increased potencies.

9.
J Med Chem ; 58(17): 6844-63, 2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26275028

RESUMO

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.


Assuntos
Antineoplásicos/química , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Bases de Dados de Compostos Químicos , Desenho de Fármacos , Receptores ErbB/genética , Humanos , Cinética , Neoplasias Pulmonares , Modelos Moleculares , Conformação Molecular , Mutação , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Bibliotecas de Moléculas Pequenas , Solubilidade , Relação Estrutura-Atividade , Quinases da Família src/química , Quinases da Família src/genética
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