RESUMO
Herein, we describe a systematic SAR- and SPR-investigation of the peptidomimetic hydroxy-proline based VHL-ligand VH032, from which most to-date published VHL-targeting PROTACs have been derived. This study provides for the first time a consistent data set which allows for direct comparison of structural variations including those which were so far hidden in patent literature. The gained knowledge about improved VHL binders was used to design a small library of highly potent BRD4-degraders comprising different VHL exit vectors. Newly designed degraders showed favorable molecular properties and significantly improved degradation potency compared to MZ1.
Assuntos
Proteínas Nucleares , Proteína Supressora de Tumor Von Hippel-Lindau , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Ligantes , Proteínas Nucleares/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Bintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-ß (TGF-ß) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment (TME). We hypothesized that targeting PD-L1 to the tumor by BA colocalizes the TGF-ß trap (TGF-ßRII) to the TME, enabling it to sequester TGF-ß in the tumor more effectively than systemic TGF-ß blockade, thereby enhancing antitumor activity. METHODS: Multiple technologies were used to characterize the TGF-ß trap binding avidity. BA versus combinations of anti-PD-L1 and TGF-ß trap or the pan-TGF-ß antibody fresolimumab were compared in proliferation and two-way mixed lymphocyte reaction assays. Immunophenotyping of tumor-infiltrating lymphocytes (TILs) and RNA sequencing (RNAseq) analysis assessing stromal and immune landscape following BA or the combination therapy were performed in MC38 tumors. TGF-ß and PD-L1 co-expression and their associated gene signatures in MC38 tumors and human lung carcinoma tissue were studied with single-cell RNAseq (scRNAseq) and immunostaining. BA-induced internalization, degradation, and depletion of TGF-ß were investigated in vitro. RESULTS: BA and fresolimumab had comparable intrinsic binding to TGF-ß1, but there was an ~80× avidity-based increase in binding affinity with BA. BA inhibited cell proliferation in TGF-ß-dependent and PD-L1-expressing cells more potently than TGF-ß trap or fresolimumab. Compared with the combination of anti-PD-L1 and TGF-ß trap or fresolimumab, BA enhanced T cell activation in vitro and increased TILs in MC38 tumors, which correlated with efficacy. BA induced distinct gene expression in the TME compared with the combination therapy, including upregulation of immune-related gene signatures and reduced activities in TGF-ß-regulated pathways, such as epithelial-mesenchymal transition, extracellular matrix deposition, and fibrosis. Regulatory T cells, macrophages, immune cells of myeloid lineage, and fibroblasts were key PD-L1/TGF-ß1 co-expressing cells in the TME. scRNAseq analysis suggested BA modulation of the macrophage phenotype, which was confirmed by histological assessment. PD-L1/TGF-ß1 co-expression was also seen in human tumors. Finally, BA induced TGF-ß1 internalization and degradation in the lysosomes. CONCLUSION: BA more effectively blocks TGF-ß by targeting TGF-ß trap to the tumor via PD-L1 binding. Such colocalized targeting elicits distinct and superior antitumor responses relative to single agent combination therapy.
Assuntos
Neoplasias Pulmonares , Fator de Crescimento Transformador beta , Antígeno B7-H1 , Humanos , Fatores Imunológicos , Receptor de Morte Celular Programada 1 , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Microambiente TumoralRESUMO
Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Descoberta de Drogas , Doenças do Sistema Imunitário/tratamento farmacológico , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Administração Oral , Tirosina Quinase da Agamaglobulinemia/metabolismo , Relação Dose-Resposta a Droga , Humanos , Doenças do Sistema Imunitário/metabolismo , Estrutura Molecular , Piperidinas/administração & dosagem , Piperidinas/química , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Sensory rhodopsin II, the photophobic receptor from Natronomonas pharaonis (NpSRII)5, forms a 2:2 complex with its cognate transducer (N. pharaonis halobacterial transducer of rhodopsins II (NpHtrII)) in lipid membranes. Light activation of NpSRII leads to a displacement of helix F, which in turn triggers a rotation/screw-like motion of TM2 in NpHtrII. This conformational change is thought to be transmitted through the membrane adjacent conserved signal transduction domain in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases (HAMP domain) to the cytoplasmic signaling domain of the transducer. The architecture and function of the HAMP domain are still unknown. In order to obtain information on the structure and dynamics of this region, EPR experiments on a truncated transducer (NpHtrII(157)) and NpSRII, site-directed spin-labeled and reconstituted into purple membrane lipids, have been carried out. A nitroxide scanning involving residues in the transducer helix TM2, in the predicted AS-1 region, and at selected positions in the following connector and AS-2 regions of the HAMP domain has been performed. Accessibility and dynamics data allowed us to identify a helical region up to residue Ala(94) in the AS-1 amphipathic sequence, followed by a highly dynamic domain protruding into the water phase. Additionally, transducer-transducer and transducer-receptor proximity relations revealed the overall architecture of the AS-1 sequences in the 2:2 complex, which are suggested to form a molten globular type of a coiled-coil bundle.
Assuntos
Halorrodopsinas/química , Rodopsinas Sensoriais/química , Sequência de Aminoácidos , Archaea/metabolismo , Membrana Celular/metabolismo , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/metabolismo , Histidina Quinase , Luz , Lipídeos/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Óxido Nítrico/química , Ligação Proteica , Conformação Proteica , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Temperatura , TermodinâmicaRESUMO
Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.
Assuntos
Proteínas Arqueais/química , Carotenoides/química , Halorrodopsinas , Rodopsinas Sensoriais , Animais , Calorimetria , Dimerização , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Concentração de Íons de Hidrogênio , Cinética , Luz , Micelas , Modelos Moleculares , Oócitos , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Prótons , RNA Mensageiro/metabolismo , Transdução de Sinais , Temperatura , Termodinâmica , Fatores de Tempo , XenopusRESUMO
The photocycle of the photophobic receptor from Natronobacterium pharaonis, NpSRII, is studied by static and time-resolved step-scan Fourier transform infrared spectroscopy. Both low-temperature static and time-resolved spectra resolve a K-like intermediate, and the corresponding spectra show little difference within the noise of the time-resolved data. As compared to intermediate K of bacteriorhodopsin, relatively large amide I bands indicate correspondingly larger distortions of the protein backbone. The time-resolved spectra identify an intermediate L-like state with surprisingly small additional molecular alterations. With the formation of intermediate M, the Schiff-base proton is transferred to the counterion Asp-75. This state is characterized by larger amide bands indicating larger distortions of the protein. We can identify a second M state that differs only in small-protein bands. Reisomerization of the chromophore to all-trans occurs with the formation of intermediate O. The accelerated decay of intermediate M caused by azide results in another red-shifted intermediate with a protonated Schiff base. The chromophore in this state, however, still has 13-cis geometry. Nevertheless, the reisomerization is still as slow as under the conditions without azide. The results are discussed with respect to mechanisms of the observed proton pumping and the possible roles of the intermediates in receptor activation.