RESUMO
Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60-65â¯%) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization.
Assuntos
Di-Hidrouracila Desidrogenase (NADP) , Fenótipo , Ligação Proteica , Timina , Uracila , Timina/metabolismo , Uracila/análogos & derivados , Uracila/metabolismo , Uracila/sangue , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Di-Hidrouracila Desidrogenase (NADP)/genética , Humanos , Cromatografia Líquida/métodos , Fluoruracila/metabolismo , Fluoruracila/sangue , Genótipo , Deficiência da Di-Hidropirimidina Desidrogenase/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.
Assuntos
Biomarcadores Tumorais , Espectrometria de Massas , Fosfopiruvato Hidratase , Fosfopiruvato Hidratase/sangue , Fosfopiruvato Hidratase/isolamento & purificação , Humanos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Acetilação , Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Imunoprecipitação/métodos , Cromatografia Líquida de Alta Pressão/métodosRESUMO
OBJECTIVE: The peroxisome proliferator-activated receptor α (PPARα) is a transcription factor driving target genes involved in fatty acid ß-oxidation. To what extent various PPARα interacting proteins may assist its function as a transcription factor is incompletely understood. An ORFeome-wide unbiased mammalian protein-protein interaction trap (MAPPIT) using PPARα as bait revealed a PPARα-ligand-dependent interaction with the orphan nuclear receptor estrogen-related receptor α (ERRα). The goal of this study was to characterize the nature of the interaction in depth and to explore whether it was of physiological relevance. METHODS: We used orthogonal protein-protein interaction assays and pharmacological inhibitors of ERRα in various systems to confirm a functional interaction and study the impact of crosstalk mechanisms. To characterize the interaction surfaces and contact points we applied a random mutagenesis screen and structural overlays. We pinpointed the extent of reciprocal ligand effects of both nuclear receptors via coregulator peptide recruitment assays. On PPARα targets revealed from a genome-wide transcriptome analysis, we performed an ERRα chromatin immunoprecipitation analysis on both fast and fed mouse livers. RESULTS: Random mutagenesis scanning of PPARα's ligand-binding domain and coregulator profiling experiments supported the involvement of (a) bridging coregulator(s), while recapitulation of the interaction in vitro indicated the possibility of a trimeric interaction with RXRα. The PPARα·ERRα interaction depends on 3 C-terminal residues within helix 12 of ERRα and is strengthened by both PGC1α and serum deprivation. Pharmacological inhibition of ERRα decreased the interaction of ERRα to ligand-activated PPARα and revealed a transcriptome in line with enhanced mRNA expression of prototypical PPARα target genes, suggesting a role for ERRα as a transcriptional repressor. Strikingly, on other PPARα targets, including the isolated PDK4 enhancer, ERRα behaved oppositely. Chromatin immunoprecipitation analyses demonstrate a PPARα ligand-dependent ERRα recruitment onto chromatin at PPARα-binding regions, which is lost following ERRα inhibition in fed mouse livers. CONCLUSIONS: Our data support the coexistence of multiple layers of transcriptional crosstalk mechanisms between PPARα and ERRα, which may serve to finetune the activity of PPARα as a nutrient-sensing transcription factor.
Assuntos
Receptor ERRalfa Relacionado ao Estrogênio , PPAR alfa , Receptores de Estrogênio , PPAR alfa/metabolismo , PPAR alfa/genética , Animais , Camundongos , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Humanos , Regulação da Expressão Gênica , Células HEK293 , Masculino , Camundongos Endogâmicos C57BL , Ligação Proteica , Fígado/metabolismoRESUMO
OBJECTIVES: Timely diagnosis of lung cancer (LC) is crucial to achieve optimal patient care and outcome. Moreover, the number of procedures required to obtain a definitive diagnosis can have a large influence on the life expectancy of a patient. Here, adherence with existing Dutch guidelines for timeliness and type and number of invasive and imaging procedures was assessed. MATERIALS AND METHODS: 1096 patients with suspected LC were enrolled in this multicenter prospective study (NL9146). The overall survival, time from referral to the first appointment with the pulmonologist, time to diagnosis and treatment, and the number of imaging and invasive procedures were evaluated. Patients were divided into different diagnostic groupsearly- and advanced stage non-small-cell lung cancer (NSCLC), small-cell lung cancer (SCLC), large cell neuroendocrine carcinoma of the lung (LCNEC), patients without LC and patients without a definitive diagnosis. RESULTS: The majority of patients (66 %) received a definitive diagnosis within 5 weeks, although the time to diagnosis of early-stage LC patients and patients without LC was significantly longer comparted to advanced stage LC. An increase in invasive procedures was seen for early-stage LC compared to advanced stage LC and for 13 % of the advanced stage non-squamous NSCLC patients up to three additional invasive procedures were performed solely to obtain sufficient material for NGS. For patients without a definitive diagnosis, 50 % did undergo at least one invasive procedure, while 11 % did not wish to undergo any invasive procedures. CONCLUSION: These insights could aid in improved LC diagnostics and efficient implementation of new techniques like liquid biopsy and artificial intelligence. This may lead to more timely LC care, a decreased number of invasive procedures, less variability between the diagnostic trajectory of different patients and aid in obtaining a definitive diagnosis for all patients.
Assuntos
Carcinoma Neuroendócrino , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/terapia , Inteligência Artificial , Estudos Prospectivos , Hospitais , PulmãoRESUMO
SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein-protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain-PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain-PRM interactions and be utilized as a general approach for other domain-peptide interactions.
Assuntos
Peptídeos , Domínios de Homologia de src , Humanos , Sequência de Aminoácidos , Proteína Adaptadora GRB2/metabolismo , Ligação Proteica , Filogenia , Peptídeos/metabolismo , Prolina/metabolismoRESUMO
OBJECTIVES: Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. METHODS: Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. RESULTS: Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100â¯ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. CONCLUSIONS: A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.
Assuntos
Antígenos de Neoplasias , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Cromatografia Líquida/métodos , Queratina-19 , Espectrometria de Massas em Tandem/métodos , Neoplasias Pulmonares/diagnóstico , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Protein-protein interaction (PPI) modulation is a promising approach in drug discovery with the potential to expand the 'druggable' proteome and develop new therapeutic strategies. While there have been significant advancements in methodologies for developing PPI inhibitors, there is a relative scarcity of literature describing the 'bottom-up' development of PPI stabilizers (Molecular Glues). The hub protein 14-3-3 and its interactome provide an excellent platform for exploring conceptual approaches to PPI modulation, including evolution of chemical matter for Molecular Glues. In this study, we employed a fragment extension strategy to discover stabilizers for the complex of 14-3-3 protein and an Estrogen Receptor alpha-derived peptide (ERα). A focused library of analogues derived from an amidine-substituted thiophene fragment enhanced the affinity of the 14-3-3/ERα complex up to 6.2-fold. Structure-activity relationship (SAR) analysis underscored the importance of the newly added, aromatic side chain with a certain degree of rigidity. X-ray structural analysis revealed a unique intermolecular π-π stacking binding mode of the most active analogues, resulting in the simultaneous binding of two molecules to the PPI binding pocket. Notably, analogue 11 displayed selective stabilization of the 14-3-3/ERα complex.
Assuntos
Proteínas 14-3-3 , Receptor alfa de Estrogênio , Proteínas 14-3-3/química , Ligação Proteica , Descoberta de Drogas/métodos , Relação Estrutura-AtividadeRESUMO
The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 µM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development.
Assuntos
Produtos Biológicos , Receptor alfa de Estrogênio , Humanos , Receptores de Estrogênio , Aminoácidos , BioensaioRESUMO
Small-molecule stabilization of protein-protein interactions (PPIs) is a promising strategy in chemical biology and drug discovery. However, the systematic discovery of PPI stabilizers remains a largely unmet challenge. Herein we report a fragment-linking approach targeting the interface of 14-3-3 and a peptide derived from the estrogen receptor alpha (ERα) protein. Two classes of fragments-a covalent and a noncovalent fragment-were co-crystallized and subsequently linked, resulting in a noncovalent hybrid molecule in which the original fragment interactions were largely conserved. Supported by 20 crystal structures, this initial hybrid molecule was further optimized, resulting in selective, 25-fold stabilization of the 14-3-3/ERα interaction. The high-resolution structures of both the single fragments, their co-crystal structures and those of the linked fragments document a feasible strategy to develop orthosteric PPI stabilizers by linking to an initial tethered fragment.
Assuntos
Proteínas 14-3-3 , Receptor alfa de Estrogênio , Proteínas 14-3-3/química , Receptor alfa de Estrogênio/metabolismo , Ligação Proteica , Descoberta de Drogas/métodosRESUMO
Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.
Assuntos
Proteínas 14-3-3 , Receptor alfa de Estrogênio , Humanos , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Ligantes , Tamoxifeno/farmacologia , Ligação Proteica/efeitos dos fármacos , Descoberta de Drogas , Antagonistas de Estrogênios/farmacologiaRESUMO
The natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells. Among the identified 14-3-3 target proteins are THEMIS2, receptor interacting protein kinase 2 (RIPK2), EIF2AK2, and several members of the LDB1 complex. Biophysical and structural biology studies confirm these 14-3-3 PPIs as physical targets of FC stabilization, and transcriptome as well as pathway analyses suggest possible explanations for the observed synergistic effect of IFNα/FC treatment on cancer cells. This study elucidates the polypharmacological effects of FCs in cancer cells and identifies potential targets from the vast interactome of 14-3-3s for therapeutic intervention in oncology.
Assuntos
Interferon-alfa , Neoplasias Ovarianas , Humanos , Feminino , Interferon-alfa/farmacologia , Apoptose , Linhagem Celular Tumoral , Morte CelularRESUMO
Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein-protein or protein-nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein-protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Células Artificiais , Proteínas 14-3-3/química , Peptídeos/química , FosforilaçãoRESUMO
Neuron-specific enolase (NSE) is a promising small-cell lung cancer (SCLC) biomarker composed of αγ and γγ isozyme dimers. As the conventional immunoassays are prone to interferences and cannot differentiate between the isozymes, we developed a multiplex immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of NSEα and NSEγ in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed αα and γγ enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1αγ:1γγ:1αα. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSEγ. The isolated αγ and γγ dimers were denatured and trypsin digested to allow quantification of the selected signature peptides and their corresponding isotopically labelled peptide internal standard. The obtained linear dynamic ranges were determined to be 1.5-56 ng/mL and 0.64-167 ng/mL for NSEα and NSEγ (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSEα and NSEγ. The method was successfully applied to patient serum in which both isozymes were detected. Compared to the conventional immunoassay, substantially lower total NSE concentrations were measured in IA LC-MS/MS. With this multiplex IA LC-MS/MS assay, the clinical value of quantifying the individual isozymes can be explored. In addition, together with the calibrator described here, it may be applied to standardize NSE immunoassays across different platforms.
Assuntos
Isoenzimas , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Peptídeos , Fosfopiruvato Hidratase , Reprodutibilidade dos TestesRESUMO
Molecules that stabilize protein-protein interactions (PPIs) are invaluable as tool compounds for biophysics and (structural) biology, and as starting points for molecular glue drug discovery. However, identifying initial starting points for PPI stabilizing matter is highly challenging, and chemical optimization is labor-intensive. Inspired by chemical crosslinking and reversible covalent fragment-based drug discovery, we developed an approach that we term "molecular locks" to rapidly access molecular glue-like tool compounds. These dual-covalent small molecules reversibly react with a nucleophilic amino acid on each of the partner proteins to dynamically crosslink the protein complex. The PPI between the hub protein 14-3-3 and estrogen-related receptor γ (ERRγ) was used as a pharmacologically relevant case study. Based on a focused library of dual-reactive small molecules, a molecular glue tool compound was rapidly developed. Biochemical assays and X-ray crystallographic studies validated the ternary covalent complex formation and overall PPI stabilization via dynamic covalent crosslinking. The molecular lock approach is highly selective for the specific 14-3-3/ERRγ complex, over other 14-3-3 complexes. This selectivity is driven by the interplay of molecular reactivity and molecular recognition of the composite PPI binding interface. The long lifetime of the dual-covalent locks enabled the selective stabilization of the 14-3-3/ERRγ complex even in the presence of several other competing 14-3-3 clients with higher intrinsic binding affinities. The molecular lock approach enables systematic, selective, and potent stabilization of protein complexes to support molecular glue drug discovery.
Assuntos
Descoberta de Drogas , Receptores de Estrogênio , Humanos , Ligação Proteica , Proteínas 14-3-3/química , Aminoácidos/metabolismoRESUMO
OBJECTIVES: Pathologic subtyping of tissue biopsies is the gold standard for the diagnosis of lung cancer (LC), which could be complicated in cases of e.g. inconclusive tissue biopsies or unreachable tumors. The diagnosis of LC could be supported in a minimally invasive manner using protein tumor markers (TMs) and circulating tumor DNA (ctDNA) measured in liquid biopsies (LBx). This study evaluates the performance of LBx-based decision-support algorithms for the diagnosis of LC and subtyping into small- and non-small-cell lung cancer (SCLC and NSCLC) aiming to directly impact clinical practice. MATERIALS AND METHODS: In this multicenter prospective study (NL9146), eight protein TMs (CA125, CA15.3, CEA, CYFRA 21-1, HE4, NSE, proGRP and SCCA) and ctDNA mutations in EGFR, KRAS and BRAF were analyzed in blood of 1096 patients suspected of LC. The performance of individual and combined TMs to identify LC, NSCLC or SCLC was established by evaluating logistic regression models at pre-specified positive predictive values (PPV) of ≥95% or ≥98%. The most informative protein TMs included in the multi-parametric models were selected by recursive feature elimination. RESULTS: Single TMs could identify LC, NSCLC and SCLC patients with 46%, 25% and 40% sensitivity, respectively, at pre-specified PPVs. Multi-parametric models combining TMs and ctDNA significantly improved sensitivities to 65%, 67% and 50%, respectively. CONCLUSION: In patients suspected of LC, the LBx-based decision-support algorithms allowed identification of about two-thirds of all LC and NSCLC patients and half of SCLC patients. These models therefore show clinical value and may support LC diagnostics, especially in patients for whom pathologic subtyping is impossible or incomplete.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos Prospectivos , Biomarcadores Tumorais , Fosfopiruvato Hidratase , Biópsia LíquidaRESUMO
Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809) and the triple combination Trikafta®. This macrocycle is a useful tool to study the CFTR/14-3-3 interaction and the potential of molecular glues in cystic fibrosis therapeutics.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Aminofenóis/metabolismo , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Membrana Celular/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , MutaçãoRESUMO
Scaffold proteins operate as organizing hubs to enable high-fidelity signaling, fulfilling crucial roles in the regulation of cellular processes. Bottom-up construction of controllable scaffolding platforms is attractive for the implementation of regulatory processes in synthetic biology. Here, we present a modular and switchable synthetic scaffolding system, integrating scaffold-mediated signaling with switchable kinase/phosphatase input control. Phosphorylation-responsive inhibitory peptide motifs were fused to 14-3-3 proteins to generate dimeric protein scaffolds with appended regulatory peptide motifs. The availability of the scaffold for intermolecular partner protein binding could be lowered up to 35-fold upon phosphorylation of the autoinhibition motifs, as demonstrated using three different kinases. In addition, a hetero-bivalent autoinhibitory platform design allowed for dual-kinase input regulation of scaffold activity. Reversibility of the regulatory platform was illustrated through phosphatase-controlled abrogation of autoinhibition, resulting in full recovery of 14-3-3 scaffold activity.
Assuntos
Proteínas 14-3-3 , Peptídeos , Proteínas 14-3-3/química , Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Ligação ProteicaRESUMO
The modulation of protein-protein interactions (PPIs) has developed into a well-established field of drug discovery. Despite the advances achieved in the field, many PPIs are still deemed as 'undruggable' targets and the design of PPIs stabilizers remains a significant challenge. The application of fragment-based methods for the identification of drug leads and to evaluate the 'tractability' of the desired protein target has seen a remarkable development in recent years. In this study, we explore the molecular characteristics of the 14-3-3/Amot-p130 PPI and the conceptual possibility of targeting this interface using X-ray crystallography fragment-based screening. We report the first structural elucidation of the 14-3-3 binding motif of Amot-p130 and the characterization of the binding mode and affinities involved. We made use of fragments to probe the 'ligandability' of the 14-3-3/Amot-p130 composite binding pocket. Here we disclose initial hits with promising stabilizing activity and an early-stage selectivity toward the Amot-p130 motifs over other representatives 14-3-3 partners. Our findings highlight the potential of using fragments to characterize and explore proteins' surfaces and might provide a starting point toward the development of small molecules capable of acting as molecular glues.
RESUMO
Supernatant pleural effusions (PE) have shown to be a valuable source for the detection of driver mutations in circulating tumor DNA (ctDNA). In this prospective study, the clinical value of ctDNA analysis in supernatant PE to support therapy selection and disease monitoring in lung cancer patients is assessed. Paired PE and plasma samples were collected from lung cancer patients before initiation of therapy (N = 2) and from EGFR positive patients during therapy (N = 3). Supernatant PE and plasma were tested for mutations in EGFR, KRAS and BRAF by droplet digital PCR. In PE of two patients with suspected lung cancer, a KRAS mutation was detected with a 5- and 8-fold higher fractional abundance (FA) compared to plasma. For three patients with progressive disease during therapy, both the EGFR L858R and T790M mutations were detected in PE. However, in plasma only for two of these patients the L858R mutation was detected with a 46- and 14- fold lower FA, and only for one patient the T790M mutation was detected with a 8-fold lower FA. For one patient, longitudinal ctDNA analysis in PE revealed the T790M and L858R mutations already two months prior to detection of progressive disease by CT-scan. In this study, a higher ctDNA concentration and FA was obtained from PE compared to the corresponding blood samples, which enables more sensitive mutation analysis. Thus, PE is a valuable liquid biopsy, complementing plasma, for ctDNA analysis to support therapy selection and disease monitoring in lung cancer patients.
Assuntos
DNA Tumoral Circulante/sangue , Técnicas de Genotipagem/métodos , Neoplasias Pulmonares/patologia , Derrame Pleural/patologia , Feminino , Humanos , MasculinoRESUMO
Rational design of protein-protein interaction (PPI) inhibitors is challenging. Connecting a general supramolecular protein binder with a specific peptidic ligand provides a novel conceptual approach. Thus, lysine-specific molecular tweezers were conjugated to a peptide-based 14-3-3 ligand and produced a strong PPI inhibitor with 100-fold elevated protein affinity. X-ray crystal structure elucidation of this supramolecular directed assembly provides unique molecular insight into the binding mode and fully aligns with Molecular Dynamics (MD) simulations. This new supramolecular chemical biology concept opens the path to novel chemical tools for studying PPIs.