14 research outputs found
Chemotherapy for patients with <i>EGFR</i>-mutated NSCLC after progression on EGFR-TKI's:Exploration of efficacy of unselected treatment in a multicenter cohort study
A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase
Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source
Design, Synthesis and Structural Analysis of Glucocerebrosidase Imaging Agents
Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in β-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activity-based probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N- functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA’s preference for O6- and N -functionalised reagents, we synthesised a bi-functional aziridine ABP which we hoped would offer a more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes
Status and Perspectives of Detector Databases in the CMS Experiment at the LHC
This note gives an overview at a high conceptual level of the various databases that capture the information concerning the CMS detector. The detector domain has been split up into four, partly overlapping parts that cover phases in the detector life cycle: construction, integration, configuration and condition, and a geometry part that is common to all phases. The discussion addresses the specific content and usage of each part, and further requirements, dependencies and interfaces
Fasting-induced myocardial lipid accumulation in long-chain acyl-CoA dehydrogenase knockout mice is accompanied by impaired left ventricular function
Lipotoxicity may be a key contributor to the pathogenesis of cardiac abnormalities in mitochondrial long-chain fatty acid β-oxidation (FAO) disorders. Few data are available on myocardial lipid levels and cardiac performance in FAO deficiencies. The purpose of this animal study is to assess fasting-induced changes in cardiac morphology, function, and triglyceride (TG) storage as a consequence of FAO deficiency in a noninvasive fashion. MRI and proton magnetic resonance spectroscopy ((1)H-MRS) were applied in vivo in long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mice and wild-type (WT) mice (n=8 per genotype). Fasting was used to increase the heart's dependency on FAO for maintenance of energy homeostasis. In vivo data were complemented with ex vivo measurements of myocardial lipids. Left ventricular (LV) mass was higher in LCAD KO mice compared with WT mice (P <0.05), indicating LV myocardial hypertrophy. Myocardial TG content was higher in LCAD KO mice at baseline (P <0.001) and further increased in fasted LCAD KO mice (P <0.05). Concomitantly, LV ejection fraction (P <0.01) and diastolic filling rate (P <0.01) decreased after fasting, whereas these functional parameters did not change in fasted WT mice. Myocardial ceramide content was higher in fasted LCAD KO mice compared with fasted WT mice (P <0.05). Using a noninvasive approach, this study reveals accumulation of myocardial TG in LCAD KO mice. Toxicity of accumulating lipid metabolites such as ceramides may be responsible for the fasting-induced impairment of cardiac function observed in the LCAD KO mous
Tobacco Smoking-Related Mutational Signatures in Classifying Smoking-Associated and Nonsmoking-Associated NSCLC
Introduction: Patient-reported smoking history is frequently used as a stratification factor in NSCLC-directed clinical research. Nevertheless, this classification does not fully reflect the mutational processes in a tumor. Next-generation sequencing can identify mutational signatures associated with tobacco smoking, such as single-base signature 4 and indel-based signature 3. This provides an opportunity to redefine the classification of smoking- and nonsmoking-associated NSCLC on the basis of individual genomic tumor characteristics and could contribute to reducing the lung cancer stigma. Methods: Whole genome sequencing data and clinical records were obtained from three prospective cohorts of metastatic NSCLC (N = 316). Relative contributions and absolute counts of single-base signature 4 and indel-based signature 3 were combined with relative contributions of age-related signatures to divide the cohort into smoking-associated (“smoking high”) and nonsmoking-associated (“smoking low”) clusters. Results: The smoking high (n = 169) and smoking low (n = 147) clusters differed considerably in tumor mutational burden, signature contribution, and mutational landscape. This signature-based classification overlapped considerably with smoking history. Yet, 26% of patients with an active smoking history were included in the smoking low cluster, of which 52% harbored an EGFR/ALK/RET/ROS1 alteration, and 4% of patients without smoking history were included in the smoking high cluster. These discordant samples had similar genomic contexts to the rest of their respective cluster. Conclusions: A substantial subset of metastatic NSCLC is differently classified into smoking- and nonsmoking-associated tumors on the basis of smoking-related mutational signatures than on the basis of smoking history. This signature-based classification more accurately classifies patients on the basis of genome-wide context and should therefore be considered as a stratification factor in clinical research
Effect of Pembrolizumab after Stereotactic Body Radiotherapy vs Pembrolizumab Alone on Tumor Response in Patients with Advanced Non-Small Cell Lung Cancer: Results of the PEMBRO-RT Phase 2 Randomized Clinical Trial
Importance: Many patients with advanced non-small cell lung cancer (NSCLC) receiving immunotherapy show primary resistance. High-dose radiotherapy can lead to increased tumor antigen release, improved antigen presentation, and T-cell infiltration. This radiotherapy may enhance the effects of checkpoint inhibition. Objective: To assess whether stereotactic body radiotherapy on a single tumor site preceding pembrolizumab treatment enhances tumor response in patients with metastatic NSCLC. Design, Setting, and Participants: Multicenter, randomized phase 2 study (PEMBRO-RT) of 92 patients with advanced NSCLC enrolled between July 1, 2015, and March 31, 2018, regardless of programmed death-ligand 1 (PD-L1) status. Data analysis was of the intention-to-treat population. Interventions: Pembrolizumab (200 mg/kg every 3 weeks) either alone (control arm) or after radiotherapy (3 doses of 8 Gy) (experimental arm) to a single tumor site until confirmed radiographic progression, unacceptable toxic effects, investigator decision, patient withdrawal of consent, or a maximum of 24 months. Main Outcomes and Measures: Improvement in overall response rate (ORR) at 12 weeks from 20% in the control arm to 50% in the experimental arm with P <.10. Results: Of the 92 patients enrolled, 76 were randomized to the control arm (n = 40) or the experimental arm (n = 36). Of those, the median age was 62 years (range, 35-78 years), and 44 (58%) were men. The ORR at 12 weeks was 18% in the control arm vs 36% in the experimental arm (P =.07). Median progression-free survival was 1.9 months (95% CI, 1.7-6.9 months) vs 6.6 months (95% CI, 4.0-14.6 months) (hazard ratio, 0.71; 95% CI, 0.42-1.18; P =.19), and median overall survival was 7.6 months (95% CI, 6.0-13.9 months) vs 15.9 months (95% CI, 7.1 months to not reached) (hazard ratio, 0.66; 95% CI, 0.37-1.18; P =.16). Subgroup analyses showed the largest benefit from the addition of radiotherapy in patients with PD-L1-negative tumors. No increase in treatment-related toxic effects was observed in the experimental arm. Conclusions and Relevance: Stereotactic body radiotherapy prior to pembrolizumab was well tolerated. Although a doubling of ORR was observed, the results did not meet the study's prespecified end point criteria for meaningful clinical benefit. Positive results were largely influenced by the PD-L1-negative subgroup, which had significantly improved progression-free survival and overall survival. These results suggest that a larger trial is necessary to determine whether radiotherapy may activate noninflamed NSCLC toward a more inflamed tumor microenvironment. Trial Registration: ClinicalTrials.gov identifier: NCT02492568
Synthesis of 6-Hydroxysphingosine and alpha-Hydroxy Ceramide Using a Cross-Metathesis Strategy
In this paper, a new synthetic route toward 6-hydroxysphingosine and alpha-hydroxy ceramide is described. The synthesis employs a cross-metathesis to unite a sphingosine head allylic alcohol with a long-chain fatty acid alkene that also bears an allylic alcohol group. To allow for a productive CM coupling, the sphingosine head allylic alcohol was protected with a cyclic carbonate moiety and a reactive CM catalyst system, consisting of Grubbs II catalyst and CuI, was employe
Ultrasensitive in situ visualization of active glucocerebrosidase molecules
Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.
Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors
This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutic