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The T-cell fingerprint of MALT1 paracaspase revealed by selective inhibition
No full textMALT1 is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T cell activation and fate after engagement of the T cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted survival after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the high affinity IL-2 receptor (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T cell signaling pathways. Altogether, this first report with a potent and selective MALT1 inhibitor elucidates that MALT1 paracaspase activity indirectly controls gamma-chain receptor dependent signaling, which is required for T cell proliferation and clonal expansion
Fingolimod inhibits brain atrophy and promotes brain-derived neurotrophic factor in an animal model of multiple sclerosis
No full textLongitudinal brain atrophy quantification is a critical efficacy measurement in multiple sclerosis (MS) clinical trials and the determination of No Evidence of Disease Activity (NEDA). Utilising fingolimod as a clinically validated therapy we evaluated the use of repeated brain tissue volume measures during chronic experimental autoimmune encephalomyelitis (EAE) as a new preclinical efficacy measure. Brain volume changes were quantified using magnetic resonance imaging (MRI) at 7 Tesla and correlated to treatment-induced brain derived neurotrophic factor (BDNF) measured in blood, cerebrospinal fluid, spinal cord and brain. Serial brain MRI measurements revealed slow progressive brain volume loss in vehicle treated EAE mice despite a stable clinical score. Fingolimod (1 mg/kg) significantly ameliorated brain tissue atrophy in the cerebellum and striatum when administered from established EAE disease onwards. Fingolimod-dependent tissue preservation was associated with induction of BDNF specifically within the brain and co-localized with neuronal soma. In contrast, therapeutic teriflunomide (3 mg/kg) treatment failed to inhibit CNS autoimmune mediated brain degeneration. Finally, weekly anti-IL-17A antibody (15 mg/kg) treatment was highly efficacious and preserved whole brain, cerebellum and striatum volume. Fingolimod-mediated BDNF increases within the CNS may contribute to limiting progressive tissue loss during chronic neuroinflammation
A slow cycling Lgr5 tumor cell population mediates resistance to Smoothened inhibitor in Basal Cell Carcinoma
No full textBasal cell carcinoma (BCC) is the most common human cancer. Smoothened inhibitor (Smoi) is used for the treatment of locally advanced and metastatic BCC. The mechanism by which Smoi mediates BCC regression is currently unknown. Here, we used two different genetically engineered mouse models to investigate the mechanisms by which Smoi mediates tumor regression. We found that Smoi mediates BCC regression by inhibiting their reprogramming into hair follicle like fate and promoting their differentiation towards interfollicular epidermis, infundibulum and sebaceous gland fates depending on their cellular origin. During the course of Smoi administration, some BCC became resistant to therapy mimicking the situation found in humans. We demonstrated that the resistant tumor cells express Lgr5 and were characterized by active Wnt signalling in mouse and human BCCs. Smoi in combination with Lgr5 lineage ablation or Wnt signalling inhibition leads to BCC eradication. Altogether, our study reveals that Smoi induces tumor regression by promoting tumor differentiation, and demonstrates that the synergy between Wnt and Smo inhibitors constitutes a clinically relevant strategy to overcome resistance to therapy in BCCs
Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing
No full textmRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered
Dual Allosteric Inhibition of SHP2 Phosphatase
No full textSHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene involved in proliferation, differentiation, and survival. Recently we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor. SHP099 binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies and tactics that enabled the identification of a second, distinct small molecule allosteric site of SHP2 inhibition. SHP244 (2) was identified and characterized as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that like 1, 2 binds and stabilizes the inactive, closed conformation of SHP2 but at a distinct, unexplored binding site- a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 via structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies
Synthesis of 4,5-Diazaspiro[2.3]hexanes and 1,2-Diazaspiro[3.3]heptanes as Hexahydropyridazine Analogues
No full text4,5-Diazaspiro[2.3]hexanes are made by dihalocarbene addition across the exocyclic double bond of readily accessible 3-alkylidene-1,2-diazetidines. Using difluorocarbene, generated from TMSCF3/NaI, these spirocycles were produced in yields up to 97% by stereospecific addition across the alkene. Lower yields (up to 64%) were observed using more reactive dichlorocarbene, due to competitive insertion of the carbene into the N-N bond. Larger 1,2-diazaspiro[3.3]heptanes are produced by [2 + 2] cycloaddition of 3-alkylidene-1,2-diazetidines with tetracyanoethylene (TCNE) in up to 99% yield
Developing and Implementing Performance Outcome Assessments: Evidentiary, Methodological, and Operational Considerations
No full textThis is a white paper summarizing the key points made at a two-day meeting organized by the Duke University-Robert J. Margolis Center for Health Policy and the FDA on Performance Outcome Measures. The information from this meeting and this article will provide the basis for a FDA Guidance on the topic to be published in 2018
Water-promoted chlorination of 2-mercaptobenzothiazoles
No full textSubstituted benzothiazoles play an important role in medicinal
chemistry due to their pharmacological properties. Their 2-substituted
derivatives are often prepared from 2-chlorobenzothiazoles, which in turn can
be made from the 2-mercapto precursor using sulfuryl chloride. In practice,
this seemingly straightforward, and widely used reaction can be impeded by
poor reproducibility and reaction yields. In this communication we report the
importance of acid as catalyst for the parent reaction. The addition of water to
the reaction mixture represents a facile and practical method for generating
acid in situ. This simple solution reproducibly ensured excellent yields of a
variety of chlorinated products
Development of an automated, interference-free, immunoaffinity-based mass spectrometric assay for quantification a therapeutic monoclonal antibody in human sera
No full textHybrid ligand binding-mass spectrometry assays are increasingly being applied to quantitative measurement of proteins in biological matrices. These assays combine the high specificity and enrichment capabilities of selective affinity reagents with the unmatched specificity and unequivocal analyte identification provided by mass spectrometry. Often these assays involve initial analyte enrichment at the protein level. Although these assays can work well, they are limited by the lack of appropriate well-characterized affinity reagents and, as with some immunoassays, may suffer from interferences caused by off-target protein binding, autoantibodies and anti-immunoglobulin antibodies. To address such problems, the method known as stable isotope standards and capture by anti-peptide antibodies (SISCAPA) was developed, which involves the use of well characterized, high affinity antibodies to enrich proteotypic peptide surrogates of the target protein followed by their identification and quantitation by mass spectrometry. The SISCAPA method involves tryptic digestion of the proteins in the sample matrix which destroys unwanted antibodies and other interfering proteins. Here we report the development of an automated, SISCAPA-based two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) assay for measurement of total levels a monoclonal antibody currently in clinical development. An affinity purified polyclonal antibody specific for a peptide in the complementarity determining region (CDR) of the antibody was used to enrich the surrogate peptide from trypsin-digested human plasma, followed by 2D-LC-MS/MS quantitation. The assay was compared to a standard ligand binding assay currently being used for the antibody quantitation. The data indicate that the peptide-based SISCAPA-2D-LC-MS/MS assay is a valuable alternative to specific protein quantification assays devoid of target or binding protein interference
N-aryl-piperidine-4-carboxamides as a novel class of potent inhibitors of MALT1 proteolytic activity
No full textStarting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties