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Continuous Flow as Enabling Technology: Synthesis of Heteroaromatic Sulfinates as Bench Stable Cross-Coupling Partners
No full textAn enabling continuous flow setup for handling of unstable organolithium intermediates and synthesis of heteroaryl sulfinates on multigram scale is described. The developed continuous flow process allows for the synthesis and simple isolation of heteroaryl sulfinates which otherwise are challenging to access in classical batch mode. The lithium sulfinate salts prepared by this method were shown to be efficient reaction partners in palladium catalyzed C(sp2)−C(sp2) cross-coupling to access medicinally relevant bis-heteroaryl motifs
ReSCoSS: A Flexible Quantum Chemistry Workflow Identifying Relevant Solution Conformers of Drug-like Molecules
No full textConformational equilibria are at the heart of drug design, yet their energetic description is often hampered by the insufficient accuracy of low-cost methods. Here we present a flexible and semi-automatic workflow based on quantum chemistry, ReSCoSS, designed to identify relevant conformers and predict their equilibria across different solvent environments in the Conductor-like Screening Model for Real Solvents (COSMO-RS) framework. We demonstrate the utility and accuracy of the workflow through conformational case studies on several drug-like molecules from literature where relevant conformations are known. We further show that including ReSCoSS conformers significantly improves COSMO-RS based predictions of physicochemical properties over single-conformation approaches. ReSCoSS has found broad adoption in the in-house drug discovery and development work streams and has contributed to establishing quantum-chemistry methods as a strategic pillar in ligand discovery
High-sensitivity Flow Cytometric Assays: Considerations for Design Control and Sensitivity Validation for Identification of Rare Events
No full textThe current consensus recommendation papers dealing with the unique requirements for the validation of assays performed by flow cytometry address the validation of assay sensitivity only in general terms (1-3). In this paper, a detailed approach for designing and validating the sensitivity of rare event methods is described. The impact of panel design and optimization on the Lower Limit of Quantification (LLOQ) as well as suggestions for reporting data near, or below, the LLOQ will be addressed. Thus, this paper will serve to provide best practices for the development, optimization, and validation of flow cytometric assays designed to assess rare events
The European Federation for Medicinal Chemistry (EFMC) Best Practice Initiative: Validating Chemical Probes
No full textAs part of an initiative aimed to share best practices in Medicinal Chemistry, the European Federation for Medicinal Chemistry (EFMC) is preparing a series of webinars and slide sets focused on the early phase of drug discovery. This educational material is freely accessible through the EFMC. The main target audiences are students or early career scientists and we also believe it will be valuable for experienced practitioners. The first of the series is focused on the generation and validation of high-quality chemical probes, which are critical for drug discovery and more broadly to further our understanding of human biology and disease
Development of a Practical Synthesis of an FXI (Factor XI) Inhibitor
No full textThe scalable synthesis of an FXI (Factor XI) inhibitor 12 employing multiple emerging technologies is described. The reduction of ketone to chiral alcohol was established through a biocatalysis approach. The Suzuki-Miyaura cross coupling reaction was facilitated by surfactant chemistry. A harsh hydrolysis of the nitrile was performed in a continuous manufacturing mode. Extensive reaction optimization and process development led to a well-controlled protocol for scale-up. The alternative approach described here addressed issues from the discovery route and was utilized to deliver the desired target for preclinical studies
Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice
No full textPost-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies
Inhibition of the Wnt/β-catenin pathway enhances anti-tumor immunity in ovarian cancer
No full textBackground: The Wnt/β-catenin pathway is linked to tumorigenesis in a variety of tumors and
promotes T cell exclusion and resistance to checkpoint inhibitors. We sought to determine whether a small molecule inhibitor of this pathway, WNT974, would impair tumor growth, affect gene expression patterns, and improve the immune response in human and murine ovarian cancer models.
Methods: Human ovarian cancer cells were treated with WNT974 in vitro. RNAseq libraries were constructed and differences in gene expression patterns between responders and nonresponders were compared to The Cancer Genome Atlas (TCGA). Mice with subcutaneous or intraperitoneal ID8 ovarian cancer tumors were treated with WNT974, paclitaxel, combination, or control. Tumor growth and survival were measured. Flow cytometry and β-TCR repertoire analysis were used to determine the immune response.
Results: Gene expression profiling revealed distinct signatures in responders and nonresponders,
which strongly correlated with T cell infiltration patterns in the TCGA analysis of ovarian cancer.
WNT974 inhibited tumor growth, prevented ascites formation, and prolonged survival in mouse models. WNT974 increased the ratio of CD8+ T cells to T regulatory cells (Tregs) in tumors and enhanced the effector functions of infiltrating CD4+ and CD8+ T cells. Treatment also decreased the expression of inhibitory receptors on CD8+ T cells. Combining WNT974 with paclitaxel further reduced tumor growth, prolonged survival, and expanded the T cell repertoire.
Conclusions: These findings suggest that inhibiting the Wnt/β-catenin pathway may have a potent immunomodulatory effect in the treatment of ovarian cancer, particularly when combined with paclitaxel
Recommendations for the Development of Cell-Based Anti-Viral Vector Neutralizing Antibody Assays
No full textViral vector–based gene therapies (GTx) have received significant attention in the recent years and the number of ongoing GTx clinical trials is increasing. A platform of choice for many of these studies is adeno-associated virus (AAV). All humans may be exposed to natural AAV infections and could mount an immune response against the virus. Consequently, there can be a high prevalence of pre-existing anti-AAV immunity. This presents a potential limitation for AAV-based GTx due to the potential for AAV-specific antibodies to reduce the efficacy of the GTx. Therefore, appropriate assessment of potential subjects enrolled in these studies should include evaluation for the presence and degree of anti-AAV immunity, including anti-AAV neutralizing antibodies (NAb). Recommendations for the development and validation of cell-based anti-AAV NAb detection methods, including considerations related to selection of appropriate cell line, surrogate vector/reporter gene, assay matrix and controls, and methodologies for calculating assay cut-point are discussed herein. General recommendations for the key assay validation parameters are provided as well as considerations for the development of NAb diagnostic tests. This manuscript is produced by a group of scientists involved in GTx therapeutic development representing various companies. It is our intent to provide recommendations and guidance to industrial and academic laboratories working on viral vector based GTx modalities with the goal of achieving a more consistent approach to anti-AAV NAb assessment
Molecular Profile of Gastrointestinal Stromal Tumors in Sixty-Eight Patients from a Single Swiss Institution.
No full textGastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. It has distinct molecular features and primarily affects the KIT and PDGFRA genes
Successful Development of a Method for the Incorporation of Fmoc-Arg(Pbf)-OH in Solid-Phase Peptide Synthesis using N Butylpyrrolidinone (NBP) as a Green Solvent
No full textNBP has proved an excellent alternative solvent to the hazardous DMF for SPPS. Here we studied the incorporation of Fmoc-Arg(Pbf)-OH, one of the most problematic amino acids, into a growing peptide chain.. The poor performance of this amino acid is attributed to the formation of a fully inactive -lactam, which causes a reduction in yield and very often the concomitant formation the corresponding des-Arg peptides. This problem is exacerbated when NBP is used as solvent, presumably because of its high viscosity, which impairs the penetration of the coupling cocktail into the resin. To tackle this issue, we propose the following strategy for the safe introduction of Fmoc-Arg(Pbf)-OH in SPPS at 45C, keeping excesses to a minimum: 1.75 equiv. of the protected amino acids, 1.8 equiv. of DIC, and 1.5 equiv. of OxymaPure. The cornerstone of the strategy is to carry out in situ activation. In this regard, Fmoc-Arg(Pbf)-OH and OxymaPure dissolved in NBP were added to peptidyl-resin, allow to reach the 45C, then half the DIC was added and left for 30 min, followed by the other half and some extra Fmoc-Arg(Pbf)-OH. During the entire process, the temperature was kept at 45C, with the double purpose of reducing the viscosity of NBP, thus facilitating the penetration of the coupling cocktail into the resin, and speeding up the coupling itself. It is envisaged that this strategy could be widely used to improve the performance of SPPS, including the industrial preparation of peptides using this approach