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A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor
No full textThe mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of
rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure−activity relationship (SAR) studies led to the identification of compound 12b with a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment
Manumycin Polyketides Act as Molecular Glues Between UBR7 and P53 to Impair Breast Cancer Pathogenicity
No full textMolecular glues are an intriguing therapeutic modality that harness small-molecules to induce interactions between proteins that typically do not interact, thus enabling the creation of novel protein function not naturally encoded in biology. While molecular glues such as thalidomide and rapamycin have catalyzed drug discovery efforts, such molecules are rare and have often been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention of disease. Historically, natural products have proven to be important sources of molecular glues and we postulated that natural products bearing multiple electrophilic sites may be an unexplored source of such molecules, potentially through multi-covalent attachment. Using activity-based protein profiling (ABPP)-based chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells to impair breast cancer pathogenicity through engaging in molecular glue interactions with the neo-substrate tumor-suppressor TP53, leading to the activation of p53 transcriptional activity and cell death. Our results reveal a previously undiscovered anti-cancer mechanism of this natural product family and highlight the potential for combining chemoproteomics and multi-covalent natural products for the discovery and characterization of new molecular glues with non-natural function
Process Analytical Technology for continuous manufacturing tableting processing : A case study
No full textThe use of Near Infrared Spectroscopy (NIRS) as a fast and non-destructive technique was employed for the control and monitoring of the tableting step during a continuous manufacturing process.
Two NIRS methods were optimized in order to in-line control the blend uniformity in the tablet feed frame and the API concentration on freshly pressed tablets prior the ejection. The novelty of this work first lies in the acquisition speed of NIR spectra reaching up to 70 000 tablets/h. Partial Least Square (PLS) regression was used as chemometric tool for the computation that resulted in excellent predictive calibration results. A coefficient of correlation (r) value of 0.99 was obtained for both probes. The root mean square error of calibration (RMSEC) and the root mean square error of prediction (RMSEP) were respectively 1.8% and 1.8% for active content in the tablet feeder and 2.2% and 2.3% for the tablet content. In addition, calibration performance and robustness of the methods were evaluated. Moreover several qualitative methods were proposed to monitor the tableting process in different stages of development (single wavelength, Principal Component Analysis, and Independent Component Analysis). In early phase development, the requirement/quality of the input material is not established yet; hence the use of a qualitative approach allows to confirm the suitability of the PAT methodology for in-process material monitoring & control. Later, the qualitative approach constitutes the foundation for the quantitative approach when input materials are fixed and larger production size occurs. The proposed strategy is a performant PAT tool for continuous manufacturing and a step forward to real time releas
Site-Directed Fragment-Based Screening for the Discovery of Protein-Protein Interaction Stabilizers
No full textModulation of protein-protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery
Capmatinib (INC280) is active against models of non–small cell lung cancer and other cancer types with defined mechanisms of MET activation
No full textPurpose: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection. Experimental Design: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line– or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models. Results: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations. Conclusions: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance
A CRISPR-Cas9 screen identifies essential CTCF anchor sites for the mitogenic function of ER-alpha in breast cancer
No full textEstrogen receptor α (ERα) is an enhancer activating transcriptional factor, a key driver of breast cancer, and a main target for cancer therapy. ER-mediated gene regulation requires proper chromatin-conformation to stimulate physical linkage between ER-bound enhancers and their target promoters. A major determinant of chromatin structure is CTCF that anchors chromatin domains. However, whether CTCF-binding elements (CBEs) are essential for ER mitogenic activity is unknown. To address this question in a global manner, we implemented a CRISPR-based functional genetic screening approach targeting CBEs located in the vicinity of ERα-bound enhancers. We identified four functional CBEs and demonstrated the role of one in inducing chromatin conformation changes in favor of activation of PREX1, a key ERα target gene in breast cancer. Indeed, high PREX1 expression predicted sensitivity to loss of ER, and good survival to anti-hormonal treatment. Altogether, our data show that CTCF-mediated chromatin structure is required for ER mitogenic function
Prediction of fraction unbound in microsomal and hepatocyte incubations – a comparison of methods across industry data sets (by the IQ in silico ADME working group)
No full textFraction unbound in the incubation, fu,inc, is an important parameter to evaluate intrinsic clearance from in vitro CLint measurements in hepatocytes or microsomes. Reliable estimates of fu,inc from structure have the potential to positively impact the screening timelines in drug discovery. Previous works suggest that fu,inc is primarily driven by passive processes and can be described using physico-chemical properties such as lipophilicity and protonation state of the molecule. While models based on these principles proved predictive in relatively small datasets including marketed drugs, their applicability domain has not been extensively explored. The presented work from the in silico ADME discussion group (part of the International Consortium for Innovation through Quality in Pharmaceutical Development, the IQ consortium) investigates the accuracy of these models in larger proprietary datasets considering several thousand compounds across chemical space. Overall, the equations do well for compounds with low lipophilicity, i.e., they correctly predict that fu,inc is in general above 0.5 for compounds with calculated LogP≤3. When applied to lipophilic compounds, the models failed to produce quantitatively accurate predictions of fu,inc with a high risk to underestimate binding properties. Therefore, these models could be used quantitatively for non-lipophilic compounds. Proprietary in-house machine-learning models consistently predict more than 70% of the values within 2-fold of the experimental result regardless of lipophilicity or ionization state. Additionally, data shown indicates that microsomal binding is in general a good proxy for hepatocyte binding
Modulation of Microglia by Voluntary Exercise or CSF1R Inhibition Prevents Age-Related Loss of Functional Motor Units
No full textAge-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss
Performance characterization of static mixers in precipitating environments
No full textStatic mixers are extensively used in various industries (pharmaceutical, food processing, chemical, pulp and paper, polymer synthesis, water treatment and petrochemical). They offer several advantages with regard to continuous operation (e.g., intensified mixing, byproduct reduction and sharp residence time distribution, no moving parts) and are frequently used in harsh chemical environments, in which fouling could cause serious problems or to a shut-down of the entire production line. Fouling has diverse mechanisms: precipitation fouling, particulate fouling, corrosion fouling, chemical reaction fouling, solidification, biofouling and composite fouling. This work investigates the application of static mixers in pharmaceutical purification processes that involve solvent exchange washing. Various equipment configurations were investigated and fouling probability and stability was analyzed. It was established that co-axial mixing of feed and anti-solvent before the static mixer, with an application of ultrasound, resulted in the most stable process. Furthermore, the particle size in various feed and product suspensions was analyzed and correlations between the amount of suspended particles and the particle growth were determined