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Resistance to allosteric SHP2 inhibition in FGFR-driven cancers through rapid feedback activation of FGFR
No full textSHP2 mediates RAS activation downstream of multiple receptor tyrosine kinases (RTKs) and cancer cell lines dependent on RTKs are in general dependent on SHP2. Profiling of the allosteric SHP2 inhibitor SHP099 across cancer cell lines harboring various RTK dependencies reveals that FGFR-dependent cells are often insensitive to SHP099 when compared to EGFR-dependent cells. We find that FGFR-driven cells depend on SHP2 but exhibit resistance to SHP2 inhibitors in vitro and in vivo. Treatment of such models with SHP2 inhibitors results in an initial decrease in phosphorylated ERK1/2 (p-ERK) levels, however p-ERK levels rapidly rebound within two hours. This p-ERK rebound is blocked by FGFR inhibitors or high doses of SHP2 inhibitors. Mechanistically, compared with EGFR-driven cells, FGFR-driven cells tend to express high levels of RTK negative regulators such as the SPRY family proteins, which are rapidly downregulated upon ERK inhibition. Moreover, over-expression of SPRY4 in FGFR-driven cells prevents MAPK pathway reactivation and sensitizes them to SHP2 inhibitors. We also identified two novel combination approaches to enhance the efficacy of SHP2 inhibitors, either with a distinct site 2 allosteric SHP2 inhibitor or with a RAS-SOS1 interaction inhibitor. Our findings suggest the rapid FGFR feedback activation following initial pathway inhibition by SHP2 inhibitors may promote the open conformation of SHP2 and lead to resistance to SHP2 inhibitors. These findings may assist to refine patient selection and predict resistance mechanisms in the clinical development of SHP2 inhibitors and to suggest strategies for discovering SHP2 inhibitors that are more effective against upstream feedback activation
Patient-derived glioblastoma cultures as a tool for small-molecule drug discovery
No full textThere is a compelling need for new therapeutic strategies for glioblastoma multiforme (GBM). Preclinical target and therapeutic discovery for GBMs is primarily conducted using cell lines grown in serum-containing media, such as U-87 MG, which do not reflect the gene expression profiles of tumors found in GBM patients. To address this lack of representative models, we sought to develop a panel of patient-derived GBM models and characterize their genomic features, using RNA sequencing (RNA-seq) and growth characteristics, both when grown as neurospheres in culture, and grown orthotopically as xenografts in mice. When we compared these with commonly used GBM cell lines in the Cancer Cell Line Encyclopedia (CCLE), we found these patient-derived models to have greater diversity in gene expression and to better correspond to GBMs directly sequenced from patient tumor samples. We also evaluated the potential of these models for targeted therapy, by using the genomic characterization to identify small molecules that inhibit the growth of distinct subsets of GBMs, paving the way for precision medicines for GBM
Predictive model-based process start-up in pharmaceutical continuous manufacturing
No full textContinuous Manufacturing (CM) is a promising strategy to achieve cost reduction in pharmaceutical production, as it can improve flexibility, efficiency and safety of manufacturing processes. One of the main technical disadvantages of CM is that the process needs a certain start-up phase before steady-state operation is reached, which can potentially cause out-of-specification (OOS) material. In this context, the presented paper aims to demonstrate that suitable process control strategies during start-up of a continuous drying operation can avoid OOS material production and hence can ensure that the provided benefits of CM are not outweighed by poor production yields. In detail, heat-up of the drying chamber prior the start of production can lead to excessive heat being stored inside of the stainless-steel housing, that is known to cause over-dried granules in the first few minutes of the drying process. To compensate this issue, an automatic ramping procedure of dryer rotation speed (and hence drying time) was introduced into the plant’s process control system, that counteracts the excessive drying capacity during start-up. As a result, dry granules exiting the dryer complied with the targeted Critical Quality Attribute loss-on-drying (LOD) from the very beginning of production
Organic anion transporting polypeptide 2B1 (OATP2B1), an expanded substrate profile, does it align with OATP2B1's hypothesized function?
No full text1. An expanded view of the substrate landscape of organic anion transporting polypeptide (OATP) 2B1 was pursued with the goal of understanding if the identification of novel in vitro substrates could shed additional light on the impact of OATP2B1 on intestinal absorption and brain penetration.
2. To examine this hypothesis, a series of experiments measured the cellular accumulation of a diverse array of compounds. Representative angiotensin II receptor blockers (ARBs) and other compounds of interest were subsequently investigated for inhibition, time dependence, and kinetics.
3. The study identified ARBs as a class of OATP2B1 substrates and found balsalazide, olsalzine, and gavestinel to be novel substrates of OATP2B1 too. Some compounds previously reported to be OATP2B1 substrates in the literature, aliskiren, erlotinib, montelukast, fexofenadine, and taurocholate could not be confirmed as substrates here.
4. Literature describing in vivo outcomes for OATP2B1 substrates, coproporphyrin III, ARBs, balsalazide, olsalzine, and gavestinel highlight the absence of a substantial impact of OATP2B1 on the oral absorption and/or brain penetration of OATP2B1 substrates. Suggestions of including OATP2B1 assessment as part of the drug approval process are likely premature and further mechanistic work with more robust OATP2B1 substrates, which may include some of those described here, is desirable
Summary of major conclusions from the 7th International Workshop on Genotoxicity Testing (IWGT), Tokyo, Japan
No full text• The 7th IWGT was held in Tokyo, Japan, in November 2017
• It consisted of 5 Working Groups (WGs) and a plenary session (summarized here)
• Three WGs provided guidance on the conduct and interpretation of individual test systems
• Two WGs focused on more strategic questions in genotoxicity testing
• The plenary session focused on high-dimensional genotoxicity dat
Effects of a patient-derived de novo coding alteration of CACNA1I in mice connect a schizophrenia risk gene with sleep spindle deficits.
No full textCACNA1I, a schizophrenia risk gene, encodes a subtype of voltage-gated T-type calcium channel CaV3.3. We previously reported that a patient-derived missense de novo mutation (R1346H) of CACNA1I impaired CaV3.3 channel function. Here, we generated CaV3.3-RH knock-in animals, along with mice lacking CaV3.3, to investigate the biological impact of R1346H (RH) variation. We found that RH mutation altered cellular excitability in the thalamic reticular nucleus (TRN), where CaV3.3 is abundantly expressed. Moreover, RH mutation produced marked deficits in sleep spindle occurrence and morphology throughout non-rapid eye movement (NREM) sleep, while CaV3.3 haploinsufficiency gave rise to largely normal spindles. Therefore, mice harboring the RH mutation provide a patient derived genetic model not only to dissect the spindle biology but also to evaluate the effects of pharmacological reagents in normalizing sleep spindle deficits. Importantly, our analyses highlighted the significance of characterizing individual spindles and strengthen the inferences we can make across species over sleep spindles. In conclusion, this study established a translational link between a genetic allele and spindle deficits during NREM observed in schizophrenia patients, representing a key step toward testing the hypothesis that normalizing spindles may be beneficial for schizophrenia patients
Discovery of a Covalent Ligand Targeting an Intrinsically Disordered Cysteine Within MYC
No full textMYC is a major oncogenic transcriptional driver of most human cancers. Yet, MYC has remained intractable to direct targeting because much of MYC is intrinsically disordered and there are no known obvious pockets within MYC that can be pharmacologically interrogated. Here, we have performed a cysteine-reactive covalent ligand screen to identify compounds that could disrupt the binding of MYC to its DNA consensus sequence in vitro and also impair MYC transcriptional activity in situ in cells. We have identified a covalent ligand hit EN4 that uniquely and covalently targets cysteine 171 (C171) of MYC within a predicted intrinsically disordered region of the protein. We show EN4 treatment directly targets MYC in cells and inhibits MYC transcriptional activity, downregulates multiple MYC transcriptional targets, and impairs breast tumor xenograft growth in vivo. We further show that mutation of C171 to a tyrosine attenuates the MYC inhibitory activity of EN4 in cells. We also show initial structure-activity relationships of EN4 and identify compounds that show improved potency. Overall, we identify a novel ligandable site within an intrinsically disordered region of MYC that leads to inhibition of MYC transcriptional activity
Target-Based Identification and Optimization of 5-Indazol-5-yl Pyridones as Toll-like Receptor 7 and 8 Antagonists using a Biochemical TLR8 Antagonist Competition Assay
No full textInappropriate activation of the endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 co-crystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (1) a core morph guided by a TLR7 homology model in order to achieve a dual TLR7/8 antagonism profile and (2), introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral PK properties and improved TLR8 binding affinity
Genetic variation in twins with vastly different phenotypical expression of recurrent respiratory papillomatosis
No full textN/
Identification of bioisosteric analogs by deep neural network
No full textBioisosteric design is a classical technique used in medicinal chemistry to improve potency, drug-like properties or the synthetic accessibility of a compound or to find similar potent compounds that exist in novel chemical space. Bioisosteric design involves replacing part of a molecule by another part that has similar properties. The replacements may be identified by applying medicinal chemistry knowledge, by mining chemical databases or by choosing analogs similar in molecular physicochemical properties. In this article a novel approach to identify bioisosteric analogs is described where the suggestions are made by a deep neural network trained on data collected from a large corpus of medicinal chemistry literature. Thanks to this the trained network is able to mimic the decision making of experienced medicinal chemists and identify standard as well as non-classical bioisosteric analogs even for structures outside the training set. Examples of the results are provided and application possibilities discussed