7196 research outputs found
Sort by
PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer.
No full textPHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins
Genome-wide screening in human kidney organoids identifies novel aspects of nephrogenesis
No full textHuman organoids allow studying proliferation, lineage specification, and three-dimensional tissue development. Here, we present the first genome-wide CRISPR screen in iPSC-derived kidney organoids. The combination of inducible genome editing, longitudinal sampling and endpoint sorting of tubular and stromal cells generated a complex, high quality dataset uncovering a broad spectrum of novel biology from early development to ‘adult’ epithelial morphogenesis. Our functional dataset allows improving mesoderm induction by ROCK inhibition, contains monogenetic and complex trait kidney disease genes, confirms two novel CAKUT genes (CCDC170 and MYH7B), and provides a large candidate list of ciliopathy-related genes. Finally, the identification of a cis-inhibitory effect of Jagged1 controlling epithelial proliferation shows how mosaic knockouts in pooled CRISPR screening can discover novel ways of communication between heterogeneous cell populations in complex tissues. Collectively, these data serve both as a rich resource for the kidney community and as a benchmark for future iPSC-derived organoid CRISPR screens
The effect of mAb and excipient cryoconcentration on long-term frozen storage stability - Part 1: Higher molecular weight species and subvisible particle formation.
No full textCryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at -80 °C, -20 °C, and - 10 °C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure Tg' of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine Tg' in formulations without glass forming sugars. Storage below Tg' did not lead to mAb aggregation. Above Tg', at -20 °C and - 10 °C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below Tg' completely prevents mAb aggregation and particle formation
Species-dependent hepatic and intestinal metabolism of selective oestrogen receptor degrader LSZ102 by sulphation and glucuronidation.
No full textLSZ102 is an orally bioavailable selective oestrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass.To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase (SULT) enzymes potentially involved in metabolism of LSZ102 was characterised.Sulphate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolised by several human SULTs expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulphation in human but glucuronidation in rat.As LSZ102 is metabolised by a number of different SULTs, drug-drug interactions resulting from the inhibition of one SULT are unlikely.Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulphate M5, glucuronide M4, and secondary glucuronide/sulphate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites
Deubiquitinase-targeting chimeras for targeted protein stabilization.
No full textMany diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS
Immobilization of 3,5-Dimethylphenyl Carbamate of Cellulose and Amylose on Silica by Photochemical and Thermal Radical Processes
No full textThe immobilization of cellulose 3,5-dimethylphenyl carbamate and amylose 3,5-
dimethylphenyl carbamate on silica gel carrier was achieved by using photochemical and
thermal processes. Both approaches provide an easy access to materials which were applied
as chiral stationary phases (CSPs) for the chromatographic resolution of racemic molecules.
The influence of parameters such as irradiation time and solvent on immobilization
effectiveness were investigated. For the thermal process, azo-bis-isobutyrontrile and di-tertbutyl
peroxide were evaluated as radical initiators. The influence of parameters such as
amount of radical initiator, solvent, temperature and further handling operations on the
immobilization rate were examined. The chiral recognition ability and the overall
performance of the prepared immobilized phases was evaluated by injection of a series of
racemic compounds onto packed HPLC columns. As there is almost no limitation of organic
solvent types that can be used as mobile phases with the immobilized CSP, they can be
applied under chromatographic conditions which are prohibited with the corresponding nonbonded
CSP. This extended applicability considerably broaden the options for improving
enantioselectivity and resolving chiral compounds which are not or only poorly soluble in the
classical mobile phases
Scale-up of Diazonium Salts and Azides in a Three-step Continuous Flow Sequence
No full textRapid synthesis and scale-up of active molecules to support the development process of new drug candidates is key in the pharmaceutical industry. Herein we describe the development of a scalable continuous flow procedure for two key steps in the synthesis of 2H-indazoles which were identified as highly potent and selective TLR7 and TLR8 antagonists. Transformation of hazardous azide chemistries from batch to continuous flow mode helped to mitigate and limit the risks associated with the handling of large amounts of hazardous reagents and intermediates in batch. In a two-step approach, we first screened and optimized reaction parameter for an azidation and cyclization reaction using a commercial research scale plug-flow reactor. In a second step we demonstrated the robustness and scalability of both reactions which finally enabled us to rapidly prepare and deliver the required amount of material in high quality
Selection of datasets for FAIRification in Drug Discovery and Development: Which, Why, and How?
No full textAbstract
Despite the intuitive value of adopting the Findable, Accessible, Interoperable, and Reusable (FAIR) principles in both academic and industrial sectors, challenges exist in resourcing, balancing long- versus short-term priorities, and achieving technical implementation. This situation is exacerbated by the unclear mechanisms by which costs and benefits can be assessed when decisions on FAIR are made. Scientific and research and development (R&D) leadership need reliable evidence of the potential benefits and information on effective implementation mechanisms and remediating strategies. In this article, we describe procedures for cost-benefit evaluation, and identify best-practice approaches to support the decision-making process involved in FAIR implementation
Broad-spectrum cyclic boronate β-lactamase inhibitors featuring an intramolecular prodrug for oral bioavailability.
No full textEarly efforts to broaden the spectrum and potency of cyclic boronic acid β-lactamase inhibitor vaborbactam included a series of 7-membered ring boronates. Exploration of stereoisomers and incorporation of heteroatoms allowed identification of the all-carbon cyclic boronate with substituents trans as the preferred core structure, showing inhibition of Class A and C enzymes. Crystal structures of one analog bound to important β-lactamase enzymes were obtained. When isolated under acidic conditions, these compounds spontaneously formed a neutral cyclic anhydride (intramolecular prodrug) which was shown to have much-improved oral bioavailability (52-69%) compared to the ring-opened carboxylate salt (9%)
STING agonists/antagonists: their potential as therapeutics and future developments
No full textThe cGAS STING pathway has received much attention in the last years and it has been recognized as an important trigger of the innate immune response. Since the discovery of STING (in 2008) and later of cGAS (in 2013) there has been many reports suggesting that faulty regulation of this pathway is involved in many Type I IFN autoinflammatory disorders. Evidences have been accumulating that cGAS/STING might play an important role other pathologies beyond the classical immune diseases. Human genetic mutations, which result in the activation of STING, have been demonstrated as the driver of SAVI, a rare interferonopathy affecting young children. Nevertheless, no evidence is available in the clinics for the role of this pathway in a broader disease space, due to the lack of STING antagonists/antagonists that are qualified for clinical exploration. Hopes of quickly learning from STING agonists, which have reached clinical trials in the recent years for oncology indications (with mixed success) and of transforming these compounds in potent safe antagonists did not yet fulfill. Nevertheless, there has been progress in identifying novel compounds, showing in some cases unexpected mode of action, that might move to early clinical trials in the very near future. We will summarize current efforts in developing STING antagonists, describe their strength/weakness and discuss their potential for a STING drug