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PET Imaging of Autotaxin in Thyroid and Breast Cancer Models using [18F]PRIMATX
No full textAutotaxin (ATX) is a secreted enzyme responsible for producing lysophosphatidic acid (LPA). The ATX/LPA signalling axis is typically activated in wound healing and tissue repair processes. It is highjacked and upregulated in the progression and persistence of several chronic inflammatory diseases, including cancer. As ATX inhibitors are now progressing to clinical testing, innovative diagnostic tools such as positron emission tomography (PET) are needed for the exact and accurate measurement of ATX expression in vivo. Recently, a radiotracer, [18F]PRIMATX, was developed and tested for PET imaging of ATX in vivo in a murine melanoma model. The goal of the present work was to further validate [18F]PRIMATX as a PET imaging agent by analyzing its in vivo metabolic stability and suitability for PET imaging of ATX in models of human 8305C thyroid tumour and murine 4T1 breast cancer. [18F]PRIMATX displayed favourable metabolic stability in vivo (65% of intact radiotracer after 60 min p.i.) and provided sufficient tumour uptake profiles in both tumour models. Radiotracer uptake could be blocked by 8-12% in 8305C thyroid tumours in the presence of ATX inhibitor AE-32-NZ70 as determined with PET and ex vivo biodistribution analyses. [18F]PRIMATX also showed high brain uptake, which was reduced by 50% through the administration of ATX inhibitor AE-32-NZ70. [18F]PRIMATX is a suitable radiotracer for PET imaging of ATX in the brain and peripheral tumour tissues
Chemical Investigations of Gymnastatin and Dankastatin Alkaloid Biosynthesis
No full textElectrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an especially interesting natural library of halogenated cytotoxic agents derived from tyrosine which feature an array of reactive functional groups. Herein we explore biosynthetic relationships between architecturally complex gymnastatin and dankastatin members, finding factors that favor formation of a given scaffold from a common intermediate. Additionally, we find that multiple natural products can be formed from aranorosin, a non-halogenated natural product also produced by gymnascella sp. fungi, using simple chloride salts. Finally, growth inhibitory activity of multiple members against challenging human triple negative breast cancer cells is reported
LLF580, an FGF21 Analog, Reduces Triglycerides and Hepatic Fat in obese persons with modestly elevated triglycerides: A 12 Week Randomized Double Blind Placebo Controlled Study
No full textPurpose: To evaluate the safety and potential efficacy of LLF580, a genetically engineered variant of human fibroblast growth factor-21, for triglyceride lowering, weight loss, and hepatic fat reduction.
Methods: A multicenter, double-blind, parallel design trial in obese, mildly hypertriglyceridemic and otherwise apparently healthy adults randomized (1:1) to LLF580 300 mg or placebo subcutaneously every four weeks for three doses.
Results: Of 64 randomized study participants, 61 (mean±SD: age 45±11 years, 49% male, 80/15/5% Caucasian/African American/Other, BMI 36.1±3.8 kg/m2) received LLF580 (n=30) or placebo (n=31) at 7 research sites in the USA. LLF580 lowered serum triglycerides by 54% (least square mean placebo adjusted change from baseline), total cholesterol 7%, LDL-cholesterol 12%, and increased HDL-cholesterol 36% compared to placebo (all P<0.001) over 12 weeks. Substantial reduction of liver fat of 52% over placebo (P<0.001) was also demonstrated, in the setting of improved liver function tests including alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, the composite enhanced liver fibrosis score, and N-terminal type III collagen propeptide (all P<0.05). Insulin and C-peptide levels and insulin resistance by HOMA-IR were all lower, and adiponectin higher with LLF580 treatment compared to placebo, while fasting glucose and HbA1c were unchanged. Reductions in biomarkers of bone formation without differences in markers of bone resorption were observed. LLF580 was generally safe and well tolerated, except for higher incidence of generally mild to moderate gastrointestinal adverse effects.
Conclusions: In obese, mildly hypertriglyceridemic adults, LLF580 was generally safe and demonstrated beneficial effects on serum lipids, liver fat and biomarkers of liver injury, suggesting it may be effective for treatment of select metabolic disorders including hypertriglyceridemia and non-alcoholic fatty liver disease. Assessments of longer-term safety and efficacy are warranted
Impact of Artificial Intelligence on Compound Discovery, Design, and Synthesis
No full textAs in other areas, artificial intelligence (AI) is heavily promoted in different scientific fields, including chemistry. Although chemistry traditionally tends to be a conservative field and slower than others to adapt new concepts, AI is increasingly being investigated across chemical disciplines. In medicinal chemistry, supported by computer-aided drug design and cheminformatics, computational methods have long been employed to aid in the search for and optimization of active compounds. We are currently witnessing a multitude of AI-related publications in the medicinal-chemistry-relevant literature and anticipate that the numbers will further increase. Often, advances through AI promoted in such reports are difficult to reconcile or remain questionable, which hampers the acceptance of computational work in interdisciplinary environments. Herein we attempt to highlight selected investigations in which AI has shown promise to impact medicinal chemistry in areas such as compound design and synthesis
A comprehensive review on non-clinical methods to study transfer of medication into breast milk – A contribution from the ConcePTION project
No full textBreastfeeding plays a major role in the health and wellbeing of mother and infant. However, information on the safety of maternal medication during breastfeeding is lacking for most medications. This leads to discontinuation of either breastfeeding or maternal therapy, although many medications are likely to be safe. Since human lactation studies are costly and challenging, validated non-clinical methods would offer an attractive alternative. This review gives an extensive overview of the non-clinical methods (in vitro, in vivo and in silico) to study the transfer of maternal medication into the human breast milk, and subsequent neonatal systemic exposure. Several in vitro models are available, but model characterization, including quantitative medication transport data across the in vitro blood-milk barrier, remains rather limited. Furthermore, animal in vivo models have been used successfully in the past. However, these models don't always mimic human physiology due to species-specific differences. Several efforts have been made to predict medication transfer into the milk based on physicochemical characteristics. However, the role of transporter proteins and several physiological factors (e.g., variable milk lipid content) are not accounted for by these methods. Physiologically-based pharmacokinetic (PBPK) modelling offers a mechanism-oriented strategy with bio-relevance. Recently, lactation PBPK models have been reported for some medications, showing at least the feasibility and value of PBPK modelling to predict transfer of medication into the human milk. However, reliable data as input for PBPK models is often missing. The iterative development of in vitro, animal in vivo and PBPK modelling methods seems to be a promising approach. Human in vitro models will deliver essential data on the transepithelial transport of medication, whereas the combination of animal in vitro and in vivo methods will deliver information to establish accurate in vitro/in vivo extrapolation (IVIVE) algorithms and mechanistic insights. Such a non-clinical platform will be developed and thoroughly evaluated by the Innovative Medicines Initiative ConcePTION
Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy
No full textThe monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods
Application of lectin immobilized on polyHIPE monoliths for bioprocess monitoring of Fc-fusion surface glycosylated protein
No full textIn-process monitoring of glycosylated protein concentration becomes very important with introduction of perfusion bioprocesses. Affinity chromatography based on lectines allows selective monitoring of surface glycosylated proteins. In this work we immobilized lectine on polyHIPE type of monoliths and implemented it for bioprocess monitoring. Spacer was introduced to lectine, which increased binding kinetics toward fusion protein, demonstrated by biolayer interferometry. Furthermore, complete desorption using 0.25 M galactose was demonstrated. Affinity column exhibited linearity in the range between 0.5 and 8 mg/ml and flow-unaffected binding for the flow-rates between 0.5 and 8 ml/min. Long-term stability over at least 4 months period was demonstrated. No unspecific binding of culture media components, including host cell proteins and DNA, was detected. Results obtained by affinity column matched concentration values obtained by a reference method
Use of Intramolecular 1,5-sulfur-oxygen and 1,5-sulfur-halogen interactions in the design of N-methyl-5-aryl-N-(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,4-thiadiazol-2-amine SMN2 splicing modulators
No full textSpinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from deletion or loss of function mutation of the survival motor neuron 1 (SMN1) gene. Branaplam (1) elevates levels of full-length SMN protein in vivo by modulating splicing of the related gene SMN2 to enhance exon-7 inclusion and increase levels of SMN. The intramolecular hydrogen bond present in the 2-hydroxyphenyl pyridazine core of 1 enforces a planar conformation of the biaryl system and is critical for compound activity. Scaffold morphing revealed that the pyridazine could be replaced by a 1,3,4-thiadiazole which provided additional opportunities for conformational constraint of the biaryl through intramolecular 1,5-sulfur-oxygen (S···O) or 1,5-sulfur-halogen (S···X) noncovalent interactions. Compound 26, which incorporates a 2-fluorophenyl thiadiazole motif, demonstrated a greater than fifty percent increase in production of full-length SMN protein in a mouse model of SMA
CARD10 cleavage by MALT1 restricts lung carcinoma growth in vivo
No full textCARD-CC complexes involving BCL10 and MALT1 are major cellular signaling hubs. They govern NF-κB activation through their scaffolding properties as well as MALT1 paracaspase function, which cleaves substrates involved in NF-κB regulation. In human lymphocytes, gain-of-function defects in this pathway lead to lymphoproliferative disorders. CARD10, the prototypical CARD-CC protein in non-hematopoietic cells, is overexpressed in several cancers and has been associated with poor prognosis. However, regulation of CARD10 remains poorly understood. Here, we identified CARD10 as the first MALT1 substrate in non-hematopoietic cells and showed that protein-kinase-C-induced CARD10 cleavage by MALT1 at R587 dampens its capacity to activate NF-κB. Preventing CARD10 cleavage in the lung tumor A549 cell line increased basal levels of IL-6 and extracellular matrix components in vitro, and led to increased tumor growth in a mouse xenograft model, suggesting that CARD10 cleavage by MALT1 might be a built-in mechanism controlling tumorigenicity
Mass Cytometry of CSF Identifies an MS-Associated B-cell Population
No full textOBJECTIVE: To identify an MS-specific immune cell population by deep immune phenotyping and relate it to soluble signaling molecules in CSF. METHODS: We analyzed surface expression of 22 markers in paired blood/CSF samples from 39 patients using mass cytometry (cytometry by time of flight). We also measured the concentrations of 296 signaling molecules in CSF using proximity extension assay. Results were analyzed using highly automated unsupervised algorithmic informatics. RESULTS: Mass cytometry objectively identified a B-cell population characterized by the expression of CD49d, CD69, CD27, CXCR3, and human leukocyte antigen (HLA)-DR as clearly associated with MS. Concentrations of the B cell-related factors, notably FCRL2, were increased in MS CSF, especially in early stages of the disease. The B-cell trophic factor B cell activating factor (BAFF) was decreased in MS. Proteins involved in neural plasticity were also reduced in MS. CONCLUSION: When analyzed without a priori assumptions, both the soluble and the cellular compartments of the CSF in MS were characterized by markers related to B cells, and the strongest candidate for an MS-specific cell type has a B-cell phenotype