Accurate NMR chemical shift assignments are essential for atomic-resolution characterization of proteins. Especially for intrinsically disordered proteins (IDPs) and regions (IDRs), however, the assignment remains a labor-intensive task due to spectral overlap and conformational heterogeneity. Consequently, complete side-chain assignments are rare. Here, we present a comprehensive reference dataset, comprising the complete NMR chemical shift assignments for 275 of the most prevalent dipeptides in the IDPome, covering 93% of it. In addition, we report side-chain protonation-dependent chemical shifts for dipeptides containing aspartic or glutamic acid. The dataset contains all NMR-accessible backbone and side-chain nuclei, in total 11 571 validated data points, as well as the 1D (1H, 13C) and 2D (1H-15N HSQC, 1H-13C HSQC, TOCSY, NOESY, 1H-13C HMBC) spectra used for the assignment, making it a rich resource for the training, testing, and benchmarking of tools for data-driven protein assignment, peak picking, and synthetic spectrum generation. To facilitate such machine learning applications, all data are delivered in standardized, machine-readable formats.

Phosphorylation is a central regulatory post-translational modification whose accurate representation is essential for molecular simulations of biomolecular systems. Although CHARMM includes parameters for phosphorylated residues, their nonbonded interactions were largely estimated decades ago and have remained insufficiently validated due to the scarcity of experimental data for highly charged phosphate groups and the inherent chemical instability of the phosphoester linkage in model compounds. Here, we reparametrize the amino acid side-chain analog methylphosphate, a model for phosphorylated serine, in multiple charge states using force matching to density functional theory reference data obtained from our own quantum chemical calculations in an aqueous environment. The resulting parameters are validated against new experimental measurements, including osmotic pressure measured as osmotic concentration and nuclear magnetic resonance (NMR) relaxation data for phosphorylated dipeptides. This combined computational-experimental approach enables a systematic refinement of the nonbonded parameters for all relevant phosphate charge states, yielding physically accurate hydration and ion-interaction behavior while maintaining compatibility with the CHARMM36m force field.

Urea cycle disorders (UCDs) are inherited diseases causing recurrent life-threatening metabolic decompensations due to impaired hepatic ammonia detoxification and decreased ureagenesis. Ornithine transcarbamylase (OTC) deficiency (OTCD) is X-linked and the most common and often fatal UCD. In male hemizygous patients, disease severity primarily depends on the pathogenic sequence variant, while in heterozygous females, disease severity also depends on the X-chromosomal inactivation (XCI) pattern. Females with unfavorable XCI predominantly expressing the mutant OTC protein may be severely affected. Here, we investigated a novel treatment strategy for OTCD since there is an unmet need for better therapies. In the first step, we performed a high throughput screening (HTS) using a diversity library with 10 000 chemical compounds to identify pharmacological chaperone (PC) candidates that stabilize purified wild-type OTC. Stratification of our HTS results revealed five potential PCs, which were selected for further experimentation in cellular systems using primary human hepatocytes (PHHs) and human induced pluripotent stem cell (hiPSC)-derived hepatocytes (hiPSC-Heps) from healthy controls and OTCD patients. Two PCs-PC1 and PC4-increased OTC protein stability and activity in control hiPSC-Heps, while PC4 in addition increased OTC activity in patient-derived PHHs from a female OTCD patient with unfavorable XCI. Finally, PC1 and PC4 both significantly increased ureagenesis in patient-derived PHHs. To conclude, we identified two PCs that stabilized wild-type OTC and enhanced enzyme activity and ureagenesis. Our work suggests that PCs could provide a novel treatment strategy for OTCD specifically in females with unfavorable XCI.

AbstractPrecise, fast, and reliable identification and quantification of microplastic contamination are essential for determining their environmental concentrations for risk assessments. This study investigates the use of nuclear magnetic resonance (NMR) spectroscopy to quantify microplastics by analysing dilution series of polystyrene (PS), polyisoprene-cis (PI), polybutadiene-cis (PB), polylactic acid (PLA), polyvinyl chloride (PVC) and polyurethane (PU). Each polymer type was dissolved in a suitable solvent and an internal standard was utilized for quantification. Detection and quantification limits for each polymer type were established in two ways: (1) by using an equation based on proton signals and an internal standard with known concentration and (2) by using the LOQ based on the signal-to-noise ratio. Both data sets were compared and showed that using the internal standard (method 1) results in more accurate and lower concentration limits in the range of 0.2–8 µg mL−1 for all six polymer types, while the LOQ based on the SNR (method 2) gives consistently higher concentration limits (1–10 µg mL−1). The research shows the accuracy, efficacy, and reliability of quantitative NMR spectroscopy for polymer analysis in these concentration ranges compared to established quantifying methods, such as, PyGC/MS, FTIR, or Raman spectroscopy.

This case study introduces a green, 1 h single-step method using water-rich natural deep eutectic solvent (WRNADES) for ultrasound-assisted extraction (UAE) of polyphenols fromSaccharina latissima, a commercially cultivated brown seaweed. The extraction efficiency was evaluated using a selective quantitative NMR method (s-qNMR) and the traditional nonselective colorimetric total phenolic content assay (TPC). Initial 6 h extractions in traditional solvents (methanol, ethanol, acetone, and ethyl acetate) showed a 40-60% increase in polyphenolic yields in 50% aqueous solutions measured by the TPC method. Six different water-rich (50%) NADES (WRNADES) combinations were tested (choline chloride/betaine with lactic acid, citric acid, and 1,3-butanediol), with betaine and 1,3-butanediol (1:1) proving most effective. Parameters for the WRNADES were optimized using Box-Behnken design response surface methodology, resulting in a 1:20 w/w biomass to solvent ratio and a 1 h extraction time at 50 degrees C. The WRNADES extraction process was refined into a scalable, single-step procedure and compared with traditional solvent extractions (6 h, 50% aqueous methanol and acetone). A final XAD-7 polyphenol recovery step was included in all extractions. The optimized WRNADES extraction yielded 15.97 mg GAE/g of the dry weight recovered polyphenolic extract (s-qNMR), exceeding the 6 h 50% aqueous methanol (12.4 mg GAE/g) and acetone (11.4 mg GAE/g) extractions. Thus, the UAE-WRNADES method presented in this case study provides a cost-effective, sustainable, and eco-friendly alternative for the extraction of phenolic compounds from seaweed. It promotes the development of environmentally friendly production processes within the seaweed biorefinery.

Natural products obtained from marine organisms continue to be a rich source of novel structural architecture and of importance in drug discovery, medicine, and health. However, the success of such endeavors depends on the exact structural elucidation and access to sufficient material, often by stereoselective total synthesis, of the isolated natural product of interest. (−)-Mucosin (1), a fatty acid derivative, previously presumed to contain a rare cis-bicyclo[4.3.0]non-3-ene moiety, has since been shown to be the trans-congener. Analytically, the fused bicyclic ring system in (−)-1 constitutes a particular challenge in order to establish its relative and absolute stereochemistry. Herein, data from biological evaluations, NMR and molecular modeling studies of (−)-1 are presented. An overview of the synthetic strategies enabling the exact structural elucidation of (−)-mucosin (1) is also presented.

Four different nuclear magnetic resonance (NMR) predictors have been evaluated for their ability to predict 600-MHz H-1 spectra of free fatty acids and fatty acid methyl esters of 20 common fatty acids. The predictors were evaluated on two main criteria: (1) their accuracy in direct prediction of the spectra (absolute accuracy) and (2) the ability to reveal trends or predict the change that occurs in the spectra as a result of a change in the fatty acid carbon chain, or by esterification of the free fatty acids to methyl esters (relative accuracy). The absolute accuracy in chemical shift prediction for fatty acids was good, compared with previous reports on a broader range of compounds. All four predictors had median prediction errors for chemical shifts of the signals in fatty acid methyl esters well below 0.1 ppm and as low as 0.015 ppm for one of the predictors. However, all predictors also had outliers with errors far above the upper interquartile range. In general, they also fail to reproduce trends of diagnostic value that were observed in the experimental data or properly predict the result of a minor change in molecular structure. All four predictors depend on experimental data from different origins. This may be a limiting factor for the relative accuracy of the predictors.

Metabolically induced cancer heterogeneity creates a large source of novel potential targets towards an enhanced therapeutic window alone and in combination with classic chemo- and radiotherapy [1, 2]. This is of particular interest for non-small cell lung cancer (NSCLC), which accounts for more than 80% of all lung tumor types characterized by limited responses to current treatment options [3-5]. Genetically-defined KRAS/LKB1-mutant NSCLC tumors exploit the proximal urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) as an intermediate step for pyrimidine biosynthesis, thereby contrasting its crucial hepatic role in ammonia detoxification [6-8]. Thus, CPS1 could serve as a novel target for NSCLC susceptibility (Figure 1A). In this study, we investigated the metabolic changes observed in both NSCLC and healthy hepatic systems following the identification, characterization and application of a small molecule inhibitor of ectopic CPS1 functionality. To search for CPS1 ligands, we performed a high-throughput screening approach as part of a thermal stability assay with recombinant human CPS1 and identified 5 out of more than 11,000 compounds as potential CPS1 inhibitory ligands (Figure 1B, Supplementary Figure S1 and Supplementary Table S1; see also Supplementary Information for all assays). The compound 1-(3-chlorophenyl)-3-(2-[2,2,3,3-tetrafluoropropoxy]phenyl)urea (herein referred to as AT067-H09, Figure 1C) exhibited the highest inhibitory effect on recombinant CPS1 activity towards citrulline synthesis and an IC50 below 5 × 10−3 mmol/L in the CPS1 activity assay (with full and weak strength concentrations of relevant cofactors) at which adenosine diphosphate (ADP) production was monitored at increasing concentrations of this compound (Figure 1D). Here, we aimed to predict the binding site for AT067-H09 on the crystal structure of CPS1 (apo form) reported thus far at the highest resolution (PDB file: 6UEL). SwissDock identified 39 clusters with 256 docked conformers in this apo form of CPS1 and confidently positioned AT067-H09 within the same pocket of the previously identified CPS1 inhibitor H3B-120 [9] (Supplementary Table S2). This binding pocket was located between the integrating and bicarbonate phosphorylation domains of CPS1, where hydrophobic interactions of AT067-H09 with residues Trp776, Leu778, His817 and Ile851 occurred, while its highly polar perfluorinated chain faced outwards, away from the protein backbone (Figure 1E). The propoxyphenyl and chlorophenyl groups of AT067-H09 facilitate stacking interactions with amino acids His817 and Ile851 of the apoenzyme, further stabilizing the protein-ligand conformation. The proximity of this binding pocket to the bicarbonate phosphorylation domain of CPS1 may concur with CPS1-inhibition exhibiting a low IC50 value. To validate metabolic rewiring and enhanced dependence of KRAS/LKB1-mutant NSCLC tumors on CPS1, cell viability was probed in several NSCLC cell lines, which either expressed CPS1 (H460, A549, based on a perturbed KRAS/LKB1 genetic background) or lacked CPS1-expression (H1299, H358) (Supplementary Table S3). H460 and A549 cells exhibited a strong concentration-dependent reduction in cell viability with half-maximal growth inhibition (GIC50) of 3.4 × 10−3 mmol/L and 6.7 × 10−3 mmol/L, respectively (Figure 1F), which was significantly lower than the GIC50 for H1299 wildtype and H358 KRAS mutant-only NSCLC cells. Likewise, primary fibroblasts from CPS1-deficient (CPS1B/Z, CPS1mut) or healthy individuals (K3/7, CPS1wt) exhibited considerably increased resistance to AT067-H09 treatment. Furthermore, siRNA-mediated CPS1 knockdown reproduced this effect in KRAS/LKB1-mutant NSCLC cells but not on siRNA transfection of H1299 cells, also supporting CPS1-driven tropism of KRAS/LKB1-mutated tumor cells for AT067-H09 (Figure 1G). Combination treatment with ionizing radiation (IR) was examined; however, no relevant synergism was observed for the CPS1-expressing H460 cells (Supplementary Figure S2). Of note, the ammonia concentration escalated over time in the supernatant of the CPS1-expressing H460 cells on incubation with 1 × 10−2 mmol/L AT067-H09, while AT067-H09 had little if any effect on ammonia content measured in the culture medium derived from H1299 cells (Figure 1H). In agreement with this, transfection of both cell types with siRNAs targeting CPS1 resulted in significantly increased ammonia levels only in H460 cells (Supplementary Figure S3), suggesting that CPS1 is an important element for ammonia homeostasis in H460 cells. A mechanism supporting CPS1 involvement in ammonia utilization in KRAS/LKB1-mutant NSCLCs may be the incorporation of CPS1-derived carbamoyl phosphate into the pyrimidine biosynthetic pathway [6]. In line, metabolic profiling on AT067-H09-treated H460 cells identified several pyrimidine pathway intermediates that were significantly downregulated on cellular incubation with AT067-H09, including (S)-dihydroorotate, uridine monophosphate (UMP), uridine-5'-diphosphate (UDP) and cytidine-5'-monophosphate (CMP) (Supplementary Figure S4). A previous study suggested the complete absence of the urea cycle in various NSCLC subtypes independent of the KRAS/LKB1-mutant status [6]. We characterized the expression levels of all urea cycle enzymes and transporters in our cultured NSCLCs. Ornithine transcarbamylase (OTC) and arginase 1 (ARG1) were consistently undetectable in the four lung carcinoma cell lines, with argininosuccinate lyase (ASL) being expressed in very low abundance (Figure 1I). Surprisingly, N-acetylglutamate synthase (NAGS) was strongly expressed in all cultured NSCLCs. The expression level of this enzyme has not been previously studied in lung carcinomas, but it is highly relevant for CPS1 ectopic activation since it provides the essential CPS1 allosteric cofactor NAG. The two transporters, ornithine transporter 1 (ORNT1) and citrin, were consistently expressed in all NSCLCs tested, with higher abundance in KRAS/LKB1-mutant cells (Figure 1I). Lastly, argininosuccinate synthetase 1 (ASS1) was the most abundant urea cycle protein noted in H460 and H1299 cell lines, also supported by previous arginine and citrulline depletion studies [6]. Collectively, these results corroborate previous indications that a functional urea cycle as naturally occurring in hepatic tissue is not present in NSCLC. To investigate the potential impact of CPS1 inhibition on native hepatic metabolism and towards a therapeutic window, ureagenesis was investigated in differentiated HepaRG cells that faithfully model metabolic processes found in human hepatocytes [10]. When exposed to 1 × 10−2 mmol/L AT067-H09, differentiated HepaRG cells showed impaired stable isotope incorporation reflected on the significantly reduced (15N)-Urea enrichment value (Figure 1J) and a dose-dependent increase in ammonia accumulation compared to vehicle-treated cells (Figure 1K). Importantly, HepaRG viability was unaltered for AT067-H09 concentrations up to at least 2 × 10−2 mmol/L, suggesting that the observed metabolic changes are not a toxicity-related outcome (Figure 1L). Likewise, human control iPSCs, which were differentiated into hepatic-like cells and treated with increasing concentrations of AT067-H09, well-tolerated this compound up to concentrations of at least 1 × 10−2 mmol/L (Supplementary Figure S5). No significant alterations in mitochondrial membrane potential were detectable in control experiments with HepG2 cells exposed to the same AT067-H09 concentration range (Supplementary Figure S6). These findings indicate that liver metabolism and specifically the urea cycle were sensitive to transient pharmacological cues imposed by AT067-H09 but that the viability of hepatocytes was less affected by AT067-H09 and that hepatocytes with functionally active CPS1 tolerated AT067-H09 at higher concentrations than KRAS/LKB1-mutant NSCLC tumor cells. In conclusion, compound AT067-H09 could inhibit CPS1 activity and exert robust antiproliferative effects in CPS1-expressing NSCLC cells. AT067-H09 led to ammonia accumulation in CPS1-expressing KRAS/LKB1-mutated tumor cells and untransformed hepatocyte-like normal cells without causing intolerable urea cycle flux impairment in hepatocytes. Metabolic interference via inhibition of CPS1 in tumor cells demonstrates enhanced cytotoxicity compared to untransformed cells, which might indicate differential oncogenic signaling or additionally affected downstream metabolic pathways in these targeted tumor cells. In this regard, our investigation is of great relevance for common malignant tumors caused by concurrent mutations in KRAS and LKB1, including NSCLC, where AT067-H09 can act as a novel therapeutic approach, alone or in combination with state-of-the-art treatment modalities, namely the immune checkpoint inhibitors. Future experiments focusing on preclinical testing could further explore the in vivo efficacy of AT067-H09 toward CPS1-dependent tumor sensitization. Georgios Makris, Carmen Diez-Fernandez, Martin Pruschy and Johannes Häberle designed the study. Georgios Makris, Semih Kayhan, Marvin Kreuzer, Véronique Rüfenacht, Erica Faccin, Jarl Underhaug, Carmen Diez-Fernandez, Philip A. Knobel, Martin Poms and Nadine Gougeard performed experiments and analyzed data. Georgios Makris, Vicente Rubio, Aurora Martinez, Martin Pruschy and Johannes Häberle analyzed data and revised the manuscript. Georgios Makris, Martin Pruschy and Johannes Häberle wrote the manuscript. All authors read and approved the final manuscript. We acknowledge the Functional Genomics Center Zurich (FGCZ) facility for the support with the metabolomic analysis and the Biophysics, Structural Biology and Screening (BiSS) core facility, University of Bergen, for the support with the high-throughput screening. The authors declare that they have no competing interests. This work was supported by the Swiss National Science Foundation, Switzerland (grant 320030_176088 to Johannes Häberle) and the Wolfermann-Nägeli-Stiftung, Switzerland (grant 2020/28 to Martin Pruschy). The high-throughput screening was supported by the Research Council of Norway (NOR-OPENSCREEN 245922/F50). The part of the work done by Vicente Rubio and Nadine Gougeard was supported by The Fundación Ramón Areces (grant CIVP20A6610). The work was also supported by a grant from European Union's Framework Program for Research and Innovation Horizon 2020 (2014-2020) under Marie Skłodowska-Curie (Grant Agreements No. 860245 (ITN THERADNET) to Marvin Kreuzer and Martin Pruschy. All data generated or analyzed during this study are included in this published article (and its supplementary information files). The study protocol was approved by the Swiss Ethics Committee (permit number: BASEC-No. 2021-02314). The patients' skin fibroblasts were obtained with the written informed consent of the respective individuals or their guardians. Consent for publication was obtained from Swissethics (BASEC-No. 2021-02314). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Sustainable production based on renewable biomass and efficient bioprocesses are important elements in the growing blue bioeconomy. The traditional Laminaria hyperborea alginate production disposes approximately 80 % of the raw material, ignoring large amounts of potential high-value products from the alga. Particularly, the leaf fraction of the seaweed is often disregarded. This study aimed to characterize high value products from the alginate production side-stream - focusing on the leaf biomass and particularly targeting the phenolic content. After extraction and solvent optimization, 60 % methanol was used for the extraction. The extract was further purified with PuriFlash and semi-preparative chromatography and increasing phenolic selectivity and purity was observed with TPC and qNMR, as well as antioxidant activity (ORAC). In the purified fractions, the LR LC-MS analyses displayed several masses, where 96 % (n = 1376) were of lower molecular weights (< 800 Da). Fifteen high value compounds were further identified using HR LC-MS (MS/MS) and/or NMR. This also included non-phenolics such as fucoxanthin, aliphatic acids and mannitol. Nonetheless, most compounds were identified as the targeted phenolics, consisting of lower molecular weight phenolic acids (salicylic acid, veratric acid, 5caboxyvanillic acid, sinapic acid, 5-sulfosalicylic acid, vanillic acid 4-sulfate, and dihydrocaffeic acid 3-sulfate) and phlorotannins (trimer, tetramer, hexamer, and a sulfated dimer). None of the identified phenolics have previously been reported in L. hyperborea. In general, a high occurrence of sulfated phenolic compounds was observed and a sulfated diphlorethol/difucol was characterized for the first time. The isolation and characterization of high value components in the leaf biomass of L. hyperborea strongly supports the development of a total utilization of commercial alginate production. The characterization also adds information on the phenolic content of seaweeds at a molecular level, valuable to research on seaweed biosynthesis and development, chemical ecology, and ocean monitoring.

The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site—the two tryptophans and the α1-helix form and maintain the binding pocket— were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues.

The identification of new drugs for novel therapeutic targets requires the screening of libraries containing tens of thousands of compounds. While experimental screenings are assisted by high-throughput technologies, in target-based biophysical assays, such as differential scanning fluorimetry (DSF), the analysis steps must be calculated manually, often combining several software packages. To simplify the determination of the melting temperature (Tm) of the target and the change induced by ligand binding (ΔTm), we developed the HTSDSF explorer, a versatile, all-in-one, user-friendly application suite. Implemented as a server-client application, in the primary screenings, HTSDSF explorer pre-analyzes and displays the Tm and ΔTm results interactively, thereby allowing the user to study hundreds of conditions and select the primary hits in minutes. This application also allows the determination of preliminary binding constants (KD) through a series of subsequent dose-response assays on the primary hits, thereby facilitating the ranking of validated hits and the advance of drug discovery efforts.

Enzymatic protein hydrolysates based on side stream materials from the fish-filleting industry are increasingly explored as food ingredients. However, intense sensory properties, and high salt contents, are often a limiting factor. Most of the sensory attributes, such as fish flavor and salty taste, can be ascribed to low-molecular-weight, water-soluble components, whereas bitterness is associated with small hydrophobic peptides. In this study, protein hydrolysates based on head and backbone residuals from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) were produced using two different enzymes. The effects of micro- and nanofiltration on the chemical composition, protein recovery, and sensory properties of the final products were investigated. The choice of raw material and enzyme had negligible effects, whereas nanofiltration caused a considerable reduction in metabolites, ash, and the intensity of several sensory attributes. The intensity of bitterness increased after nanofiltration, indicating that small peptides associated with bitter taste were retained by the membrane. Total protein yield after microfiltration was 24%-29%, whereas 19%-24% were recovered in the nanofiltration retentate. PRACTICAL APPLICATION: Enzymatic protein hydrolysates can be included in food products to increase the protein content, and as a nutritional supplement and/or functional ingredient; however, unpalatable and intense flavors limit applications. This study investigated the use of membrane filtration to improve flavor quality and reduce salt content in fish protein hydrolysates. Although some protein loss is unavoidable in micro- and nanofiltration, this study demonstrates the production of fish protein hydrolysates with >90% protein and peptide content, which is suitable for inclusion in foods.

FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio-layer interferometry as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure-based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening of our in-house library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl-binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure-based exploration of PaFabF.

Heat shock protein (Hsp) synthesis is upregulated in a wide range of cancers to provide the appropriate environment for tumor progression. The Hsp110 and Hsp70 families have been associated to cancer cell survival and resistance to chemotherapy. In this study, we explore the strategy of drug repurposing to find new Hsp70 and Hsp110 inhibitors that display toxicity against melanoma cancer cells. We found that the hits discovered using Apg2, a human representative of the Hsp110 family, as the initial target bind also to structural regions present in members of the Hsp70 family, and therefore inhibit the remodeling activity of the Hsp70 system. One of these compounds, the spasmolytic agent pinaverium bromide used for functional gastrointestinal disorders, inhibits the intracellular chaperone activity of the Hsp70 system and elicits its cytotoxic activity specifically in two melanoma cell lines by activating apoptosis. Docking and molecular dynamics simulations indicate that this compound interacts with regions located in the nucleotide-binding domain and the linker of the chaperones, modulating their ATPase activity. Thus, repurposing of pinaverium bromide for cancer treatment appears as a promising novel therapeutic approach.

Methods for thermochemical conversion of biomass into renewable energy and materials rapidly increase in range and outreach. A focus on the target product streams for valorization is natural, yet several pretreatment steps and conversion methods also result in an aqueous byproduct, which has been given less attention. This paper aims to fill this knowledge gap in the existing literature on identification and quantification of organic components in such aqueous phases by reporting a fast and direct workup protocol combined with application of quantitative analytical nuclear magnetic resonance (NMR) spectroscopy. Laboratory workup procedures combined with subsequent proton NMR spectroscopy with water signal suppression using presaturation pulses during relaxation delay, noesygppr1d, have been established, evaluated, and approved by testing on three different Bruker BioSpin NMR spectrometers; an 850 MHz AVANCE III HD with a 5 mm TCI CryoProbe, a 600 MHz AVANCE NEO with a QCI CryoProbe, and a 500 MHz AVANCE with a 5 mm BBO room-temperature probe additionally confirmed the quantification method to be applicable. The analytical procedure identified furfural, methanol, acetic acid, and formic acid as the dominating compounds in the analyzed aqueous samples, which were process effluents generated by the patented Arbacore pellet production process using steam explosion of wood shavings. A selected range of quantitative results in the aqueous phase from large-scale steam explosion is included in the study. The described procedure provides excellent quantitative reproducibility with experimental series standard deviations of <1% (mM), is nondestructive, and can be automated on demand.

Nuclear magnetic resonance (NMR) metabolomics profiling was evaluated as a new tool in sensory assessment of protein hydrolysates. Hydrolysates were produced on the basis of different raw materials (cod, salmon, and chicken), enzymes (Food Pro PNL and Bromelain), and hydrolysis time (10 and 50 min). The influence of raw material and hydrolysis parameters on sensory attributes was determined by traditional descriptive sensory analysis and H-1 NMR spectroscopy. The raw material had a major influence on the attribute intensity and metabolite variation, followed by enzyme and hydrolysis time. However, the formation of bitter taste was not affected by the raw material. Partial least-squares regression (PLSR) on H-1 NMR and sensory data provided good models (Q(2) = 0.55-0.89) for 11 of the 17 evaluated attributes, including bitterness. Significant metabolite-attribute associations were identified. The study confirms the potential prediction of the sensory properties of protein hydrolysates from cod, salmon, and chicken based on H-1 NMR metabolomics profiling.

Chromatin post‐translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N‐terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock–key models, missing important aspects of histone‐tail CW interactions. We here present an analysis of the ASHH2 CW‐H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. β‐augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C‐terminal coils, but also in the β1 and β2 strands and the C‐terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post‐translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome.DatabasesStructural data are available in the PDB database under the accession code 6QXZ. Resonance assignments for CW42 in its apo‐ and holo‐forms are available in the BMRB database under the accession code 27251.

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells over-expressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients' specific mutation.