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Amino groups in proteins can react with aldehyde groups in aldoses or keto groups in ketoses, e.g., d-glucose and d-fructose, yielding Schiff bases that rearrange to more stable Amadori and Heyns products, respectively. Analytical strategies to identify and quantify each glycation product in the presence of the corresponding isomer are challenged by similar physicochemical properties, impeding chromatographic separations, and by identical masses including very similar fragmentation patterns in tandem mass spectrometry. Thus, we studied the separation of seven peptide families, each consisting of unmodified, glucated, and fructated 15mer to 22mer peptides using reversed-phase (RP) and hydrophilic interaction chromatography (HILIC). In RP-HPLC using acidic acetonitrile gradients, unglycated peptides eluted ~ 0.1 to 0.8 min after the corresponding glycated peptides with four of seven peptides being baseline separated. Isomeric glucated and fructated peptides typically coeluted, although two late-eluting peptides were partially separated. Neutral eluents (pH 7.2) improved the chromatographic resolution (Rs), especially in the presence of phosphate, providing good and often even baseline separations for six of the seven isomeric glycated peptide pairs with fructated peptides eluting earlier (Rs = 0.7 to 1.5). Some glucated and unmodified peptides coeluted, but they can be distinguished by mass spectrometry. HILIC separated glycated and unmodified peptides well, whereas glucated and fructated peptides typically coeluted. In conclusion, HILIC efficiently separated unmodified and the corresponding glycated peptides, while isomeric Amadori and Heyns peptides were best separated by RP-HPLC using phosphate buffered eluents.

The inherent poor sampling of fragment ions in time-of-flight mass analyzers was recently improved for data-dependent acquisition (DDA) by considering their drift times in traveling wave ion mobility spectrometry (TWIMS). Here, we extend this TWIMS-DDA approach to the data-independent acquisition (DIA) mode MS

Nanomedicines including lipid- and polymer-based nanoparticles and polymer-drug conjugates enable targeted drug delivery for the treatment of numerous diseases. Quantitative analysis of components in nanomedicines is routinely performed to characterize the products to ensure quality and property consistency but has been mainly focused on the active pharmaceutical ingredients (APIs) in academic publications. It has been increasingly recognized that excipients in nanomedicines are critical in determining the product quality, stability, consistency, and safety. APIs are often analyzed by high-performance liquid chromatography (HPLC), and it would be convenient if the same method can be applied to excipients to robustly quantify all components in nanomedicines. Here, we report the development of a HPLC method that combined an evaporative light scattering (ELS) detector with an UV-vis detector to simultaneously analyze drugs and excipients in nanomedicines. This method was tested on diverse nanodrug delivery systems, including a niosomal nanoparticle encapsulating a phytotherapeutic, a liposome encapsulating an immune boosting agent, and a PEGylated peptide. This method can be utilized for a variety of applications, such as monitoring drug loading, studying drug release, and storage stability. The information obtained from the analyses is of importance for nanomedicine formulation development.

Supplementary Figure 8 (Relates to Figure 6) - SIK2 regulates a cell cycle network and a ER stress network related to cancer.

Supplementary Figure 2 (Relates to Figure 2) - SIK2 knock-down reduces proliferation and viability of LNCaP-Bic, C4-2 and DuCaP cells.

Supplementary Figure 1 (Relates to Figure 2) - Kinase-dead SIK2 and SIK2 knock-down reduce proliferation of LNCaP cells.

Supplementary Figure 7 (Relates to Figure 6) - Validation of microarray hits.

Supplementary Figure Legend

Supplementary Table S1. qRT-PCR primer sequences. When primer sequences were not from published studies, they were designed using Integrated DNA Technologies software.

Supplementary Figure 4 (Relates to Figure 4) - SIK2 localises at the centrosomes of SKOv3 and LNCaP cells.

Supplementary Figure 6 (Relates to Figure 5) - SIK2 regulates CREB1 activity via the phosphorylation of TORC2 and TORC3 and their sequestration in the cytoplasm.

Supplementary Figure 5. Validation of SIK2, TORC1, TORC2 and TORC3 antibodies by Western-Blot.

Supplementary Figure 3 (Relates to Figure 4) - SIK2 knock-down induces a cell cycle arrest in G1 phase and delays mitotic progression of LNCaP cells.

The GAIN domain is a hallmark of adhesion G-protein coupled receptors (aGPCRs) as this extracellular domain contains an integral agonistic sequence (Stachel) for activation via binding to the 7-transmembrane helical (7TM) domain of the receptor. Many aGPCRs are autoproteolytically cleaved at the GPCR proteolysis site (GPS) site within the GAIN domain formed HXS/T sequence motif. However, other aGPCR can be activated without GPS cleavage. We determined the crystal structure of the human ADGRB2/BAI2 hormone receptor (HormR) and GPCR autoproteolysis-inducing (GAIN) domains and found that this aGPCR is resistant to autoproteolysis despite the presence of a canonical HLS sequence motif at the GPS. We used structural comparisons and molecular dynamics (MD) simulations to identify structural determinants that are important for autocleavage beyond the canonical HXS/T motif. These studies characterized a conserved glycine residue and an edge-π interaction of the histidine base of the GPS sequence with a phenylalanine residue that is highly conserved in cleavage-competent aGPCRs. The MD simulations showed that this interaction is important to position the imidazole group of the histidine for deprotonation of the serine or threonine nucleophile. Removal of this interaction reduced autoprote-olytic activity in the ADGRL1 receptor and restored cleavage competence of the ADGRB3 receptor in a R866H/L821F double mutant. Conservation analysis indicates that wild-type ADGRB2 and ADGRB3 are auto-cleavage-incompetent receptors.

Androgen signalling remains the seminal therapeutic approach for management of advanced prostate cancer. However, most tumours eventually shift towards an aggressive phenotype, characterised by androgen-independence and treatment resistance. The cyclic adenosine monophosphate (cAMP) pathway plays a crucial role in regulating various cellular processes, with the phosphodiesterase PDE4D7 being a vital modulator of cAMP signalling in prostate cancer cells. Our study provides evidence that loss of PDE4D7 expression represents a pivotal switch driving the transition from an androgen-sensitive state to hormone unresponsiveness and neuroendocrine differentiation. Additionally, we demonstrate that PDE4D7 loss results affects DNA repair pathways, conferring resistance to poly ADP ribose polymerase (PARP) inhibitors. Reinstating PDE4D7 expression sensitises prostate cancer cells to anti-androgens, DNA damage response inhibitors, and cytotoxic therapies. These findings provide significant insight into the regulatory role of PDE4D7 in the development of lethal prostate cancer and the potential of its modulation as a novel therapeutic strategy.

Objectives: To investigate the association of the prognostic risk score CAPRA&PDE4D5/7/9 as measured on pre-surgical diagnostic needle biopsy tissue with pathological outcomes after radical prostatectomies in a clinically low–intermediate-risk patient cohort. Patients and Methods: RNA was extracted from biopsy punches of diagnostic needle biopsies. The patient cohort comprises n = 151 patients; of those n = 84 had low–intermediate clinical risk based on the CAPRA score and DRE clinical stage 2, or pathological pT stage > pT3a, or tumor penetrated prostate capsular status, or pN1 disease); (ii) any ISUP pathological Gleason >2; (iii) any ISUP pathological Gleason >1. In the n = 84 patients with low to intermediate clinical risk profiles, the clinical-genomics CAPRA&PDE4D5/7/9_BCR risk score was significantly lower in patients with favorable vs. unfavorable outcomes. In univariable logistic regression modeling the genomics PDE4D5/7/9_BCR as well as the clinical-genomics CAPRA&PDE4D5/7/9_BCR combination model were significantly associated with all three post-surgical pathology outcomes (p = 0.02, p = 0.0004, p = 0.04; and p = 0.01, p = 0.0002, p = 0.01, respectively). The clinically used PRIAS criteria for the selection of low-risk candidate patients for active surveillance (AS) were not significantly associated with any of the three tested post-operative pathology outcomes (p = 0.3, p = 0.1, p = 0.1, respectively). In multivariable analysis adjusted for the CAPRA score, the genomics PDE4D5/7/9_BCR risk score remained significant for the outcomes of adverse pathology (p = 0.04) and ISUP pathological Gleason >2 (p = 0.004). The negative predictive value of the CAPRA&PDE4D5/7/9_BCR risk score using the low-risk cut-off (0.1) for the three pathological endpoints was 82.0%, 100%, and 59.1%, respectively for a selected low-risk cohort of n = 22 patients (26.2% of the entire cohort) compared to 72.1%, 94.4%, and 55.6% for n = 18 low-risk patients (21.4% of the total cohort) selected based on the PRIAS inclusion criteria. Conclusion: In this study, we have shown that the previously reported clinical-genomics prostate cancer risk model CAPRA&PDE4D5/7/9_BCR which was developed to predict biological outcomes after surgery of primary prostate cancer is also significantly associated with post-surgical pathology outcomes. The risk score predicts adverse pathology independent of the clinical risk metrics. Compared to clinically used active surveillance inclusion criteria, the clinical-genomics CAPRA&PDE4D5/7/9_BCR risk model selects 22% (n = 8) more low-risk patients with higher negative predictive value to experience unfavorable post-operative pathology outcomes.

A 19-amino acid longproline-richantimicrobialpeptide (PrAMP) Drosocin (Dro) is encoded in the fruit fly genome. Native Dro is glycosylated at a specific threonine residue, but the non-glycosylated peptide retains antibacterial activity. Dro shows sequence similarity to several other PrAMPs that bind in the ribosomal nascent peptide exit tunnel and inhibit protein synthesis by varying mechanisms. However, the target and mechanism of action of Dro remain unknown. Here we show that the primary mode of Dro action is inhibition of termination of protein synthesis. Our in vitro and in vivo experiments demonstrate that Dro stalls ribosomes at stop codons, likely sequestering class 1 release factors associated with the terminating ribosome. As the result, Dro strongly promotes readthrough of stop codons at subinhibitory concentrations. The elucidated mode of Dro action allows assigning it as the second member of the type II PrAMPs, of which only one representative, the antimicrobial peptide apidaecin (Api) produced by honeybees, was previously known. However, despite its functional similarity with Api, Dro interacts with the target in a markedly distinct way. The analysis of a comprehensive single-amino acid substitution library of endogenously expressed Dro variants shows that binding to the ribosome involves interactions of multiple amino acid residues distributed through the entire length of the PrAMP. Our data further show that the ribosome-targeting activity of non-glycosylated Dro can be significantly enhanced by single amino acid substitutions illuminating directions for improving its antibacterial properties.

The liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03118-9.

Aldoses and ketoses can glycate proteins yielding isomeric Amadori and Heyns products, respectively. Evidently, d-fructose is more involved in glycoxidation than d-glucose favoring the formation of advanced glycation endproducts (AGEs). While Amadori products and glucation have been studied extensively, the in vivo effects of fructation are largely unknown. The characterization of isomeric Amadori and Heyns peptides requires sufficient quantities of pure peptides. Thus, the glycated building block Nα-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl),Nε-Boc]-OH (Fmoc-Lys(Glc,Boc)-OH), which was synthesized in two steps starting from unprotected d-fructose and Fmoc-l-lysine hydrochloride, was site-specifically incorporated during solid-phase peptide synthesis. The building block allowed the synthesis of a peptide identified in tryptic digests of human serum albumin containing the reported glycation site at Lys233. The structure of the glycated amino acid derivatives and the peptide was confirmed by mass spectrometry and NMR spectroscopy. Importantly, the unprotected sugar moiety showed neither notable epimerization nor undesired side reactions during peptide elongation, allowing the incorporation of epimerically pure glucosyllysine. Upon acidic treatment, the building block as well as the resin-bound peptide formed one major byproduct due to incomplete Boc-deprotection, which was well separated by reversed-phase chromatography. Expectedly, the tandem mass spectra of the fructated amino acid and peptide were dominated by signals indicating neutral losses of 18, 36, 54, 84 and 96 m/z-units generating pyrylium and furylium ions.

Proline‐rich antimicrobial peptides expressed in insects are primarily active against Enterobacteriaceae. Mechanistically, they target the bacterial (70S) ribosome after partially transporter‐based cellular uptake, as revealed for Api137 and Onc112 on Escherichia coli. Following molecular modeling indicating that the Onc112 contact site is conserved among the ribosomes of high‐priority pathogens, the ribosome binding of Api137 and Onc112 was studied. The dissociation constants (K d) of Onc112 were ∼75 nmol/L for Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, 36 nmol/L for Pseudomonas aeruginosa, and 102 nmol/L for Staphylococcus aureus, thus indicating a very promising lead structure for developing broad‐spectrum antibiotics. Api137 bound weaker with K d values ranging from 155 nmol/L to 13 μmol/L. For most bacteria, the antibacterial activities were lower than predicted from the K d values, which was only partially explained by their ability to enter bacterial cells. Other factors limiting the activity expected from the ribosome binding might be off‐target binding., Resisting resistance: Proline‐rich antimicrobial peptides Api137 and especially Onc112 bind strongly to the bacterial (70S) ribosome, even to ribosomes of pathogens that appear to be resistant in standards antibiotic tests. The presented data indicate that Onc112 in particular might represent a valid lead structure to develop broad‐spectrum antibiotics relying on a novel bactericidal mechanism.

PDE4D7 expression is diminished during progression of PCa and the phosphodiesterase has been proposed as a prognostic biomarker. RNA sequencing of PCa tissue identified sequences in the PDE4D coding region on Chr5q12 exhibiting similarities in mRNA expression profile to PDE4D7. As previously identified, PART1 had a significantly similar expression pattern to PDE4D7 across samples. However, other genes also matched expression to PDE4D7, including miRNAs and lncRNAs. These novel PDE4D7 associated genes represent putative PCa biomarkers and could have mechanistic roles in PCa progression.

d-glucose and d-fructose present in blood, tissues, and organs of all mammals can react with amino groups, leading to glucated (Amadori) and fructated (Heyns) products, i.e., proteins glycated at lysine residues. While typically present at low concentration in humans, metabolic diseases including diabetes elevate sugar levels, favoring glycation and consecutive reactions leading to advanced glycation end products (AGEs) linked to diabetic complications and cardiovascular diseases. Analytical methods able to differentiate and to individually quantify Amadori- and Heyns-modified proteins in complex sample mixtures, e.g., serum, are still very limited. Here, we show that the reported and supposedly specific neutral losses displayed in tandem mass spectra of Heyns peptides cannot be used for a reliable differentiation as they were also observed for Amadori peptides. However, the combination of several neutral loss signals in fragment ion ratios at both precursor and fragment ion signals allowed the differentiation and relative quantitation of coeluting isomeric Amadori and Heyns peptides at different concentrations and peptide ratios. This was also true for digested human plasma. Thus, the presented strategy allows the quantitation of Amadori and Heyns peptides in complex samples, especially by spiking isotope-labeled peptides. This will allow searching for glucated and fructated biomarkers in clinical samples.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.

ObjectivesTo investigate the impact on efficiency and quality of pre-prostatectomy multidisciplinary therapy conferences (MDT) at Karolinska University Hospital related to the use of a digital solution compared to standard of care. Further, to explore whether gains in MDT efficiency and quality impact oncological or functional patient outcomes.MethodsWe conducted a prospective, observational study of preoperative prostate-cancer MDT at Karolinska between February 2017 and March 2021. We compared efficiency and quality of the standard MDT and the MDT using the digital solution IntelliSpace Precision Medicine Multidisciplinary Team Orchestrator (ISPM) based on the previously used MDT-MODe approach. Clinical and patient-reported functional outcomes were derived from the medical records and the Swedish National Prostate Cancer Register (NPCR).ResultsWhile ISPM was used during the multidisciplinary therapy conference, the time spent per patient was reduced by 24 % (pDiscussion & ConclusionTo increase the value of the MDT, all data relevant for treatment decision need to be purposefully presented and compiled, which also enables secondary use of the data.The use of a digital solution during preoperative MDTs for prostate-cancer decision-making at Karolinska University Hospital improved the efficiency and quality of this multidisciplinary team meeting without impacting patient outcomes.What is already known?Multidisciplinary therapy conferences are widely used in modern cancer care.Patients discussed in a multidisciplinary therapy conference are more likely to receive appropriate staging and treatment.The multidisciplinary therapy conference is time consuming and rarely digitalized or adequately structured.What does this paper add?The use of a digital clinical decision support system during preoperative prostate-cancer multidisciplinary therapy conferences improved the efficiency and quality of the meetings.The introduction of this digital clinical decision support system was not associated with changes in oncological and functional outcomes after surgery.

In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.

Background Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non–poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. Results We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA–minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG–positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. Conclusion By using the full potential of non–poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.

Bovine milk plays an important role in human nutrition and is one of the main products of dairy industry. Its composition changes in response to various factors including forage, which are rapidly reflected by the milk lipidome. Most cows receive a silage-based diet despite a recent trend towards more traditional husbandry relying on hay-feeding. Here, changes in the lipidome upon different animal diets were addressed by studying milk of cows from two different feeding regimes and associated seasonal variations over one year. Extracted lipids were analyzed by reversed-phase liquid chromatography coupled on-line to high resolution mass spectrometry. Overall, 1302 lipid molecular species were identified including 1038 triacylglycerides (∼80%), whereas the remaining 20% were represented by a variety of species from twelve lipid classes. A semi-absolute quantitation of 264 lipid species showed diet- and season-induced variations in the milk lipidome with many odd chain triacylglycerides upregulated in hay milk.

The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (Kd = 34 ± 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z’-value of 0.85 ± 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by high-priority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram-negative and Gram-positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram-negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off-target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.

The antimicrobial peptide (AMP) ARV-1502 was designed based on naturally occurring short proline-rich AMPs, including pyrrhocoricin and drosocin. Identification of chaperone DnaK as a therapeutic target in Escherichia coli triggered intense research on the ligand-DnaK-interactions using fluorescence polarization and X-ray crystallography to reveal the binding motif and characterize the influence of the chaperone on protein refolding activity, especially in stress situations. In continuation of this research, 182 analogs of ARV-1502 were designed by substituting residues involved in antimicrobial activity against Gram-negative pathogens. The peptides synthesized on solid-phase were examined for their binding to E. coli and S. aureus DnaK providing 15 analogs with improved binding characteristics for at least one DnaK. These 15 analogs were distinguished from the original sequence by their increased hydrophobicity parameters. Additionally, the influence of the entire DnaK chaperone system, including co-chaperones DnaJ and GrpE on refolding and ATPase activity, was investigated. The increasingly hydrophobic peptides showed a stronger inhibitory effect on the refolding activity of E. coli chaperones, reducing protein refolding by up to 64%. However, these more hydrophobic peptides had only a minor effect on the ATPase activity. The most dramatic changes on the ATPase activity involved peptides with aspartate substitutions. Interestingly, these peptides resulted in a 59% reduction of the ATPase activity in the E. coli chaperone system whereas they stimulated the ATPase activity in the S. aureus system up to 220%. Of particular note is the improvement of the antimicrobial activity against S. aureus from originally >128 µg/mL to as low as 16 µg/mL. Only a single analog exhibited improved activity over the original value of 8 µg/mL against E. coli. Overall, the various moderate-throughput screenings established here allowed identifying (un)favored substitutions on 1) DnaK binding, 2) the ATPase activity of DnaK, 3) the refolding activity of DnaK alone or together with co-chaperones, and 4) the antimicrobial activity against both E. coli and S. aureus.

α-Aminoxy peptides represent an interesting group of peptidomimetics with high proteolytic stability and the ability to fold into specific, predictable secondary structures. Here, we present a series of hybrid peptides consisting of α-aminoxy acids and α-amino acids with cationic and aromatic, hydrophobic side chains in an alternating manner synthesized using an efficient protocol that combines solution- and solid-phase synthesis. 2D ROESY experiments with a representative hexamer suggested the presence of a 7/8 helical conformation in solution. Biological evaluation revealed a significant impact of the peptide chain length and the N-terminal cap on the antimicrobial and anticancer properties of this series of hybrid peptides. The Fmoc-capped peptide 6e displayed the most potent antimicrobial activity against a panel of Gram-negative and Gram-positive bacterial strains (e. g. against E. Coli: MIC=8 mg/L; S. aureus: MIC=4 mg/L).

Aldoses and ketoses can glycate proteins yielding isomeric Amadori and Heyns products, respectively. Evidently, d-fructose is more involved in glycoxidation than d-glucose favoring the formation of advanced glycation endproducts (AGEs). While Amadori products and glucation have been studied extensively, the in vivo effects of fructation are largely unknown. The characterization of isomeric Amadori and Heyns peptides requires sufficient quantities of pure peptides. Thus, the glycated building block Nα-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl),Nε-Boc]-OH (Fmoc-Lys(Glc,Boc)-OH), which was synthesized in two steps starting from unprotected d-fructose and Fmoc-l-lysine hydrochloride, was site-specifically incorporated during solid-phase peptide synthesis. The building block allowed the synthesis of a peptide identified in tryptic digests of human serum albumin containing the reported glycation site at Lys233. The structure of the glycated amino acid derivatives and the peptide was confirmed by mass spectrometry and NMR spectroscopy. Importantly, the unprotected sugar moiety showed neither notable epimerization nor undesired side reactions during peptide elongation, allowing the incorporation of epimerically pure glucosyllysine. Upon acidic treatment, the building block as well as the resin-bound peptide formed one major byproduct due to incomplete Boc-deprotection, which was well separated by reversed-phase chromatography. Expectedly, the tandem mass spectra of the fructated amino acid and peptide were dominated by signals indicating neutral losses of 18, 36, 54, 84 and 96 m/z-units generating pyrylium and furylium ions. Supplementary Information The online version contains supplementary material available at 10.1007/s00726-021-02989-7.