Studies about Cecropin P1 - an antimicrobial peptide

Antimikrobielle Peptide sind in allen Lebewesen verbreitete Peptide, welche ein breites Wirkspekt-rum gegen Bakterien, aber auch Pilze, Parasiten und Viren besitzen. Ihre Toxizität ist meist spezifisch und die Gefahr der Resistenzbildung im Vergleich zu medizinisch angewendeten Antibiotika gering. Dies macht sie für die medizinische Forschung und Anwendung sehr interessant. Um diese Peptide rational pharmakologisch zu optimieren ohne die Vorteile der AMPs gegenüber der Antibiotika ein-zubüßen, müssen der grundlegende Mechanismus der AMP-Bakterien-Interaktion entschlüsselt und die für die Toxizität nötigen Eigenschaften der Peptide identifiziert werden. Innerhalb dieser Arbeit wurde das Peptid Cecropin P1 aus dem Spulwurm des Schweins ausgewählt, welches spezifisch mit bakteriellen Membranen interagiert und Bakterien lysiert. Für die Charakteri-sierung der Struktur und Dynamik mittels NMR-Spektroskopie wurde das synthetische Cecro-pin P1-Gen in einen pET-SUMO-Vektor mit His-tag kloniert, in E. coli C43 15N-markiert exprimiert und das Peptid anschließende über eine His-tag-Affinitätschromatografie gereinigt. Die erstmals durchge-führte NMR-spektroskopische Analyse des Cecropin P1 in wässriger Umgebung ergab das Bild eines monomeren Peptides mit einer überwiegend α-helikalen amphipathischen N-terminalen Hälfte und einer im Vergleich dazu flexibleren unstrukturierten C-terminalen Hälfte mit einer hydrophoben Se-quenz. Auffällig ist dabei, dass der flexible Bereich nahe des Glycins an Position 21 beginnt, was da-rauf hindeutet, dass Cecropin P1 den Insektencecropinen mit ihrer α-Helix-„Gly-Pro-Gelenk“-α-Helix-Struktur in Membranumgebung ähnlicher ist als bisher angenommen. Weiterhin wurden Cecropin P1-Varianten entworfen um systematisch die Rolle der Hydrophobizität und Länge des hydrophoben Bereichs im C-terminalen Abschnitt von Cecropin P1 für die Toxizität in E. coli zu untersuchen. Die Toxizität der Cecropin P1 Varianten wurde dafür über die Ermittlung der minimalen inhibitorischen Konzentration (MIC) im laborintern etablierten Microplate Broth Dilution-Test abgeschätzt und zusätzlich mit einer Dreifach-Fluoreszenzfärbung näher charakterisiert. Diese Art der Charakterisierung lässt Aussagen über die totale Zellzahl, Viabilität und Membranintegrität der E. coli-Zellen nach Inkubation mit den Peptiden zu. Die größte Toxizität hatten hier Peptide mit einem 45 bis 55 % hydrophoben C-Terminus. Die Substitution der hydrophoben Aminosäuren an Position 22 bis 27 durch andere hydrophobe Aminosäuren ergab zwar eine identische MIC, veränder-te jedoch die Kinetik der Toxizität. Eine Verlängerung und Verkürzung der hydrophoben Sequenz im C-terminalen Bereich des Cecropin P1 um weitere hydrophobe Aminosäuren führte zu einer Vermin-derung der Toxizität. Dabei war eine Verkürzung um 2 Aminosäuren noch genauso toxisch wie das Wildtyp-Cecropin P1, die Verlängerung um 2 Aminosäuren verringerte die Toxizität signifikant. Die Arbeiten zeigen, dass weiterführende Studien zur Kinetik der Toxizitätswirkung der einzelnen Cecro-pin P1-Varianten und Visualisierung mit Hilfe der Fluoreszenzmikroskopie einen noch detaillierteren Einblick in den Toxizitätsmechanismus des Cecropin P1 geben könnten. Die erstmalige Expression von Isotopen-markiertem Cecropin P1 erlaubt zudem eine strukturelle und auch dynamische Analyse des Cecropin P1 mit Hilfe der NMR-Spektroskopie in verschiedenen Membransystemen und ermög-licht den Interaktionsmechanismus von Cecropin P1 mit Biomembranen detailliert zu untersuchen.Antimicrobial peptides are common in all living organisms. They have broad-spectrum activity against bacteria, fungi, parasites and viruses. Their toxicity is specific and the development of resistances less probable compared to conventional antibiotics. These characteristics make the medical use of anti-microbial peptides a matter of interest. For this reason the peptides have to be pharmacologically optimized without losing their benefits. Therefore it is reasonable to get insights into the molecular basis of peptide-membrane interaction mechanism and the determinants of the toxicity. Here Cecropin P1 from the porcine round worm Ascaris suum was chosen, which is known to interact with bacterial membranes and finally causes lysis of bacteria. To enable the structural and dynamical characterization of the peptide the synthetic gene encoding Cecropin P1 was cloned into a pET-SUMO vector with a His-tag, afterwards the 15N-labeled peptide was expressed in E. coli C43 and purified via immobilized metal ion affinity chromatography. First NMR-spectroscopy studies on Cecropin P1 in an aqueous environment indicate a monomeric peptide with an N-terminal α-helical structured part and a comparatively more flexible unstructured C-terminal half including a hydrophobic sequence. The enhanced flexibility starts around the con-served glycine at position 21, which is part of the flexible hinge in the helix-hinge-helix structure well known for the Cecropins of insects. This suggests a higher structural similarity of Cecropin P1 to the Cecropins of insects as expected. Furthermore Cecropin P1 variants were designed to analyze the relevance of hydrophobicity and length of the hydrophobic sequence of the C-terminal part of the peptide for the toxicity in E. coli. The toxicity of the variants was quantified with an established microplate broth dilution assay, which results in a minimal inhibitory concentration (MIC) of the peptide needed to completely kill the bac-teria. Additionally a triple fluorescence staining of bacteria incubated with peptide variants which gives information about total cell number, viability and membrane integrity was used to characterize the toxicity in detail. Cecropin P1 variants with 45-55 % hydrophobic C-terminus were most toxic, while the substitution of the hydrophobic amino acids 22-27 with different hydrophobic amino acids led to an identic MIC but altered kinetics of toxicity. Elongation with additional hydrophobic amino acids and shortening of the hydrophic C-terminal sequence of Cecropin P1 resulted in a decreased toxicity. These experiments indicate, that future studies on the kinetics of toxicity of the Cecropin P1 variants and visualization of bacteria threatened with peptide via fluorescence microscopy could give deeper insights into the mechanism of toxicity of Cecropin P1. For the first time 15N-labeled Cecropin P1 was produced and enables a structural and dynamical characterization of the peptide with different membrane models, which facilitates the detailed analysis of peptide-membrane interaction

Estimating affinities of calcium ions to proteins

Ca2+-ions have a range of affinities to different proteins, depending on the various functions of these proteins. This makes the determination of Ca2+-protein affinities an interesting subject for functional studies. We have investigated the performance of two methods – Fold-X and AutoDock vina – in the prediction of Ca2+-protein affinities. Both methods, although based on different energy functions, showed virtually the same correlation with experimental affinities. Guided by insight from experiment, we further derived a simple linear model based on the solvent accessible surface of Ca2+ that had practically the same performance in terms of absolute errors as the more complex docking methods

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

BackgroundSparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis.MethodsPROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850.FindingsBetween Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals.InterpretationOver 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function

Sparsentan in patients with IgA nephropathy: a prespecified interim analysis from a randomised, double-blind, active-controlled clinical trial

Background: Sparsentan is a novel, non-immunosuppressive, single-molecule, dual endothelin and angiotensin receptor antagonist being examined in an ongoing phase 3 trial in adults with IgA nephropathy. We report the prespecified interim analysis of the primary proteinuria efficacy endpoint, and safety. Methods: PROTECT is an international, randomised, double-blind, active-controlled study, being conducted in 134 clinical practice sites in 18 countries. The study examines sparsentan versus irbesartan in adults (aged ≥18 years) with biopsy-proven IgA nephropathy and proteinuria of 1·0 g/day or higher despite maximised renin-angiotensin system inhibitor treatment for at least 12 weeks. Participants were randomly assigned in a 1:1 ratio to receive sparsentan 400 mg once daily or irbesartan 300 mg once daily, stratified by estimated glomerular filtration rate at screening (30 to 1·75 g/day). The primary efficacy endpoint was change from baseline to week 36 in urine protein-creatinine ratio based on a 24-h urine sample, assessed using mixed model repeated measures. Treatment-emergent adverse events (TEAEs) were safety endpoints. All endpoints were examined in all participants who received at least one dose of randomised treatment. The study is ongoing and is registered with ClinicalTrials.gov, NCT03762850. Findings: Between Dec 20, 2018, and May 26, 2021, 404 participants were randomly assigned to sparsentan (n=202) or irbesartan (n=202) and received treatment. At week 36, the geometric least squares mean percent change from baseline in urine protein-creatinine ratio was statistically significantly greater in the sparsentan group (-49·8%) than the irbesartan group (-15·1%), resulting in a between-group relative reduction of 41% (least squares mean ratio=0·59; 95% CI 0·51-0·69; p<0·0001). TEAEs with sparsentan were similar to irbesartan. There were no cases of severe oedema, heart failure, hepatotoxicity, or oedema-related discontinuations. Bodyweight changes from baseline were not different between the sparsentan and irbesartan groups. Interpretation: Once-daily treatment with sparsentan produced meaningful reduction in proteinuria compared with irbesartan in adults with IgA nephropathy. Safety of sparsentan was similar to irbesartan. Future analyses after completion of the 2-year double-blind period will show whether these beneficial effects translate into a long-term nephroprotective potential of sparsentan. Funding: Travere Therapeutics

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

Background Sparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis. Methods PROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850. Findings Between Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals. Interpretation Over 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function.</p

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to &lt; 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of &amp; GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P &lt; 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo