Structure-guided design of peptides as tools to probe the protein-protein interaction between Cullin-2 and Elongin BC substrate adaptor in Cullin RING E3 ubiquitin ligases

Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi-subunit complexes that control the fate of many proteins in cells. CRLs constitute attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. Here we describe a structure-guided biophysical approach to probe the protein-protein interaction (PPI) between the Cullin-2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel-Lindau complex (CRL2VHL) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the “EloC site”, with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2-adaptor PPI within CRLs

Design und Synthese Peptid-basierter Inhibitoren für Protein–Protein Interaktionen

Das Adressieren von Protein–Protein Interaktionen (PPIs), die über ausgedehnte und gering-profilierte Oberflächen wechselwirken, ist ein anspruchsvolles Problem der aktuellen Medizinalforschung. Insbesondere kleine GTPasen der Ras–Superfamilie kontrollieren über PPIs zahlreiche essentielle zelluläre Funktionen und sind als molekulare Schalter attraktive Zielproteine für die Wirkstoffentwicklung. Rab–Proteine, die größte Familie kleiner GTPasen, sind Schlüsselregulatoren des zellulären Vesikeltransports, und ihre Fehlregulation wird in den letzten Jahren vermehrt mit diversen menschlichen Erkrankungen in Verbindung gebracht. Im Rahmen dieser Arbeit wurde die Synthese von Peptid-basierten Rab–Inhibitoren untersucht. Darüber hinaus wurden die entwickelten Methoden zur Adressierung anderer komplexer PPIs angewendet. Es wurden α-helikale Bindemotive auf Grundlage struktureller Daten von Rab–Protein-Komplexen ausgewählt und mittels chemischer Modifikationen stabilisiert. Mit Hilfe von α-methylierten Kohlenwasserstoffverbrückungen (hydrocarbon peptide stapling) konnten erstmals stabilisierte α-Helices zur Bindung an Rab–GTPasen identifiziert werden. Der höchstaffine Binder ist in der Lage eine Rab–Effektor-Wechselwirkung in vitro zu inhibieren und konnte durch Sequenzoptimierung sowie Variation der Verbrückung in seiner biologischen Verfügbarkeit verbessert werden. Das optimierte Peptid zeigt größere Affinität gegenüber Rab8a als das Ausgangspeptid, ist besser zellgängig und verfügt über eine herausragende Stabilität gegenüber proteolytischem Verdau. Weiterhin wurde in dieser Arbeit die Alkin-Ringschlussmetathese (RCAM) für die Synthese von makrozyklischen Peptiden an fester Phase etabliert. Die Alkinverbrückung lässt sich in unterschiedlichen Architekturen in die Kohlenwasserstoffverbrückung von Peptiden einbauen und anschließend gezielt funktionalisieren. Der Einbau eines Alkin-Makrozyklus in das 14-3-3 Bindemotiv von Exoenzym S lieferte reversible Binder mit submikromolarer Affinität. Eine Analyse der Kristallstruktur des affinsten Binders im Komplex mit 14-3-3 zeigt, dass das Alkin-makrozyklisierte Peptid das Zielprotein unter Ausbildung einer irregulären Sekundärstruktur bindet. Zudem ermöglicht die Alkin-Makrozyklisierung die Synthese bizyklischer Peptide mittels orthogonaler RCAM/RCM (Olefin-Ringschlussmetathese) mit unterschiedlichen Verbrückungsgeometrien. Auf diese Weise konnte ein monozyklischer Binder von Rab8a zu einem bizyklischen Peptid mit erhöhter Bindungsaffinität weiterentwickelt werden. Des Weiteren wurden in Rahmen dieser Arbeit die ersten Peptid-basierten Inhibitoren von UNC119 entwickelt. Das Lipid-bindende Protein UNC119 kontrolliert die zelluläre Lokalisation N-myristoylierter Proteine und transportiert sie zwischen den Membranen. Mittels α-methylierter, Kohlenwasserstoffverbrückung konnte ein nanomolarer UNC119a Binder identifiziert werden, der im Zellexperiment den vollständigen Ablauf der Zytokinese verhindert

Cyclic and macrocyclic peptides as chemical tools to recognise protein surfaces and probe protein-protein interactions

Targeting protein surfaces and protein–protein interactions (PPIs) with small molecules is a frontier goal of chemical biology and provides attractive therapeutic opportunities in drug discovery. The molecular properties of protein surfaces, including their shallow features and lack of deep binding pockets, pose significant challenges, and as a result have proved difficult to target. Peptides are ideal candidates for this mission due to their ability to closely mimic many structural features of protein interfaces. However, their inherently low intracellular stability and permeability and high in vivo clearance have thus far limited their biological applications. One way to improve these properties is to constrain the secondary structure of linear peptides by cyclisation. Herein we review various classes of cyclic and macrocyclic peptides as chemical probes of protein surfaces and modulators of PPIs. The growing interest in this area and recent advances provide evidence of the potential of developing peptide‐like molecules that specifically target these interactions

CONVINCE in the context of existing evidence on haemodiafiltration

Haemodiafiltration (HDF) provides a greater removal of larger solutes and protein-bound compounds than conventional high-flux haemodialysis (HD). There are indications that the patients receiving the highest convection volumes of HDF result in improved survival compared with HD. However, the comparative efficacy of HDF versus HD remains unproven. Here we provide a comparative account of the methodology and aims of ‘the comparison of high-dose HDF with high-flux HD’ (CONVINCE) study in the context of the totality of evidence and how this study will contribute to reaching a higher level of certainty regarding the comparative efficacy of HDF versus HD in people with end-stage kidney disease

Frequent hemodialysis versus standard hemodialysis for people with kidney failure: Systematic review and meta-analysis of randomized controlled trials

Background: Frequent hemodialysis provided more than three times per week may lower mortality and improve health-related quality of life. Yet, the evidence is inconclusive. We evaluated the benefits and harms of frequent hemodialysis in people with kidney failure compared with standard hemodialysis. Methods: We performed a systematic review of randomized controlled trials including adults on hemodialysis with highly sensitive searching in MEDLINE, Embase, CENTRAL, and Google Scholar on 3 January 2024. Data were pooled using random-effects meta-analysis. Risk of bias was assessed using the Cochrane Risk of Bias 2 tool. We adjudicated evidence certainty using GRADE. Results: From 11,142 unique citations, only seven studies involving 518 participants proved eligible. The effects of frequent hemodialysis on physical and mental health were imprecise due to few data. Frequent hemodialysis probably had uncertain effect on death from all cause compared with standard hemodialysis (relative risk 0.79, 95% confidence interval 0.33–1.91, low certainty evidence). Data were not reported for death from cardiovascular causes, major cardiovascular events, fatigue or vascular access. Conclusion: The evidentiary basis for frequent hemodialysis is incomplete due to clinical trials with few or no events reported for mortality and cardiovascular outcome measures and few participants in which patient-reported outcomes including health-related quality of life and symptoms were reported

High-Target Hemodiafiltration Convective Dose Achieved in Most Patients in a 6-Month Intermediary Analysis of the CONVINCE Randomized Controlled Trial

Introduction: High convection volumes in hemodiafiltration (HDF) result in improved survival; however, it remains unclear whether it is achievable in all patients. Methods: CONVINCE, a randomized controlled trial, randomized patients with end-stage kidney disease 1:1 to high-dose HDF versus high-flux hemodialysis (HD) continuation. We evaluated the proportion of patients achieving high-dose HDF target: convection volume per visit of ≥23 l (range ±1 l) at baseline, month 3, and month 6. We compared baseline characteristics in the following 2 ways: (i) patients on target for all 3 visits versus patients who missed target on ≥1 visits and (ii) patients on target for all 3 visits or missing it once versus patients who missed target on ≥2 visits. Results: A total of 653 patients were randomized to HDF. Their mean age was 62.2 (SD 13.5) years, 36% were female, 81% had fistula vascular access, and 33% had diabetes. Across the 3 visits, 75 patients (11%), 27 patients (4%), and 11 patients (2%) missed the convection volume target once, twice, and thrice, respectively. Apart from diabetes, there were no apparent differences in patient characteristics between patients who always achieved the high-dose target (83%) and those who missed the target either once or more (17%) or twice or more (6%). Conclusion: Achieving high-dose HDF is feasible for nearly all patients in CONVINCE and could be maintained during the 6-month follow-up period. Apart from diabetes, there were no other indications for confounding by indication on multivariable analyses that may explain the potential survival advantage for patients receiving high-dose HDF

Acute and rapid degradation of endogenous proteins by Trim-Away.

Protein depletion is a key approach to understanding the functions of a protein in a biological system. We recently developed the Trim-Away approach in order to rapidly degrade endogenous proteins without prior modification. Trim-Away is based on the ubiquitin ligase and Fc receptor TRIM21, which recognizes antibody-bound proteins and targets them for degradation by the proteasome. In a typical Trim-Away experiment, protein degradation is achieved in three steps: first, introduction of an antibody against the target protein; second, recruitment of endogenous or exogenous/overexpressed TRIM21 to the antibody-bound target protein; and third, proteasome-mediated degradation of the target protein, antibody and TRIM21 complex. Protein degradation by Trim-Away is acute and rapid, with half-lives of ~10-20 min. The major advantages of Trim-Away over other protein degradation methods are that it can be applied to any endogenous protein without prior modification; that it uses conventional antibodies that are widely available; and that it can be applied to a wide range of cell types, including nondividing primary human cells, for which other loss-of-function assays are challenging. In this protocol, we describe the detailed procedures for antibody preparation and delivery in mouse oocytes and cultured cells via microinjection and electroporation. In addition, we provide recommendations for antibody selection and validation, and for the generation of TRIM21-overexpressing cell lines for cases in which endogenous TRIM21 is limited. A typical Trim-Away experiment takes just a few hours.The research leading to these results received financial support from the Medical Research Council (MC_U105192711 and MC_U105181010), the Max Planck Society, the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 241548, European Research Council (ERC) Starting Grant no. 337415 and a Wellcome Trust Investigator Award

Physical performance tasks were linked to the PROMIS physical function metric in patients undergoing hemodialysis

OBJECTIVES: To investigate whether a multi-item performance outcome measure, the physical performance test (PPT), can be calibrated to a common scale with patient-reported outcome measures, using the Patient-Reported Outcomes Measurement Information System (PROMIS) physical function (PF) metric. STUDY DESIGN AND SETTING: We analyzed baseline data (N = 1,113) from the CONVINCE study, an international trial in end-stage kidney disease patients comparing high-dose hemodiafiltration with high-flux hemodialysis. Assumptions of item response theory (IRT) modelling were investigated for the combined set of the nine-item PPT and a four-item PROMIS PF short form (PROMIS-PF4a). We applied unidimensional IRT linking for calibrating the PPT to the PROMIS PF metric. RESULTS: Although some evidence for multidimensionality was found, classical test statistics (Cronbach's Alpha = 0.93), Mokken (Loevinger's H = 0.50), and bifactor analysis (explained common variance = 0.65) indicated that PPT and PROMIS-PF4a items can be used to assess a common PF construct. On the group level, the agreement between PROMIS-PF4a and linked PPT scores was stable across several subsamples. On the individual level, scores differed considerably. CONCLUSION: We found preliminary evidence that the PPT can be linked to the PROMIS PF metric in hemodialysis patients, enabling group comparisons across patient-reported outcome and performance outcome measures. Alternative linking methods should be applied in future studies using a more comprehensive PROMIS PF item set

Using a measurement type-independent metric to compare patterns of determinants between patient-reported versus performance-based physical function in hemodialysis patients

PURPOSE: We applied a previously established common T-score metric for patient-reported and performance-based physical function (PF), offering the unique opportunity to directly compare measurement type-specific patterns of associations with potential laboratory-based, psychosocial, sociodemographic, and health-related determinants in hemodialysis patients. METHODS: We analyzed baseline data from the CONVINCE trial (N = 1,360), a multinational randomized controlled trial comparing high-flux hemodialysis with high-dose hemodiafiltration. To explore the associations of potential determinants with performance-based versus patient-reported PF, we conducted multiple linear regression (backward elimination with cross-validation and Lasso regression). We used standardized T-scores as estimated from the PROMIS PF short-form 4a (patient-reported PF) and the Physical Performance Test (performance-based PF) as dependent variables. RESULTS: Performance-based and patient-reported PF were both significantly associated with a laboratory marker-based indicator of muscle mass (simplified creatinine index), although the effects were relatively small (partial f 2  = 0.04). Age was negatively associated with PF; the effect size was larger for performance-based (partial f 2  = 0.12) than for patient-reported PF (partial f 2  = 0.08). Compared to performance-based PF, patient-reported PF showed a stronger association with self-reported health domains, particularly pain interference and fatigue. When using the individual difference between patient-reported and performance-based T-scores as outcome, we found that younger age and more fatigue were associated with lower patient-reported PF compared to performance-based PF (small effect size). CONCLUSION: Patient-reported and performance-based assessments were similarly associated with an objective marker of physical impairment in hemodialysis patients. Age and fatigue may result in discrepancies when comparing performance-based and patient-reported scores on the common PF scale. Trial Registration CONVINCE is registered in the Dutch Trial Register (Register ID: NL64750.041.18). The registration can be accessed at: https://onderzoekmetmensen.nl/en/trial/52958