The peptide-break technology : a novel approach for protein post-translational modification assays in drug discovery

The work presented in this thesis describes the development of a universal platform for the biochemical detection of protein post-translational modifications (PTM) using the quenching resonance energy transfer (QRET) technique in an antibody-free system. The QRET technique employed is based on the difference in the luminescence signal produced by the target bound and unbound to a Ln3+-ligand in the presence of soluble quenchers. In the project, PTM monitoring was first demonstrated in a simple QRET tyrosine EGFR assay using antibodies to recognize the modified substrate peptide, a commonality in FRET assays, yet in a single-label approach. On the basis of this, a universal PTM detection method called “the peptide-break technology” was further developed as an antibody-free system. The approach relied on the peptide dimerization concept of leucine zipper (LZ) coiled-coils. The dimer was composed of an enzyme-substrate peptide and a detection Eu3+-peptide. In the assay, the PTM addition to the substrate peptide disrupted the dimer, leading to a low luminescence signal, whereas without PTM addition the dimer formation proceeds and provides Eu3+-chelate protection leading to a high luminescence. The peptide-break technology was successfully developed for a variety of enzymes for phosphorylation, dephosphorylation, deacetylation, and citrullination, using nanomolar concentrations without the need for antibodies or enzyme reporters. Later, the peptide-break technology was simplified and optimized to increase the freedom for substrate peptide selection and design. The peptide dimerization was mediated by the interaction between oppositely charged peptides. Finally, the technology was further studied in a thermal shift assay, allowing the use of high substrate peptide concentrations that are needed in assays with low affinity or activity enzymes. The reported data suggest that this technology is potentially applicable to other challenging PTM types, possibly providing new assay solutions within academia as well as for the pharmaceutical industry.Peptide-break teknologia: Uusi lääkeseulontaan soveltuva menetelmä proteiinien jälkitranslationaalisten modifikaatioiden Tämän tutkimuksen tavoitteena oli kehittää universaali biokemiallinen ja ilman vasta-aineita toteutettu määritysalusta proteiinien jälkitranslaationaalisten modifikaatioiden (PTM) havainnointiin. PTM-reaktion havainnoinnissa käytettiin sammutus-resonanssi-energiansiirtoa (QRET), joka perustuu sammuttajamolekyylin kykyyn erotella kohteeseen sitoutunut ja sitoutumaton lantanidikelaatin sisältämä reportterimolekyyli ja luoda tähän perustuva luminesenssi-signaalin muutos. Ensimmäisenä QRET teknologian soveltuvuus PTM havainnointiin osoitettiin käyttäen lyhyttä repotteripeptidiä ja leimaamatonta vasta-ainetta, joka tunnisti epidermaalisen kasvutekijäreseptorin (EGFR) luoman fosfotyrosiinin. Universaalin määritysalustan luomiseksi, PTM havainnointiin kehitettiin vasta-aineista riippumaton ”peptide-break” teknologia, joka perustuu luontaisiin kahden peptidin muodostamiin leusiinivetoketju-rakenteisiin. Perustilassaan sitoutuneet muokkaamattomat peptidit tuottavat korkean luminesenssi-signaalin, mutta aktiivisen entsyymin ollessa läsnä, muokkaamattoman reportteripeptidin ja entsyymin muokkaaman vasta-peptidin sitoutuminen estyy ja luminesenssi-signaali laskee. Menetelmän toiminnallisuus ja nanomolaarinen herkkyys havainnollistettiin lukuisien eri entsyymien ja entsyymiryhmien kanssa. Tämän jälkeen peptide-break teknologian toiminnallisuus ja monipuolisuus osoitettiin käyttämällä varauksellisia peptidejä leusiinivetoketju-peptidien sijasta. Näin helpotettiin peptidien suunnittelua, tinkimättä menetelmän suorituskyvystä. Lisäksi peptide-break teknologiasta kehitettiin lämpötilaan perustuva muunnos, jossa nostamalla entsymaattisesti muokattavan peptidin määrää kyettiin havainnoimaan huonon toiminnallisuuden omaavia entsyymejä. Tämän tutkimuksen perusteella, peptide-break teknologia soveltuu lukuisten PTM-ryhmien havainnointiin ja luo uudentyyppisen lähestymistavan, joka on hyödynnettävissä niin akateemisesti kuin teollisuudessakin

Toward universal protein post-translational modification detection in high throughput format

Post-translational modification (PTM) of proteins plays essential regulatory roles in a variety of pathological conditions. Reliable and practical assays are required to accelerate the discovery of inhibitors and activators for PTM related diseases. Today, methodologies are based on specific or group-specific PTM recognition of e.g. phosphate for kinase activity without extending to other type of PTMs. Here we have established a universal time-resolved luminescence assay on a peptide-break platform for the direct detection of wide variety of PTMs. The developed assay is based on the leucine zipper concept wherein a europium-chelate labeled detection peptide and a non-labeled peptide substrate form a highly luminescent dimer. As an active PTM enzyme at sub or low nanomolar concentration modifies the substrate peptide, the luminescent signal of the detached detection peptide is quenched in the presence of soluble quenchers. The functionality of this universal assay technique has been demonstrated for the monitoring of phosphorylation, dephosphorylation, deacetylation, and citrullination with high applicability also to other PTMs in a high throughput format.Peer reviewe

Toward universal protein post-translational modification detection in high throughput format

Post-translational modification (PTM) of proteins plays essential regulatory roles in a variety of pathological conditions. Reliable and practical assays are required to accelerate the discovery of inhibitors and activators for PTM related diseases. Today, methodologies are based on specific or group-specific PTM recognition of e.g. phosphate for kinase activity without extending to other type of PTMs. Here we have established a universal time-resolved luminescence assay on a peptide-break platform for the direct detection of wide variety of PTMs. The developed assay is based on the leucine zipper concept wherein a europium-chelate labeled detection peptide and a non-labeled peptide substrate form a highly luminescent dimer. As an active PTM enzyme at sub or low nanomolar concentration modifies the substrate peptide, the luminescent signal of the detached detection peptide is quenched in the presence of soluble quenchers. The functionality of this universal assay technique has been demonstrated for the monitoring of phosphorylation, dephosphorylation, deacetylation, and citrullination with high applicability also to other PTMs in a high throughput format

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Global expression of AMACR transcripts predicts risk for prostate cancer - a systematic comparison of AMACR protein and mRNA expression in cancerous and noncancerous prostate.

The high false negative rates for initial prostate biopsies refer a large number of the men for repeat biopsies each year. Therefore, biomarkers associated with high risk of the presence of malignancy in histologically benign biopsies could provide a tool to discriminate the patients who need repeat biopsy or intensive follow-up from those who do not. Here we examined the diagnostic applicability of alpha-methylacyl CoA racemase (AMACR) and androgen receptor (AR) mRNA expression and AMACR protein levels in benign and cancerous prostatic tissue