Associations between depressive symptoms and disease progression in older patients with chronic kidney disease: results of the EQUAL study

Background Depressive symptoms are associated with adverse clinical outcomes in patients with end-stage kidney disease; however, few small studies have examined this association in patients with earlier phases of chronic kidney disease (CKD). We studied associations between baseline depressive symptoms and clinical outcomes in older patients with advanced CKD and examined whether these associations differed depending on sex. Methods CKD patients (>= 65 years; estimated glomerular filtration rate <= 20 mL/min/1.73 m(2)) were included from a European multicentre prospective cohort between 2012 and 2019. Depressive symptoms were measured by the five-item Mental Health Inventory (cut-off <= 70; 0-100 scale). Cox proportional hazard analysis was used to study associations between depressive symptoms and time to dialysis initiation, all-cause mortality and these outcomes combined. A joint model was used to study the association between depressive symptoms and kidney function over time. Analyses were adjusted for potential baseline confounders. Results Overall kidney function decline in 1326 patients was -0.12 mL/min/1.73 m(2)/month. A total of 515 patients showed depressive symptoms. No significant association was found between depressive symptoms and kidney function over time (P = 0.08). Unlike women, men with depressive symptoms had an increased mortality rate compared with those without symptoms [adjusted hazard ratio 1.41 (95% confidence interval 1.03-1.93)]. Depressive symptoms were not significantly associated with a higher hazard of dialysis initiation, or with the combined outcome (i.e. dialysis initiation and all-cause mortality). Conclusions There was no significant association between depressive symptoms at baseline and decline in kidney function over time in older patients with advanced CKD. Depressive symptoms at baseline were associated with a higher mortality rate in men

On-DNA hit validation methodologies for ligands identified from DNA-encoded chemical libraries

DNA-encoded chemical libraries (DECLs) are large compound collections attached to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of pharmaceutical interest. In DECL selections, ligands are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified using this procedure need to be validated by resynthesis and by performing affinity measurements. Here we report novel on-DNA hit validation strategies, which enable the facile confirmation of ligand-protein interaction as well as the determination of equilibrium and kinetic binding constants. The experimental procedures, which had been inspired by enzyme-linked immunosorbent assays (ELISA), were validated using ligands of different affinity to carbonic anhydrase II and IX. © 2020 Elsevier Inc.ISSN:0006-291XISSN:1090-210

Site-Specific Fluorophore Labeling of Guanosines in RNA G‑Quadruplexes

RNA G-quadruplexes are RNA secondary structures that are implicated in many cellular processes. Although conventional biophysical techniques are widely used for their in vitro characterization, more advanced methods are needed to study complex equilibria and the kinetics of their folding. We have developed a new Förster resonance energy-transfer-based method to detect the folding of RNA G-quadruplexes, which is enabled by labeling the 2′-positions of participating guanosines with fluorophores. Importantly, this does not interfere with the required anti conformation of the nucleobase in a quadruplex with parallel topology. Sequential click reactions on the solid phase and in solution using a stop-and-go strategy circumvented the issue of unselective cross-labeling. We exemplified the method on a series of sequences under different assay conditions. In contrast to the commonly used end-labeling approach, our internal labeling strategy would also allow the study of G-quadruplex formation in long functional RNAs

Synthesis of triazolo-fused benzoxazepines and benzoxazepinones via Passerini reactions followed by 1,3-dipolar cycloadditions

Azidobenzaldehydes can be used in Passerini three-component condensations to synthesize small collections of triazolo-fused heterocycles in an efficient and combinatorial fashion upon post-condensation azide-alkyne cycloadditions. Triazolo-fused benzoxazepinones were obtained in moderate to good overall yields with a concise two-step protocol. Triazolo-fused benzoxazepines were instead prepared by means of a longer, yet straightforward route comprising a Passerini reaction, hydrolysis of the ester moiety, O-alkylation with propargylic bromides, and 1,3-dipolar cycloaddition

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries

ISSN:1439-4227ISSN:1439-763

OPHA (Oxidation–Passerini–Hydrolysis–Alkylation) Strategy: a Four-Step, One-Pot Improvement of the Alkylative Passerini Reaction

Multicomponent reactions are often recognized for their efficiency and convergency, if compared with multistep organic synthesis. Nevertheless, we here demonstrate that a four-step-one-pot approach (named OPHA strategy for the initials of the four steps involved) is not only able to afford compounds that could not be obtained by an alkylative Passerini reaction but also capable of generating them with minimal loss of atoms and high operational simplicity, as in a typical multicomponent approach

Affinity Selections of DNA-Encoded Chemical Libraries on Carbonic Anhydrase IX-Expressing Tumor Cells Reveal a Dependence on Ligand Valence

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; KD=8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise. © 2021 Wiley-VCH GmbHISSN:0947-6539ISSN:1521-376

Selective Fragments for the CREBBP Bromodomain Identified from an Encoded Self‐assembly Chemical Library

DNA‐encoded chemical libraries (DECLs) are collections of chemical moieties individually coupled to distinctive DNA barcodes. Compounds can be displayed either at the end of a single DNA strand (i. e., single‐pharmacophore libraries) or at the extremities of two complementary DNA strands (i. e., dual‐pharmacophore libraries). In this work, we describe the use of a dual‐pharmacophore encoded self‐assembly chemical (ESAC) library for the affinity maturation of a known 4,5‐dihydrobenzodiazepinone ring (THBD) acetyl‐lysine (KAc) mimic for the cyclic‐AMP response element binding protein (CREB) binding protein (CREBBP or CBP) bromodomain. The new pair of fragments discovered from library selection showed a sub‐micromolar affinity for the CREBBP bromodomain in fluorescence polarization and ELISA assays, and selectivity against BRD4(1). © 2020 Wiley-VCH GmbH.ISSN:1860-7179ISSN:1860-718

Kidney Failure Prediction Models: A Comprehensive External Validation Study in Patients with Advanced CKD

Background: Various prediction models have been developed to predict the risk of kidney failure in patients with CKD. However, guideline-recommended models have yet to be compared head to head, their validation in patients with advanced CKD is lacking, and most do not account for competing risks.Methods: To externally validate 11 existing models of kidney failure, taking the competing risk of death into account, we included patients with advanced CKD from two large cohorts: the European Quality Study (EQUAL), an ongoing European prospective, multicenter cohort study of older patients with advanced CKD, and the Swedish Renal Registry (SRR), an ongoing registry of nephrology-referred patients with CKD in Sweden. The outcome of the models was kidney failure (defined as RRT-treated ESKD). We assessed model performance with discrimination and calibration.Results: The study included 1580 patients from EQUAL and 13,489 patients from SRR. The average c statistic over the 11 validated models was 0.74 in EQUAL and 0.80 in SRR, compared with 0.89 in previous validations. Most models with longer prediction horizons overestimated the risk of kidney failure considerably. The 5-year Kidney Failure Risk Equation (KFRE) overpredicted risk by 10%-18%. The four- and eight-variable 2-year KFRE and the 4-year Grams model showed excellent calibration and good discrimination in both cohorts.Conclusions: Some existing models can accurately predict kidney failure in patients with advanced CKD. KFRE performed well for a shorter time frame (2 years), despite not accounting for competing events. Models predicting over a longer time frame (5 years) overestimated risk because of the competing risk of death. The Grams model, which accounts for the latter, is suitable for longer-term predictions (4 years)