Four of a Kind: A Complete Collection of ADP-Ribosylated Histidine Isosteres Using Cu(I)- and Ru(II)-Catalyzed Click Chemistry

This is the final version. Available from American Chemical Society via the DOI in this record. The data underlying this study are available in the published article and its online Supporting Material. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.3c00827.Adenosine diphosphate ribosylation (ADP-ribosylation) is a crucial post-translational modification involved in important regulatory mechanisms of numerous cellular pathways including histone maintenance and DNA damage repair. To study this modification, well-defined ADP-ribosylated peptides, proteins, and close analogues thereof have been invaluable tools. Recently, proteomics studies have revealed histidine residues to be ADP-ribosylated. We describe here the synthesis of a complete set of triazole-isosteres of ADP-ribosylated histidine to serve as probes for ADP-ribosylating biomachinery. By exploiting Cu(I)- and Ru(II)-catalyzed click chemistry between a propargylglycine building block and an α- or β-configured azidoribose, we have successfully assembled the α- and β-configured 1,4- and 1,5-triazoles, mimicking N(τ)- and N(π)-ADP-ribosylated histidine, respectively. The ribosylated building blocks could be incorporated into a peptide sequence using standard solid-phase peptide synthesis and transformed on resin into the ADP-ribosylated fragments to provide a total of four ADP-ribosyl triazole conjugates, which were evaluated for their chemical and enzymatic stability. The 1,5-triazole analogues mimicking the N(π)-substituted histidines proved susceptible to base-induced epimerization and the ADP-ribosyl α-1,5-triazole linkage could be cleaved by the (ADP-ribosyl)hydrolase ARH3.Biotechnology and Biological Sciences Research Council (BBSRC)Wellcome TrustWellcome TrustOvarian Cancer Research Allianc

Solid-phase synthesis and biological evaluation of peptides ADP-Ribosylated at histidine

The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for the spatiotemporal regulation of vital processes such as apoptosis and DNA repair. Recent mass-spectrometry based analyses of the "ADP-ribosylome" have identified histidine as ADP-ribose acceptor site. In order to study this modification, a fully synthetic strategy towards α-configured N(τ)- and N(π)-ADP-ribosylated histidine-containing peptides has been developed. Ribofuranosylated histidine building blocks were obtained via Mukaiyama-type glycosylation and the building blocks were integrated into an ADP-ribosylome derived peptide sequence using fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. On-resin installation of the ADP moiety was achieved using phosphoramidite chemistry, and global deprotection provided the desired ADP-ribosylated oligopeptides. The stability under various chemical conditions and resistance against (ADP-ribosyl) hydrolase-mediated degradation has been investigated to reveal that the constructs are stable under various chemical conditions and non-degradable by any of the known ADP-ribosylhydrolases.Bio-organic Synthesi

Mimetics of ADP-ribosylated histidine through copper(I)-catalyzed click chemistry

A convergent synthesis provided nearly perfect tau-ADP-ribosylated histidine isosteres (His*-tau-ADPr) via a copper(I)-catalyzed cycloaddition between an azido-ADP-ribosyl analogue and an oligopeptide carrying a propargyl glycine. Both alpha- and beta-configured azido-ADP-ribosyl analogues have been synthesized. The former required participation of the C-2 ester functionality during glycosylation, while the latter was obtained in high stereoselectivity from an imidate donor with a nonparticipating para-methoxy benzyl ether. Four His*-tau-ADPr peptides were screened against a library of human ADP-ribosyl hydrolases.Bio-organic Synthesi

Aorto-Iliac Artery Calcification Prior to Kidney Transplantation

As vascular calcification is common in kidney transplant candidates, aorto-iliac vessel imaging is performed for surgical planning. The aim of the present study was to investigate whether a novel non-contrast enhanced computed tomography-based quantification technique for aorto-iliac calcification can be used for cardiovascular risk stratification prior to kidney transplantation. In this dual-center cohort study, we measured the aorto-iliac calcium score (CaScore) of 547 patients within three years prior to transplantation (2005-2018). During a median (interquartile range) follow-up of 3.1 (1.4, 5.2) years after transplantation, 80 (14.7%) patients died, of which 32 (40.0%) died due to cardiovascular causes, and 84 (15.5%) patients had a cardiovascular event. Kaplan-Meier survival curves showed significant differences between the CaScore tertiles for cumulative overall-survival (Log-rank testp<0.0001), cardiovascular survival (p<0.0001), and cardiovascular event-free survival (p<0.001). In multivariable Cox regression, the aorto-iliac CaScore was associated with all-cause mortality (hazard ratio 1.53, 95%CI 1.14-2.06,p= 0.005), cardiovascular mortality (2.04, 1.20-3.45,p= 0.008), and cardiovascular events (1.35, 1.01-1.80,p= 0.042). These independent associations of the aorto-iliac CaScore with the outcome measures can improve the identification of patients at risk for (cardiovascular) death and those who could potentially benefit from stringent cardiovascular monitoring to improve their prognosis after transplantation

Aorto-Iliac Artery Calcification and Graft Outcomes in Kidney Transplant Recipients

While the association of vascular calcification with inferior patient outcomes in kidney transplant recipients is well-established, the association with graft outcomes has received less attention. With this dual-centre cohort study, we aimed to determine the clinical impact of recipient pre-transplant aorto-iliac calcification, measured on non-contrast enhanced computed tomography (CT)-imaging within three years prior to transplantation (2005&ndash;2018). We included 547 patients (61.4% male, age 60 (interquartile range 51&ndash;68) years), with a median follow-up of 3.1 (1.4&ndash;5.2) years after transplantation. The aorto-iliac calcification score (CaScore) was inversely associated with one-year estimated-glomerular filtration rate (eGFR) in univariate linear regression analysis (standard &beta; &minus;3.3 (95% CI &minus;5.1 to &minus;1.5, p &lt; 0.0001), but not after adjustment for potential confounders, including donor and recipient age (p = 0.077). In multivariable Cox regression analyses, a high CaScore was associated with overall graft failure (p = 0.004) and death with a functioning graft (p = 0.002), but not with death-censored graft failure and graft function decline. This study demonstrated that pre-transplant aorto-iliac calcification is associated with one-year eGFR in univariate, but not in multivariable linear regression analyses. Moreover, this study underlines that transplantation in patients with a high CaScore does not result in earlier transplant function decline or worse death censored graft survival, although ongoing efforts for the prevention of death with a functioning graft remain essential

Stenting the ureteroneocystostomy reduces urological complications in kidney transplantation: a noninferiority randomized controlled trial, SPLINT trial

The role of ureteral stents in living-donor kidney transplantation remains uncertain. In this randomized controlled trial (SPLINT), we compared urological complications in living-donor kidney transplantations performed with or without stents. We included 200 consecutive patients that received living-donor kidney transplantations at the Erasmus MC, University Medical Center, Rotterdam. Patients (124 males, 76 females, mean age 54 ± 13) were randomized for suprapubic externalized single J stents (N = 100) or no stent (N = 100). The primary outcome was the probability of a percutaneous nephrostomy insertion (PCN) during a 12-month follow-up. To assess whether no stenting is noninferior to stenting, we allowed the probability of a PCN to increase by at most 5% (this is the noninferiority margin). Baseline characteristics were comparable between groups. In the no-stent group, there were more PCN insertions, 14% (95% CI 4.3–23.7%); urinary leakages, 12% (95% CI 5.4–21.3%); and surgical re-interventions because of urological complications, 8% (95% CI 1.5–14.5%). The stent group had more hematuria, 26% (95% CI 13.1–38.9%); and graft rejections, 15% (95% CI 2.7–27.3%). Patients in both groups had similar mean GFRs at several time points. Besides a better Euro-Qol-5D in the no-stent group at 2 and 6 weeks postoperative, similar quality of life was reported based on SF-36 and Euro-Qol-5D scores. In this trial, noninferiority has not been demonstrated for no-stent placement in relation to the number urological complications

Fasting before living-kidney donation:effect on donor well-being and postoperative recovery: study protocol of a multicenter randomized controlled trial

BACKGROUND: One of the main effectors on the quality of life of living-kidney donors is postoperative fatigue. Caloric restriction (CR) and short-term fasting (STF) are associated with improved fitness and increased resistance to acute stress. CR/STF increases the expression of cytoprotective genes, increases immunomodulation via increased anti-inflammatory cytokine production, and decreases the expression of pro-inflammatory markers. As such, nutritional preconditioning by CR or STF represents a non-invasive and cost-effective method that could mitigate the effects of acute surgery-induced stress and postoperative fatigue. To investigate whether preoperative STF contributes to a reduction in fatigue after living-kidney donation, a randomized clinical trial is indicated. METHODS: We aim to determine whether 2.5 days of fasting reduces postoperative fatigue score in subjects undergoing living-kidney donation. In this randomized study, the intervention group will follow a preoperative fasting regime for 2.5 days with a low-dose laxative, while the control group will receive standard care. The main study endpoint is postoperative fatigue, 4 weeks after living-kidney donation. Secondary endpoints include the effect of preoperative fasting on postoperative hospital admission time, the feasibility of STF, and the postoperative recovery of donor and recipient kidney function. This study will provide us with knowledge of the feasibility of STF and confirm its effect on postoperative recovery. DISCUSSION: Our study will provide clinically relevant information on the merits of caloric restriction for living-kidney donors and recipients. We expect to reduce the postoperative fatigue in living-kidney donors and improve the postoperative recovery of living-kidney recipients. It will provide evidence on the clinical merits and potential caveats of preoperative dietary interventions. TRIAL REGISTRATION: Netherlands Trial Register NL9262. EudraCT 2020-005445-16. MEC Erasmus MC MEC-2020-0778. CCMO NL74623.078.21 SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13063-021-05950-x

Label-free detection of prostaglandin transporter (SLCO2A1) function and inhibition: insights by wound healing and TRACT assays

The prostaglandin transporter (PGT, SLCO2A1) mediates transport of prostanoids (a.o. prostaglandin E2 (PGE2)) into cells and thereby promotes their degradation. Overexpression of PGT leads to low extracellular PGE2 levels and has been linked to impaired wound healing of diabetic foot ulcers. Inhibition of PGT could thus be beneficial, however, no PGT inhibitors are currently on the market and drug discovery efforts are hampered by lack of high-through screening assays for this transporter. Here we report on a label-free impedance-based assay for PGT that measures transport activity through receptor activation (TRACT) utilizing prostaglandin E2 receptor subtype EP3 and EP4 that are activated by PGE2. We found that induction of PGT expression on HEK293-JumpIn-SLCO2A1 cells that also express EP3 and EP4 leads to an over 10-fold reduction in agonistic potency of PGE2. PGE2 potency could be recovered upon inhibition of PGT-mediated PGE2 uptake with PGT inhibitors olmesartan and T26A, the potency of which could be established as well. Moreover, the TRACT assay enabled the assessment of transport function of PGT natural variants. Lastly, HUVEC cells endogenously expressing prostanoid receptors and PGT were exploited to study wound healing properties of PGE2 and T26A in real-time using a novel impedance-based scratch-induced wound healing assay. These novel impedance-based assays will advance PGT drug discovery efforts and pave the way for the development of PGT-based therapies

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer