Gasdermin D-deficient mice are hypersensitive to acute kidney injury.

Signaling pathways of regulated necrosis, such as necroptosis and ferroptosis, contribute to acute kidney injury (AKI), but the role of pyroptosis is unclear. Pyroptosis is mediated by the pore-forming protein gasdermin D (GSDMD). Here, we report a specific pattern of GSDMD-protein expression in the peritubular compartment of mice that underwent bilateral ischemia and reperfusion injury (IRI). Along similar lines, the GSDMD-protein expression in whole kidney lysates increased during the first 84 h following cisplatin-induced AKI. Importantly, unlike whole kidney lysates, no GSDMD-protein expression was detectable in isolated kidney tubules. In IRI and cisplatin-induced AKI, GSDMD-deficient mice exhibited hypersensitivity to injury as assessed by tubular damage, elevated markers of serum urea, and serum creatinine. This hypersensitivity was reversed by a combined deficiency of GSDMD and the necroptosis mediator mixed lineage kinase domain-like (MLKL). In conclusion, we demonstrate a non-cell autonomous role for GSDMD in protecting the tubular compartment from necroptosis-mediated damage in IRI.

COVID-19 and Diabetic Nephropathy.

Diabetic nephropathy is the most common condition that requires a chronic renal replacement therapy, such as hemodialysis, peritoneal dialysis, kidney transplantation, or simultaneous kidney-pancreas transplantation. Chronic kidney disease progression, that is the loss of nephrons, which causes the continuous decline of the eGFR, underlies the pathogenesis of diabetic nephropathy. During the COVID-19 pandemic, it became clear that diabetic nephropathy is amongst the independent risk factors that predicts unfavourable outcome upon SARS-CoV2 infection. While we still lack conclusive mechanistic insights into how nephrons are rapidly lost upon SARS-CoV2 infection and why patients with diabetic nephropathy are more susceptible to severe outcomes upon SARS-CoV2 infection, here, we discuss several aspects of the interface of COVID-19 with diabetic nephropathy. We identify the shortage of reliable rodent models of diabetic nephropathy, limited treatment options for human diabetic nephropathy and the lack of knowledge about virus-induced signalling pathways of regulated necrosis, such as necroptosis, as key factors that explain our failure to understand this system. Finally, we focus on immunosuppressed patients and discuss vaccination efficacy in these and diabetic patients. We conclude that more basic science and mechanistic understanding will be required both in diabetic nephropathy as well as in host immune responses to the SARS-CoV2 virus if novel therapeutic strategies are desired.

Dexamethasone sensitizes to ferroptosis by glucocorticoid receptor-induced dipeptidase-1 expression and glutathione depletion.

Dexamethasone is widely used as an immunosuppressive therapy and recently as COVID-19 treatment. Here, we demonstrate that dexamethasone sensitizes to ferroptosis, a form of iron-catalyzed necrosis, previously suggested to contribute to diseases such as acute kidney injury, myocardial infarction, and stroke, all of which are triggered by glutathione (GSH) depletion. GSH levels were significantly decreased by dexamethasone. Mechanistically, we identified that dexamethasone up-regulated the GSH metabolism regulating protein dipeptidase-1 (DPEP1) in a glucocorticoid receptor (GR)-dependent manner. DPEP1 knockdown reversed the phenotype of dexamethasone-induced ferroptosis sensitization. Ferroptosis inhibitors, the DPEP1 inhibitor cilastatin, or genetic DPEP1 inactivation reversed the dexamethasone-induced increase in tubular necrosis in freshly isolated renal tubules. Our data indicate that dexamethasone sensitizes to ferroptosis by a GR-mediated increase in DPEP1 expression and GSH depletion. Together, we identified a previously unknown mechanism of glucocorticoid-mediated sensitization to ferroptosis bearing clinical and therapeutic implications.

Shuttling of Peptide-Drug Conjugates by G Protein-Coupled Receptors Is Significantly Improved by Pulsed Application.

G protein-coupled receptors (GPCRs) can be used to shuttle peptide-drug conjugates into cells. But, for efficient therapy, a high concentration of cargo needs to be delivered. To explore this, we studied the pharmacologically interesting neuropeptide Y1 receptor (Y1 R) in one recombinant and three oncogenic cell systems that endogenously express the receptor. We demonstrate that recycled receptors behave identically to newly synthesized receptors with respect to ligand binding and internalization pathways. Depending on the cell system, biosynthesis, recycling efficiency, and peptide uptake differ partially, but shuttling was efficient in all systems. However, by comparing continuous application of the ligand for four hours to four cycles of internalization and recycling in between, a significantly higher amount of peptide uptake was achieved in the pulsed application (150-250 % to 300-400 %). Accordingly, in this well-suited drug shuttle system pulsed application is superior under all investigated conditions and should be considered for innovative, targeted drug delivery in general.

Peptide-Drug Conjugates and Their Targets in Advanced Cancer Therapies.

Cancer became recently the leading cause of death in industrialized countries. Even though standard treatments achieve significant effects in growth inhibition and tumor elimination, they cause severe side effects as most of the applied drugs exhibit only minor selectivity for the malignant tissue. Hence, specific addressing of tumor cells without affecting healthy tissue is currently a major desire in cancer therapy. Cell surface receptors, which bind peptides are frequently overexpressed on cancer cells and can therefore be considered as promising targets for selective tumor therapy. In this review, the benefits of peptides as tumor homing agents are presented and an overview of the most commonly addressed peptide receptors is given. A special focus was set on the bombesin receptor family and the neuropeptide Y receptor family. In the second part, the specific requirements of peptide-drug conjugates (PDC) and intelligent linker structures as an essential component of PDC are outlined. Furthermore, different drug cargos are presented including classical and recent toxic agents as well as radionuclides for diagnostic and therapeutic approaches. In the last part, boron neutron capture therapy as advanced targeted cancer therapy is introduced and past and recent developments are reviewed.

Selective Neuropeptide Y Conjugates with Maximized Carborane Loading as Promising Boron Delivery Agents for Boron Neutron Capture Therapy.

G-protein-coupled receptors like the human Y1 receptor (hY1R) are promising targets in cancer therapy due to their high overexpression on cancer cells and their ability to internalize together with the bound ligand. This mechanism was exploited to shuttle boron atoms into cancer cells for the application of boron neutron capture therapy (BNCT), a noninvasive approach to eliminate cancer cells. A maximized number of carboranes was introduced to the hY1R-preferring ligand [F7,P34]-NPY by solid phase peptide synthesis. Branched conjugates loaded with up to 80 boron atoms per peptide molecule exhibited a maintained receptor activation profile, and the selective uptake into hY1R-expressing cells was demonstrated by internalization studies. In order to ensure appropriate solubility in aqueous solution, we proved the need for eight hydroxyl groups per carborane. Thus, we suggest the utilization of bis-deoxygalactosyl-carborane building blocks in solid phase peptide synthesis to produce selective boron delivery agents for BNCT.

A Selective Carborane-Functionalized Gastrin-Releasing Peptide Receptor Agonist as Boron Delivery Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) allows the selective elimination of malignant tumor cells without affecting healthy tissue. Although this binary radiotherapy approach has been known for decades, BNCT failed to reach the daily clinics to date. One of the reasons is the lack of selective boron delivery agents. Using boron loaded peptide conjugates, which address G protein-coupled receptors overexpressed on tumor cells allow the intracellular accumulation of boron. The gastrin-releasing peptide receptor (GRPR) is a well-known target in cancer diagnosis and can potentially be used for BNCT. Here, we present the successful introduction of multiple bis-deoxygalactosyl-carborane building blocks to the GRPR-selective ligand [d-Phe6, ß-Ala11, Ala13, Nle14]Bn(6-14) (sBB2L) generating peptide conjugates with up to 80 boron atoms per molecule. Receptor activation was retained, metabolic stability was increased, and uptake into PC3 cells was proven without showing any intrinsic cytotoxicity. Furthermore, undesired uptake into liver cells was suppressed by using l-deoxygalactosyl modified carborane building blocks. Due to its high boron loading and excellent GRPR selectivity, this conjugate can be considered as a promising boron delivery agent for BNCT.

Tuning a modular system - synthesis and characterisation of a boron-rich s-triazine-based carboxylic acid and amine bearing a galactopyranosyl moiety.

Introduction of a bis(isopropylidene)-protected galactopyranosyl moiety in s-triazine-based boron-rich carboxylic acids and amines results in soluble and suitable coupling partners for tumour-selective biomolecules with applications as selective agents for boron neutron capture therapy (BNCT). Bearing either a carboxylic acid or primary amine as a functional group, these compounds are highly versatile and thus largely extend the possible coupling strategies with suitable biomolecules. Modification of the gastrin-releasing peptide receptor (GRPR) selective agonist [d-Phe6, ß-Ala11, Ala13, Nle14]Bn(6-14) with the carboxylic acid derivative yielded a bioconjugate with an optimal receptor activation and internalisation profile. This demonstrates the great potential of this approach for the development of novel boron delivery agents.

Identification and stabilization of a highly selective gastrin-releasing peptide receptor agonist.

The gastrin-releasing peptide receptor (GRPR) is part of the bombesin receptor family and a well-known target in cancer diagnosis and therapy. In the last decade, promising results have been achieved by using peptide-drug conjugates, which allow selective targeting of GRPR expressing tumor cells. Most ligands, however, have been antagonists even though agonists can lead to higher tumor uptake owing to their internalization. So far, only a few studies focused on the identification of small GRPR-selective agonists that are metabolically stable. Here, we developed novel bombesin analogs with high selectivity for the GRPR and improved blood plasma stability. The most promising analog [d-Phe6 , ß-Ala11 , NMe-Ala13 , Nle14 ]Bn(6-14) displays an activity of 0.3nM at the GRPR, a more than 4000-fold selectivity over the other two bombesin receptors and more than 75% stability in human blood plasma after 24 hours. This analog is proposed as a promising drug shuttle for the intracellular delivery of different payloads in targeted tumor therapy approaches.

Modular triazine-based carborane-containing carboxylic acids - synthesis and characterisation of potential boron neutron capture therapy agents made of readily accessible building blocks.

Based on a modular combination of s-triazine, the well-known 9-mercapto-1,7-dicarba-closo-dodecaborane(12) and commercially available carboxylic acids, namely thioglycolic acid, glycine, and Nα-Boc-l-lysine, several carboxylic acid derivatives were synthesised and fully characterised. The thioglycolic acid derivative was introduced into a peptide hormone by solid phase peptide synthesis. High activity and selective internalisation into peptide receptor-expressing cells was observed. With a very high boron content of twenty boron atoms, these derivatives are interesting as selective Boron Neutron Capture Therapy (BNCT) agents.

Unusual features of the high light acclimation of Chromera velia.

In the present study, the high light (HL) acclimation of Chromera velia (Chromerida) was studied. HL-grown cells exhibited an increased cell volume and dry weight compared to cells grown at medium light (ML). The chlorophyll (Chl) a-specific absorption spectra ([Formula: see text]) of the HL cells showed an increased absorption efficiency over a wavelength range from 400 to 750 nm, possibly due to differences in the packaging of Chl a molecules. In HL cells, the size of the violaxanthin (V) cycle pigment pool was strongly increased. Despite a higher concentration of de-epoxidized V cycle pigments, non-photochemical quenching (NPQ) of the HL cells was slightly reduced compared to ML cells. The analysis of NPQ recovery during low light (LL) after a short illumination with excess light showed a fast NPQ relaxation and zeaxanthin epoxidation. Purification of the pigment-protein complexes demonstrated that the HL-synthesized V was associated with the chromera light-harvesting complex (CLH). However, the difference absorption spectrum of HL minus ML CLH, together with the 77 K fluorescence excitation spectra, suggested that the additional V was not protein bound but localized in a lipid phase associated with the CLH. The polypeptide analysis of the pigment-protein complexes showed that one out of three known LHCr proteins was associated in higher concentration with photosystem I in the HL cells, whereas in ML cells, it was enriched in the CLH fraction. In conclusion, the acclimation of C. velia to HL illumination shows features that are comparable to those of diatoms, while other characteristics more closely resemble those of higher plants and green algae.