Illuminating a Dark Kinase: Structure-Guided Design, Synthesis, and Evaluation of a Potent Nek1 Inhibitor and Its Effects on the Embryonic Zebrafish Pronephros.

NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in ciliogenesis, apoptosis, and the DNA-damage response. Despite its involvement in various diseases and its potential as a cancer drug target, no directed medicinal chemistry efforts toward inhibitors against this dark kinase are published. Here, we report the structure-guided design of a potent small-molecule Nek1 inhibitor, starting from a scaffold identified by kinase cross-screening analysis. Seven lead compounds were identified in silico and evaluated for their inhibitory activity. The top compound, 10f, was further profiled for efficacy, toxicity, and bioavailability in a zebrafish polycystic kidney disease model. Administration of 10f caused the expansion of fluorescence-labeled proximal convoluted tubules, supporting our hypothesis that Nek1-inhibition causes cystic kidneys in zebrafish embryos. Compound 10f displayed insignificant inhibition in 48 of 50 kinases in a selectivity test panel. The findings provide a powerful tool to further elucidate the function and pharmacology of this neglected kinase.

Investigating the RAS can be a fishy business: interdisciplinary opportunities using Zebrafish.

The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since the discovery of renin, its biological role continues to broaden with the identification and characterization of new peptides. The evolutionarily distant zebrafish is a remarkable model for studying the kidney due to its genetic tractability and accessibility for in vivo imaging. The zebrafish pronephros is an especially useful kidney model due to its structural simplicity yet complex functionality, including capacity for glomerular and tubular filtration. Both the pronephros and mesonephros contain renin-expressing perivascular cells, which respond to RAS inhibition, making the zebrafish an excellent model for studying the RAS. This review summarizes the physiological and genetic tools currently available for studying the zebrafish kidney with regards to functionality of the RAS, using novel imaging techniques such as SPIM microscopy coupled with targeted single cell ablation and synthesis of vasoactive RAS peptides.

ppargc1a controls nephron segmentation during zebrafish embryonic kidney ontogeny.

Nephron segmentation involves a concert of genetic and molecular signals that are not fully understood. Through a chemical screen, we discovered that alteration of peroxisome proliferator-activated receptor (PPAR) signaling disrupts nephron segmentation in the zebrafish embryonic kidney (Poureetezadi et al., 2016). Here, we show that the PPAR co-activator ppargc1a directs renal progenitor fate. ppargc1a mutants form a small distal late (DL) segment and an expanded proximal straight tubule (PST) segment. ppargc1a promotes DL fate by regulating the transcription factor tbx2b, and restricts expression of the transcription factor sim1a to inhibit PST fate. Interestingly, sim1a restricts ppargc1a expression to promote the PST, and PST development is fully restored in ppargc1a/sim1a-deficient embryos, suggesting Ppargc1a and Sim1a counterbalance each other in an antagonistic fashion to delineate the PST segment boundary during nephrogenesis. Taken together, our data reveal new roles for Ppargc1a during development, which have implications for understanding renal birth defects.

The tbx2a/b transcription factors direct pronephros segmentation and corpuscle of Stannius formation in zebrafish.

The simplified and genetically conserved zebrafish pronephros is an excellent model to examine the cryptic processes of cell fate decisions during the development of nephron segments as well as the origins of associated endocrine cells that comprise the corpuscles of Stannius (CS). Using whole mount in situ hybridization, we found that transcripts of the zebrafish genes t-box 2a (tbx2a) and t-box 2b (tbx2b), which belong to the T-box family of transcription factors, were expressed in the caudal intermediate mesoderm progenitors that give rise to the distal pronephros and CS. Deficiency of tbx2a, tbx2b or both tbx2a/b reduced the size of the distal late (DL) segment, which was accompanied by a proximal convoluted segment (PCT) expansion. Further, tbx2a/b deficiency led to significantly larger CS clusters. These phenotypes were also observed in embryos with the from beyond (fby)c144 mutation, which encodes a premature stop codon in the tbx2b T-box sequence. Conversely, overexpression of tbx2a and tbx2b in wild-type embryos expanded the DL segment where cells were comingled with the adjacent DE, and also decreased CS cell number, but notably did not alter PCT development-providing independent evidence that tbx2a and tbx2b are each necessary and sufficient to promote DL fate and suppress CS genesis. Epistasis studies indicated that tbx2a acts upstream of tbx2b to regulate the DL and CS fates, and likely has other targets as well. Retinoic acid (RA) addition and inhibition studies revealed that tbx2a and tbx2b are negatively regulated by RA signaling. Interestingly, the CS cell expansion that typifies tbx2a/b deficiency also occurred when blocking Notch signaling with the chemical DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). Ectopic activation of Notch in Tg(hsp70::Gal4; UAS::NICD)(NICD) embryos led to a reduced CS post heat-shock induction. To further examine the link between the tbx2a/b genes and Notch during CS formation, DAPT treatment was used to block Notch activity in tbx2a/b deficient embryos, and tbx2a/b knockdown was performed in NICD transgenic embryos. Both manipulations caused similar CS expansions, indicating that Notch functions upstream of the tbx2a/b genes to suppress CS ontogeny. Taken together, these data reveal for the first time that tbx2a/b mitigate pronephros segmentation downstream of RA, and that interplay between Notch signaling and tbx2a/b regulate CS formation, thus providing several novel insights into the genetic regulatory networks that influence these lineages.

Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney.

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems.

Effects of neurotoxin - anatoxin-a on common carp (Cyprinus carpio L.) innate immune cells in vitro.

OBJECTIVES: The aim of this study was to determine if cyanoneurotoxin - anatoxin-a (ANTX-a) alters the essential functions of innate immune cells such as free radicals generation in phagocytic cells and phagocytosis. DESIGN: In the experiments pure ANTX-a was used at concentrations of 0.01, 0.05, 0.1 and 1 µg/ml RPMI-1640 medium. Phagocytes were isolated from carp blood and pronephros. Relative changes in intracellular total free radical presence in fish phagocytes were monitored using a fluorescent probe, dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ) which detects hydrogen peroxide (H2O2), nitric oxide (NO), peroxyl radical and peroxynitrite anion. Phagocytic activity of fish leukocytes was analyzed with a Vybrant phagocytosis assay kit. RESULTS: The H2O2 level generated in response to ANTX-a at the highest used concentration was significantly suppressed in pronephros but not in blood phagocytes. Moreover, it was observed that generation of superoxide radicals and nitrite formation was significantly increased in blood and pronephros phagocytes after incubation with lower concentrations of the neurotoxin. The phagocytosis of fish leukocytes was significantly reduced at the two highest used toxin concentrations (0.1 and 1 µg/ml medium). CONCLUSION: This findings suggests that ANTX-a could change innate immunity and reduced adaptive immunity after stress induced by cyanobacterial blooms.