Search Results (115)

Infectious spleen and kidney necrosis virus (ISKNV) causes systemic infections and high mortality in various freshwater and marine fish species, posing a serious threat to aquaculture and ornamental fish industries. In this study, 72 antiviral compounds were screened against five ISKNV target proteins, DNA polymerase, transcription elongation factor (TFIIS), adenosine triphosphatase (ATPase), ankyrin repeat-containing protein, and major capsid protein (MCP) using molecular docking, followed by in vitro evaluation in dwarf gourami fin (DGF) cells. Fludarabine, liquiritin, and lycorine consistently ranked among the top 20 compounds across all five targets and were selected for further evaluation. Fludarabine and lycorine strongly inhibited MCP, ATPase, and DNA polymerase expression with inhibition rates exceeding 85%. Infectious viral titers were reduced by approximately 39- and 81-fold in the fludarabine- and lycorine-treated groups, respectively, compared with those in the ISKNV-infected control group. Time-course analysis revealed that both compounds suppressed extracellular viral release and delayed the progression of cytopathic effects. These results suggest that multi-target docking combined with in vitro evaluation may be a useful strategy for prioritizing antiviral candidates against ISKNV. Full article

Tankyrases (TNKS1 and TNKS2) are multifunctional enzymes of the poly(ADP-ribose) polymerase (PARP) family that regulate cellular homeostasis by catalyzing poly(ADP-ribosyl)ation and stabilizing protein–protein interactions through their ankyrin repeat clusters. By engaging with diverse sets of proteins, TNKSs act as central hubs that coordinate signaling and metabolic pathways. In this review, we discuss how TNKS –protein interactions underpin their roles across multiple biological pathways, including Wnt/β-catenin, YAP and SRC signaling, mTORC1 signaling, DNA damage repair (via PARP crosstalk and recruitment of repair factors), telomere maintenance, cell-cycle regulation, glucose metabolism, cytoskeleton rearrangement, autophagy, proteasomal degradation, and apoptosis. We highlight the structural basis of these interactions, emphasizing ankyrin repeat domain recognition motifs and the consequences of TNKS-mediated PARylation on protein stability and localization. By integrating findings from oncology, virology, and metabolism, we illustrate how TNKS functions as a nodal regulator linking genome stability, signaling fidelity, and metabolic control. The interplay between TNKS and these varied pathways is essential for the well-being of the organism, with its dysregulation having severe biological and clinical consequences, which are discussed here. Finally, we consider therapeutic implications of disrupting TNKS–protein interactions, with particular attention paid to selective small-molecule inhibitors and their translational potential in cancer, viral infections, and degenerative diseases. Full article

Cathepsin B (CatB) is a lysosomal cysteine protease that plays a major role in various pathologies and is therefore considered a valuable therapeutic target. To address species-specific inhibitor challenges, we characterized the selective binding of designed ankyrin repeat protein (DARPin) 4m3 toward mouse cathepsin B (mCatB) over human CatB (hCatB). The mCatB–DARPin 4m3 complex was validated by size-exclusion chromatography (SEC), nano-differential scanning fluorimetry (nano-DSF), and surface plasmon resonance (SPR), revealing high affinity binding (K_D = 65.7 nM) and potent inhibition (Ki = 26.7 nM; mixed competitive/noncompetitive). DARPin 4m3 showed no binding/inhibition toward hCatB. The 1.67 Å crystal structure of the complex—the first for mCatB—identified key interaction residues (e.g., I65/Q66 in mCatB vs. S65/M66 in hCatB) conferring selectivity. Proteomic analysis of endogenous substrates using a support vector machine (SVM) revealed greater similarity between mCatB and hCatB cleavages (Area Under the Curve (AUC) = 0.733) than between mCatB and other human cathepsins (AUC = 0.939–0.965). Clustering and SVM methods offer broadly applicable tools for protease specificity profiling in drug discovery. This study demonstrates the utility of DARPins for species-selective targeting and highlights the importance of integrated structural and proteomic approaches for dissecting protein–protein interactions. Full article

Radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression might permit the selection of patients for EGFR-targeting therapies. Designed ankyrin repeat protein (DARPin) E01 with a high affinity to the ectodomain III of the EGFR is a possible EGFR imaging probe. The goal of this study was to evaluate the potential of radiolabeled DARPin E01 for in vivo imaging of EGFR. DARPin E01 containing the (HE)₃-tag was site-specifically labeled with a residualizing ^99mTc (using ^99mTc]Tc(CO)₃). Two methods providing non-residualizing ¹²³I labels, direct electrophilic radioiodination and indirect radioiodination using [¹²³I]I-para-iodobenzoate (PIB), were tested. [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB preserved specific binding to EGFR-expressing cells and affinity in the single-digit nanomolar range. Direct labeling with ¹²³I resulted in a substantial loss of binding. In vitro cellular processing studies showed that both [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB had rapid binding and relatively slow internalization. Evaluation of [^99mTc]Tc-(HE)₃-E01 biodistribution in normal CD1 mice showed that its hepatic uptake was non-saturable, suggesting that this tracer does not bind to murine EGFR. A side-by-side comparison of biodistribution and tumor targeting of [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB was performed in Nu/j mice bearing EGFR-positive A-431 and EGFR-negative Ramos human cancer xenografts. Both radiolabeled DARPins demonstrated EGFR-specific tumor uptake. However, [¹²³I]I-(HE)₃-E01-PIB had appreciably lower uptake in normal organs compared to [^99mTc]Tc-(HE)₃-E01, which provided significantly (p < 0.05) higher tumor-to-organ ratios. Gamma-camera imaging confirmed that [¹²³I]I-(HE)₃-E01-PIB demonstrated a higher imaging contrast in preclinical models than [^99mTc]Tc-(HE)₃-E01. In conclusion, DARPin (HE)₃-E01 labeled using a non-residualizing [¹²³I]I-para-iodobenzoate (PIB) label is the preferred radiotracer for in vivo imaging of EGFR expression in cancer. Full article

Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the ASB family of proteins, which act as a substrate recognition component of E3 ubiquitin ligases and regulate various reproductive processes. ASB9 was previously identified as being induced in bovine granulosa cells (GCs) by LH/hCG, and its binding partners, including protease-activated receptor 1 (PAR1), were reported. The aim of this study was to decipher ASB9’s mechanisms of action in GCs and determine whether ASB9 induction by LH/hCG is necessary for the regulation of PAR1 and the signaling pathways involved in GC function and activity. Cultured GCs were treated with different doses of FSH, LH, and thrombin. RT-qPCR analyses revealed that thrombin increased PAR1 expression, while FSH had no effect on PAR1. Treatment with LH significantly downregulated PAR1, even in the presence of thrombin, possibly via ASB9. The phosphorylation profile of MAPK3/1 in thrombin-treated GCs suggests PAR1-mediated control. ASB9 induction appeared to have a negative effect on the MAPK pathway, although thrombin treatment briefly (within an hour) blocked the negative effect of ASB9 on PAR1. Proliferation assays showed that ASB9 negatively regulated the GC number while increasing apoptosis. These data provide evidence of ASB9’s mode of action and its potent functional effects on PAR1 regulation, GC proliferation, and, potentially, the ovulatory process in bovine species. Full article

Lumpy Skin Disease Virus (LSDV) is a transboundary pathogen that affects cattle, causing significant economic losses, particularly in Africa and Asia. While the virus was originally endemic to sub-Saharan Africa, it has rapidly spread to Europe, the Middle East, and Asia, necessitating comprehensive genomic surveillance. Despite LSDV’s African origins, genomic data from West and Central Africa remain scarce, limiting insights into regional viral evolution and vaccine compatibility. In this study, molecular detection of LSDV was carried out on cattle samples from Nigeria, Cameroon, and Benin. However, comparative genomic analysis was performed using two near-complete LSDV genomes obtained from Cameroon. Phylogenetic evaluation revealed that LSDV strains from Nigeria and Cameroon cluster within the classical 1.2 lineage. Furthermore, the two sequences from this study cluster with the only publicly available sequence from West and Central Africa, supporting earlier findings of the presence of a West/Central African sub-lineage. Functional genomic analysis identified mutations in genes encoding ankyrin repeat Kelch-like proteins, and envelope proteins involved in immune evasion and viral virulence, raising concerns about vaccine effectiveness. Furthermore, the detection of LSDV in flesh flies (Sarcophaga spp.) underlines their potential role in virus transmission. These findings highlight the importance of genomic monitoring and targeted surveillance. Full article

Designed ankyrin repeat protein (DARPin) Ec1, a small scaffold protein (18 kDa), binds with high affinity the epithelial cell adhesion molecule (EpCAM) that is overexpressed in several carcinomas. To enhance the targeted delivery of cytotoxic drugs using Ec1, we investigated the potential of fusing Ec1 with an albumin-binding domain (ABD) to improve its circulation time and decrease renal uptake. Two fusion proteins were created, Ec1-ABD, with the ABD at the C-terminus, and ABD-Ec1, with the ABD at the N-terminus. Both variants were labeled with ¹¹¹In. ABD-fused variants bound specifically to EpCAM-expressing cells with picomolar affinity. Adding human albumin reduced the affinity. This effect was more pronounced for Ec1-ABD; however, the affinity remained in the subnanomolar range. The position of the ABD did not influence the internalization rate of both variants by human cancer cells. In mouse models with human cancer xenografts, both variants demonstrated over 10-fold lower renal uptake compared to the Ec1. Tumor uptake of the ABD-fused variants was higher than the uptake of Ec1. ABD-Ec1 provided two-fold higher tumor uptake, indicating fusion with an ABD as a promising way to modulate the targeting properties of an Ec1-based construct. However, the effect of fusion depends on the order of the domains. Full article

Scrub typhus is an emerging chigger-borne disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. Necroptosis is a form of programmed cell death (PCD) mediated by RIPK3 (serine/threonine kinase receptor interacting protein 3) and its downstream effector MLKL (mixed-lineage kinase domain-like). While O. tsutsugamushi modulates apoptosis, another form of PCD, its interplay with necroptosis is unknown. Much of Orientia pathobiology is linked to its ankyrin repeat (AR)-containing effectors (Anks). Two of these, Ank1 and Ank6, share similarities with the cowpox AR protein, vIRD (viral inducer of RIPK3 degradation) that prevents necroptosis. Here, we show that Ank1 and Ank6 reduce RIPK3 cellular levels although not as robustly as and mechanistically distinct from vIRD. Orientia infection lowers RIPK3 amounts and does not elicit necroptosis in endothelial cells. In HeLa cells ectopically expressing RIPK3, Orientia fails to inhibit RIPK3 and MLKL phosphorylation as well as cell death. MLKL colocalization with Orientia or Listeria monocytogenes, another intracytoplasmic pathogen, was not observed. Thus, O. tsutsugamushi reduces cellular levels of RIPK3 and does not elicit necroptosis but cannot inhibit this PCD pathway once it is induced. This study is a first step toward understanding how the relationship between Orientia and necroptosis contributes to scrub typhus pathogenesis. Full article

Ankyrin repeat domain 1 (Ankrd1), a transcriptional target of Yes-associated protein (YAP), is linked to cardiomyopathy. However, its role in cancer, particularly in clear cell renal cell carcinoma (ccRCC), remains vague. In this study, we examined the expression, regulation, and function of Ankrd1 in ccRCC. High Ankrd1 expression was related to poor prognosis in patients with ccRCC in The Cancer Genome Atlas cohort. Ankrd1 expression was regulated by YAP in all ccRCC cell lines examined and also by ERK5 in a subset of ccRCC cell lines. Moreover, silencing of Ankrd1 in ccRCC cell lines resulted in decreased cell motility, whereas its overexpression increased the cell motility. Ankrd1 colocalized with F-actin in lamellipodia upon phorbol ester stimulation. Ankrd1 silencing resulted in alterations in the shape of RCC cells and caused a decrease in lamellipodia formation. Ankrd1 also colocalized with talin-1 in lamellipodia. Ankrd1 depletion repressed talin-1-mediated activation of the integrin pathway. Immunohistochemical examination of surgical specimens revealed high expression of Ankrd1 in metastatic RCC tissues compared with that in primary RCC tissues from the same patients. Collectively, these findings suggest that Ankrd1 plays a critical role in the motility of ccRCC cells through lamellipodia formation. Full article

The asb5 gene, a member of the Asb protein subfamily characterized by six ankyrin repeat domains, is highly conserved and comprises two subtypes, asb5a and asb5b, in zebrafish. Our previous research has demonstrated that a deficiency of the asb5 gene significantly impairs early cardiac contractile function, highlighting its close relationship with heart development. Zebrafish asb5 expression was disrupted by both morpholino (MO) antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. A high-throughput RNA-Seq analysis was used to analyze the possible molecular regulatory mechanism of asb5 gene deletion leading to left–right (L-R) asymmetry defects in the heart. Whole-mount in situ hybridization (WISH) was conducted to evaluate gene expression patterns of Nodal signaling components and the positions of heart organs. Heart looping was defective in zebrafish asb5 morphants. Rescue experiments in the asb5-deficiency group (inactivating both asb5a and asb5b) demonstrated that the injection of either asb5a-mRNA or asb5b-mRNA alone was insufficient to rectify the abnormal L-R asymmetry of the heart. In contrast, the simultaneous injection of both asb5a-mRNA and asb5b-mRNA successfully rescued the morphological phenotype. A high-throughput RNA-Seq analysis of embryos at 48 h post fertilization (hpf) revealed that numerous genes associated with L-R asymmetry exhibited expression imbalances in the asb5-deficiency group. WISH further confirmed that the expression of genes such as fli1a, acta1b, hand2, has2, prrx1a, notch1b, and foxa3 were upregulated, while the expression of mei2a and tal1 was downregulated. These results indicated that loss of the asb5 gene in zebrafish led to the disordered development of L-R asymmetry in the heart, resulting in an imbalance in the expression of genes associated with the regulation of L-R asymmetry. Subsequently, we examined the expression patterns of classical Nodal signaling pathway-related genes using WISH. The results showed that the midline barrier factor gene lefty1 was downregulated at early stages in the asb5-deficiency group, and the expression of spaw and lefty2, which are specific to the left lateral plate mesoderm (LPM), was disrupted. This study reveals that the two subtypes of the asb5 gene in zebrafish, asb5a and asb5b, interact and jointly regulate the establishment of early cardiac L-R asymmetry through the Nodal-spaw-lefty signaling pathway. Full article

Targeting HER2-positive cancer cells with precision therapies is a critical challenge in oncology. Here, we present a study on gold nanoparticles (AuNPs) conjugated with DARPin_9-29, a designed ankyrin repeat protein with high specificity and affinity for HER2 receptors. In this study, we investigate the therapeutic potential of AuNP-DARPin_9-29 conjugates, which was synthesized and characterized by us earlier, for photothermal therapy (PTT). By combining AuNP-DARPin treatment with visible light illumination, we show selective inhibition of HER2-positive cancer cell proliferation and tumor progression in a murine model. The results highlight the effectiveness of AuNP-DARPin in disrupting cancer cell viability and reducing tumor growth, providing a cost-effective and targeted approach for combating HER2-positive cancers. Full article

Non-immunoglobulin-based scaffold proteins (SPs) represent one of the key therapeutic target-specific and high-affinity binders in modern medicine. Among their cellular targets are signaling receptors, in particular, receptor tyrosine kinases, whose dysfunction leads to the development of cancer and other serious diseases. Successful applications of SPs have been reported for HER receptor type 2 (HER2), a member of the human epidermal growth factor receptor family that regulates cell growth and differentiation. To extend the blood residence of SPs and prevent their high accumulation in the kidneys, these proteins are often fused with serum albumin. Promising results for HER2-binding activity were obtained for SP G3 from the DARPins (Designed Ankyrin Repeat Proteins) family fused with an albumin-binding domain (ABD). Interestingly, the detected HER2–G3 binding strongly depended on the position of the G3 module in the sequence of the constructs. Further improvement of these constructs for biomedical applications requires deciphering the molecular mechanism responsible for this effect. Here, we investigate the structural and dynamic aspects of ABD–G3 and G3–ABD chimeras using NMR spectroscopy and molecular modeling. Based on biophysical data, we come to the conclusion that extensive inter-domain contacts form in both constructs, although their binding interfaces and complex stability are somewhat different. Also, it is shown that the domain linker plays an important role—it limits the accessibility of the detected protein–protein binding sites, depending on the order of the domains in the chimeric molecules. These results create a solid structural basis for the rational design of new effective SP constructs targeting the signaling receptors in cells. Full article

The TaqIA polymorphism is a marker of both the Ankyrin Repeat and Kinase Domain containing I gene (ANKK1) encoding a RIP-kinase, and the DRD2 gene for the dopamine receptor D2. Despite a large number of studies of TaqIA in addictions and other psychiatric disorders, there is difficulty in interpreting this genetic phenomenon due to the lack of knowledge about ANKK1 function. In SH-SY5Y neuroblastoma models, we show that ANKK1 interacts with the synapse protein FERM ARH/RhoGEF and Pleckstrin Domain 1 (FARP1), which is a guanine nucleotide exchange factor (GEF) of the RhoGTPases RAC1 and RhoA. ANKK1–FARP1 colocalized in F-ACTIN-rich structures for neuronal maturation and migration, and both proteins activate the Wnt/PCP pathway. ANKK1, but not FARP1, promotes neuritogenesis, and both proteins are involved in neuritic spine outgrowth. Notably, the knockdown of ANKK1 or FARP1 affects RhoGTPases expression and neural differentiation. Additionally, ANKK1 binds WGEF, another GEF of Wnt/PCP, regulating its interaction with RhoA. During neuronal differentiation, ANKK1–WGEF interaction is downregulated, while ANKK1–FARP1 interaction is increased, suggesting that ANKK1 recruits Wnt/PCP components for bidirectional control of F-ACTIN assembly. Our results suggest a brain structural basis in TaqIA-associated phenotypes. Full article

Preeclampsia is a complex pregnancy-related hypertensive disorder which poses significant risks for both maternal and fetal health. Preeclampsia affects 5–8% of pregnancies in the United States, causing a significant public health and economic burden. Despite extensive research, the etiology and pathogenesis of preeclampsia remain elusive, but have been correlated with maternal conditions such as obesity. In recent decades, the incidence of preeclampsia increased along with the prevalence of obesity among women of reproductive age. Maternal obesity has been shown to negatively affect pregnancy in almost all aspects. However, the precise mechanisms by which obesity influences preeclampsia are unclear. Ankyrin repeat and SOCS Box Containing protein 4 (ASB4) is an E3 ubiquitin ligase that can promote the degradation of a wide range of target proteins. ASB4-null mice display a full spectrum of preeclampsia-like phenotypes during pregnancy including hypertension, proteinuria, and decreased litter size. Furthermore, maternal obesity induced by a high-fat diet aggravates preeclampsia-like phenotypes in pregnant mice lacking ASB4. Variants in the ASB4 gene have been associated with obesity in humans, and a functional connection between the ASB4 gene and obesity has been established in mice. This review discusses the connections between preeclampsia, obesity, and ASB4. Full article

A high level of EpCAM overexpression in lung cancer makes this protein a promising target for targeted therapy. Radionuclide visualization of EpCAM expression would facilitate the selection of patients potentially benefiting from such treatment. Single-photon computed tomography (SPECT) using ^99mTc-labeled engineered scaffold protein DARPin Ec1 has shown its effectiveness in imaging tumors with overexpression of EpCAM in preclinical studies, providing high contrast just a few hours after injection. This first-in-human study aimed to evaluate the safety and distribution of [^99mTc]Tc(CO)₃-(HE)₃-Ec1 in patients with primary lung cancer. Twelve lung cancer patients were injected with 300.7 ± 103.2 MBq of [^99mTc]Tc(CO)₃-(HE)₃-Ec1. Whole-body planar imaging (at 2, 4, 6 and 24 h after injection) and SPECT/CT of the lung (at 2, 4, and 6 h) were performed. The patients’ vital signs and possible side effects were monitored up to 7 days after injection. The patients tolerated the injection of [^99mTc]Tc(CO)₃-(HE)₃-Ec1 well, and their somatic condition remained normal during the entire follow-up period. There were no abnormalities in blood and urine tests after injection of [^99mTc]Tc(CO)₃-(HE)₃-Ec1. The highest absorbed doses were in the kidneys, liver, pancreas, thyroid, gallbladder wall, and adrenals. There was also a relatively high accumulation of [^99mTc]Tc(CO)₃-(HE)₃-Ec1 in the small and large intestines, pancreas and thyroid. According to the SPECT/CT, accumulation of [^99mTc]Tc(CO)₃-(HE)₃-Ec1 in the lung tumor was found in all patients included in the study. Intensive accumulation of [^99mTc]Tc(CO)₃-(HE)₃-Ec1 was also noted in regional metastases. [^99mTc]Tc(CO)₃-(HE)₃-Ec1 can potentially be considered a diagnostic tracer for imaging EpCAM expression in lung cancer patients and other tumors with overexpression of EpCAM. Full article