Topoisomerase II alpha and beta (TOP2A and TOP2B) isoenzymes perform essential and non-redundant cellular functions. Anthracyclines induce their potent anti-cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. Here we describe the development of the obex class of TOP2 inhibitors that bind to a previously unidentified druggable pocket in the TOP2 ATPase domain to act as allosteric catalytic inhibitors by locking the ATPase domain conformation with the capability of isoform-selective inhibition. Through rational drug design we have developed topobexin, which interacts with residues that differ between TOP2A and TOP2B to provide inhibition that is both selective for TOP2B and superior to dexrazoxane. Topobexin is a potent protectant against chronic anthracycline cardiotoxicity in an animal model. This demonstration of TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.

• MeSH
• Anthracyclines * adverse effects   pharmacology   MeSH
• DNA Topoisomerases, Type II * metabolism   chemistry   MeSH
• Topoisomerase II Inhibitors * pharmacology   chemistry   MeSH
• Cardiotonic Agents * pharmacology   chemistry   MeSH
• Cardiotoxicity * prevention & control   MeSH
• Humans MeSH
• Mice MeSH
• Poly-ADP-Ribose Binding Proteins antagonists & inhibitors   metabolism   chemistry   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.

• MeSH
• Antiviral Agents * pharmacology   chemistry   MeSH
• Fluorometry methods   MeSH
• Humans MeSH
• Piperidines pharmacology   MeSH
• Pyridines MeSH
• Pyrimidines MeSH
• Pyrroles MeSH
• RNA Caps metabolism   MeSH
• RNA-Dependent RNA Polymerase antagonists & inhibitors   metabolism   MeSH
• High-Throughput Screening Assays * methods   MeSH
• Viral Proteins * antagonists & inhibitors   metabolism   MeSH
• Influenza A virus drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Neurotropic pathogens, notably, herpesviruses, have been associated with significant neuropsychiatric effects. As a group, these pathogens can exploit molecular mimicry mechanisms to manipulate the host central nervous system to their advantage. Here, we present a systematic computational approach that may ultimately be used to unravel protein-protein interactions and molecular mimicry processes that have not yet been solved experimentally. Toward this end, we validate this approach by replicating a set of pre-existing experimental findings that document the structural and functional similarities shared by the human cytomegalovirus-encoded UL144 glycoprotein and human tumor necrosis factor receptor superfamily member 14 (TNFRSF14). We began with a thorough exploration of the Homo sapiens protein database using the Basic Local Alignment Search Tool (BLASTx) to identify proteins sharing sequence homology with UL144. Subsequently, we used AlphaFold2 to predict the independent three-dimensional structures of UL144 and TNFRSF14. This was followed by a comprehensive structural comparison facilitated by Distance-Matrix Alignment and Foldseek. Finally, we used AlphaFold-multimer and PPIscreenML to elucidate potential protein complexes and confirm the predicted binding activities of both UL144 and TNFRSF14. We then used our in silico approach to replicate the experimental finding that revealed TNFRSF14 binding to both B- and T-lymphocyte attenuator (BTLA) and glycoprotein domain and UL144 binding to BTLA alone. This computational framework offers promise in identifying structural similarities and interactions between pathogen-encoded proteins and their host counterparts. This information will provide valuable insights into the cognitive mechanisms underlying the neuropsychiatric effects of viral infections.

• MeSH
• Cognition physiology   MeSH
• Humans MeSH
• Molecular Mimicry * MeSH
• Models, Molecular MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Viral Proteins metabolism   chemistry   MeSH
• Computational Biology methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Lens epithelium-derived growth factor p75 (LEDGF/p75), member of the hepatoma-derived growth-factor-related protein (HRP) family, is a transcriptional co-activator and involved in several pathologies including HIV infection and malignancies such as MLL-rearranged leukemia. LEDGF/p75 acts by tethering proteins to the chromatin through its integrase binding domain. This chromatin interaction occurs between the PWWP domain of LEDGF/p75 and nucleosomes carrying a di- or trimethylation mark on histone H3 Lys36 (H3K36me2/3). Our aim is to rationally devise small molecule drugs capable of inhibiting such interaction. To bootstrap this development, we resorted to X-ray crystallography-based fragment screening (FBS-X). Given that the LEDGF PWWP domain crystals were not suitable for FBS-X, we employed crystals of the closely related PWWP domain of paralog HRP-2. As a result, as many as 68 diverse fragment hits were identified, providing a detailed sampling of the H3K36me2/3 pocket pharmacophore. Subsequent structure-guided fragment expansion in three directions yielded multiple compound series binding to the pocket, as verified through X-ray crystallography, nuclear magnetic resonance and differential scanning fluorimetry. Our best compounds have double-digit micromolar affinity and optimally sample the interactions available in the pocket, judging by the Kd-based ligand efficiency exceeding 0.5 kcal/mol per non-hydrogen atom. Beyond π-stacking within the aromatic cage of the pocket and hydrogen bonding, the best compounds engage in a σ-hole interaction between a halogen atom and a conserved water buried deep in the pocket. Notably, the binding pocket in LEDGF PWWP is considerably smaller compared to the related PWWP1 domains of NSD2 and NSD3 which feature an additional subpocket and for which nanomolar affinity compounds have been developed recently. The absence of this subpocket in LEDGF PWWP limits the attainable affinity. Additionally, these structural differences in the H3K36me2/3 pocket across the PWWP domain family translate into a distinct selectivity of the compounds we developed. Our top-ranked compounds are interacting with both homologous LEDGF and HRP-2 PWWP domains, yet they showed no affinity for the NSD2 PWWP1 and BRPF2 PWWP domains which belong to other PWWP domain subfamilies. Nevertheless, our developed compound series provide a strong foundation for future drug discovery targeting the LEDGF PWWP domain as they can further be explored through combinatorial chemistry. Given that the affinity of H3K36me2/3 nucleosomes to LEDGF/p75 is driven by interactions within the pocket as well as with the DNA-binding residues, we suggest that future compound development should target the latter region as well. Beyond drug discovery, our compounds can be employed to devise tool compounds to investigate the mechanism of LEDGF/p75 in epigenetic regulation.

• MeSH
• Small Molecule Libraries chemistry   pharmacology   chemical synthesis   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Intercellular Signaling Peptides and Proteins metabolism   chemistry   MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Protein Domains MeSH
• Drug Design * MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The Pax6 gene is essential for eye and brain development across various animal species. Here, we investigate the function of Pax6 in the development of the anterior central nervous system (CNS) of the invertebrate chordate amphioxus using CRISPR/Cas9-induced genome editing. Specifically, we examined Pax6 mutants featuring a 6 bp deletion encompassing two invariant amino acids in the conserved paired domain, hypothesized to impair Pax6 DNA-binding capacity and gene regulatory functions. Although this mutation did not result in gross morphological changes in amphioxus larvae, it demonstrated a reduced ability to activate Pax6-responsive reporter gene, suggesting a hypomorphic effect. Expression analysis in mutant larvae revealed changes in gene expression within the anterior CNS, supporting the conserved role of Pax6 gene in brain regionalization across chordates. Additionally, our findings lend support to the hypothesis of a zona limitans intrathalamica (ZLI)-like region in amphioxus, suggesting evolutionary continuity in brain patterning mechanisms. ZLI region, found in both hemichordates and vertebrates, functions as a key signaling center and serves as a restrictive boundary between major thalamic regions.

• Publication type
• Journal Article MeSH

Methylation of histone H3 at lysine 36 (H3K36me3) marks active chromatin. The mark is interpreted by epigenetic readers that assist transcription and safeguard the integrity of the chromatin fiber. The chromodomain protein MSL3 binds H3K36me3 to target X-chromosomal genes in male Drosophila for dosage compensation. The PWWP-domain protein JASPer recruits the JIL1 kinase to active chromatin on all chromosomes. Unexpectedly, depletion of K36me3 had variable, locus-specific effects on the interactions of those readers. This observation motivated a systematic and comprehensive study of K36 methylation in a defined cellular model. Contrasting prevailing models, we found that K36me1, K36me2 and K36me3 each contribute to distinct chromatin states. A gene-centric view of the changing K36 methylation landscape upon depletion of the three methyltransferases Set2, NSD and Ash1 revealed local, context-specific methylation signatures. Set2 catalyzes K36me3 predominantly at transcriptionally active euchromatin. NSD places K36me2/3 at defined loci within pericentric heterochromatin and on weakly transcribed euchromatic genes. Ash1 deposits K36me1 at regions with enhancer signatures. The genome-wide mapping of MSL3 and JASPer suggested that they bind K36me2 in addition to K36me3, which was confirmed by direct affinity measurement. This dual specificity attracts the readers to a broader range of chromosomal locations and increases the robustness of their actions.

• MeSH
• Chromatin * metabolism   MeSH
• DNA-Binding Proteins metabolism   genetics   MeSH
• Drosophila melanogaster genetics   metabolism   MeSH
• Heterochromatin metabolism   genetics   MeSH
• Histone-Lysine N-Methyltransferase * metabolism   genetics   MeSH
• Histones * metabolism   MeSH
• Lysine metabolism   MeSH
• Methyltransferases metabolism   genetics   MeSH
• Methylation MeSH
• Protein Serine-Threonine Kinases MeSH
• Drosophila Proteins * metabolism   genetics   MeSH
• Transcription Factors metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Armadillo repeat-containing proteins (ARMCs) are a large family found throughout eukaryotes, which play prominent roles in cell adhesion, signaling and cytoskeletal regulation. The ARMC6 protein is highly conserved in primates, including humans, but to date does not have a clear function beyond initial hints of a link to cancer and telomerase activity. We report here in vitro experiments showing ARMC6 binding to DNA promoter sequences from several cancer-related genes (e.g., EGFR, VEGF and c-MYC), and also to the telomeric RNA repeat (TERRA). ARMC6 binding activity appears to recognize G-quadruplex motifs, which are being increasingly implicated as structure-based protein binding sites in chromosome maintenance and repair. In vivo investigation of ARMC6 function revealed that when this protein is overexpressed in human cell lines, there is different expression of genes connected with oncogenic pathways and those implicated in downstream non-canonical telomerase pathways (e.g., VEGF, hTERT, c-MYC, ESM1, MMP3). ARMC6 is already known to interact with human shelterin protein TRF2 and telomerase. The protein binds G-quadruplex structures and does so preferentially to RNA over DNA. As such, this protein may be an example of how a non-canonical nucleic acid structural motif allows mediation between gene regulation and telomeric chromatin rearrangement pathways.

• MeSH
• DNA-Binding Proteins MeSH
• G-Quadruplexes * MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Neoplasms genetics   metabolism   MeSH
• Promoter Regions, Genetic * MeSH
• Armadillo Domain Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• RNA metabolism   genetics   MeSH
• Telomerase metabolism   genetics   MeSH
• Telomere * metabolism   MeSH
• Transcription Factors MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Adipose tissue is significantly involved in inflammatory bowel disease (IBD). Vitamin D can affect both adipogenesis and inflammation. The aim of this study was to compare the production of selected adipokines, potentially involved in the pathogenesis of IBD - adiponectin, resistin, retinol binding protein 4 (RBP-4), adipocyte fatty acid binding protein and nesfatin-1 in children with IBD according to the presence of 25-hydroxyvitamin D (25(OH)D) deficiency. METHODS: The study was conducted as a case-control study in pediatric patients with IBD and healthy children of the same sex and age. In addition to adipokines and 25(OH)D, anthropometric parameters, markers of inflammation and disease activity were assessed in all participants. RESULTS: Children with IBD had significantly higher resistin levels regardless of 25(OH)D levels. IBD patients with 25(OH)D deficiency only had significantly lower RBP-4 compared to healthy controls and also compared to IBD patients without 25(OH)D deficiency. No other significant differences in adipokines were found in children with IBD with or without 25(OH)D deficiency. 25(OH)D levels in IBD patients corelated with RBP-4 only, and did not correlate with other adipokines. CONCLUSIONS: Whether the lower RBP-4 levels in the 25(OH)D-deficient group of IBD patients directly reflect vitamin D deficiency remains uncertain. The production of other adipokines does not appear to be directly related to vitamin D deficiency.

• MeSH
• Adipokines * blood   MeSH
• Adiponectin blood   deficiency   MeSH
• Biomarkers blood   MeSH
• Child MeSH
• DNA-Binding Proteins blood   MeSH
• Inflammatory Bowel Diseases blood   complications   MeSH
• Humans MeSH
• Adolescent MeSH
• Vitamin D Deficiency * complications   blood   MeSH
• Nucleobindins blood   MeSH
• Retinol-Binding Proteins, Plasma metabolism   analysis   MeSH
• Fatty Acid-Binding Proteins blood   MeSH
• Calcium-Binding Proteins blood   MeSH
• Resistin blood   MeSH
• Case-Control Studies MeSH
• Vitamin D * blood   analogs & derivatives   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

INTRODUCTION: Hepatocyte nuclear factor 1-beta (HNF1B) gene variants or the chromosome 17q12 deletion (17q12del) represent the most common monogenic cause of developmental kidney disease. Although neurodevelopmental disorders have been associated with the 17q12del, specific genotype-phenotype associations with respect to kidney function evolution have not yet been fully defined. Here, we aimed to determine whether 17q12del or specific HNF1B variants were associated with kidney survival in a large patient population with HNF1B disease. METHODS: This was a retrospective observational study involving 521 patients with HNF1B disease from 14 countries using the European Reference Network for rare kidney diseases with detailed information on the HNF1B genotype (HNF1B variants or the 17q12del). Median follow-up time was 11 years with 6 visits per patient. The primary end point was progression to chronic kidney disease (CKD) stage 3 (estimated glomerular filtration rate [eGFR] < 60 ml/min per 1.73 m2). Secondary end points were the development of hypomagnesemia or extrarenal disorders, including hyperuricemia and hyperglycemia. RESULTS: Progression toward CKD stage 3 was significantly delayed in patients with the 17q12del compared to patients with HNF1B variants (hazard ratio [HR]: 0.29, 95% confidence interval [CI]: 0.19-0.44, P < 0.001). Progression toward CKD stage 3 was also significantly delayed when HNF1B variants involved the HNF1B Pit-1, Oct-1, and Unc-86 homeodomain (POUh) DNA-binding and transactivation domains rather than the POU-specific domain (POUs) DNA-binding domain (HR: 0.15 [95% CI: 0.06-0.37), P < 0.001 and HR: 0.25 (95% CI: 0.11-0.57), P = 0.001, respectively). Finally, the 17q12del was positively associated with hypomagnesemia and negatively associated with hyperuricemia, but not with hyperglycemia. CONCLUSION: Patients with the 17q12del display a significantly better kidney survival than patients with other HNF1B variants; and for the latter, variants in the POUs DNA-binding domain lead to the poorest kidney survival. These are clinically relevant HNF1B kidney genotype-phenotype correlations that inform genetic counseling.

• Publication type
• Journal Article MeSH

Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.

• MeSH
• Enzyme Inhibitors * pharmacology   chemistry   chemical synthesis   MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Pentosyltransferases * antagonists & inhibitors   metabolism   MeSH
• Drug Design * MeSH
• Trypanosoma brucei brucei drug effects   enzymology   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH