Tryptanthrin Analogs Substoichiometrically Inhibit Seeded and Unseeded Tau4RD Aggregation.

Microtubule-associated protein tau is an intrinsically disordered protein (IDP) that forms characteristic fibrillar aggregates in several diseases, the most well-known of which is Alzheimer's disease (AD). Despite keen interest in disrupting or inhibiting tau aggregation to treat AD and related dementias, there are currently no FDA-approved tau-targeting drugs. This is due, in part, to the fact that tau and other IDPs do not exhibit a single well-defined conformation but instead populate a fluctuating conformational ensemble that precludes finding a stable "druggable" pocket. Despite this challenge, we previously reported the discovery of two novel families of tau ligands, including a class of aggregation inhibitors, identified through a protocol that combines molecular dynamics, structural analysis, and machine learning. Here we extend our exploration of tau druggability with the identification of tryptanthrin and its analogs as potent, substoichiometric aggregation inhibitors, with the best compounds showing potencies in the low nanomolar range even at a ~100-fold molar excess of tau4RD. Moreover, conservative changes in small molecule structure can have large impacts on inhibitory potency, demonstrating that similar structure-activity relationship (SAR) principles as used for traditional drug development also apply to tau and potentially to other IDPs.

Defining the condensate landscape of fusion oncoproteins.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future.

An Image Analysis Pipeline for Quantifying the Features of Fluorescently-Labeled Biomolecular Condensates in Cells.

Biomolecular condensates are cellular organelles formed through liquid-liquid phase separation (LLPS) that play critical roles in cellular functions including signaling, transcription, translation, and stress response. Importantly, condensate misregulation is associated with human diseases, including neurodegeneration and cancer among others. When condensate-forming biomolecules are fluorescently-labeled and examined with fluorescence microscopy they appear as illuminated foci, or puncta, in cells. Puncta features such as number, volume, shape, location, and concentration of biomolecular species within them are influenced by the thermodynamics of biomolecular interactions that underlie LLPS. Quantification of puncta features enables evaluation of the thermodynamic driving force for LLPS and facilitates quantitative comparisons of puncta formed under different cellular conditions or by different biomolecules. Our work on nucleoporin 98 (NUP98) fusion oncoproteins (FOs) associated with pediatric leukemia inspired us to develop an objective and reliable computational approach for such analyses. The NUP98-HOXA9 FO forms hundreds of punctate transcriptional condensates in cells, leading to hematopoietic cell transformation and leukemogenesis. To quantify the features of these puncta and derive the associated thermodynamic parameters, we developed a live-cell fluorescence microscopy image processing pipeline based on existing methodologies and open-source tools. The pipeline quantifies the numbers and volumes of puncta and fluorescence intensities of the fluorescently-labeled biomolecule(s) within them and generates reports of their features for hundreds of cells. Using a standard curve of fluorescence intensity versus protein concentration, the pipeline determines the apparent molar concentration of fluorescently-labeled biomolecules within and outside of puncta and calculates the partition coefficient (Kp) and Gibbs free energy of transfer (ΔGTr), which quantify the favorability of a labeled biomolecule partitioning into puncta. In addition, we provide a library of R functions for statistical analysis of the extracted measurements for certain experimental designs. The source code, analysis notebooks, and test data for the Punctatools pipeline are available on GitHub: https://github.com/stjude/punctatools. Here, we provide a protocol for applying our Punctatools pipeline to extract puncta features from fluorescence microscopy images of cells.

7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome.

Defective 3ß-hydroxysterol-Δ7 -reductase (DHCR7) in the developmental disorder, Smith-Lemli-Opitz syndrome (SLOS), results in a deficiency in cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC). Here, we show that loss of DHCR7 causes accumulation of 7-DHC-derived oxysterol metabolites, premature neurogenesis from murine or human cortical neural precursors, and depletion of the cortical precursor pool, both in vitro and in vivo. We found that a major oxysterol, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), mediates these effects by initiating crosstalk between glucocorticoid receptor (GR) and neurotrophin receptor kinase TrkB. Either loss of DHCR7 or direct exposure to DHCEO causes hyperactivation of GR and TrkB and their downstream MEK-ERK-C/EBP signaling pathway in cortical neural precursors. Moreover, direct inhibition of GR activation with an antagonist or inhibition of DHCEO accumulation with antioxidants rescues the premature neurogenesis phenotype caused by the loss of DHCR7. These results suggest that GR could be a new therapeutic target against the neurological defects observed in SLOS.

Structure-Activity Relationships of Novel Tau Ligands: Passive Fibril Binders and Active Aggregation Inhibitors.

Intrinsically disordered proteins (IDPs) are core components of many biological processes and are central players in several pathologies. Despite being important drug targets, attempts to design small-molecule ligands that would help understand and attenuate their behavior are frustrated by the structural diversity exhibited by these flexible proteins. To accommodate the dynamic nature of IDPs, we developed a procedure that efficiently identifies active small-molecule ligands for disordered proteins. By exploring the chemical space around these ligands, we refined their effect on aggregation and identified molecular features critical for activity and affinity. Notably, the discovery of this new family of disordered protein ligands was achieved more quickly and with less expense than conventional high-throughput screening (HTS) or docking alone would have allowed. The resulting ligands include tau aggregation inhibitors as well as at least one compound that binds fibrils potently but does not appear to perturb the extent of kinetics of aggregation.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, and NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs. SIGNIFICANCE: We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

The Rational Discovery of a Tau Aggregation Inhibitor.

Intrinsically disordered proteins play vital roles in biology, and their dysfunction contributes to many major disease states. These proteins remain challenging targets for rational ligand discovery or drug design because they are highly dynamic and fluctuate through a diverse set of conformations, frustrating structure-based approaches. To meet this challenge, we have developed protocols to efficiently identify active small molecule ligands of disordered proteins. Our approach utilizes enhanced sampling molecular dynamics and conformational analysis approaches optimized for disordered targets, coupled with computational docking and machine learning-based screens of compound libraries. By applying this protocol to an amyloid-forming segment of microtubule-associated protein tau, we successfully identified novel, chemically diverse tau ligands, including an inhibitor that delays the aggregation reaction in vitro without affecting the amount of aggregate formed at the steady state. Our results indicate that we have expanded the toolkit of protein aggregation inhibitors into new areas of chemical space and demonstrate the feasibility of our ligand discovery strategy.

Bulk preparation of holey graphene via controlled catalytic oxidation.

Structural manipulation of the two dimensional graphene surface has been of significant interest as a means of tuning the properties of the nanosheets for enhanced performance in various applications. In this report, a straightforward and highly scalable method is presented to prepare bulk quantities of "holey graphenes", which are graphene sheets with holes ranging from a few to tens of nm in average diameter. The approach to their preparation takes advantage of the catalytic properties of silver (Ag) nanoparticles toward the air oxidation of graphitic carbon. In the procedure, Ag nanoparticles were first deposited onto the graphene sheet surface in a facile, controllable, and solvent-free process. The catalyst-loaded graphene samples were then subjected to thermal treatment in air. The graphitic carbons in contact with the Ag nanoparticles were selectively oxidized into gaseous byproducts, such as CO or CO2, leaving holes in the graphene surface. The Ag was then removed by refluxing in diluted nitric acid to obtain the final holey graphene products. The average size of the holes on the graphene was found to correlate with the size of the Ag nanoparticles, which could be controlled by adjusting the silver precursor concentration. In addition, the temperature and time of the air oxidation step, and the catalyst removal treatment conditions were found to strongly affect the morphology of the holes. Characterization results of the holey graphene products suggested that the hole generation might have started from defect-rich regions present on the starting graphene sheets. As a result, the remaining graphitic carbon structures on the holey graphene sheets were highly crystalline, with no significant increase of the overall defect density despite the presence of structural holes. Preliminary experiments are also presented on the use of holey graphene sheets as fillers for polymeric composites. The results indicated that these sheets might be better reinforcing fillers than the starting graphene sheets due to their perforated structure. Other unique potentials of these materials, such as for energy storage applications, are also discussed.

Instantaneous formation of metal and metal oxide nanoparticles on carbon nanotubes and graphene via solvent-free microwave heating.

Microwave irradiation was shown to be an effective energy source for the rapid decomposition of organic metal salts (such as silver acetate) in a solid mixture with various carbon and noncarbon substrates under completely solvent-free conditions. The rapid and local Joule heating of microwave absorbing substrates (i.e., carbon-based) resulted in the instantaneous formation of metal and metal oxide nanoparticles on the substrate surfaces within seconds of microwave exposure. Other less absorbing substrates (such as hexagonal boron nitride) required longer exposure times for the salt decomposition to occur. Details of the effects of microwave reaction time, temperature, power, and other experimental parameters were investigated and discussed. The solvent-free microwave method was shown to be widely applicable to various organic metal salts with different substrates including single- and multiwalled carbon nanotubes, graphene, expanded graphite, hexagonal boron nitride and silica-alumina particles, forming substrate-supported metal (e.g., Ag, Au, Co, Ni, Pd, Pt) or metal oxide (e.g., Fe3O4, MnO, TiO2) nanoparticles in high yields within short duration of microwave irradiation. The method was also successfully applied to large structural substrates such as nanotube yarns, further suggesting its application potential and versatility. To demonstrate one potential application, we successfully used both carbon nanotube powder and yarn samples decorated with Ag nanoparticles prepared via the above method to improve data acquisition in surface enhanced Raman spectroscopy.