Background and objectives The PWWP domain of lens epithelium-derived growth factor p LEDGF p mediates chromatin engagement through recognition of histone H lysine di- and trimethylation H K me and nucleosomal DNA LEDGF p plays a role in multiple human diseases In particular its interaction with HIV- integrase enables viral ...More |Related Solutions: Rock Imager^®

Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed by X-ray crystallography. Functional effects on chromatin engagement were evaluated using the AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. Crystal structures of two Nbs (NbC03 and NbH10) confirmed canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and demonstrates their ability to functionally disrupt the LEDGF-chromatin interaction. These Nbs serve as valuable tools towards functional studies and structure-based drug design. Less |Related Solutions: Rock Imager^®

Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye Melanin binding of drugs in vitro correlates with in vivo binding but current workflows for binding affinity require multiple slow steps and analytical method development and or they ...More |Related Solutions: μPulse^®

Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye. Melanin binding of drugs in vitro correlates with in vivo binding, but current workflows for binding affinity require multiple slow steps, and analytical method development, and/or they may result in high data variability. We developed tangential flow filtration-based methodology to produce size-specific fractions of water-soluble melanin nanoparticles (MNPs), reducing production time from 2-3 days to just a few hours and yielding MNPs with enhanced fluorescence signal. Improved MNPs enabled modifications to a previously published microscale thermophoresis-based melanin binding protocol, shifting analytical focus toward thermophoretic behavior, reducing data variability, and improving reproducibility. The process was tested with nine compounds with varying melanin binding affinities, and the results were consistent with literature, confirming the ability of the method to differentiate compounds based on melanin binding. Fast and reliable workflow will be useful in screening binding affinity for therapeutics and new drug candidates to melanin thereby facilitating ocular drug discovery and construction of predictive pharmacokinetic simulation models. Less |Related Solutions: μPulse^®

SummaryBACH is a transcriptional regulator that modulates various cytoprotective pathways Among these pathways BACH regulates the cellular oxidative stress responses by suppressing the expression of cytoprotective genes Dysregulated BACH activity has been implicated in a range of pathologies including chronic inflammatory diseases fibrosis and cancer making it a promising therapeutic ...More |Related Solutions: Tempest^®

SummaryBACH1 is a transcriptional regulator that modulates various cytoprotective pathways. Among these pathways BACH1 regulates the cellular oxidative stress responses by suppressing the expression of cytoprotective genes. Dysregulated BACH1 activity has been implicated in a range of pathologies, including chronic inflammatory diseases, fibrosis, and cancer, making it a promising therapeutic target. However, BACH1 remains an underexploited drug target, with limited pharmacological inhibitors available. We have developed a novel luciferase-based reporter cell line enabling quantitative, high-throughput assessment of BACH1 inhibition. Using this platform, we rigorously screened two small-molecule libraries with 2,046 compounds and identified four structurally distinct compounds that robustly inhibit BACH1 function. Notably, these compounds simultaneously activate transcription factor NRF2, suggesting the potential for a broader modulation of oxidative stress pathways.Importantly, we demonstrate that commonly used 2D migration assays may fail to detect phenotypes consistent with BACH1 inhibition, resulting in false negatives. In contrast, we establish that 3D invasion assays more robustly capture anti-invasive effects of BACH1 functional inhibition. Using this 3D system, we validate the identified compounds as potent suppressors of lung cancer cell invasion in vitro.This study delivers a novel screening platform for BACH1-targeted drug discovery, and challenges current in vitro standards by establishing 3D invasion assays as a more accurate functional readout for BACH1-targeting compounds. Additionally, it identifies new dual functional BACH1 inhibitors/NRF2 activators, offering novel chemical scaffolds for the development of anti-metastatic therapies and potentially treatments for diseases driven by oxidative stress and inflammation. Less |Related Solutions: Tempest^®

Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease factor f Xa and the cofactor fVa on activated cell surfaces to form the prothrombinase complex Prothrombinase cleaves prothrombin at two sites in a processive manner -times ...More |Related Solutions: Rock Imager^®

Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease, factor (f) Xa, and the cofactor fVa on activated cell surfaces to form the prothrombinase complex. Prothrombinase cleaves prothrombin at two sites in a processive manner ∼500,000-times faster than fXa on its own. How fVa confers rapid and processive cleavage of prothrombin is an enzymatical mystery with profound consequence. We created a variant of fXa that binds to fVa with high affinity in the absence of phospholipids that preserves the activity of wild-type prothrombinase, and recently reported on the cryo-EM structure of the complex. It revealed an extensive interface between the two proteins, including a critical interaction between the first acidic region C-terminal to the A2 domain of fVa (the N-terminal portion of the a2-loop) with the heparin binding site of fXa. Here we present the cryo-EM structures of prothrombinase bound to prothrombin and the intermediate meizothrombin, both to 3.1 Å resolution. The prothrombin complex revealed a surprising interaction between the second acidic region of the a2-loop with exosite I of prothrombin, accounting for 70% of the total buried surface area. Cleavage at Arg320 triggers the zymogen-to-protease conformational change in meizothrombin which alters all domain-domain and fVa interactions, and results in the presentation of the second cleavage site (Arg271) for processing. Together, these structures reveal a remarkable enzymatic mechanism that depends on the active participation of the substrate itself, and introduce the new paradigm of substrate allostery. Less |Related Solutions: Rock Imager^®

The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules protein sidechains and backbone carbonyls As in aqueous solution protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores To engineer ...More |Related Solutions: NT8^®

The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules, protein sidechains, and backbone carbonyls. As in aqueous solution, protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores. To engineer proton-conductive pathways, therefore, we must explicitly account for the dynamic behavior of these networks. In previous work, we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient, single-file water wires that enable proton-selective transport. Here, we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions to extend connectivity across the pore. We find that the position of Ser relative to Gln modulates sidechain dynamics and, in turn, channel hydration. Although increased polarity reduces hydrophobic length and enhances hydration, these effects alone do not explain the observed conduction rates. Instead, asymmetry in the arrangement and dynamics of polar sidechains emerges as a key determinant of proton conductivity. Together, these results demonstrate that proton conduction is governed not only by pore polarity and hydration, but also by the dynamic and asymmetric organization of hydrogen-bonding networks. This work establishes design principles for engineering proton-selective channels and reveals how asymmetry enables efficient proton transport across biological membranes. Less |Related Solutions: NT8^®