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Abstract: Purpose : Mutations in myocilin comprise the strongest genetic link to glaucoma, the leading cause of irreversible blindness worldwide. Whereas glaucoma typically affects the population over 40, those with myocilin mutations are on an accelerated timeline and lose vision often while still in childhood. It is estimated that 3 out of the 70 million glaucoma patients worldwide harbor pathogenic myocilin mutations. In this study we sought to identify small molecules that bind the olfactomedin (OLF) domain of myocilin, the relevant portion of myocilin for its association with glaucoma. Methods : The crystallization protocol for the OLF domain of myocilin was modified to suit the requirements for high throughput screening at the XCHEM facility (Diamond Light Source). Crystals were ranked using TeXRank. Per the SoakDB pipeline at XCHEM, after identifying suitable compound soaking parameters, the crystals were subjected to soaking with compounds from the in-house DSiP compound library (~800 compounds), which were dispensed using a Labcyte Echo acoustic liquid handling system. Crystals were harvested manually with the assistance with the Oxford Lab Shifter. Diffraction data were collected and processed using automated unmanned protocols at the beamline. Structures were solved using the XChem Explorer software pipeline. Structures of ligands bound to OLF were analyzed using Coot. Results : Crystallization of OLF was adapted to 384-well low volume plates. Rod-shaped diffraction quality crystals grew in ~80% of wells. DMSO was tolerated up to 20% for 3 h. Approximately 750 crystals were each soaked with a compound and harvested for diffraction data collection. Crystals diffracted variably depending on the compound soaked, and ~600 datasets were collected. Structures were refined and analyzed using XChem Explorer and PanDDAs. Approximately 170 datasets were further inspected for bound ligands, ~60 datasets were refined further. Bound ligands were identified at interfaces related to crystal packing as well as the molecular surface of OLF. Conclusions : High throughput crystallographic fragment screening has identified distinct sites for small molecules to bind to the myocilin OLF domain. These hit molecules serve as starting points for drug development for small molecules that can interfere with the misfolding pathogenic mechanism. In the long term, these efforts could lead to new treatments that target the underlying cause of myocilin-associated glaucoma.

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Abstract: We have developed sample-efficient delivery and reaction initiation strategies that use room temperature microcrystal slurries and serial crystallography methods for time-resolved studies [1-3]. However, interpreting electron density maps from reaction cycle intermediates can be challenging when mixtures of species are present in the data. Therefore, to help reduce ambiguity we and our collaborators have also pioneered strategies to simultaneously collect time-resolved serial crystallography (tr- SSX/tr-SFX) diffraction data in the forward direction, and X-ray emission spectroscopy (tr-XES) data at ∼ 90°, using either XFEL (tr-SFX) or synchrotron (tr-SSX) sources. The resulting atomic and electronic structures are fully correlated and have been applied to a range of enzymes [1, 2, 4-8]. For instance, isopenicillin N synthase (IPNS) uses nonheme iron to catalyse the O2- dependent conversion of its tripeptide substrate delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into isopenicillin N (IPN, the precursor of all penicillin/cephalosporin beta-lactam antibiotics). The unique four electron oxidation reaction leading to the beta-lactam bicyclic ring proceeds via two high-valent iron species, an Fe(III)-superoxo and a high-spin Fe(IV)=O oxyferryl species. These enable two sequential C-H bond cleavage steps that each exhibit large kinetic isotope effects (KIE). Our recent tr- SFX and tr-XES studies have characterised the Fe(III)-superoxo species and revealed unexpected, correlated motions throughout the whole protein caused by O2 binding [4].

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Abstract: PIF1 is a conserved helicase and G4 DNA binding and unwinding enzyme, with roles in genome stability. Human PIF1 (hPIF1) is poorly understood, but its functions can become critical for tumour cell survival during oncogene-driven replication stress. Here we report the discovery, via an X-ray crystallographic fragment screen (XChem), of hPIF1 DNA binding and unwinding inhibitors. A structure was obtained with a 4-phenylthiazol-2-amine fragment bound in a pocket between helicase domains 2A and 2B, with additional contacts to Valine 258 from domain 1A. The compound makes specific interactions, notably through Leucine 548 and Alanine 551, that constrain conformational adjustments between domains 2A and 2B, previously linked to ATP hydrolysis and DNA unwinding. We next synthesized a range of related compounds and characterized their effects on hPIF1 DNA-binding and helicase activity in vitro, expanding the structure activity relationship (SAR) around the initial hit. A systematic analysis of clinical cancer databases is also presented here, supporting the notion that hPIF1 upregulation may represent a specific cancer cell vulnerability. The research demonstrates that hPIF1 is a tractable target through 4-phenylthiazol-2-amine derivatives as inhibitors of its helicase action, setting a foundation for creation of a novel class of anti-cancer therapeutics.

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Abstract: Microbial growth often occurs within multicellular communities called biofilms, where cells are enveloped by a protective extracellular matrix. Bacillus subtilis serves as a model organism for biofilm research and produces two crucial secreted proteins, BslA and TasA, vital for biofilm matrix formation. BslA exhibits surface-active properties, spontaneously self-assembling at hydrophobic/hydrophilic interfaces to form an elastic protein film, which renders B. subtilis biofilm surfaces water-repellent. TasA is traditionally considered a fiber-forming protein with multiple matrix-related functions. In our current study, we investigate whether TasA also possesses interfacial properties and whether it has any impact on BslA’s ability to form an interfacial protein film. Our research demonstrates that TasA indeed exhibits interfacial activity, partitioning to hydrophobic/hydrophilic interfaces, stabilizing emulsions, and forming an interfacial protein film. Interestingly, TasA undergoes interface-induced restructuring similar to BslA, showing an increase in β-strand secondary structure. Unlike BslA, TasA rapidly reaches the interface and forms nonelastic films that rapidly relax under pressure. Through mixed protein pendant drop experiments, we assess the influence of TasA on BslA film formation, revealing that TasA and other surface-active molecules can compete for interface space, potentially preventing BslA from forming a stable elastic film. This raises a critical question: how does BslA self-assemble to form the hydrophobic “raincoat” observed in biofilms in the presence of other potentially surface-active species? We propose a model wherein surface-active molecules, including TasA, initially compete with BslA for interface space. However, under lateral compression or pressure, BslA retains its position, expelling other molecules into the bulk. This resilience at the interface may result from structural rearrangements and lateral interactions between BslA subunits. This combined mechanism likely explains BslA’s role in forming a stable film integral to B. subtilis biofilm hydrophobicity.

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Abstract: canSAR (https://cansar.ai) is the largest public cancer drug discovery and translational research knowledgebase. Now hosted in its new home at MD Anderson Cancer Center, canSAR integrates billions of experimental measurements from across molecular profiling, pharmacology, chemistry, structural and systems biology. Moreover, canSAR applies a unique suite of machine learning algorithms designed to inform drug discovery. Here, we describe the latest updates to the knowledgebase, including a focus on significant novel data. These include canSAR’s ligandability assessment of AlphaFold; mapping of fragment-based screening data; and new chemical bioactivity data for novel targets. We also describe enhancements to the data and interface.