Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes affected in neural diseases. Monitoring this process, particularly synaptic vesicle recycling, in living cells has been instrumental in revealing the mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from limitations, such as large probe sizes or limited compatibility for human neurons, hampering the quantitative analysis of synaptic pathophysiology. Here, we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes that target the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, noninvasive imaging and functional interrogation of Syt1 exo-endocytosis and trafficking in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed- and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for studying synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.

Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (microED), is an emerging method for determining structures from submicron-sized crystals. With the development of rapid and convenient data collection protocols, acquiring dozens of datasets in a single 3D ED/microED session has become routine. A fast and automated workflow for processing, scaling and merging a large number of 3D ED/microED datasets can significantly accelerate the structure determination process. Herein, we present an XDS-based pipeline with a graphical user interface for automated real-time and offline batch 3D ED/microED data processing (AutoLEI). We demonstrate the functionality and applications of the pipeline through four examples, using both offline and real-time data processing capabilities. The samples include small organic molecules, metal–organic frameworks (MOFs) and proteins, showcasing the versatility and efficiency of AutoLEI in various applications.

Bifunctional DNA glycosylases employ an active site lysine or the N-terminus to form a Schiff base with an abasic (AP) site base excision repair intermediate. For 8-oxoguanine DNA glycosylase 1 (OGG1), cleaving this reversible structure is the rate-determining step in the initiation of 8-oxoguanine (8-oxoG) repair in DNA. Evolution has led OGG1 to use a product-assisted catalysis approach, where the excised 8-oxoG acts as a Brønsted base for cleavage of a Schiff base intermediate. However, the physicochemical properties of 8-oxoG significantly limit the inherent enzymatic turnover leading to a weak, cellularly absent, AP lyase activity. We hypothesized that chemical synthesis of purine analogues enables access to complex structures that are suitable as product-like catalysts. Herein, the nucleobase landscape is profiled for its potential to increase OGG1 Schiff base cleavage. 8-Substituted 6-thioguanines emerge as potent and selective scaffolds enabling OGG1 to cleave AP sites opposite any canonical nucleobase by β-elimination. This effectively broadens the enzymatic substrate scope of OGG1, shaping a complete, artificial AP-lyase function. In addition, a second class of compounds, 6-substituted pyrazolo-[3,4-d]-pyrimidines, stimulate OGG1 function at high pH, while thioguanines govern enzymatic control at acidic pH. This enables up to 20-fold increased enzyme turnover and a de novo OGG1 β-elimination in conditions commonly not tolerated. The tool compounds employed here are non-toxic in cells and stimulate the repair of AP sites through a natural, APE1 dependent pathway, as opposed to previously reported β,δ-lyase stimulator TH10785.

Human single-strand-selective monofunctional uracil DNA glycosylase 1 (hSMUG1) removes uracil, 5-hydroxymethyluracil (5hmU) and 5-fluorouracil (5FU) from DNA, thereby initiating the base excision repair (BER) process. hSMUG1 is important for maintaining genomic integrity and plays a significant role in cancer biology. Here, we present the structures of hSMUG1, including complexes with products (uracil and 5FU) and an enzyme-product complex of hSMUG1 with double-stranded DNA (dsDNA). Analysis of our hSMUG1-dsDNA complex reveals how uracil is flipped out of the dsDNA for excision and identifies key residues that we confirm to be critical for both DNA binding and enzymatic activity. Furthermore, our hSMUG1 substrate complexes, molecular dynamics simulations and neutron diffraction data suggest a mechanism by which the substrate uracil rotates following base excision. The structural and functional information presented here will be highly useful for the future development of inhibitors and/or activators targeting hSMUG1.

The base excision repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) plays a central role in maintaining genome integrity and mediating cellular responses to oxidative stress. As such, it represents an attractive target for pharmaceutical modulation. Small-molecule organocatalytic switches (ORCAs) greatly enhance the rate of OGG1-catalyzed cleavage of DNA abasic sites, thereby accelerating DNA repair. Here, we present the discovery and hit-to-lead optimization of a novel class of highly potent serotonin-derived ORCAs with greatly improved pharmacokinetic properties. Biochemical assays, X-ray crystallography, and molecular dynamics simulations point toward a water-mediated mechanism of activation, distinct from previously proposed Brønsted base-assisted models. These findings establish serotonin-based ORCAs as promising chemical probes and potential leads for therapeutic modulation of OGG1 in oxidative stress-driven diseases.

Galactolipids are characteristic lipids of the photosynthesis membranes of higher plants and cyanobacteria. Due to their close relationship to the stability of the photosystem protein complexes, the biogenesis of galactolipids has been intensively studied on the genetic and molecular levels. There are two major types of galactolipids in chloroplastic membranes: monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG). Under phosphate-limiting conditions, the amount of DGDG increases dramatically to allow for phosphate salvage from phospholipids. In Arabidopsis thaliana, the membrane-associated glycosyltransferase digalactosyldiacylglycerol synthase 2 (atDGD2) is highly responsive to phosphate starvation and is significantly upregulated during such conditions. The lipid galactosylation reactions are also fundamentally interesting as they require a catalyst that is capable of bringing a hydrophilic and lipophilic substrate together at the solution-membrane phase border. Here, we present the X-ray crystal structure of atDGD2, which is the first reported DGDG synthase structure. AtDGD2 is most structurally similar to functionally unrelated GT-B enzymes. Interestingly, in spite of significant donor substrate binding differences, we identified four amino acids (Gly22, His151, Lys243, and Glu321, atDGD2 numbering) which were entirely conserved between the structurally similar enzymes. We also investigated the membrane interaction kinetics and membrane anchoring mechanism of atDGD2. This demonstrated that atDGD2 is membrane-bound but also showed that membrane binding is highly dynamic. Furthermore, our structural information in context of previous biophysical studies highlights regions of the enzyme exhibiting a high degree of structural plasticity, which we propose to be important for allowing atDGD2 to quickly adapt its activity based on the membrane lipid environment.

Human MutT homolog 1 (hMTH1) removes damaged nucleotides from the nucleotide pool, preventing their incorporation into DNA. Due to its potential as an anticancer drug target, hMTH1 has been the focus of several inhibitor development studies. Unexpectedly, we show that the anabolic steroid stanozolol (Stz) is a potent nanomolar inhibitor of hMTH1. We present the structure of hMTH1 in complex with Stz, which indicates a unique core scaffold that could be exploited for future inhibitor development. Comparison with human protein structures bound with dihydrotestosterone (DHT) shows hMTH1 is entirely unrelated in terms of its structure. As these DHT binding proteins are all involved in steroid regulation, this makes the identification of Stz as a potent hMTH1 inhibitor all the more unusual.

Fragment-based screening can catalyze drug discovery by identifying novel scaffolds, but this approach is limited by the small chemical libraries studied by biophysical experiments and the challenging optimization process. To expand the explored chemical space, we employ structure-based docking to evaluate orders-of-magnitude larger libraries than those used in traditional fragment screening. We computationally dock a set of 14 million fragments to 8-oxoguanine DNA glycosylase (OGG1), a difficult drug target involved in cancer and inflammation, and evaluate 29 highly ranked compounds experimentally. Four of these bind to OGG1 and X-ray crystallography confirms the binding modes predicted by docking. Furthermore, we show how fragment elaboration using searches among billions of readily synthesizable compounds identifies submicromolar inhibitors with anti-inflammatory and anti-cancer effects in cells. Comparisons of virtual screening strategies to explore a chemical space of 10²² compounds illustrate that fragment-based design enables enumeration of all molecules relevant for inhibitor discovery. Virtual fragment screening is hence a highly efficient strategy for navigating the rapidly growing combinatorial libraries and can serve as a powerful tool to accelerate drug discovery efforts for challenging therapeutic targets.

Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 105 m−1·s−1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 104 M−1·s−1. LC–MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism.

8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G : C→T : A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N-piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of the most potent member of the class, TH8535. Here, the N-Piperidinyl-linker adopts a chair instead of a boat conformation which was found for weaker analogues. We further demonstrate cellular target engagement and efficacy of TH8535 against a number of cancer cell lines.