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WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms 1-5 . Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Alzheimer's Disease (AD) is the most widespread form of dementia, with one of the pathological hallmarks being the formation of neurofibrillary tangles (NFTs). These tangles consist of phosphorylated Tau fragments. Asparagine endopeptidase (AEP) is a key Tau cleaving enzyme that generates aggregation-prone Tau fragments. Inhibition of AEP to reduce the level of toxic Tau fragment formation could represent a promising therapeutic strategy. Here, we report the first orthosteric, selective, orally bioavailable, and brain penetrant inhibitors with an irreversible binding mode. We outline the development of the series starting from reversible molecules and demonstrate the link between inhibition of AEP and reduction of Tau N368 fragment both in vitro and in vivo .

The conduit flow process (CFP) for MODFLOW's groundwater flow model is an advanced approach for investigating complex groundwater systems, such as karst, with coupled discrete-continuum models. CFP represents laminar and turbulent flow in a discrete pipe network coupled to a matrix continuum. However, the preprocessing demand is comparatively high to generate the conduit network and is usually performed with graphical user interfaces. To overcome this limitation and allow a scalable, reproducible, and comprehensive workflow, existing and new routines were aggregated to a Python package named CFPy, to allow script-based modeling that harmonizes well with the available and widely used FloPy package. CFPy allows information about the location and geometry of the conduit network to be considered by user-specific approaches or by sophisticated methods such as stochastic conduit network generators. The latter allows the automatic generation of many model variants with differing conduit networks for advanced investigations like multi-model approaches in combination with automatic parameter estimation. Additional postprocessing routines provide powerful control and valuable insights for CFP applications. In this methods note, a general technical description of the approach is complemented with two examples that guide users and demonstrate the main capabilities of CFPy., (© 2023 The Authors. Groundwater published by Wiley Periodicals LLC on behalf of National Ground Water Association.)

Successful structure-based drug design (SBDD) requires the optimization of interactions with the target protein and the minimization of ligand strain. Both factors are often modulated by small changes in the chemical structure which can lead to profound changes in the preferred conformation and interaction preferences of the ligand. We draw from examples of a Roche project targeting phosphodiesterase 10 to highlight that details matter in SBDD. Data mining in crystal structure databases can help to identify these sometimes subtle effects, but it is also a great resource to learn about molecular recognition in general and can be used as part of molecular design tools. We illustrate the use of the Cambridge Structural Database for identifying preferred structural motifs for intramolecular hydrogen bonding and of the Protein Data Bank for deriving propensities for protein-ligand interactions., (Copyright 2023 Bernd Kuhn, Jens-Uwe Peters, Markus G. Rudolph, Peter Mohr, Martin Stahl, Andreas Tosstorff. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

The rise of multidrug-resistant (MDR) Gram-negative bacteria is a major global health problem necessitating the discovery of new classes of antibiotics. Novel bacterial topoisomerase inhibitors (NBTIs) target the clinically validated bacterial type II topoisomerases with a distinct binding site and mechanism of action to fluoroquinolone antibiotics, thus avoiding cross-resistance to this drug class. Here we report the discovery of a series of NBTIs incorporating a novel indane DNA binding moiety. X-ray cocrystal structures of compounds 2 and 17a bound to Staphylococcus aureus DNA gyrase-DNA were determined, revealing specific interactions with the enzyme binding pocket at the GyrA dimer interface and a long-range electrostatic interaction between the basic amine in the linker and the carboxylate of Asp83. Exploration of the structure-activity relationship within the series led to the identification of lead compound 18c , which showed potent broad-spectrum activity against a panel of MDR Gram-negative bacteria., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a β-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the β-bulge loop is required for supercoiling activity. It has previously been shown that the β-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a β-bulge loop as a minimal latch is reported. It is shown that the β-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk., (open access.)

Karst aquifers are important sources of fresh water on a global scale. The hydrological modelling of karst spring discharge, however, still poses a challenge. In this study we apply a transfer function noise (TFN) model in combination with a bucket-type recharge model to simulate karst spring discharge. The application of the noise model for the residual series has the advantage that it is more consistent with assumptions for optimization such as homoscedasticity and independence. In an earlier hydrological modeling study, named Karst Modeling Challenge (KMC; Jeannin et al., J Hydrol 600:126-508, 2021), several modelling approaches were compared for the Milandre Karst System in Switzerland. This serves as a benchmark and we apply the TFN model to KMC data, subsequently comparing the results to other models. Using different data-model-combinations, the most promising data-model-combination is identified in a three-step least-squares calibration. To quantify uncertainty, the Bayesian approach of Markov-chain Monte Carlo (MCMC) sampling is subsequently used with uniform priors for the previously identified best data-model combination. The MCMC maximum likelihood solution is used to simulate spring discharge for a previously unseen testing period, indicating a superior performance compared to all other models in the KMC. It is found that the model gives a physically feasible representation of the system, which is supported by field measurements. While the TFN model simulated rising limbs and flood recession especially well, medium and baseflow conditions were not represented as accurately. The TFN approach poses a well-performing data-driven alternative to other approaches that should be considered in future studies., Competing Interests: Conflict of interestThe authors have no competing interests to declare that are relevant to the content of this article., (© The Author(s) 2023.)

We release a new, high quality data set of 1162 PDE10A inhibitors with experimentally determined binding affinities together with 77 PDE10A X-ray co-crystal structures from a Roche legacy project. This data set is used to compare the performance of different 2D- and 3D-machine learning (ML) as well as empirical scoring functions for predicting binding affinities with high throughput. We simulate use cases that are relevant in the lead optimization phase of early drug discovery. ML methods perform well at interpolation, but poorly in extrapolation scenarios-which are most relevant to a real-world application. Moreover, we find that investing into the docking workflow for binding pose generation using multi-template docking is rewarded with an improved scoring performance. A combination of 2D-ML and 3D scoring using a modified piecewise linear potential shows best overall performance, combining information on the protein environment with learning from existing SAR data., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The autotaxin-lysophosphatidic acid (ATX-LPA) signaling pathway plays a role in a variety of autoimmune diseases, such as rheumatoid arthritis or neurodegeneration. A link to the pathogenesis of glaucoma is suggested by an overactive ATX-LPA axis in aqueous humor samples of glaucoma patients. Analysis of such samples suggests that the ATX-LPA axis contributes to the fibrogenic activity and resistance to aqueous humor outflow through the trabecular meshwork. In order to inhibit or modulate this pathway, we developed a new series of ATX-inhibitors containing novel bicyclic and spirocyclic structural motifs. A potent lead compound (IC 50 against ATX: 6 nM) with good in vivo PK, favorable in vitro property, and safety profile was generated. This compound leads to lowered LPA levels in vivo after oral administration. Hence, it was suitable for chronic oral treatment in two rodent models of glaucoma, the experimental autoimmune glaucoma (EAG) and the ischemia/reperfusion models. In the EAG model, rats were immunized with an optic nerve antigen homogenate, while controls received sodium chloride. Retinal ischemia/reperfusion (I/R) was induced by elevating the intraocular pressure (IOP) in one eye to 140 mmHg for 60 min, followed by reperfusion, while the other untreated eye served as control. Retinae and optic nerves were evaluated 28 days after EAG or 7 and 14 days after I/R induction. Oral treatment with the optimized ATX-inhibitor lead to reduced retinal ganglion cell (RGC) loss in both glaucoma models. In the optic nerve, the protective effect of ATX inhibition was less effective compared to the retina and only a trend to a weakened neurofilament distortion was detectable. Taken together, these results provide evidence that the dysregulation of the ATX-LPA axis in the aqueous humor of glaucoma patients, in addition to the postulated outflow impairment, might also contribute to RGC loss. The observation that ATX-inhibitor treatment in both glaucoma models did not result in significant IOP increases or decreases after oral treatment indicates that protection from RGC loss due to inhibition of the ATX-LPA axis is independent of an IOP lowering effect., Competing Interests: DH, PM, PS, PD, JH, MGR, JB, MS, BG, SM, RG, and CU are employees at F. Hoffmann-La Roche Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hunziker, Reinehr, Palmhof, Wagner, Biniasch, Stute, Mattei, Schmitz, DiGiorgio, Hert, Rudolph, Benz, Stihle, Gsell, Müller, Gasser, Schonhoven, Ullmer and Joachim.)

Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor-associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Richter et al.)

The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3 -/- mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3 -/- mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population.

Doublecortin, a microtubule-associated protein that is only produced during neurogenesis, cooperatively binds to microtubules and stimulates microtubule polymerization and cross-linking by unknown mechanisms. A domain swap is observed in the crystal structure of the C-terminal domain of doublecortin. As determined by analytical ultracentrifugation, an open conformation is also present in solution. At higher concentrations, higher-order oligomers of the domain are formed. The domain swap and additional interfaces observed in the crystal lattice can explain the formation of doublecortin tetramers or multimers, in line with the analytical ultracentrifugation data. Taken together, the domain swap offers a mechanism for the observed cooperative binding of doublecortin to microtubules. Doublecortin-induced cross-linking of microtubules can be explained by the same mechanism. The effect of several mutations leading to lissencephaly and double-cortex syndrome can be traced to the domain swap and the proposed self-association of doublecortin.

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The Drug Design Data Resource (D3R) ran Grand Challenge 2 (GC2) from September 2016 through February 2017. This challenge was based on a dataset of structures and affinities for the nuclear receptor farnesoid X receptor (FXR), contributed by F. Hoffmann-La Roche. The dataset contained 102 IC50 values, spanning six orders of magnitude, and 36 high-resolution co-crystal structures with representatives of four major ligand classes. Strong global participation was evident, with 49 participants submitting 262 prediction submission packages in total. Procedurally, GC2 mimicked Grand Challenge 2015 (GC2015), with a Stage 1 subchallenge testing ligand pose prediction methods and ranking and scoring methods, and a Stage 2 subchallenge testing only ligand ranking and scoring methods after the release of all blinded co-crystal structures. Two smaller curated sets of 18 and 15 ligands were developed to test alchemical free energy methods. This overview summarizes all aspects of GC2, including the dataset details, challenge procedures, and participant results. We also consider implications for progress in the field, while highlighting methodological areas that merit continued development. Similar to GC2015, the outcome of GC2 underscores the pressing need for methods development in pose prediction, particularly for ligand scaffolds not currently represented in the Protein Data Bank ( http://www.pdb.org ), and in affinity ranking and scoring of bound ligands.

Microbial transglutaminases (MTGs) catalyze the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications ( e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates ( i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies. KalbTG was produced in Escherichia coli as soluble and active enzyme in the presence of its natural inhibitor ammonium to prevent potentially toxic cross-linking activity. The crystal structure of KalbTG revealed a conserved core similar to other MTGs but very short surface loops, making it the smallest MTG characterized to date. Ultra-dense peptide array technology involving a pool of 1.4 million unique peptides identified specific recognition motifs for KalbTG in these peptides. We determined that the motifs YRYRQ and RYESK are the best Gln and Lys substrates of KalbTG, respectively. By first reacting a bifunctionalized peptide with the more specific KalbTG and in a second step with the less specific MTG from Streptomyces mobaraensis , a successful bio-orthogonal labeling system was demonstrated. Fusing the KalbTG recognition motif to an antibody allowed for site-specific and ratio-controlled labeling using low label excess. Its site specificity, favorable kinetics, ease of use, and cost-effective production render KalbTG an attractive tool for a broad range of applications, including production of therapeutic antibody-drug conjugates., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.

Fructose-1,6-bisphosphatase (FBPase) is a key regulator of gluconeogenesis and a potential drug target for type 2 diabetes. FBPase is a homotetramer of 222 symmetry with a major and a minor dimer interface. The dimers connected via the minor interface can rotate with respect to each other, leading to the inactive T-state and active R-state conformations of FBPase. Here, the first crystal structure of human liver FBPase in the R-state conformation is presented, determined at a resolution of 2.2 Å in a tetragonal setting that exhibits an unusual arrangement of noncrystallographic symmetry (NCS) elements. Self-Patterson function analysis and various intensity statistics revealed the presence of pseudo-translation and the absence of twinning. The space group is P4 1 2 1 2, but structure determination was also possible in space groups P4 3 2 1 2, P4 1 22 and P4 3 22. All solutions have the same arrangement of three C 2 -symmetric dimers spaced by 1/3 along an NCS axis parallel to the c axis located at (1/4, 1/4, z), which is therefore invisible in a self-rotation function analysis. The solutions in the four space groups are related to one another and emulate a body-centred lattice. If all NCS elements were crystallographic, the space group would be I4 1 22 with a c axis three times shorter and a single FBPase subunit in the asymmetric unit. I4 1 22 is a minimal, non-isomorphic supergroup of the four primitive tetragonal space groups, explaining the space-group ambiguity for this crystal.

Dual inhibition of fatty acid binding proteins 4 and 5 (FABP4 and FABP5) is expected to provide beneficial effects on a number of metabolic parameters such as insulin sensitivity and blood glucose levels and should protect against atherosclerosis. Starting from a FABP4 selective focused screening hit, biostructure information was used to modulate the selectivity profile in the desired way and to design potent dual FABP4/5 inhibitors with good selectivity against FABP3. With very good pharmacokinetic properties and no major safety alerts, compound 12 was identified as a suitable tool compound for further in vivo investigations., (Copyright © 2016. Published by Elsevier Ltd.)

Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.

DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

Nuclear hormone receptors are cytoplasm-based transcription factors that bind a ligand, translate to the nucleus and initiate gene transcription in complex with a co-activator such as TIF2 (transcriptional intermediary factor 2). For structural studies the co-activator is usually mimicked by a peptide of circa 13 residues, which for the largest part forms an α-helix when bound to the receptor. The aim was to co-crystallize the glucocorticoid receptor in complex with a ligand and the TIF2 co-activator peptide. The 1.82 Å resolution diffraction data obtained from the crystal could not be phased by molecular replacement using the known receptor structures. HPLC analysis of the crystals revealed the absence of the receptor and indicated that only the co-activator peptide was present. The self-rotation function displayed 13-fold rotational symmetry, which initiated an exhaustive but unsuccessful molecular-replacement approach using motifs of 13-fold symmetry such as α- and β-barrels in various geometries. The structure was ultimately determined by using a single α-helix and the software ARCIMBOLDO, which assembles fragments placed by PHASER before using them as seeds for density modification model building in SHELXE. Systematic variation of the helix length revealed upper and lower size limits for successful structure determination. A beautiful but unanticipated structure was obtained that forms superhelices with left-handed twist throughout the crystal, stabilized by ligand interactions. Together with the increasing diversity of structural elements in the Protein Data Bank the results from TIF2 confirm the potential of fragment-based molecular replacement to significantly accelerate the phasing step for native diffraction data at around 2 Å resolution.

Methylation catalysed by catechol-O-methyltransferase (COMT) is the main pathway of catechol neurotransmitter deactivation in the prefrontal cortex. Low levels of this class of neurotransmitters are held to be causative of diseases such as schizophrenia, depression and Parkinson's disease. Inhibition of COMT may increase neurotransmitter levels, thus offering a route for treatment. Structure-based drug design hitherto seems to be based on the closed enzyme conformation. Here, a set of apo, semi-holo, holo and Michaelis form crystal structures are described that define the conformational space available to COMT and that include likely intermediates along the catalytic pathway. Domain swaps and sizeable loop movements around the active site testify to the flexibility of this enzyme, rendering COMT a difficult drug target. The low affinity of the co-substrate S-adenosylmethionine and the large conformational changes involved during catalysis highlight significant energetic investment to achieve the closed conformation. Since each conformation of COMT is a bona fide target for inhibitors, other states than the closed conformation may be promising to address. Crystallographic data for an alternative avenue of COMT inhibition, i.e. locking of the apo state by an inhibitor, are presented. The set of COMT structures may prove to be useful for the development of novel classes of inhibitors.

Lysobacter enzymogenes lysyl endoproteinase (LysC) is a trypsin-type serine protease with a high pH optimum that hydrolyses all Lys-Xaa peptide bonds. The high specificity of LysC renders it useful for biotechnological purposes. The K30R variant of a related lysyl endoproteinase from Achromobacter lyticus has favourable enzymatic properties that might be transferrable to LysC. To visualize structural differences in the substrate-binding sites, the crystal structures of wild-type and the K30R variant of LysC were determined. The mutation is located at a distance of 12 Å from the catalytic triad and subtly changes the surface properties of the substrate-binding site. The high pH optimum of LysC can be attributed to electrostatic effects of an aromatic Tyr/His stack on the catalytic aspartate and is a general feature of this enzyme subfamily. LysC crystals in complex with the covalent inhibitor N(α)-p-tosyl-lysyl chloromethylketone yielded data to 1.1 and 0.9 Å resolution, resulting in unprecedented precision of the active and substrate-binding sites for this enzyme subfamily. Error estimates on bond lengths and difference electron density indicate that instead of the expected oxyanion a hydroxyl group binds to the partially solvent-exposed oxyanion hole. Protonation of the alkoxide catalytic intermediate might be a recurring feature during serine protease catalysis.

Human β-galactoside α-2,6-sialyltransferase I (ST6Gal-I) establishes the final glycosylation pattern of many glycoproteins by transferring a sialyl moiety to a terminal galactose. Complete sialylation of therapeutic immunoglobulins is essential for their anti-inflammatory activity and protein stability, but is difficult to achieve in vitro owing to the limited activity of ST6Gal-I towards some galactose acceptors. No structural information on ST6Gal-I that could help to improve the enzymatic properties of ST6Gal-I for biotechnological purposes is currently available. Here, the crystal structures of human ST6Gal-I in complex with the product cytidine 5'-monophosphate and in complex with cytidine and phosphate are described. These complexes allow the rationalization of the inhibitory activity of cytosine-based nucleotides. ST6Gal-I adopts a variant of the canonical glycosyltransferase A fold and differs from related sialyltransferases by several large insertions and deletions that determine its regiospecificity and substrate specificity. A large glycan from a symmetry mate localizes to the active site of ST6Gal-I in an orientation compatible with catalysis. The glycan binding mode can be generalized to any glycoprotein that is a substrate of ST6Gal-I. Comparison with a bacterial sialyltransferase in complex with a modified sialyl donor lends insight into the Michaelis complex. The results support an SN2 mechanism with inversion of configuration at the sialyl residue and suggest substrate-assisted catalysis with a charge-relay mechanism that bears a conceptual similarity to serine proteases.

Type II DNA topoisomerases alter the supercoiling state of DNA in an ATP-dependent fashion that requires large conformational changes. The directionality of DNA strand transfer is controlled by three transient protein interfaces, termed the N-gate, DNA-gate, and C-gate. Bacterial gyrase is a type II DNA topoisomerase of A2B2 composition. The N-gate is formed by the two GyrB subunits and the GyrA subunits form the DNA- and C-gates. In structures of type II topoisomerase fragments, the DNA- and C-gates delimit a cavity for DNA and can be open or closed. However, the conformational space accessible has not yet been mapped. Here, we describe the crystal structure of the Bacillus subtilis DNA gyrase A subunit lacking the C-terminal DNA-wrapping domains. Five dimeric states of the GyrA N-terminal domain are observed, with their DNA- and C-gates either closed, or open to different extents. All of these conformations can in principle accommodate double-stranded DNA in the central cavity but only one conformation has its DNA-gate open wide enough for DNA to enter. The structure thus reflects the lower limit of DNA-gate opening that must occur during gyrase catalysis. The DNA-gate is formed by two flat surfaces, with few interactions. In contrast, the C-gate exhibits a highly undulated surface and forms a large number of interactions. None of the dimers in the crystal structures display an open C-gate that would allow DNA passage, in agreement with a transient opening of this gate during the catalytic cycle of DNA supercoiling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for "heat-resistant RNA-binding ATPase") contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA-protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented.

Venom-derived peptide toxins can modify the gating characteristics of excitatory channels in neurons. How they bind and interfere with the flow of ions without directly blocking the ion permeation pathway remains elusive. Here we report the crystal structure of the trimeric chicken Acid-sensing ion channel 1 in complex with the highly selective gating modifier Psalmotoxin 1 at 3.0 Å resolution. The structure reveals the molecular interactions of three toxin molecules binding at the proton-sensitive acidic pockets of Acid-sensing ion channel 1 and electron density consistent with a cation trapped in the central vestibule above the ion pathway. A hydrophobic patch and a basic cluster are the key structural elements of Psalmotoxin 1 binding, locking two separate regulatory regions in their relative, desensitized-like arrangement. Our results provide a general concept for gating modifier toxin binding suggesting that both surface motifs are required to modify the gating characteristics of an ion channel.

The biological activity of catechol neurotransmitters such as dopamine in the synapse is modulated by transporters and enzymes. Catechol-O-methyltransferase (COMT; EC 2.1.1.6) inactivates neurotransmitters by catalyzing the transfer of a methyl group from S-adenosylmethionine to catechols in the presence of Mg²⁺. This pathway also inactivates L-DOPA, the standard therapeutic for Parkinson's disease. Depletion of catechol neurotransmitters in the prefrontal cortex has been linked to schizophrenia. The inhibition of COMT therefore promises improvements in the treatment of these diseases. The concept of bisubstrate inhibitors for COMT has been described previously. Here, ribose-modified bisubstrate inhibitors were studied. Three high-resolution crystal structures of COMT in complex with novel ribose-modified bisubstrate inhibitors confirmed the predicted binding mode but displayed subtle alterations at the ribose-binding site. The high affinity of the inhibitors can be convincingly rationalized from the structures, which document the possibility of removing and/or replacing the ribose 3'-hydroxyl group and provide a framework for further inhibitor design.

Reverse gyrase is the only enzyme known to introduce positive supercoils into DNA. Positive supercoiling is achieved by the functional cooperation of a helicase-like and a topoisomerase domain. The isolated helicase-like domain is a DNA-stimulated ATPase, and the isolated topoisomerase domain can relax supercoiled DNA. In the context of reverse gyrase, these individual activities are suppressed or attenuated. The helicase-like domain of Thermotoga maritima reverse gyrase is a nucleotide-dependent conformational switch that binds DNA and ATP cooperatively. It provides a nucleotide-dependent DNA-binding site to reverse gyrase and thus serves as a valuable model for the investigation of the effect of nucleotides on DNA processing by reverse gyrase that is key to its supercoiling activity. To improve our understanding of the structural basis for the functional cooperation of a helicase domain with a DNA topoisomerase, we have determined the structures of the isolated helicase-like domain of T. maritima reverse gyrase in five different conformations. Comparison of these structures reveals extensive domain flexibility in the absence of conformational restrictions by the topoisomerase that is consistent with single-molecule Förster resonance energy transfer experiments presented here. The structure of the first ADP-bound form provides novel details about nucleotide binding to reverse gyrase. It demonstrates that reverse gyrases use the canonical nucleotide binding mode common to superfamily 2 helicases despite large deviations in the conserved motifs. A characteristic insert region adopts drastically different structures in different reverse gyrases. Counterparts of this insert region are located at very different positions in other DNA-processing enzymes but may point toward a general role in DNA strand separation.

L-Dopa, the standard therapeutic for Parkinson's disease, is inactivated by the enzyme catechol-O-methyltransferase (COMT). COMT catalyzes the transfer of an activated methyl group from S-adenosylmethionine (SAM) to its catechol substrates, such as L-dopa, in the presence of magnesium ions. The molecular recognition properties of the SAM-binding site of COMT have been investigated only sparsely. Here, we explore this site by structural alterations of the adenine moiety of bisubstrate inhibitors. The molecular recognition of adenine is of special interest due to the great abundance and importance of this nucleobase in biological systems. Novel bisubstrate inhibitors with adenine replacements were developed by structure-based design and synthesized using a nucleosidation protocol introduced by Vorbrüggen and co-workers. Key interactions of the adenine moiety with COMT were measured with a radiochemical assay. Several bisubstrate inhibitors, most notably the adenine replacements thiopyridine, purine, N-methyladenine, and 6-methylpurine, displayed nanomolar IC(50) values (median inhibitory concentration) for COMT down to 6 nM. A series of six cocrystal structures of the bisubstrate inhibitors in ternary complexes with COMT and Mg(2+) confirm our predicted binding mode of the adenine replacements. The cocrystal structure of an inhibitor bearing no nucleobase can be regarded as an intermediate along the reaction coordinate of bisubstrate inhibitor binding to COMT. Our studies show that solvation varies with the type of adenine replacement, whereas among the adenine derivatives, the nitrogen atom at position 1 is essential for high affinity, while the exocyclic amino group is most efficiently substituted by a methyl group., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DEAD-box proteins disrupt or remodel RNA and protein/RNA complexes at the expense of ATP. The catalytic core is composed of two flexibly connected RecA-like domains. The N-terminal domain contains most of the motifs involved in nucleotide binding and serves as a minimalistic model for helicase/nucleotide interactions. A single conserved glutamine in the so-called Q-motif has been suggested as a conformational sensor for the nucleotide state. To reprogram the Thermus thermophilus RNA helicase Hera for use of oxo-ATP instead of ATP and to investigate the sensor function of the Q-motif, we analyzed helicase activity of Hera Q28E. Crystal structures of the Hera N-terminal domain Q28E mutant (TthDEAD_Q28E) in apo- and ligand-bound forms show that Q28E does change specificity from adenine to 8-oxoadenine. However, significant structural changes accompany the Q28E mutation, which prevent the P-loop from adopting its catalytically active conformation and explain the lack of helicase activity of Hera_Q28E with either ATP or 8-oxo-ATP as energy sources. 8-Oxo-adenosine, 8-oxo-AMP, and 8-oxo-ADP weakly bind to TthDEAD_Q28E but in non-canonical modes. These results indicate that the Q-motif not only senses the nucleotide state of the helicase but could also stabilize a catalytically competent conformation of the P-loop and other helicase signature motifs.

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain.

Structure-guided lead optimization of recently described benzimidazolyl acetamides addressed the key liabilities of the previous lead compound 1. These efforts culminated in the discovery of 4-{(S)-2-[2-(4-chloro-phenyl)-5,6-difluoro-benzoimidazol-1-yl]-2-cyclohexyl-acetylamino}-3-fluoro-benzoic acid 7g, a highly potent and selective FXR agonist with excellent physicochemical and ADME properties and potent lipid lowering activity after oral administration to LDL receptor deficient mice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Herein we describe the synthesis and structure activity relationship of a new class of FXR agonists identified from a high-throughput screening campaign. Further optimization of the original hits led to molecules that were highly active in an LDL-receptor KO model for dyslipidemia. The most promising candidate is discussed in more detail., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The protein kinase C and casein kinase 2 substrates in neurons (PACSINs) represent a subfamily of membrane-binding proteins characterized by an amino-terminal Bin-Amphiphysin-Rvs (F-BAR) domain. PACSINs link membrane trafficking with actin dynamics and regulate the localization of distinct cargo molecules. The F-BAR domain forms a dimer essential for lipid binding. We have obtained crystals of authentic murine PACSIN 2 that contain an ordered F-BAR domain, indicating that additional domains are flexibly connected to F-BAR. The structure shares similarity to other BAR domains and exhibits special features unique to PACSINs. These include the uneven distribution of charged residues on the concave molecular surface and a so-called wedge loop that is driven into the membrane upon binding of PACSIN. The murine PACSIN 2 F-BAR domain requires dimerization for sensing of curved membranes, and the present structure also provides a mechanism for higher-order oligomer formation. Importantly, comparison of murine with human and Drosophila PACSIN 2 F-BAR domains reveals stark differences in the orientation of distal helical segments leading to a wider crescent shape of murine PACSIN 2. We define hinge residues for these movements that may help PACSINs sense and concomitantly reinforce membrane curvature., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The human fatty acid synthase (FAS) is a key enzyme in the metabolism of fatty acids and a target for antineoplastic and antiobesity drug development. Due to its size and flexibility, structural studies of mammalian FAS have been limited to individual domains or intermediate-resolution studies of the complete porcine FAS. We describe the high-resolution crystal structure of a large part of human FAS that encompasses the tandem domain of beta-ketoacyl synthase (KS) connected by a linker domain to the malonyltransferase (MAT) domain. Hinge regions that allow for substantial flexibility of the subdomains are defined. The KS domain forms the canonical dimer, and its substrate-binding site geometry differs markedly from that of bacterial homologues but is similar to that of the porcine orthologue. The didomain structure reveals a possible way to generate a small and compact KS domain by omitting a large part of the linker and MAT domains, which could greatly aid in rapid screening of KS inhibitors. In the crystal, the MAT domain exhibits two closed conformations that differ significantly by rigid-body plasticity. This flexibility may be important for catalysis and extends the conformational space previously known for type I FAS and 6-deoxyerythronolide B synthase.

DEAD box family helicases consist of a helicase core that is formed by two flexibly linked RecA-like domains. The helicase activity can be regulated by N- or C-terminal extensions flanking the core. Thermus thermophilus heat resistant RNA-dependent ATPase (Hera) is the first DEAD box helicase that forms a dimer using a unique dimerization domain. In addition to the dimerization domain, Hera contains a C-terminal RNA binding domain (RBD) that shares sequence homology only to uncharacterized proteins of the Deinococcus/Thermus group. The crystal structure of Hera_RBD reveals the fold of an altered RNA recognition motif (RRM) with limited structural homology to the RBD of the DEAD box helicase YxiN from Bacillus subtilis. Comparison with RRM/RNA complexes shows that a RNA binding mode different than that suggested for YxiN, but similar to U1A, can be inferred for Hera. The orientation of the RBD relative to the helicase core was defined in a second crystal structure of a Hera fragment including the C-terminal RecA domain, the dimerization domain, and the RBD. The structures allow construction of a model for the entire Hera helicase dimer. A likely binding surface for large RNA substrates that spans both RecA-like domains and the RBD is identified.

Orotidine-5'-monophosphate decarboxylase (OMPD) catalyzes the decarboxylation of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate (UMP) in an extremely proficient manner. The reaction does not require any cofactors and proceeds by an unknown mechanism. In addition to decarboxylation, OMPD is able to catalyze other reactions. We show that several C6-substituted UMP derivatives undergo hydrolysis or substitution reactions that depend on a lysine residue (Lys314) in the OMPD active site. 6-Cyano-UMP is converted to UMP, and UMP derivatives with good leaving groups inhibit OMPD by a suicide mechanism in which Lys314 covalently binds to the substrate. These non-classical reactivities of human OMPD were characterized by cocrystallization and freeze-trapping experiments with wild-type OMPD and two active-site mutants by using substrate and inhibitor nucleotides. The structures show that the C6-substituents are not coplanar with the pyrimidine ring. The extent of this substrate distortion is a function of the substituent geometry. Structure-based mechanisms for the reaction of 6-substituted UMP derivatives are extracted in accordance with results from mutagenesis, mass spectrometry, and OMPD enzyme activity. The Lys314-based mechanisms explain the chemodiversity of OMPD, and offer a strategy to design mechanism-based inhibitors that could be used for antineoplastic purposes for example.

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

According to the docking studies and the analysis of a co-crystal structure of GW4064 with FXR, a series of 3-aryl heterocyclic isoxazole analogs were designed and synthesized. N-Oxide pyridine analog (7b) was identified as a promising FXR agonist with potent binding affinity and good efficacy, supporting our hypothesis that through an additional hydrogen bond interaction between the pyridine substituent of isoxazole analogs and Tyr373 and Ser336 of FXR, binding affinity and functional activity could be improved.