Engineering oxypurinol-responsive riboswitches based on bacterial xanthine aptamers for gene expression control in mammalian cell culture.

Riboswitch-mediated control of gene expression without the interference of potentially immunogenic proteins is a promising approach for the development of tailor-made tools for biological research and the advancement of gene therapies. However, the current selection of applicable ligands for synthetic riboswitches is limited and strategies have mostly relied on de novo selection of aptamers. Here, we show that the bacterial xanthine I riboswitch aptamer recognizes oxypurinol, the active metabolite of the widely prescribed anti-gout drug allopurinol (Zyloprim®).

Protons taken hostage: Dynamic H-bond networks of the pH-sensing GPR68.

Proton-sensing G Protein Coupled Receptors (GPCRs) sense changes in the extracellular pH to effect cell signaling for cellular homeostasis. They tend to be overexpressed in solid tumors associated with acidic extracellular pH, and are of direct interest as drug targets. How proton-sensing GPCRs sense extracellular acidification and activate upon protonation change is important to understand, because it may guide the design of therapeutics.

BI 1291583: a novel selective inhibitor of cathepsin C with superior in vivo profile for the treatment of bronchiectasis.

Background: Airway inflammation in chronic inflammatory lung diseases (e.g. bronchiectasis) is partly mediated by neutrophil-derived serine protease (NSP)/antiprotease imbalance.

Ligand recognition and activation of neuromedin U receptor 2.

Neuromedin U receptor 2 (NMU2), an emerging attractive target for treating obesity, has shown the capability in reducing food intake and regulating energy metabolism when activated. However, drug development of NMU2 was deferred partially due to the lack of structural information. Here, we present the cryo-electron microscopy (cryo-EM) structure of NMU2 bound to the endogenous agonist NmU-25 and G at 3.

Dichlorophenylpyridine-Based Molecules Inhibit Furin through an Induced-Fit Mechanism.

Inhibitors of the proprotein convertase furin might serve as broad-spectrum antiviral therapeutics. High cellular potency and antiviral activity against acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we characterized the binding mechanism of this inhibitor class using structural, biophysical, and biochemical methods.

Action of Dipeptidyl Peptidase-4 Inhibitors on SARS-CoV-2 Main Protease.

In a recent publication, Eleftheriou et al. proposed that inhibitors of dipeptidyl peptidase-4 (DPP-4) are functional inhibitors of the main protease (M ) of SARS-CoV-2. Their predictions prompted the authors to suggest linagliptin, a DPP-4 inhibitor and approved anti-diabetes drug, as a repurposed drug candidate against the ongoing COVID-19 pandemic.

MALDI-TOF-Based Affinity Selection Mass Spectrometry for Automated Screening of Protein-Ligand Interactions at High Throughput.

Demonstration of in vitro target engagement for small-molecule ligands by measuring binding to a molecular target is an established approach in early drug discovery and a pivotal step in high-throughput screening (HTS)-based compound triaging. We describe the setup, evaluation, and application of a ligand binding assay platform combining automated affinity selection (AS)-based sample preparation and label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The platform enables mass spectrometry (MS)-based HTS for small-molecule target interactions from single-compound incubation mixtures and is embedded into a regular assay automation environment.

A Single Second Shell Amino Acid Determines Affinity and Kinetics of Linagliptin Binding to Type 4 Dipeptidyl Peptidase and Fibroblast Activation Protein.

Drugs targeting type 4 dipeptidyl peptidase (DPP-4) are beneficial for glycemic control, whereas fibroblast activation protein alpha (FAP-α) is a potential target for cancer therapies. Unlike other gliptins, linagliptin displays FAP inhibition. We compared biophysical and structural characteristics of linagliptin binding to DPP-4 and FAP to better understand what differentiates linagliptin from other gliptins.

A small-molecule inhibitor of lectin-like oxidized LDL receptor-1 acts by stabilizing an inactive receptor tetramer state.

The C-type lectin family member lectin-like oxidized LDL receptor-1 (LOX-1) has been object of intensive research. Its modulation may offer a broad spectrum of therapeutic interventions ranging from cardiovascular diseases to cancer. LOX-1 mediates uptake of oxLDL by vascular cells and plays an important role in the initiation of endothelial dysfunction and its progression to atherosclerosis.

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A.

The in meso in situ serial X-ray crystallization method (Huang et al., (2015) Acta Crystallogr D Biol Crystallogr 71, 1238) combines lipid cubic phase crystallization, direct freezing of the crystallization droplet without handling of the crystals, and data collection in situ. Recently, this method was used to overcome the mechanical fragility of crystals which enabled the X-ray structure determination of chemokine receptor 2A (Apel et al.

MALDI-TOF Mass Spectrometry-Based High-Throughput Screening for Inhibitors of the Cytosolic DNA Sensor cGAS.

Comprehensive and unbiased detection methods are a prerequisite for high-throughput screening (HTS) campaigns within drug discovery research. Label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an HTS-compatible readout for biochemical test systems to support the drug discovery process. So far, reported HTS applications were based on surface-modified systems or proof-of-concept studies.

Enhancing Drug Residence Time by Shielding of Intra-Protein Hydrogen Bonds: A Case Study on CCR2 Antagonists.

The target residence time (RT) for a given ligand is one of the important parameters that have to be optimized during drug design. It is well established that shielding the receptor-ligand hydrogen bond (H-bond) interactions from water has been one of the factors in increasing ligand RT. Building on this foundation, here we report that shielding an intra-protein H-bond, which confers rigidity to the binding pocket and which is not directly involved in drug-receptor interactions, can strongly influence RT for CCR2 antagonists.

Crystal Structure of CC Chemokine Receptor 2A in Complex with an Orthosteric Antagonist Provides Insights for the Design of Selective Antagonists.

We determined two crystal structures of the chemokine receptor CCR2A in complex with the orthosteric antagonist MK-0812. Full-length CCR2A, stabilized by rubredoxin and a series of five mutations were resolved at 3.3 Å.

Engineering Salt Bridge Networks between Transmembrane Helices Confers Thermostability in G-Protein-Coupled Receptors.

Introduction of specific point mutations has been an effective strategy in enhancing the thermostability of G-protein-coupled receptors (GPCRs). Our previous work showed that a specific residue position on transmembrane helix 6 (TM6) in class A GPCRs consistently yields thermostable mutants. The crystal structure of human chemokine receptor CCR5 also showed increased thermostability upon mutation of two positions, A233D and K303E.

Comparative Analysis of Binding Kinetics and Thermodynamics of Dipeptidyl Peptidase-4 Inhibitors and Their Relationship to Structure.

The binding kinetics and thermodynamics of dipeptidyl peptidase (DPP)-4 inhibitors (gliptins) were investigated using surface plasmon resonance and isothermal titration calorimetry. Binding of gliptins to DPP-4 is a rapid electrostatically driven process. Off-rates were generally slow partly because of reversible covalent bond formation by some gliptins, and partly because of strong and extensive interactions.

Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib.

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma.

Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease characterised by fibrosis of the lung parenchyma and loss of lung function. Although the pathogenic pathways involved in IPF have not been fully elucidated, IPF is believed to be caused by repetitive alveolar epithelial cell injury and dysregulated repair, in which there is uncontrolled proliferation of lung fibroblasts and differentiation of fibroblasts into myofibroblasts, which excessively deposit extracellular matrix (ECM) proteins in the interstitial space. A number of profibrotic mediators including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor-β are believed to play important roles in the pathogenesis of IPF.

Fast native-SAD phasing for routine macromolecular structure determination.

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

Crystallizing Membrane Proteins in the Lipidic Mesophase. Experience with Human Prostaglandin E2 Synthase 1 and an Evolving Strategy.

The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type.

Crystal structure of glucokinase regulatory protein.

Glucokinase (GK) plays a major role in the regulation of blood glucose homeostasis in both the liver and the pancreas. In the liver, GK is controlled by the GK regulatory protein (GKRP). GKRP in turn is activated by fructose 6-phosphate (F6P) and inactivated by fructose 1-phosphate (F1P).

A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems.

High amounts of membrane protein samples are needed for structural or functional analysis and a first bottleneck is often to obtain sufficient production efficiencies. The reduced complexity of protein production in cell-free expression systems results in a frequent correlation of efficiency problems with the essential transcription/translation process. We present a systematic tag variation strategy for the rapid improvement of cell-free expression efficiencies of membrane proteins based on the optimization of translation initiation.

Design, synthesis, and evaluation of indolinones as inhibitors of the transforming growth factor β receptor I (TGFβRI).

Inhibition of transforming growth factor β (TGFβ) type I receptor (Alk5) offers a novel approach for the treatment of fibrotic diseases and cancer. Indolinones substituted in position 6 were identified as a new chemotype inhibiting TGFβRI concomitant with a low cross-reactivity among the human kinome. A subset of compounds showed additional inhibition of platelet-derived growth factor receptor alpha (PDGFRα), contributing to an interesting pharmacological profile.