Molecular basis of TASL recruitment by the peptide/histidine transporter 1, PHT1.

PHT1 is a histidine /oligopeptide transporter with an essential role in Toll-like receptor innate immune responses. It can act as a receptor by recruiting the adaptor protein TASL which leads to type I interferon production via IRF5. Persistent stimulation of this signalling pathway is known to be involved in the pathogenesis of systemic lupus erythematosus (SLE). Understanding how PHT1 recruits TASL at the molecular level, is therefore clinically important for the development of therapeutics against SLE and other autoimmune diseases. Here we present the Cryo-EM structure of PHT1 stabilized in the outward-open conformation. By combining biochemical and structural modeling techniques we propose a model of the PHT1-TASL complex, in which the first 16 N-terminal TASL residues fold into a helical structure that bind in the central cavity of the inward-open conformation of PHT1. This work provides critical insights into the molecular basis of PHT1/TASL mediated type I interferon production.

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. Lack of publicly available experimental structures make it challenging to discriminate between conflicting mechanisms proposed for proton-binding, as main roles have been assigned to either an extracellular histidine cluster or to an internal carboxylic triad. Here we present a protocol to derive and evaluate structural models of the proton-sensing GPR68. This approach integrates state-of-the-art homology modeling with microsecond-timescale atomistic simulations, and with a detailed assessment of the compatibility of the structural models with known structural features of class A GPCRs. To decipher structural elements of potential interest for protonation-coupled conformational changes of GPR68, we used the best-compatible model as a starting point for independent atomistic simulations of GPR68 with different protonation states, and graph computations to characterize the response of GPR68 to changes in protonation. We found that GPR68 hosts an extended hydrogen-bond network that inter-connects the extracellular histidine cluster to the internal carboxylic triad, and which can even reach groups at the cytoplasmic G-protein binding site. Taken together, results suggest that GPR68 relies on dynamic, hydrogen-bond networks to inter-connect extracellular and internal proton-binding sites, and to elicit conformational changes at the cytoplasmic G-protein binding site.

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. NSPs are activated during neutrophil myelopoiesis in bone marrow by cathepsin C (CatC; DPP1). CatC is therefore an attractive target to reduce NSP activity in the lungs of patients with bronchiectasis, restoring the protease/antiprotease balance. We report results from the preclinical pharmacological assessment of the novel CatC inhibitor BI 1291583. METHODS: Binding kinetics of BI 1291583 to human CatC were determined by surface plasmon resonance. In vitro inhibition of human CatC activity was determined by CatC-specific fluorescent assay, and selectivity was assessed against related cathepsins and unrelated proteases. Inhibition of NSP neutrophil elastase (NE) production was assessed in a human neutrophil progenitor cell line. In vivo inhibition of NE and NSP proteinase 3 (PR3) in bronchoalveolar lavage fluid (BALF) neutrophils after lipopolysaccharide (LPS) challenge and distribution of BI 1291583 was determined in a mouse model. RESULTS: BI 1291583 bound human CatC in a covalent, reversible manner, selectively and fully inhibiting CatC enzymatic activity. This inhibition translated to concentration-dependent inhibition of NE activation in U937 cells and dose-dependent, almost-complete inhibition of NE and PR3 activity in BALF neutrophils in an in vivo LPS-challenge model in mice. BI 1291583 exhibited up to 100 times the exposure in the target tissue bone marrow compared with plasma. CONCLUSION: BI 1291583-mediated inhibition of CatC is expected to restore the protease-antiprotease balance in the lungs of patients with chronic airway inflammatory diseases such as bronchiectasis.

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 Gi1 at 3.3 Å resolution. Combined with functional and computational data, the structure reveals the key factors that govern the recognition and selectivity of peptide agonist as well as non-peptide antagonist, providing the structural basis for design of novel and highly selective drugs targeting NMU2. In addition, a 25-degree rotation of Gi protein in reference to NMU2 is also observed compared in other structures of class A GPCR-Gi complexes, suggesting heterogeneity in the processes of G protein-coupled receptors (GPCRs) activation and G protein coupling.

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. We established a MALDI-TOF-MS-based furin activity assay, determined IC50 values, and solved X-ray structures of (3,5-dichlorophenyl)pyridine-derived compounds in complex with furin. The inhibitors induced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. These changes formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into a newly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strong structural stabilization in surface plasmon resonance and differential scanning fluorimetry experiments, respectively. The discovered furin conformation offers new opportunities for structure-based drug discovery.

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. Efficient separation of target-ligand complexes is achieved by in-plate size exclusion chromatography (SEC), and small-molecule ligands are subsequently identified by MALDI-TOF analysis. In contrast to alternative HTS-capable binding assay formats, MALDI-TOF AS-MS is capable of identifying orthosteric and allosteric ligands, as shown for the model system protein tyrosine phosphatase 1B (PTP1B), irrespective of protein function. Furthermore, determining relative binding affinities (RBAs) enabled ligand ranking in accordance with functional inhibition and reference data for PTP1B and a number of diverse protein targets. Finally, we present a validation screen of more than 23,000 compounds within 24 h, demonstrating the general applicability of the platform for the HTS-compatible assessment of protein-ligand interactions.

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. Linagliptin exhibited high binding affinity (KD ) and a slow off-rate (koff ) when dissociating from DPP-4 (KD 6.6 pM; koff 5.1×10-5  s-1 ), and weaker inhibitory potency to FAP (KD 301 nM; koff >1 s-1 ). Co-structures of linagliptin with DPP-4 or FAP were similar except for one second shell amino acid difference: Asp663 (DPP-4) and Ala657 (FAP). pH dependence of enzymatic activities and binding of linagliptin for DPP-4 and FAP are dependent on this single amino acid difference. While linagliptin may not display any anticancer activity at therapeutic doses, our findings may guide future studies for the development of optimized inhibitors.

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 (Mpro ) 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. We used an enzymatic assay measuring the inhibition of Mpro catalytic activity in the presence of four different commercially available gliptins (linagliptin, sitagliptin, alogliptin and saxagliptin) and several structural analogues of linagliptin to study the binding of DPP-4 inhibitors to Mpro and their functional activity. We show here that DPP-4 inhibitors like linagliptin, other gliptins and structural analogues are inactive against Mpro .

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. So far only a few compounds targeting oxLDL-LOX-1 interaction are reported with a limited level of characterization. Here we describe the identification and characterization of BI-0115, a selective small molecule inhibitor of LOX-1 that blocks cellular uptake of oxLDL. Identified by a high throughput screening campaign, biophysical analysis shows that BI-0115 binding triggers receptor inhibition by formation of dimers of the homodimeric ligand binding domain. The structure of LOX-1 bound to BI-0115 shows that inter-ligand interactions at the receptor interfaces are key to the formation of the receptor tetramer thereby blocking oxLDL binding.

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. We present the utilization of a MALDI-TOF-based screening platform to identify inhibitors of human cyclic GMP-AMP synthase (cGAS), a mediator of innate immune response whose aberration has been causally correlated to a number of inflammatory disorders. In this context, the development and validation of a MALDI-TOF-based activity assay is reported to demonstrate fast, robust, and accurate detection of chemical cGAS inhibition by direct quantification of the physiological reaction product cyclic GMP-ATP (cGAMP). Results from a screen of a diverse library of more than 1 million small molecules in 1536-well format against the catalytic cGAS activity are presented with excellent assay performance and data quality. Identified hits were qualified in dose-response experiments and confirmed by RapidFire-MS measurements. Conclusively, the presented data provide the first proof of applicability of direct automated MALDI-TOF MS as a readout strategy for large-scale drug discovery HTS campaigns.

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., (2019) Structure 27, 427) at 2.7 Å resolution. The CCR2 structure provides the structural basis for ligand selectivity of CCR2 against chemokine receptor 5 and provides insights into the residence time of MK-0812 analogs based on molecular dynamics simulations. These findings offer new opportunities for drug discovery targeting chemokine receptors.

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. Based on our recently solved CCR2 structure with MK-0812 and molecular dynamics (MD) simulations, we show that the RT for this and structurally related ligands is directly dependent on the shielding of the Tyr120-Glu291 H-bond from the water. If solvated this H-bond is often broken, making the binding pocket flexible and leading to shorter RT.

Allosteric Activation of Striatal-Enriched Protein Tyrosine Phosphatase (STEP, PTPN5) by a Fragment-like Molecule.

Protein tyrosine phosphatase non-receptor type 5 (PTPN5, STEP) is a brain specific phosphatase that regulates synaptic function and plasticity by modulation of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking. Dysregulation of STEP has been linked to neurodegenerative and neuropsychiatric diseases, highlighting this enzyme as an attractive therapeutic target for drug discovery. Selective targeting of STEP with small molecules has been hampered by high conservation of the active site among protein tyrosine phosphatases. We report the discovery of the first small molecule allosteric activator for STEP that binds to the phosphatase domain. Allosteric binding is confirmed by both X-ray and 15N NMR experiments, and specificity has been demonstrated by an enzymatic test cascade. Molecular dynamics simulations indicate stimulation of enzymatic activity by a long-range allosteric mechanism. To allow the scientific community to make use of this tool, we offer to provide the compound in the course of an open innovation initiative.

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. An N- and C-terminally truncated CCR2A construct was crystallized in an alternate crystal form, which yielded a 2.7 Å resolution structure using serial synchrotron crystallography. Our structures provide a clear structural explanation for the observed key role of residue E2917.39 in high-affinity binding of several orthosteric CCR2 antagonists. By combining all the structural information collected, we generated models of co-structures for the structurally diverse pyrimidine amide class of CCR2 antagonists. Even though the representative Ex15 overlays well with MK-0812, it also interacts with the non-conserved H1213.33, resulting in a significant selectivity over CCR5. Insights derived from this work will facilitate drug discovery efforts directed toward highly selective CCR2 antagonists with potentially superior efficacy.

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, A233D6.33 and K303E7.59. With the goal of testing the transferability of these two thermostabilizing mutations in other chemokine receptors, we tested the mutations A237D6.33 and R307E7.59 in human CCR3 for thermostability and aggregation properties in detergent solution. Interestingly, the double mutant exhibited a 6-10-fold decrease in the aggregation propensity of the wild-type protein. This is in stark contrast to the two single mutants whose aggregation properties resemble the wild type (WT). Moreover, unlike in CCR5, the two single mutants separately showed no increase in thermostability compared to the wild-type CCR3, while the double-mutant A237D6.33/R307E7.59 confers an increase of 2.6 °C in the melting temperature compared to the WT. Extensive all-atom molecular dynamics (MD) simulations in detergent micelles show that a salt bridge network between transmembrane helices TM3, TM6, and TM7 that is absent in the two single mutants confers stability in the double mutant. The free energy surface of the double mutant shows conformational homogeneity compared to the single mutants. An annular n-dodecyl maltoside detergent layer packs tighter to the hydrophobic surface of the double-mutant CCR3 compared to the single mutants providing additional stability. The purification of other C-C chemokine receptors lacking such stabilizing residues may benefit from the incorporation of these two point mutations.

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. Binding of all gliptins is enthalpy-dominated due to strong ionic interactions and strong solvent-shielded hydrogen bonds. Using a congeneric series of molecules which represented the intermediates in the lead optimization program of linagliptin, the onset of slow binding kinetics and development of the thermodynamic repertoire were analyzed in the context of incremental changes of the chemical structures. All compounds rapidly associated, and therefore the optimization of affinity and residence time is highly correlated. The major contributor to the increasing free energy of binding was a strong increase of binding enthalpy, whereas entropic contributions remained low and constant despite significant addition of lipophilicity.

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. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here, we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The cocrystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions -1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs.

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-ß are believed to play important roles in the pathogenesis of IPF. Nintedanib is a potent small molecule inhibitor of the receptor tyrosine kinases PDGF receptor, FGF receptor and vascular endothelial growth factor receptor. Data from in vitro studies have shown that nintedanib interferes with processes active in fibrosis such as fibroblast proliferation, migration and differentiation, and the secretion of ECM. In addition, nintedanib has shown consistent anti-fibrotic and anti-inflammatory activity in animal models of lung fibrosis. These data provide a strong rationale for the clinical efficacy of nintedanib in patients with IPF, which has recently been demonstrated in phase III clinical trials.

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. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic α-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des.2013, 14, 2846-2857). Here, we describe its application to the human α-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 °C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method.