Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Glucocorticoid receptor Thr524 phosphorylation by MINK1 induces interactions with 14-3-3 protein regulators.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rho-associated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention.

Discovery of a selective c-MET inhibitor with a novel binding mode.

The c-MET receptor tyrosine kinase has received considerable attention as a cancer drug target yet there remains a need for inhibitors which are selective for c-MET and able to target emerging drug-resistant mutants. We report here the discovery, by screening a DNA-encoded chemical library, of a highly selective c-MET inhibitor which was shown by X-ray crystallography to bind to the kinase in an unprecedented manner. These results represent a novel mode of inhibiting c-MET with a small molecule and may provide a route to targeting drug-resistant forms of the kinase whilst avoiding potential toxicity issues associated with broad kinome inhibition.

AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins.

Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr24 and Ser256 in FOXO1 to stimulate binding of 14-3-3 proteins, causing FOXO1 inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser383 and Thr649 in FOXO1. In this study, we identified Ser22 as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser22 phosphorylation impairs Thr24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser383 and Thr649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity.

MraY-antibiotic complex reveals details of tunicamycin mode of action.

The rapid increase of antibiotic resistance has created an urgent need to develop novel antimicrobial agents. Here we describe the crystal structure of the promising bacterial target phospho-N-acetylmuramoyl-pentapeptide translocase (MraY) in complex with the nucleoside antibiotic tunicamycin. The structure not only reveals the mode of action of several related natural-product antibiotics but also gives an indication on the binding mode of the MraY UDP-MurNAc-pentapeptide and undecaprenyl-phosphate substrates.

The Structure of Murine N1-Acetylspermine Oxidase Reveals Molecular Details of Vertebrate Polyamine Catabolism.

N1-Acetylspermine oxidase (APAO) catalyzes the conversion of N1-acetylspermine or N1-acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N1-acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.

Drug discovery at the single molecule level: inhibition-in-solution assay of membrane-reconstituted ß-secretase using single-molecule imaging.

Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against ß-secretase 1 (BACE1), the assay was benchmarked with respect to sensitivity and dynamic range against SPR. We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (Kd < nM) compounds.

Preserved transmembrane protein mobility in polymer-supported lipid bilayers derived from cell membranes.

Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, ß-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms.

Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry.

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K(D) of 138 ± 55 nM, a ΔH of -9708 ± 1195 cal/mol and a ΔS of -1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K(D) of 3.2 ± 0.8 µM, a ΔH of -2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.

The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

Reprint of: Effective high-throughput overproduction of membrane proteins in Escherichia coli.

Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-ß-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2mg/l, with 18 targets producing at levels of 5mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.

Structural Basis for Targeting the Folded P-Loop Conformation of c-MET.

We report here a fragment screen directed toward the c-MET kinase from which we discovered a series of inhibitors able to bind to a rare conformation of the protein in which the P-loop adopts a collapsed, or folded, arrangement. Preliminary SAR exploration led to an inhibitor (7) with nanomolar biochemical activity against c-MET and promising cell activity and kinase selectivity. These findings increase our structural understanding of the folded P-loop conformation of c-MET and provide a sound structural and chemical basis for further investigation of this underexplored yet potentially therapeutically exploitable conformational state.

Conformational regulation of charge recombination reactions in a photosynthetic bacterial reaction center.

In bright light the photosynthetic reaction center (RC) of Rhodobacter sphaeroides stabilizes the P(+)(870).Q(-)(A) charge-separated state and thereby minimizes the potentially harmful effects of light saturation. Using X-ray diffraction we report a conformational change that occurs within the cytoplasmic domain of this RC in response to prolonged illumination with bright light. Our observations suggest a novel structural mechanism for the regulation of electron transfer reactions in photosynthesis.

Secretome-Based Screening in Target Discovery.

Secreted proteins and their cognate plasma membrane receptors regulate human physiology by transducing signals from the extracellular environment into cells resulting in different cellular phenotypes. Systematic use of secretome proteins in assays enables discovery of novel biology and signaling pathways. Several secretome-based phenotypic screening platforms have been described in the literature and shown to facilitate target identification in drug discovery. In this review, we summarize the current status of secretome-based screening. This includes annotation, production, quality control, and sample management of secretome libraries, as well as how secretome libraries have been applied to discover novel target biology using different disease-relevant cell-based assays. A workflow for secretome-based screening is shared based on the AstraZeneca experience. The secretome library offers several advantages compared with other libraries used for target discovery: (1) screening using a secretome library directly identifies the active protein and, in many cases, its cognate receptor, enabling a rapid understanding of the disease pathway and subsequent formation of target hypotheses for drug discovery; (2) the secretome library covers significant areas of biological signaling space, although the size of this library is small; (3) secretome proteins can be added directly to cells without additional manipulation. These factors make the secretome library ideal for testing in physiologically relevant cell types, and therefore it represents an attractive approach to phenotypic target discovery.

Exploring the Active Site of the Antibacterial Target MraY by Modified Tunicamycins.

The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.

Crystal structure of AcrB in complex with a single transmembrane subunit reveals another twist.

Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.

CETSA beyond Soluble Targets: a Broad Application to Multipass Transmembrane Proteins.

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.

Structural and Molecular Insight into Resistance Mechanisms of First Generation cMET Inhibitors.

Many small molecule inhibitors of the cMET receptor tyrosine kinase have been evaluated in clinical trials for the treatment of cancer and resistance-conferring mutations of cMET are beginning to be reported for a number of such compounds. There is now a need to understand specific cMET mutations at the molecular level, particularly concerning small molecule recognition. Toward this end, we report here the first crystal structures of the recent clinically observed resistance-conferring D1228V cMET mutant in complex with small molecule inhibitors, along with a crystal structure of wild-type cMET bound by the clinical compound savolitinib and supporting cellular, biochemical, and biophysical data. Our findings indicate that the D1228V alteration induces conformational changes in the kinase, which could have implications for small molecule inhibitor design. The data we report here increases our molecular understanding of the D1228V cMET mutation and provides insight for future inhibitor design.

An Angle on MK2 Inhibition-Optimization and Evaluation of Prevention of Activation Inhibitors.

The mitogen-activated protein kinase p38α pathway has been an attractive target for the treatment of inflammatory conditions such as rheumatoid arthritis. While a number of p38α inhibitors have been taken to the clinic, they have been limited by their efficacy and toxicological profile. A lead identification program was initiated to selectively target prevention of activation (PoA) of mitogen-activated protein kinase-activated protein kinase 2 (MK2) rather than mitogen- and stress-activated protein kinase 1 (MSK1), both immediate downstream substrates of p38α, to improve the efficacy/safety profile over direct p38α inhibition. Starting with a series of pyrazole amide PoA MK2 inhibitor leads, and guided by structural chemistry and rational design, a highly selective imidazole 9 (2-(3'-(2-amino-2-oxoethyl)-[1,1'-biphenyl]-3-yl)-N-(5-(N,N-dimethylsulfamoyl)-2-methylphenyl)-1-propyl-1H-imidazole-5-carboxamide) and the orally bioavailable imidazole 18 (3-methyl-N-(2-methyl-5-sulfamoylphenyl)-2-(o-tolyl)imidazole-4-carboxamide) were discovered. The PoA concept was further evaluated by protein immunoblotting, which showed that the optimized PoA MK2 compounds, despite their biochemical selectivity against MSK1 phosphorylation, behaved similarly to p38 inhibitors in cellular signaling. This study highlights the importance of selective tool compounds in untangling complex signaling pathways, and although 9 and 18 were not differentiated from p38α inhibitors in a cellular context, they are still useful tools for further research directed to understand the role of MK2 in the p38α signaling pathway.