Over the past decade there has been a revolution in the field of G protein-coupled receptor (GPCR) structural biology. Many years of innovative research from different areas have come together to ...fuel this significant change in the fortunes of this field, which for many years was characterized by the paucity of high-resolution structures. The determination to succeed has been in part due to the recognized importance of these proteins as drug targets, and although the pharmaceutical industry has been focusing on these receptors, it can be justifiably argued and demonstrated that many of the approved and commercially successful GPCR drugs can be significantly improved to increase efficacy and/or reduce undesired side effects. In addition, many validated targets in this class remain to be drugged. It is widely recognized that application of structure-based drug design approaches can help medicinal chemists a long way toward discovering better drugs. The achievement of structural biologists in providing high-resolution insight is beginning to transform drug discovery efforts, and there are a number of GPCR drugs that have been discovered by use of structural information that are in clinical development. This review aims to highlight the key developments that have brought success to GPCR structure resolution efforts and exemplify the practical application of structural information for the discovery of adenosine A2A receptor antagonists that have potential to treat multiple conditions.
Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To ...gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.
Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled ...glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.
The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, ...sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.
The adenosine A2A receptor (A2AR) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A2AR was determined recently in ...an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A2AR bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist 3H5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A2AR-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A2AR-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A2AR-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation.
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A detailed investigation of employing landfill gas together with additives such as hydrogen or propane or both as a primary low reactivity fuel in a reactivity controlled compression ignition ...combustion of a diesel engine is conducted. A 3401E caterpillar single-cylinder diesel engine with a bathtub piston bowl profile is utilized to execute the study. The engine is operated at various intake pressures of 1.6, 1.9, and 2.2 bar, and runs at a fixed engine speed of 1300 rpm. For verification purposes, the conduct of the present engine running on pure methane as a low reactivity fuel is compared to that of the same engine available in the literature. Next, a numerical simulation is made to assess the performance of the present engine running on landfill gas plus the additives. Based on the obtained results, injecting either hydrogen or propane or a combination of both up to a total amount of 10% by volume to the premixed of landfill gas and air, and advancing diesel fuel injection timing of about 20–30 deg. crank angle, render the landfill gas utilization quite competitive with using methane alone. Applying an enriched landfill gas in a reactivity controlled compression ignition diesel engine, as a power generator, drastically reduces the greenhouse gas emission to the atmosphere. Also, the CO and UHC mole fraction in the exhaust gas can be eliminated by either advancing the start of diesel injection or using hydrogen or propane or both as additives. In addition, utilizing hydrogen or propane or a combination of both with the primary fuel improves the peak pressure to about 16% in comparison with that of landfill gas alone.
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•LFG implementation in an RCCI engine eliminates methane emission to the atmosphere.•Injecting H2/Propane to LFG overcomes the LFG drawbacks in RCCI engines.•Hydrogen and/or propane improve IMEP by 10–15% and eliminate CO/UHC emissions.•Using up to 10% of additives by volume, leads to an acceptable NOx level emission.
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in ...the
gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of
ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting
might represent a viable treatment for SCA7 retinal degeneration.
Fragment screening of a thermostabilized mGlu5 receptor using a high-concentration radioligand binding assay enabled the identification of moderate affinity, high ligand efficiency (LE) pyrimidine ...hit 5. Subsequent optimization using structure-based drug discovery methods led to the selection of 25, HTL14242, as an advanced lead compound for further development. Structures of the stabilized mGlu5 receptor complexed with 25 and another molecule in the series, 14, were determined at resolutions of 2.6 and 3.1 Å, respectively.
AF Ablation After Watchman
Background
There have been an increasing number of atrial fibrillation (AF) patients with Watchman left atrial appendage occlusion (LAAO) device, requiring catheter ...ablation (CA) for maintenance of normal sinus rhythm. In this study, we describe our experience with the feasibility and safety of CA in patients with a preexisting Watchman LAAO device.
Methods
This was a retrospective multicenter AF registry of 60 patients with Watchman LAAO device who underwent CA for AF. Baseline clinical and procedural characteristics of the included subjects were retrieved from review of medical records and were analyzed.
Results
The mean age was 72.7 ± 4.9 years and the mean CHADS2 score was 2.3 ± 0.6. All patients had successful pulmonary vein isolation (PVI). The left atrial appendage (LAA) was electrically active in 34 (56%) while reentrant tachycardia and AF triggers were seen in 17 (28%) patients. Electrical isolation was attempted in these 17 patients with only 10 achieving complete LAA isolation. Repeat imaging showed new peri‐device leaks in 30% (12/40) patients, while new significant peri‐device leaks (≥5 mm) were noted in 10% (10/40) of patients after RFA, respectively, requiring continuation of oral anticoagulation. There were a higher proportion of patients with severe peri‐device leaks (≥5 mm) after LAA isolation. However, >50% of those leaks sealed off on follow‐up transesophageal echocardiogram.
Conclusion
AF ablation is a feasible and safe in patients with preexisting Watchman LAAO device. Electrical isolation of the LAA could be difficult and when attempted can result in increased risk of short‐term peri‐device leak and recurrence of AT/AF in almost all patients.
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