Structural Insights from Recent CB1 X-Ray Crystal Structures

Over the past 2 years, X-ray crystal structures of the antagonist- and agonist-bound CB1 receptor have been reported. Such structures are expected to accelerate progress in the understanding of CB1 and should provide an exceptional starting point for structure-based drug discovery. This chapter examines the consistency of these X-ray structures with the CB1 experimental literature, including mutation, NMR and covalent labeling studies. These comparisons reveal discrepancies between this literature and the TMH1-2-3 region of each CB1 crystal structure. The chapter also examines crystal packing issues with each X-ray structure and shows that the discrepancies with the experimental literature can be attributed to crystal packing problems that force the N-terminus deep in the binding pocket of the two inactive state structures and force TMH2 to bend at G2.53/S2.54 and invade the binding pocket in the activated state structure. Revision is advisable before these structures are used for structure-based drug discovery

Surge prevention for gas turbines connected with large volume size: Experimental demonstration with a microturbine

The aim of this work is the demonstration of a surge prevention technique for advanced gas turbine cycles. There is significant surge risk in dynamic operation for turbines connected with large volume size additional components, such as a fuel cell stack, a saturator, a solar receiver or a heat exchanger for external combustion. In comparison with standard gas turbines, the volume size generates different behaviour during dynamic operations (with significant surge risk), especially considering that such additional components are including important dynamic constraints. In order to prevent the surge events, a vibration analysis was carried out to develop precursors which are able to highlight the approach of this unstable operative zone. Since the sub-synchronous content of the measured vibrations is significantly increasing approaching the surge line, special attention was devoted to this parameter. The demonstration of a surge prevention system based on the sub-synchronous vibration content was carried out at the Innovative Energy Systems Laboratory of the University of Genoa. In this laboratory, a recuperated microturbine connected with a large size vessel was used. Starting from the stable operation, closing a valve in the main air line or increasing the compressor inlet temperature produced operative conditions with significant surge risk. The increase in sub-synchronous vibration content detected by the control system was used to perform an active operation (bleed valve opening) to avoid the approaching surge event

Vaccination against Tick-Borne Encephalitis (TBE) in Italy: Still a Long Way to Go

Tick-borne encephalitis (TBE) is endemic in several European countries, and its incidence has recently increased. Various factors may explain this phenomenon: social factors (changes in human behavior, duration and type of leisure activities and increased tourism in European high-risk areas), ecological factors (e.g., effects of climate change on the tick population and reservoir animals), and technological factors (improved diagnostics, increased medical awareness). Furthermore, the real burden of TBE is not completely known, as the performance of surveillance systems is suboptimal and cases of disease are under-reported in several areas. Given the potentially severe clinical course of the disease, the absence of any antiviral therapy, and the impossibility of interrupting the transmission of the virus in nature, vaccination is the mainstay of prevention and control. TBE vaccines are effective (protective effect of approximately 95% after completion of the basic vaccination\u2014three doses) and well tolerated. However, their uptake in endemic areas is suboptimal. In the main endemic countries where vaccination is included in the national/regional immunization program (with reimbursed vaccination programs), this decision was driven by a cost-effectiveness assessment (CEA), which is a helpful tool in the decision-making process. All CEA studies conducted have demonstrated the cost-effectiveness of TBE vaccination. Unfortunately, CEA is still lacking in many endemic countries, including Italy. In the future, it will be necessary to fill this gap in order to introduce an effective vaccination strategy in endemic areas. Finally, raising awareness of TBE, its consequences and the benefit of vaccination is critical in order to increase vaccination coverage and reduce the burden of the disease

Vibrational analysis for surge precursor definition in gas turbines

Compressor behaviour analysis in critical working conditions, such as incipient surge, represents a significant aspect in the turbomachinery research field. Turbines connected with large-size volumes present critical issues related to surge prevention especially during transient operations. Investigations based on acoustic and vibrational measurements appear to provide an interesting diagnostic and predictive solution by adopting suitable quantifiers calculated from microphone and accelerometer signals. For this scope a wide experimental activity has been conducted on a T100 microturbine connected with different volume sizes. A machine dynamical characterisation has been useful for better interpretation of signals during its transient to the surge. Hence, different possible methods of incipient surge identification have been developed through the use of different signal processing techniques in time, frequency and angle domain. These results will be useful for control system development to prevent compressor failures

Two-phase flow expansion: Development of an innovative test-rig for flow characterisation and CFD validation

The aim of this work is to describe the design of an innovative test rig for investigating the expansion of saturated fluids in the two-phase region. The experimental test rig was thought up and built by TPG of the University of Genoa. It will be equipped by probes and some optical accesses that permit high speed video recording and laser measurements. It will be useful for the study of the quality ratio, vapour and liquid droplet thermodynamic properties and their speed

Synthesis and Pharmacology of 1-Methoxy Analogs of CP-47,497

Three 1-methoxy analogs of CP-47,497 (7, 8 and 19) have been synthesized and their affinities for the cannabinoid CB1 and CB2 receptors have been determined. Although these compounds exhibit selectivity for the CB2 receptor none have significant affinity for either receptor. Modeling and receptor docking studies were carried out, which provide a rationalization for the weak affinities of these compounds for either receptor

(R)-N-(1-Methyl-2-hydroxyethyl)-13-(S)-methyl-arachidonamide (AMG315): A Novel Chiral Potent Endocannabinoid Ligand with Stability to Metabolizing Enzymes

The synthesis of potent metabolically stable endocannabinoids is challenging. Here we report a chiral arachidonoyl ethanolamide (AEA) analogue, namely, (13S,1'R)-dimethylanandamide (AMG315, 3a), a high affinity ligand for the CB1 receptor (Ki of 7.8 ± 1.4 nM) that behaves as a potent CB1 agonist in vitro (EC50 = 0.6 ± 0.2 nM). (13S,1'R)-dimethylanandamide is the first potent AEA analogue with significant stability for all endocannabinoid hydrolyzing enzymes as well as the oxidative enzymes COX-2. When tested in vivo using the CFA-induced inflammatory pain model, 3a behaved as a more potent analgesic when compared to endogenous AEA or its hydrolytically stable analogue AM356. This novel analogue will serve as a very useful endocannabinoid probe

Identification of CB1 Receptor Allosteric Sites Using Force-Biased MMC Simulated Annealing and Validation by Structure–Activity Relationship Studies

Positive allosteric modulation of the cannabinoid 1 receptor (CB1R) has demonstrated distinct therapeutic advantages that address several limitations associated with orthosteric agonism and has opened a promising therapeutic avenue for further drug development. To advance the development of CB1R positive allosteric modulators, it is important to understand the molecular architecture of CB1R allosteric site(s). The goal of this work was to use Force-Biased MMC Simulated Annealing to identify binding sites for GAT228 (R), a partial allosteric agonist, and GAT229 (S), a positive allosteric modulator (PAM) at the CB1R. Our studies suggest that GAT228 binds in an intracellular (IC) TMH1–2–4 exosite that would allow this compound to act as a CB1 allosteric agonist as well as a CB1 PAM. In contrast, GAT229 binds at the extracellular (EC) ends of TMH2/3, just beneath the EC1 loop. At this site, this compound can act as CB1 PAM only. Finally, these results were successfully validated through the synthesis and biochemical evaluation of a focused library of compounds

Towards a molecular understanding of the cannabinoid related orphan receptor gpr18: A focus on its constitutive activity

The orphan G-protein coupled receptor (GPCR), GPR18, has been recently proposed as a potential member of the cannabinoid family as it recognizes several endogenous, phytogenic, and synthetic cannabinoids. Potential therapeutic applications for GPR18 include intraocular pressure, metabolic disorders, and cancer. GPR18 has been reported to have high constitutive activity, i.e., activation/signaling occurs in the absence of an agonist. This activity can be reduced significantly by the A3.39N mutation. At the intracellular (IC) ends of (transmembrane helices) TMH3 and TMH6 in GPCRs, typically, a pair of oppositely charged amino acids form a salt bridge called the “ionic lock”. Breaking of this salt bridge creates an IC opening for coupling with G protein. The GPR18 “ionic lock” residues (R3.50/S6.33) can form only a hydrogen bond. In this paper, we test the hypothesis that the high constitutive activity of GPR18 is due to the weakness of its “ionic lock” and that the A3.39N mutation strengthens this lock. To this end, we report molecular dynamics simulations of wild-type (WT) GPR18 and the A3.39N mutant in fully hydrated (POPC) phophatidylcholine lipid bilayers. Results suggest that in the A3.39N mutant, TMH6 rotates and brings R3.50 and S6.33 closer together, thus strengthening the GPR18 “ionic lock”