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The first generation of CB(1) positive allosteric modulators (e.g., ZCZ011) featured a 3-nitroalkyl-2-phenyl-indole structure. Although a small number of drugs include the nitro group, it is generally not regarded as being “drug-like”, and this is particularly true for aliphatic nitro groups. There are very few case studies where an appropriate bioisostere replaced a nitro group that had a direct role in binding. This may be indicative of the difficulty of replicating its binding interactions. Herein, we report the design and synthesis of ligands targeting the allosteric binding site on the CB(1) cannabinoid receptor, in which a CF(3) group successfully replaced the aliphatic NO(2). In general, the CF(3)-bearing compounds were more potent than their NO(2) equivalents and also showed improved in vitro metabolic stability. The CF(3) analogue (1) with the best balance of properties was selected for further pharmacological evaluation. Pilot in vivo studies showed that (±)-1 has similar activity to (±)-ZCZ011, with both showing promising efficacy in a mouse model of neuropathic pain.

Objective The purpose of the study was to determine the extent and role of systemic hypoxia in the pathogenesis of spinal muscular atrophy (SMA). Methods Hypoxia was assayed in vivo in early‐symptomatic (postnatal day 5) SMA‐model mice by pimonidazole and [18F]‐Fluoroazomycin arabinoside injections, which accumulate in hypoxic cells, followed by immunohistochemistry and tracer biodistribution evaluation. Glucose uptake in hypoxic cells was assayed by [18F]‐Fluorodeoxyglucose labeling. In vitro knockdown of Survival Motor Neuron (SMN) was performed on motor neurons and lactate metabolism measured biochemically, whereas cell cycle progression and cell death were assayed by flow cytometry. Results All assays found significant levels of hypoxia in multiple organ systems in early symptomatic SMA mouse pups, except aerated tissues such as skin and lungs. This was accompanied by significantly increased glucose uptake in many affected organs, consistent with a metabolic hypoxia response. SMN protein levels were shown to vary widely between motor neuron precursors in vitro, and those with lower levels were most susceptible to cell death. In addition, SMA‐model motor neurons were particularly sensitive to hypoxia, with reduced ability to transport lactate out of the cell in hypoxic culture, and a failure in normal cell cycle progression. Interpretation Not only is there widespread tissue hypoxia and multi‐organ cellular hypoxic response in SMA model mice, but SMA‐model motor neurons are especially susceptible to that hypoxia. The data support the hypothesis that vascular defects leading to hypoxia are a significant contributor to disease progression in SMA, and offer a route for combinatorial, non‐SMN related therapy.

The bile acid analogue [18F]LCATD (LithoCholic Acid Triazole Derivative) is transported in vitro by hepatic uptake transporters such as OATP1B1 and NTCP and efflux transporter BSEP. In this in vivo “proof of principle” study, we tested if [18F]LCATD may be used to evaluate drug-drug interactions (DDIs) caused by inhibition of liver transporters. Hepatic clearance of [18F]LCATD in rats was significantly modified upon coadministration of rifamycin SV or sodium fusidate, which are known to inhibit clinically relevant uptake transporters (OATP1B1, NTCP) and canalicular hepatic transporters (BSEP) in humans. Treatment with rifamycin SV (total dose 62.5 mg·Kg−1) reduced the maximum radioactivity of [18F]LCATD recorded in the liver from 14.2 ± 0.8% to 10.2 ± 0.9% and delayed t_max by 90 seconds relative to control rats. AUCliver 0–5 min, AUCbile 0–10 min and hepatic uptake clearance CLuptake,in vivo of rifamycin SV treated rats were significantly reduced, whereas AUCliver 0–30 min was higher than in control rats. Administration of sodium fusidate (30 mg·Kg−1) inhibited the liver uptake of [18F]LCATD, although to a lesser extent, reducing the maximum radioactivity in the liver to 11.5 ± 0.3%. These preliminary results indicate that [18F]LCATD may be a good candidate for future applications as an investigational tracer to evaluate altered hepatobiliary excretion as a result of drug-induced inhibition of hepatic transporters.

Purpose Molecular imaging of αvβ3 integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of αvβ3 integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers. Procedures Experiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express αvβ3 integrin. αvβ3 integrin-specific radiotracers were used to investigate the effect of manipulating αvβ3 integrin expression or activation in cellular binding assays. β3 integrin and αvβ3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on αvβ3 integrin activation and expression was also determined. Results Radiotracer binding was proportional to αvβ3 integrin expression when it was decreased (β3 knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine–glycine–aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of αvβ3 integrin with Mn2+ or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced αvβ3 integrin activity. Conclusion Binding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of αvβ3 integrin. αvβ3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on αvβ3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies. Electronic supplementary material The online version of this article (doi:10.1007/s11307-017-1100-z) contains supplementary material, which is available to authorized users.

Prostate cancer represents a major public health threat as it is one of the most common male cancers worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer cells in a manner that correlates with both tumour stage and clinical outcome. As such, PSMA has been identified as an attractive target for both imaging and treatment of prostate cancer. In recent years the focus on urea-based peptidomimetic inhibitors of the PSMA (representing low molecular weight/high affinity binders) has intensified as they have found use in the clinical imaging of prostate tumours. Reported herein are the design, synthesis and evaluation of a new fluorinated PSMA targeting small-molecule, FDA-PEG-GUL, which possesses the Glu-NH-CO-NH-Lys pharmacophore conjugated to a 5'-fluorodeoxy- adenosine unit. Inhibition assays were performed with FDA-PEG-GUL which revealed that it inhibits the PSMA in the nanomolar range. Additionally, it has been purposely designed so that it can be produced using the fluorinase enzyme from its chlorinated precursor, allowing for the enzymatic synthesis of radiolabelled [F-18]FDA-PEG-GUL via a nucleophilic reaction that takes place in experimentally advantageous conditions (in water at neutral pH and at ambient temperature). Specific binding of [F-18]FDA-PEG-GUL to PSMA expressing cancer cells was demonstrated, validating it as a promising PSMA diagnostic tool. This work establishes a successful substrate scope expansion for the fluorinase and demonstrates its first application towards targeting the PSMA.

Cyclic CNGRC (cCNGRC) peptides are very important targeting ligands for Aminopeptidase N (APN or CD13), which is overexpressed on the surface of many cancer cells. In this work we have (1) developed an efficient solid-phase synthesis and (2) tested on purified porcine APN and APN-expressing human cells two different classes of cCNGRC peptides: the first carrying a biotin affinity tag or a fluorescent tag attached to the carboxyl Arg-Cys-COOH terminus and the second with the tags attached to the amino H2N-Cys-Asn terminus. Carboxyl-terminus functionalized cCNGRC peptides 3, 6, and 8 showed good affinity for porcine APN and very good capacity to target and be internalized into APN-expressing cells. In contrast, amino-terminus functionalized cCNGRC peptides 4, 5, and 7 displayed significantly decreased affinity and targeting capacity. These results, which are in agreement with the recently reported X-ray structure of a cCNGRC peptide bound to APN showing important stabilizing interactions between the unprotected cCNGRC amino terminus and the APN active site, indicate that the carboxyl and not the amino-terminus of cCNGRC peptides should be used as a "handle" for the attachment of toxic payloads for therapy or isotopically labeled functions for imaging and nuclear medicine.

The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth&rsquo, s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and &alpha, aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and &alpha, aminoisobutyric acid residues and demonstrated that these peptides can promote na&iuml, ve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge.

BACKGROUND Extracorporeal shockwave therapy (ESWT) is effective in reducing shoulder pain and improving function in calcific supraspinatus tendinopathy. Eccentric exercise has been introduced as an effective treatment choice for Achilles tendinopathy, but poor evidence exists about its role in the treatment of rotator cuff tendinopathy. AIM To investigate if adding a supervised eccentric training of the shoulder abductor muscles could improve the outcome of ESWT. DESIGN Pre-post intervention pilot study with matched control-group. SETTING Outpatient, University Hospital. POPULATION Twenty-two subjects affected by painful supraspinatus calcific tendinopathy. METHODS The study-group was assigned to receive focal ESWT (f-ESWT) plus a supervised eccentric training (SET) of the shoulder abductor muscles. The matched control-group received f-ESWT only. The post-treatment assessment at follow-up (T1) was performed nine weeks after the enrollment (T0). We assessed shoulder pain and function by the means of a numeric rating scale (p-NRS) and a DASH scale. As secondary outcome, we measured the isometric strength of the abductor muscles of the affected shoulder using a handheld dynamometer. RESULTS At T1, we recorded a significant decrease in pain (P

The authors thank the Engineering and Physical Sciences Research Council, UK, for a research grant. The A2A adenosine receptor belongs to a family of G-coupled protein receptors that have been subjected to extensive investigation over the last few decades. Due to their prominent role in the biological functions of the heart, lungs, CNS and brain, they have become a target for the treatment of illnesses ranging from cancer immunotherapy to Parkinson's disease. The imaging of such receptors using positron emission tomography (PET) has also been of interest, potentially providing a valuable tool to analyse and diagnose various myocardial and neurodegenerative disorders, as well as offering support to drug discovery trials. Reported herein is the design, synthesis and evaluation of two novel 5'-fluorodeoxy-adenosine (FDA) based receptor agonists (FDA-PP1 and FDA-PP2), each substituted at the C-2 position with a terminally functionalised ethynyl unit. The structures enable a synthesis of 18F-labelled analogues via direct, last-step, radiosynthesis from chlorinated precursors using the fluorinase enzyme (5'-fluoro-5'-deoxyadenosine synthase) which catalyses a transhalogenation reaction. This delivers a new class of A2A adenosine receptor agonist which can be directly radiolabelled for exploration in PET studies. Postprint

We report the synthesis of terminally fluorinated HU-210 and HU-211 analogues (HU-210F and HU-211F, respectively) and their biological evaluation as ligands of cannabinoid receptors (CB1 and CB2) and N-methyl d-aspartate receptor (NMDAR). [(18)F]-labelled HU-210F was radiosynthesised from the bromo-substituted precursor. In vitro assays showed that both HU-210F and HU-211F retain the potent pharmacological profile of HU-210 and HU-211, suggesting that [(18)F]-radiolabelled HU-210F and HU-211F could have potential as PET tracers for in vivo imaging.

With the aim of identifying a fluorinated bile acid derivative that could be used as [18F]-labeled Positron Emission Tomography (PET) tracer for imaging the in vivo functioning of liver transporter proteins, and particularly of OATP1B1, three fluorinated bile acid triazole derivatives of cholic, deoxycholic and lithocholic acid (CATD, DCATD and LCATD 4a-c, respectively) were synthesized and labeled with tritium. In vitro transport properties were studied with cell-based assays to identify the best substrate for OATP1B1. In addition, the lead compound, LCATD (4c), was tested as a substrate of other liver uptake transporters OATP1B3, NTCP and efflux transporter BSEP to evaluate its specificity of liver transport. The results suggest that 4c is a good substrate of OATP1B1 and NTCP, whereas it is a poor substrate of OATP1B3. The efflux transporter BSEP also appears to be involved in the excretion of 4c from hepatocytes. The automated radiosynthesis of [18F]-4c was accomplished in a multi-GBq scale and a pilot imaging experiment in a wild type rat was performed after i.v. administration to assess the biodistribution and clearance of the tracer. PET imaging revealed that radioactivity was primarily located in the liver (tmax=75s) and cleared exclusively through the bile, thus allowing to image the hepatobiliary excretion of bile acids in the animal model. These findings suggest that [18F]-LCATD 4c is a promising PET probe for the evaluation of hepatic transporters OATP1B1, NTCP and BSEP activity with potential for studying drug-drug interactions and drug-induced toxicity involving these transporters.

This case presentation describes a 47-year-old woman who developed complex regional pain syndrome type II with severe neuropathic pain following iatrogenic transection of the tibial nerve at the ankle. The pain and disability progressively worsened over time, markedly impaired ambulation, and were not relieved despite various analgesic treatments. After injection of botulinum toxin (abobotulinumtoxinA, BoNT-A) in the leg muscles the tendons of which pass through the tarsal tunnel (together with the tibial nerve), her pain decreased and her walking capacity improved. This case suggests a new therapeutic role for botulin toxin in treating peripheral neuropathic pain caused by movement-evoked ectopic potentials. Level of Evidence V

Objective: The aim of this study was to determine and quantify how personality traits modulate the well-known relation between neuromuscular impairment and perception of physical and social disability in patients with facial palsy. Methods: Sixty-one patients with facial palsy were evaluated through the Sunnybrook Facial Grading System (SFGS), the Facial Disability Index (FDI), the Beck depression Inventory (BDI) and the Temperament and Character Inventory (TCI). To estimate the effect of the personality traits on physical and social disability as regards neuromuscular impairment, two polynomial models were fitted and validated using specific personality variants and the SFGS composite score as explanatory variables. Results: A role of personality traits in modulating perception of both functional and social/well-being disability was enlightened. A linear effect of the Self-Directedness and the Cooperativeness traits on the perception of functional disability as regards facial impairment was estimated, equal respectively to -0.21 points [CI 95 (-0.35; -0.07)] and +0.23 points [CI 95 (0.06; 0.39)] in FDI score for any level of deviation from the average of each personality trait. As regards social/well-being disability, a linear effect of Novelty Seeking and Persistence traits was estimated, equal respectively to +3.88 points [CI 95 (1.25; 6.49)] and +6.58 points [CI 95 (0.21; 13.17)] for any level of deviation from the average. Conclusion: Personality traits differently influence the relation between disability perception and neuromuscular impairment in facial palsy. This issue might be useful to improve the relationship between physician and patient and to allow personalized therapeutic interventions.

International audience; Rhizobial soil bacteria can form a symbiosis with legumes in which the bacteria fix atmospheric nitrogen into ammonia that can be utilized by the host. The plant, in turn, supplies the rhizobia with a carbon source. After infecting the host cell, the bacteria differentiate into a distinct bacteroid form, which is able to fix nitrogen. The bacterial BacA protein is essential for bacteroid differentiation in legumes producing nodule-specific cysteine-rich peptides (NCRs), which induce the terminal differentiation of the bacteria into bacteroids. NCRs are antimicrobial peptides similar to mammalian defensins, which are important for the eukaryotic response to invading pathogens. The BacA protein is essential for rhizobia to survive the NCR peptide challenge. Similarities in the lifestyle of intracellular pathogenic bacteria suggest that host factors might also be important for inducing chronic infections associated with Brucella abortus and Mycobacterium tuberculosis. Moreover, rhizobial lipopolysaccharide is modified with an unusual fatty acid, which plays an important role in protecting the bacteria from environmental stresses. Mutants defective in the biosynthesis of this fatty acid display bacteroid development defects within the nodule. In this review, we will focus on these key components, which affect rhizobial bacteroid development and survival.

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.