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Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-D-Arg-containing autum-nalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.

After peripheral facial palsy, the onset of facial synkinesis results in aesthetic disfigurement and local muscle tension or pain, with possible deterioration of patient's well-being and social participation. The availability of valid instruments to evaluate patient-reported severity of facial synkinesis is important to capture the subjective perception of facial impairment.To generate and validate an Italian version of the Synkinesis Assessment Questionnaire, a patient-reported outcome measure to assess patient-perceived severity of facial synkinesis after peripheral facial palsy.Observational study.Outpatient clinic of a Rehabilitation Unit.Seventy-five patients with peripheral facial palsy.Through a process of translation and cross-cultural adaptation, we generated the Italian version of the questionnaire (SAQ-IT) and administered it twice to patients with peripheral facial palsy. We evaluated the clinical severity with the Sunnybrook Facial Grading System (SFGS) and the physical and social/well-being function with the two subscales of the Facial Disability Index (FDI-PHY and FDI-SWB, respectively).Cronbach's alpha was 0.87. Item-total correlations ranged from 0.30 to 0.70, while inter-item correlations ranged from 0.15 to 0.82, with an average value of 0.48. Test-retest reliability showed an Intraclass Correlation Coefficient of 0.946 (95% confidence interval: 0.916-0.966). The minimum detectable change (with a 95% confidence level, MDCinf95/inf) was 13.14 points. The correlation between SAQ-IT and the SFGS synkinesis subscore was rho=0.74, while that with the SFGS composite score was rho=0.25, with the FDI-PHY rho=-0.11 and with the FDI-SWB rho=-0.13.Our study validates the SAQ-IT in Italian-speaking individuals with peripheral facial palsy, confirming its acceptable psychometric properties, and providing the MDCinf95/inf.The availability of a valid instrument for the evaluation of patient-perceived severity of facial synkinesis plays an important role in the definition of tailored rehabilitative interventions after peripheral facial palsy.

Guanidinoglycosides are a class of non-cytotoxic molecular transporters capable of delivering high molecular weight bioactive cargos into cells at low nanomolar concentrations. Efficient bioconjugation with guanidinoglycosides has been previously demonstrated by utilizing a guanidinoneomycin decorated with a reactive but also unstable N-hydroxysuccinimmide ester-containing linker. Herein we report the synthesis, chemistry, and application of a new, stable guanidinoneomycin derivative armed with a highly specific maleimide moiety which allows for thiol-maleimide click chemistry, a highly popular bioconjugation strategy, widening the field of application of these intriguing and useful delivery vehicles.

Introduction: Colorectal cancer risk stratification traditionally focuses on tumor node metastasis staging. Seemingly equivalent tumors can differ unpredictably in prognosis; more sophisticated quantification and stratification methods are required to identify tumors with a high likelihood of becoming metastatic. Hypoxia (low oxygen concentration) is associated with aggressive phenotypes and poor prognosis. Hypoxia is associated with treatment resistance consequently there is an unmet clinical requirement to develop personalised treatment based on hypoxia. Positron emission tomography/computed tomography (PET/CT) imaging can non-invasively detect hypoxic tumors. [18F]Fluoroazomycin arabinoside ([18F]FAZA) is a leading hypoxia PET/CT radiotracer, and uptake is associated with lower disease free survival. Case Report: A 78-year-old man, diagnosed with a localised colorectal cancer, underwent [18F]FAZA PET/CT imaging pre-operatively. This confirmed hypoxic regions in the tumor with correlation demonstrated with carbonic anhydrase IX (CAIX) immunohistochemistry (IHC). He underwent a right hemicolectomy. The pathological staging for his colorectal cancer predicted a good outcome; thus, he did not receive adjuvant chemotherapy. The patient subsequently developed early metastatic disease with two lung metastases, which were resected by thoracotomy and wedge resection. He continues on follow up at present with no evidence of recurrent disease. Conclusion: Hypoxia can be an important marker in colorectal cancer when determining risk and prognosis. We present evidence of clinical correlation of FAZA uptake and CAIX IHC in colorectal cancer, a key aspect in FAZA tracer validation. PET/CT potentially provides a specific tool for stratification for hypoxia-related treatment modification and development of hypoxia biomarkers.

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.

M.M. thanks SULSA for a PhD studentship. We gratefully acknowledge financial support from the EPSRC (grant EP/I034793/1). We report the synthesis of five radiotracers incorporating different oxyamine spacers between the hypoxia‐reactive 2‐nitroimidazole moiety and the 5‐[18F]‐fluorodeoxyribose ([18F]FDR, 12 ) prosthetic group: three linear alkyl chains with 3, 5, 7 carbon atoms ( 15 a – c ), a cyclopropyl ring ( 15 d ) and a 1,4‐disubstituted‐1,2,3‐triazole ( 15 e ). Experiments in hypoxic cells showed that 15 d displays superior uptake kinetics – and similar selectivity for hypoxic cells – relative to the gold standard hypoxia tracers [18F]fluoroazomycin arabinoside ([18F]FAZA) and [18F]fluoromisonidazole ([18F]FMISO). Lipophilicity and structural rigidity have strong influence on the selectivity of tracers 15 towards hypoxic cells: the lead tracer 15 d displays a logP=0.38 and the most rigid spacer. A sixth radiotracer ( 15 f ), with a 2‐H‐imidazole replacing the 2‐nitroimidazole moiety of 15 d , was used to demonstrate that the cyclopropyl group does not play a meaningful role in the sensitivity towards hypoxia. Postprint

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click"ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.

The tetrazine/

Fatty acid oxidation (FAO) produces most of the ATP used to sustain the cardiac contractile work, although glycolysis is a secondary source of ATP under normal physiological conditions. FAO impairment has been reported in the advanced stages of heart failure (HF) and is strongly linked to disease progression and severity. Thus, from a clinical perspective, FAO dysregulation provides prognostic value for HF progression, the assessment of which could be used to improve patient monitoring and the effectiveness of therapy. Positron emission tomography (PET) imaging represents a powerful tool for the assessment and quantification of metabolic pathwaysin vivo. Several FAO PET tracers have been reported in the literature, but none of them is in routine clinical use yet. Metabolically trapped tracers are particularly interesting because they undergo FAO to generate a radioactive metabolite that is subsequently trapped in the mitochondria, thus providing a quantitative means of measuring FAOin vivo. Herein, we describe the design, synthesis, tritium labelling and radiofluorination of 4,4,16-trifluoro-palmitate (1) as a novel potential metabolically trapped FAO tracer. Preliminary PET-CT studies on [F-18]1in rats showed rapid blood clearance, good metabolic stability - confirmed by using [H-3]1in vitro - and resistance towards defluorination. However, cardiac uptake in rats was modest (0.24 +/- 0.04 % ID/g), and kinetic analysis showed reversible uptake, thus indicating that [F-18]1is not irreversibly trapped.

Fenretinide (4-HPR) is a synthetic retinoid which has been investigated for decades as an anticancer agent and more recently as a potential drug to treat metabolic syndrome. Here we synthesised a library of 4-HPR analogues - using a SAR approach - with the final goal to improve both anti-cancer activity and anti- obesity/diabetes properties of 4-HPR. Among the designed 4-HPR analogues, we selected the click-type analogue 3b, which is amenable to radiofluorination, to develop a PET tracer to further investigate the pharmacological profile of 4-HPR and analogues in vivo.

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.

PFKFB3, a glycolysis-related enzyme upregulated in inflammatory conditions and angiogenesis, is an emerging target for diagnosis and therapy of atherosclerosis. The fluorinated phenoxindazole [18F]ZCDD083was synthesized, radiolabeled in 17 ± 5% radiochemical yield and >99% radiochemical purity, and formulated for preclinical PET/CT imaging in mice. In vivo stability analysis showed no significant metabolite formation. Biodistribution studies showed high blood pool activity and slow hepatobiliary clearance. Significant activity was detected in the lung 2 h postinjection (pi) (11.0 ± 1.5%ID/g), while at 6 h pi no pulmonary background was observed. Ex vivo autoradiography at 6 h pi showed significant high uptake of [18F]ZCDD083 in the arch region and brachiocephalic artery of atherosclerotic mice, and no uptake in control mice, matching plaques distribution seen by lipid staining along with PFKFB3 expression seen by immunofluorescent staining. In vivo PET scans showed higher aortic region uptake of [18F]ZCDD083in atherosclerotic ApoE–/–Fbn1C1039G+/− than in control mice (0.78 ± 0.05 vs 0.44 ± 0.09%ID/g). [18F]ZCDD083 was detected in aortic arch and brachiocephalic artery of ApoE–/– (with moderate atherosclerosis) and ApoE–/–Fbn1C1039G+/− (with severe, advanced atherosclerosis) mice, suggesting this tracer may be useful for the noninvasive detection of atherosclerotic plaques in vivo.

PFKFB3, a glycolysis-related enzyme upregulated in inflammatory conditions and angiogenesis, is an emerging target for diagnosis and therapy of atherosclerosis. The fluorinated phenoxindazole [F-18]ZCDD083 was synthesized, radiolabeled in 17 +/- 5% radiochemical yield and >99% radiochemical purity, and formulated for preclinical PET/CT imaging in mice. In vivo stability analysis showed no significant metabolite formation. Biodistribution studies showed high blood pool activity and slow hepatobiliary clearance. Significant activity was detected in the lung 2 h postinjection (pi) (11.0 +/- 1.5%ID/g), while at 6 h pi no pulmonary background was observed. Ex vivo autoradiography at 6 h pi showed significant high uptake of [F-18]ZCDD083 in the arch region and brachiocephalic artery of atherosclerotic mice, and no uptake in control mice, matching plaques distribution seen by lipid staining along with PFKFB3 expression seen by immunofluorescent staining. In vivo PET scans showed higher aortic region uptake of [F-18]ZCDD083 in atherosclerotic ApoE(-/-)Fbn1(C1039G+/-) than in control mice (0.78 +/- 0.05 vs 0.44 +/- 0.09%ID/g). [F-18]ZCDD083 was detected in aortic arch and brachiocephalic artery of ApoE(-/-) (with moderate atherosclerosis) and ApoE(-/-)Fbn1(C1039G+/-) (with severe, advanced atherosclerosis) mice, suggesting this tracer may be useful for the noninvasive detection of atherosclerotic plaques in vivo.

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.