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Life is constructed primarily using a toolbox of 20 canonical amino acids-relying upon these building blocks for the assembly of proteins and peptides that regulate nearly every cellular task, including cell structure, function, and maintenance. While Nature continues to be a source of inspiration for drug discovery, medicinal chemists are not beholden to only 20 canonical amino acids and have begun to explore non-canonical amino acids (ncAAs) for the construction of designer peptides with improved drug-like properties. However, as our toolbox of ncAAs expands, drug hunters are encountering new challenges in approaching the iterative peptide design-make-test-analyze cycle with a seemingly boundless set of building blocks. This Microperspective focuses on new technologies that are accelerating ncAA interrogation in peptide drug discovery (including HELM notation, late-stage functionalization, and biocatalysis) while shedding light on areas where further investment could not only accelerate the discovery of new medicines but also improve downstream development., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Objective: Dissatisfaction with treatment and impaired quality of life (QOL) are reported among people with treated hypothyroidism. We aimed to gain insight into this., Design and Patients: We conducted an online survey of individuals with self-reported hypothyroidism., Results: Nine hundred sixty-nine responses were analysed. Dissatisfaction with treatment was common (77.6%), and overall QOL scores were low. Patient satisfaction did not correlate with type of thyroid hormone treatment, but treatment with combination levothyroxine (L-T4) and liothyronine (L-T3) or with desiccated thyroid extract (DTE) was associated with significantly better reported QOL than L-T4 or L-T3 monotherapies (P < .001); however, multivariate analysis inclusive of other clinical parameters failed to confirm an association between type of thyroid hormone treatment and QOL or satisfaction. Multivariate analysis showed positive correlations between satisfaction and age (P = .026), male gender (P = .011), being under the care of a thyroid specialist (P < .001), family doctor (GP) prescribing DTE or L-T4 + L-T3 or L-T3 (P < .001) and being well informed about hypothyroidism (P < .001); negative correlations were observed between satisfaction and negative experiences with L-T4 (P < .001) and expectations for more support from the GP (P < .001), for L-T4 to resolve all symptoms (P = .004), and to be referred to a thyroid specialist (P < .001). For QOL, positive correlations were with male gender (P = .011) and duration of hypothyroidism (P = .002); negative correlations were with age (P = .027), visiting the GP more than 3 times before diagnosis (P < .001), sourcing DTE or L-T3 independently (P = .014), negative experiences with L-T4 (P = .013), having expectations for L-T4 to resolve all symptoms (P < .001) and of more support from the GP (P = .006)., Conclusions: Multiple parameters including prior healthcare experiences and expectations influence satisfaction with hypothyroidism treatment and QOL. Focusing on enhancing the patient experience and clarifying expectations at diagnosis may improve satisfaction and QOL., (© 2020 John Wiley & Sons Ltd.)

At the forefront of synthetic endeavors in the pharmaceutical industry, including drug discovery and high-throughput screening, timelines are tight and large quantities of pure chemical targets are rarely available. In this regard, the development of novel and increasingly challenging chemistries requires a commensurate level of innovation to develop reliable analytical assays and purification workflows with rapid turnaround that enables accelerated pharmacological evaluation. A small-scale automation platform enabling high-throughput analysis and purification to streamline the selection of candidate leads would be a transformative advance. Herein, we introduce an automation-friendly solid-phase extraction-matrix-assisted laser desorption/ionization (SPE-MALDI) platform applied to the high-throughput purification and analysis of peptide libraries. This advance enabled us to purify peptides from microgram levels in less than a day with results comparable to traditional high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Age-associated deposition of amyloid-β in cerebral blood vessels, a condition referred to as cerebral amyloid angiopathy, can contribute to stroke and dementia. This research aimed to design new radioactive technetium-99 m complexes that bind to amyloid-β plaques that have the potential to assist in diagnosis of cerebral amyloid angiopathy using single-photon-emitted computed tomography (SPECT) imaging. Six new pyridylthiosemicarbazide ligands containing either benzofuran or styrylpyridyl functional groups that are known to selectively bind to amyloid plaques were prepared. Non-radioactive isotopes of technetium are not available so rhenium was used as a surrogate for exploratory chemistry. The new ligands were used to prepare well-defined [Re-oxo] 3+ complexes where two pyridylthiosemicarbazide ligands were coordinated to a single metal ion to give bivalent complexes with two amyloid-β targeting functional groups. The interaction of the [Re-oxo] 3+ complexes with synthetic amyloid-β 1-42 and with amyloid plaques in human brain tissue was investigated. Two ligands were selected to develop methods to prepare their [ 99m Tc-oxo] 3+ complexes at the tracer level. These technetium-99 m complexes are likely to be isostructural to their rhenium-oxo analogues.

This article aims to provide baseline data and highlight any major deficiencies in the current level of care provided for adult patients with thyroid eye disease (TED). We undertook a prospective, nonrandomized cross-sectional multicenter observational study. During a 3-month period June-August 2014, consecutive adult patients with TED who presented to nominated specialist eye clinics in the United Kingdom, completed a standardized questionnaire. Main outcome measures were: demographics, time from diagnosis to referral to tertiary centre, time from referral to review in specialist eye clinic, management of thyroid dysfunction, radioiodine and provision of steroid prophylaxis, smoking, and TED classification. 91 patients (mean age 47.88 years) were included. Female-to-male ratio was 6:1. Mean time since first symptoms of TED = 27.92 (73.71) months; from first visit to any doctor with symptoms to diagnosis = 9.37 (26.03) months; from hyperthyroidism diagnosis to euthyroidism 12.45 (16.81) months. First, 13% had received radioiodine. All those with active TED received prophylactic steroids. Seven patients who received radioiodine and did not have TED at the time went on to develop it. Then, 60% patients were current or ex-smokers. 63% current smokers had been offered smoking cessation advice. 65% patients had active TED; 4% had sight-threatening TED. A large proportion of patients (54%) were unaware of their thyroid status. Not enough patients are being provided with smoking cessation advice and information on the impact of smoking on TED and control of thyroid function.

Our previously reported structures of calpain bound to its endogenous inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated peptide macrocyclization toward the design of cyclic peptides and peptidomimetics as calpain inhibitors. Inspired by nature's hint that a β-turn loop within calpastatin forms a broad interaction around calpain's active site cysteine, we have constructed and tested a library of 45 peptidic compounds based on this loop sequence. Four molecules have shown reproducibly low micromolar inhibition of calpain-2. Further systematic sequence changes led to the development of probes that displayed increased potency and specificity of inhibition against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling other peptidomimetic calpain inhibitors and presenting an improved selectivity profile against other therapeutically relevant proteases. Competitive and mixed inhibition against calpain-2 was observed, and an allosteric inhibition site on the enzyme was identified for a noncompetitive inhibitor.

We have developed a strategy for synthesizing passively permeable peptidomimetic macrocycles. The cyclization chemistry centers on using aziridine aldehydes in a multicomponent reaction with peptides and isocyanides. The linker region in the resulting product contains an exocyclic amide positioned α to the peptide backbone, an arrangement that is not found among natural amino acids. This amide provides structural rigidity within the cyclic peptidomimetic and promotes the creation of a stabilizing intramolecular hydrogen bonding network. This exocyclic control element also contributes to the increased membrane permeability exhibited by multicomponent-derived macrocycles with respect to their homodetic counterparts. The exocyclic control element is employed along with a strategic placement of N-methyl and d-amino acids to produce passively permeable peptides, which contain multiple polar residues. This strategy should be applicable in the pursuit of synthesizing therapeutically relevant macrocycles.

Covering: up to the end of 2015.Peptides are naturally occurring compounds that play an important role in all living systems and are responsible for a range of essential functions. Peptide receptors have been implicated in disease states such as oncology, metabolic disorders and cardiovascular disease. Therefore, natural peptides have been exploited as diagnostic and therapeutic agents due to the unique target specificity for their endogenous receptors. This review discusses a variety of natural peptides highlighting their discovery, endogenous receptors, as well as their derivatization to create molecular imaging agents, with an emphasis on the design of radiolabelled peptides. This review also highlights methods for discovering new and novel peptides when knowledge of specific targets and endogenous ligands are not available.

The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease.

Aziridine aldehyde dimers, peptides, and isocyanides participate in a multicomponent reaction to yield peptide macrocycles. We have investigated the selectivity and kinetics of this process and performed a detailed analysis of its chemoselectivity. While the reactants encompass all of the elements of the traditional Ugi four-component condensation, there is a significant deviation from the previously proposed mechanism. Our results provide evidence for an imidoanhydride pathway in peptide macrocyclization and lend justification for the diastereoselectivity and high effective molarity observed in the reaction.

Mutations in the metalloprotein Cu,Zn-superoxide dismutase (SOD1) cause approximately 20% of familial cases of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease for which effective therapeutics do not yet exist. Transgenic rodent models based on over-expression of mutant SOD1 have been developed and these have provided opportunity to test new therapeutic strategies and to study the mechanisms of mutant SOD1 toxicity. Although the mechanisms of mutant SOD1 toxicity are yet to be fully elucidated, incorrect or incomplete metallation of SOD1 confers abnormal folding, aggregation and biochemical properties, and improving the metallation state of SOD1 provides a viable therapeutic option. The therapeutic effects of delivering copper (Cu) to mutant SOD1 have been demonstrated recently. The aim of the current study was to determine if delivery of zinc (Zn) to SOD1 was also therapeutic. To investigate this, SOD1G37R mice were treated with the metal complex diacetyl-bis(4-methylthiosemicarbazonato)zinc(II) [Zn(II)(atsm)]. Treatment resulted in an improvement in locomotor function and survival of the mice. However, biochemical analysis of spinal cord tissue collected from the mice revealed that the treatment did not increase overall Zn levels in the spinal cord nor the Zn content of SOD1. In contrast, overall levels of Cu in the spinal cord were elevated in the Zn(II)(atsm)-treated SOD1G37R mice and the Cu content of SOD1 was also elevated. Further experiments demonstrated transmetallation of Zn(II)(atsm) in the presence of Cu to form the Cu-analogue Cu(II)(atsm), indicating that the observed therapeutic effects for Zn(II)(atsm) in SOD1G37R mice may in fact be due to in vivo transmetallation and subsequent delivery of Cu., (Copyright © 2015. Published by Elsevier Inc.)

There is an ever-increasing interest in synthetic methods that not only enable peptide macrocyclization, but also facilitate downstream application of the synthesized molecules. We have found that aziridine amides are stereoelectronically attenuated in a macrocyclic environment such that non-specific interactions with biological nucleophiles are reduced or even shut down. The electrophilic reactivity, revealed at high pH, enables peptide sequencing by mass spectrometry, which will further broaden the utility of aziridine amide-containing libraries of macrocycles.

The first solid-phase parallel synthesis of macrocyclic peptides using three-component coupling driven by aziridine aldehyde dimers is described. The method supports the synthesis of 9- to 18-membered aziridine-containing macrocycles, which are then functionalized by nucleophilic opening of the aziridine ring. This constitutes a robust approach for the rapid parallel synthesis of macrocyclic peptides., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Context: In active Graves' orbitopathy (GO), treatment can improve the final cosmetic and visual outcome. Diagnostic delay results in significant morbidity and increases patient dissatisfaction. However, it can be challenging for endocrinologists to recognize GO and decide who should be referred for ophthalmic care., Objective: DiaGO, a clinical assessment tool, was developed for use in patients with Graves' disease (GD). The tool is designed to alert clinicians to the possibility of GO and prompt early ophthalmic assessment., Design and Setting: A 20-point assessment tool was devised and tested on 104 GD patients: 27 "positive controls" with GO and 77 people with GD attending endocrine clinics over 17 months. Those scoring positively in endocrine clinics were referred for ophthalmic assessment. Both the appropriateness of the referral and subsequent treatment were assessed., Results: Eighty-eight of the 104 patients (85%) were female (mean age, 48.5 y; range, 18-76 y). All 27 "controls" scored positively. Of the 77 people evaluated with GD, 27 (35%) scored above the threshold for referral and GO was confirmed in 24/26 (92%) who attended for specialist ophthalmic assessment. Twelve of these 24 (50%) were offered specific treatment following ophthalmology review., Conclusions: The timely diagnosis of GO is important because early intervention in active disease can improve prognosis. DiaGO alerts clinicians to the possibility of GO and prompts referral to specialist ophthalmic care. It is quick and easy to use and does not require specialist ophthalmic skills. Overall, half of those referred after use of DiaGO were offered specific treatment, suggesting its use might significantly improve the management of patients.

β-Sheets account for over 30 % of all secondary structural conformations found in proteins. The intramolecular hydrogen bonding that exists between the two peptide strands is imperative in maintaining this secondary structure. With the proper design, cyclic peptides may act as scaffolds emulating active β-sheet regions, enabling investigation of their importance in molecular recognition and protein aggregation. Starting from Fmoc-Lys(Fmoc)-OH, macrocyclic peptides were synthesized on a solid support, with peptide-chain elongation extending from both the alpha and epsilon amines of the lysine. The branching peptides were cyclized with a pyridyl tridentate chelation core followed by coordination using [(99m) Tc/Re(CO)3 (H2 O)3 ](+) . Variable temperature (1) H NMR spectroscopy studies were performed, demonstrating that intramolecular hydrogen bonding exists between the two sides of the uncoordinated macrocyclic peptide scaffolds. Additionally, computational modelling and circular dichroism spectroscopic analysis revealed that the peptide backbone exists in a similar conformation both before and after metal coordination. The ability to seamlessly incorporate a tridentate chelation core into the backbone of a macrocyclic peptide, without disrupting the secondary structure, can greatly assist in the design of metal-centric peptidomimetic imaging agents. This novel integrated imaging probe approach may facilitate the investigation into protein-protein interactions using macrocyclic β-sheet scaffolds., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cyclic peptides have wide utility in the biological sciences. As conformationally locked analogs of the parent linear peptides, they possess greater stability under physiological conditions and increased binding affinity for their targets. As investigations of biological processes often require reporter molecules and functional readouts, chemical probes are commonly appended with functional groups that allow for conjugation to biological entities. Herein we describe the functionalization of cyclic peptides prepared via aziridine aldehyde-mediated macrocyclization. These cyclic peptides contain an aziridine ring that can be further functionalized by ring opening with nucleophiles. We report on the methodology used to produce a cyclic peptide analog of Pro-Gly-Leu-Gly-Phe with either azido or sulfhydryl functionality.

The factors determining diastereoselectivity observed in the multicomponent conversion of amino acids, aziridine aldehyde dimers, and isocyanides into chiral piperazinones have been investigated. Amino acid-dependent selectivity for either trans- or cis-substituted piperazinone products has been achieved. An experimentally determined diastereoselectivity model for the three-component reaction driven by aziridine aldehyde dimers has predictive value for different substrate classes. Moreover, this model is useful in reconciling the previously reported observations in multicomponent reactions between isocyanides, α-amino acids, and monofunctional aldehydes.

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1., (Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.)

Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis in situ . Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar Cln6 nclf (Cb Cln6 nclf ) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in Cb Cln6 nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and Cb Cln6 nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in Cb Cln6 nclf cells. Importantly, a zinc-complex, Zn II (atsm), restored the nuclear-to-cytoplasmic zinc ratios in Cb Cln6 nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. Zn II (atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in Cb Cln6 nclf cells. This study highlights the complementarities of bulk and single cell analysis of metal content for understanding disease states. We demonstrate the utility and broad applicability of XFM for subcellular analysis of perturbed biometal metabolism and mechanism of action studies for novel therapeutics to target neurodegeneration.

The concept of site-specific integration of fragments into macrocyclic entities has not yet found application in the realm of synthetic chemistry. Here we show that the reduced amidicity of aziridine amide bonds provides an entry point for the site-specific integration of amino acids and peptide fragments into the homodetic cyclic peptide architecture. This new synthetic operation improves both the convergence and divergence of cyclic peptide synthesis.

Background: Aberrant biometal metabolism is a key feature of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Metal modulating compounds are promising therapeutics for neurodegeneration, but their mechanism of action remains poorly understood. Neuronal ceroid lipofuscinoses (NCLs), caused by mutations in CLN genes, are fatal childhood neurodegenerative lysosomal storage diseases without a cure. We previously showed biometal accumulation in ovine and murine models of the CLN6 variant NCL, but the mechanism is unknown. This study extended the concept that alteration of biometal functions is involved in pathology in these disorders, and investigated molecular mechanisms underlying impaired biometal trafficking in CLN6 disease., Results: We observed significant region-specific biometal accumulation and deregulation of metal trafficking pathways prior to disease onset in CLN6 affected sheep. Substantial progressive loss of the ER/Golgi-resident Zn transporter, Zip7, which colocalized with the disease-associated protein, CLN6, may contribute to the subcellular deregulation of biometal homeostasis in NCLs. Importantly, the metal-complex, ZnII(atsm), induced Zip7 upregulation, promoted Zn redistribution and restored Zn-dependent functions in primary mouse Cln6 deficient neurons and astrocytes., Conclusions: This study demonstrates the central role of the metal transporter, Zip7, in the aberrant biometal metabolism of CLN6 variants of NCL and further highlights the key contribution of deregulated biometal trafficking to the pathology of neurodegenerative diseases. Importantly, our results suggest that ZnII(atsm) may be a candidate for therapeutic trials for NCLs.

Our objective was to assess the copper(II) complex of diacetylbis(4-methylthiosemicarbazone) [Cu(II)(atsm)] for its preclinical potential as a novel therapeutic for ALS. Experimental paradigms used were designed to assess Cu(II)(atsm) efficacy relative to treatment with riluzole, as a function of dose administered, and when administered post symptom onset. Mice expressing human Cu/Zn superoxide dismutase harbouring the disease-causing G37R mutation (SOD1-G37R) were used and effects of Cu(II)(atsm) determined by assessing mouse survival and locomotor function (rotarod assay). Cu(II)(atsm) improved SOD1-G37R mouse survival and locomotor function in a dose-dependent manner. The highest dose tested improved survival by 26%. Riluzole had a modest effect on mouse survival (3.3%) but it did not improve locomotor function. Cotreatment with Cu(II)(atsm) did not alter the protective activity of Cu(II)(atsm) administered on its own. Commencing treatment with Cu(II)(atsm) after the onset of symptoms was less effective than treatments that commenced before symptom onset but still significantly improved locomotor function and survival. Improved locomotor function and survival of SOD1-G37R mice supports the potential for Cu(II)(atsm) as a novel treatment option for ALS.

One of the pathological hallmarks of Alzheimer's disease is the presence of amyloid-β plaques in the brain and the major constituent of these plaques is aggregated amyloid-β peptide. New thiosemicarbazone-pyridylhydrazine based ligands that incorporate functional groups designed to bind amyloid-β plaques have been synthesized. The new ligands form stable four coordinate complexes with a positron-emitting radioactive isotope of copper, (64)Cu. Two of the new Cu(II) complexes include a functionalized styrylpyridine group and these complexes bind to amyloid-β plaques in samples of post-mortem human brain tissue. Strategies to increase brain uptake by functional group manipulation have led to a (64)Cu complex that effectively crosses the blood-brain barrier in wild-type mice. The new complexes described in this manuscript provide insight into strategies to deliver metal complexes to amyloid-β plaques.

Cannabinoid hyperemesis syndrome (CHS) is a condition characterized by cyclical vomiting without other identifiable cause in patients with chronic cannabis use. Patients with CHS report that compulsive bathing and hot showers are the only reliable treatments to improve symptoms. Cannabinoid hyperemesis syndrome is usually unresponsive to conventional pharmacologic antiemetics, and patients often require hospital admission. We report a case of CHS that improved significantly after treatment with haloperidol in the emergency department.

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.

Molecular scaffolds have been shown to facilitate and stabilise secondary structural turn elements, with a central core-arranging functionality in a defined three-dimensional orientation. In a peptide-based molecular imaging probe, this approach is of particular value as it would essentially "hide" a metal radioisotope within the ligand framework, making the labelling element a critical component of the receptor-bound structure. Starting from a 1,2-diaminoethane loaded 2-chlorotrityl resin, a versatile set of triamine ligand systems were synthesised by using solid-phase Fmoc-based peptide chemistry. The resultant resin-bound peptides then underwent amide reduction by treatment with borane-THF at 65 °C. This provided complete conversion to the corresponding polyamine entities in high purity for the majority of the amino acids utilised. The triamines were then coordinated on solid support by using [NEt(4)](2)[Re(CO)(3)(Br)(3)] followed by resin cleavage and HPLC purification, to give the desired rhenium coordinated species. We have shown that amino acid sequences can be assembled, reduced and coordinated on-resin, resulting in a versatile set of metal-ligand constructs. These studies could be expanded to generate libraries of turn-based peptidomimetics containing Re/Tc(I) organometallic scaffolds, with the intention of developing an improved approach for finding new diagnostic and therapeutic radiopharmaceutical entities., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Histidine is a convenient tridentate chelator used in the synthesis of technetium-99m radiopharmaceuticals, as it can be pendantly attached to a biomolecule for molecular imaging applications. Once coordinated, it forms a neutral complex that is capable of forming diastereomers at the alpha amine of the histidine. This is demonstrated through the synthesis and characterization of four different histidine chelators; three small molecule chelators, which consist of a benzylated histidine at the alpha amine, and one modified dipeptide, containing a phenylalanine derivative at the C-terminus and a histidine at the N-terminus. Upon rhenium coordination, two products are observed, each having the desired exact mass of the metal-containing species. The two products have been characterized through LC-MS, (1)H, gCOSY, NOESY and ROESY NMR experiments, and the relative stereochemistry determined. The implications of diastereomer formation when using this chelation system for creating molecular imaging agents is also discussed.

Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [β-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cognitive decline associated with Alzheimer's disease appears to be related to the hyper-phosphorylation of the protein tau as a consequence of increased activity of glycogen synthase kinase 3β (GSK3β), and subsequent formation of neurotoxic neurofibrillary tangles. Abberant metal ion homeostasis, particularly involving copper has been implicitly linked to the pathogenesis of the disease. Increasing intracellular copper concentrations has been found to trigger pathways that result in inhibition of GSK3β. The syntheses and characterisation of tetradentate hybrid hydroxyquinoline-thiosemicarbazone proligands is presented. The ligands form stable complexes with Cu(II) where the copper ion is four coordinate and essentially square planar as characterised by single crystal X-ray crystallography. The reduction of the metal ion to Cu(I) has been studied by electrochemical techniques and occurs at potentials that permit intracellular reduction. The new complexes show class dependent cell membrane permeability in neuronal-like SH-SY5Y cells with subsequent increases in intracellular copper concentrations. The increased intracellular copper results in a dose-dependent inhibition (phosphorylation) of GSK3β.

The imaging of molecular markers associated with disease offers the possibility for earlier detection and improved treatment monitoring. Receptors for gastrin-releasing peptide are overexpressed on prostate cancer cells offering a promising imaging target, and analogs of bombesin, an amphibian tetradecapeptide have been previously demonstrated to target these receptors. Therefore, the pan-bombesin analog [β-Ala11, Phe13, Nle14]bombesin-(7-14) was conjugated through a linker to dye-functionalized superparamagnetic iron oxide nanoparticles for the development of a new potential magnetic resonance imaging probe. The peptide was conjugated via click chemistry, demonstrating a complementary alternative methodology to conventional peptide-nanoparticle conjugation strategies. The peptide-functionalized nanoparticles were then demonstrated to be selectively taken up by PC-3 prostate cancer cells relative to unfunctionalized nanoparticles and this uptake was inhibited by the presence of free peptide, confirming the specificity of the interaction. This study suggests that these nanoparticles have the potential to serve as magnetic resonance imaging probes for the detection of prostate cancer.

Background: Thyroid hormone replacement is one of the most commonly prescribed and cost effective treatments for a chronic disease. There have been recent changes in community prescribing policies in many areas of the UK that have changed patient access to necessary medications. This study aimed to provide a picture of thyroid hormone usage in the UK and to survey patient opinion about current community prescribing policies for levothyroxine., Methods: Data on community prescriptions for thyroid hormones in England between 1998 and 2007, provided by the Department of Health, were collated and analysed. A survey of UK members of a patient support organisation (the British Thyroid Foundation) who were taking levothyroxine was carried out., Results: The amount of prescribed thyroid hormones used in England has more than doubled, from 7 to almost 19 million prescriptions, over the last 10 years. The duration of prescriptions has reduced from 60 to 45 days, on average over the same time. Two thousand five hundred and fifty one responses to the patient survey were received. Thirty eight percent of levothyroxine users reported receiving prescriptions of 28 days' duration. 59% of respondents reported being dissatisfied with 28-day prescribing., Conclusion: Amongst users of levothyroxine, there is widespread patient dissatisfaction with 28-day prescription duration. Analysis of the full costs of 28-day dispensing balanced against the potential savings of reduced wastage of thyroid medications, suggests that this is unlikely to be an economically effective public health policy.

A family of lipophilic, cationic Au(I) complexes of N-heterocyclic carbenes (NHCs) have been designed as new mitochondria-targeted antitumor agents that combine both selective mitochondrial accumulation and selective thioredoxin reductase inhibition properties within a single molecule. Two-step ligand exchange reactions with cysteine (Cys) and selenocysteine (Sec) occur with release of the NHC ligands. At physiological pH the rate constants for the reactions with Sec are 20- to 80-fold higher than those with Cys. The complexes are selectively toxic to two highly tumorigenic breast cancer cell lines and not to normal breast cells, and the degree of selectivity and potency are optimized by modification of the substituent on the simple imidazolium salt precursor. The lead compound is shown to accumulate in mitochondria of cancer cells, to cause cell death through a mitochondrial apoptotic pathway and to inhibit the activity of thioredoxin reductase (TrxR) but not the closely related and Se-free enzyme glutathione reductase.

Six linear, two-coordinate cationic Au(I) N-heterocyclic carbene complexes of the form [(R2Im)2Au]+ (R = Me 1, Me, Et 2, i-Pr 3, n-Bu 4, t-Bu 5 and Cy 6) have been prepared by the reaction of two equivalents of the appropriate dialkylimidazol-2-ylidene (R2Im) with (Me2S)AuCl in dmf. Single crystal structural studies for 1.PF6, 2.PF6), 3.Cl and 4-6.PF6 show that for all six complexes the gold(I) centres have quasi-linear C-Au-C coordination, with quasi-parallel pairs of aromatic imidazole planes, except in 5.PF6 where they are quasi-normal; in the latter, Au-C are 2.038(3), 2.033(3) A, cf. (e.g.) 2.027(2) A. Inter-cation Au...Au are close at 3.487(2), 3.525(2) A in 1PF6 and 2.PF6. The structural studies and low temperature NMR experiments provide no supportive evidence for the presence of pi back-bonding within this series of complexes. The lipophilicities of the six compounds, as estimated from the logarithm of the n-octanol-water partition coefficients (log P), varied across the series within the range -1.09 to 1.73. To investigate their potential as possible anti-mitochondrial anti-tumour agents, five of the compounds have been evaluated for their propensities to induce mitochondrial membrane permeabilization (MMP) in isolated rat liver mitochondria. At concentrations between 1-10 microM compounds 1.Br and 3-6.Cl induced dose-dependent, Ca2+-sensitive mitochondrial swelling at rates that increased with the lipophilicities of the complexes, with the most lipophilic compounds inducing the most rapid onset of swelling. The swelling was completely inhibited by cyclosporin A, the specific inhibitor of the mitochondrial permeability transition pore.

A series of (pseudo)halo(1,3-di-tert-butylimidazol-2-ylidine)gold complexes [(But2Im)AuX](X = Cl, Br, I, CN, N3, NCO, SCN, SeCN, ONO2, OCOCH3, CH3) have been synthesized and characterised spectroscopically and structurally. 13C NMR chemical shifts for the carbene carbon vary widely with differing ancillary anion, correlating well with the sigma-donor ability of the latter and with the M-C(carbene) bond distance. These results reinforce the notion that N-heterocyclic carbene ligands are primarily sigma-donor ligands with little pi-acceptor ability.

Allowance for prolonged disease induction and latency times is an important consideration in occupational epidemiology studies of cancer and other delayed effects of exposure. Two useful approaches for assessing prolonged induction and latency periods are (1) exposure lagging and (2) considering exposures only within moving time windows. The exposure weighting scheme proposed by Jahr2 to assess exposure burdens is another method that accounts for induction and latency, although not explicitly. These three approaches, which are shown to be special cases of exposure weighting, are illustrated with an analysis of lung cancer mortality among a cohort of workers from an asbestos textile plant.

Small excesses of lung cancer and emphysema mortality had been detected among an historical cohort of 17,601 white and 4,722 nonwhite Florida phosphate industry workers. Internal mortality rate comparisons were made between worker subcohorts classified according to length of employment in 16 work areas and according to employment duration in jobs grouped by potential exposures to 10 agents, including alpha radiation. The only consistent associations seen were increased lung cancer mortality rates among long-term workers in plant-wide services and skilled crafts jobs. There was no evidence to support causal associations with exposures characteristic of the phosphate industry.