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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.)

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.

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.

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.

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.

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.

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.