Role of Amyloidogenic and Non-Amyloidogenic Protein Spaces in Neurodegenerative Diseases and their Mitigation Using Theranostic Agents.

Neurodegenerative diseases (NDDs) refer to a complex heterogeneous group of diseases which are associated with the accumulation of amyloid fibrils or plaques in the brain leading to progressive loss of neuronal functions. Alzheimer's disease is one of the major NDD responsible for 60-80 % of all dementia cases. Currently, there are no curative or disease-reversing/modifying molecules for many of the NDDs except a few such as donepezil, rivastigmine, galantamine, carbidopa and levodopa which treat the disease-associated symptoms. Similarly, there are very few FDA-approved tracers such as flortaucipir (Tauvid) for tau fibril imaging and florbetaben (Neuraceq), flutemetamol (Vizamyl), and florbetapir (Amyvid) for amyloid imaging available for diagnosis. Recent advances in the cryogenic electron microscopy reported distinctly different microstructures for tau fibrils associated with different tauopathies highlighting the possibility to develop tauopathy-specific imaging agents and therapeutics. In addition, it is important to identify the proteins that are associated with disease development and progression to know about their 3D structure to develop various diagnostics, therapeutics and theranostic agents. The current article discusses in detail the disease-associated amyloid and non-amyloid proteins along with their structural insights. We comprehensively discussed various novel proteins associated with NDDs and their implications in disease pathology. In addition, we document various emerging chemical compounds developed for diagnosis and therapy of different NDDs with special emphasis on theranostic agents for better management of NDDs.

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid-liquid phase separation of tau.

Liquid-liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.

Thiophene-Based Dual Modulators of Aß and Tau Aggregation.

Alzheimer's disease is characterized by the accumulation of amyloid beta (Aß) and Tau aggregates in the brain, which induces various pathological events resulting in neurodegeneration. There have been continuous efforts to develop modulators of the Aß and Tau aggregation process to halt or modify disease progression. A few small-molecule-based inhibitors that target both Aß and Tau pathology have been reported. Here, we report the screening of a targeted library of small molecules to modulate Aß and Tau aggregation together with their in vitro, in silico and cellular studies. In vitro ThT fluorescence assay, dot blot assay, gel electrophoresis and transmission electron microscopy (TEM) results have shown that thiophene-based lead molecules effectively modulate Aß aggregation and inhibit Tau aggregation. In silico studies performed by employing molecular docking, molecular dynamics and binding-free energy calculations have helped in understanding the mechanism of interaction of the lead thiophene compounds with Aß and Tau fibril targets. In cellulo studies revealed that the lead candidate is biocompatible and effectively ameliorates neuronal cells from Aß and Tau-mediated amyloid toxicity.

A matrix targeted fluorescent probe to monitor mitochondrial dynamics.

Mitochondria are an indispensable organelle for energy production and regulation of cellular metabolism. The structural and functional alterations to mitochondria instigate pathological conditions of cancer, and aging-associated and neurodegenerative disorders. The normal functioning of mitochondria is maintained by quality control mechanisms involving dynamic fission, fusion, biogenesis and mitophagy. Under conditions of mitophagy and neurodegenerative diseases, mitochondria are exposed to different acidic environments and high levels of reactive oxygen species (ROS). Therefore stable molecular tools and methods are required to monitor the pathways linked to mitochondrial dysfunction and disease conditions. Herein, we report a far-red fluorescent probe (Mito-TG) with excellent biocompatibility, biostability, photostability, chemical stability and turn on emission for selective targeting of the mitochondrial matrix in different live cells. The probe was successfully employed for monitoring dynamic processes of mitophagy and amyloid beta (Aß) induced mitochondrial structural changes.

Role of Post-translational Modifications in Alzheimer's Disease.

The global burden of Alzheimer's disease (AD) is growing. Valiant efforts to develop clinical candidates for treatment have continuously met with failure. Currently available palliative treatments are temporary and there is a constant need to search for reliable disease pathways, biomarkers and drug targets for developing diagnostic and therapeutic tools to address the unmet medical needs of AD. Challenges in drug-discovery efforts raise further questions about the strategies of current conventional diagnosis; drug design; and understanding of disease pathways, biomarkers and targets. In this context, post-translational modifications (PTMs) regulate protein trafficking, function and degradation, and their in-depth study plays a significant role in the identification of novel biomarkers and drug targets. Aberrant PTMs of disease-relevant proteins could trigger pathological pathways, leading to disease progression. Advancements in proteomics enable the generation of patterns or signatures of such modifications, and thus, provide a versatile platform to develop biomarkers based on PTMs. In addition, understanding and targeting the aberrant PTMs of various proteins provide viable avenues for addressing AD drug-discovery challenges. This review highlights numerous PTMs of proteins relevant to AD and provides an overview of their adverse effects on the protein structure, function and aggregation propensity that contribute to the disease pathology. A critical discussion offers suggestions of methods to develop PTM signatures and interfere with aberrant PTMs to develop viable diagnostic and therapeutic interventions in AD.

l-Dopa and dopamine conjugated naphthalenediimides modulate amyloid ß toxicity.

The process of protein misfolding and aggregation to form neurotoxic species is strongly implicated in most of the neurodegenerative disorders. In particular, amyloid beta (Aß) misfolding and aggregation is central to pathophysiological processes of Alzheimer's disease. The development of aggregation modulators has enormous implications in the discovery of effective therapeutic agents for Alzheimer's disease. Herein, we report the design and synthesis of a series of natural amino acid, l-dopa and dopamine appended derivatives of naphthalenediimide (NDI) to identify efficient aggregation modulators. Furthermore, the molecular docking studies revealed the possible binding sites and binding mode of NDI-conjugates to Aß aggregates. Among the designed NDI-conjugates, l-dopa and dopamine derivatives (NLD and NDP, respectively) showed excellent aggregation modulation efficiency (inhibition and dissolution), as shown by the thioflavin T (ThT) binding assays, dot blot analysis and in cellulo studies. The docking results from in silico studies are in good agreement with the experimental data. In addition to their significant modulation efficiency towards Aß aggregation, NLD and NDP possess antioxidant activity conducive to the development of disease-modifying therapeutic agents for the treatment of Alzheimer's disease.

Biphasic modulation of tau liquid-liquid phase separation by polyphenols.

Molecular tools that modulate tau liquid-liquid phase separation (LLPS) promise to treat tauopathies. We screened a set of polyphenols and demonstrated concentration-dependent biphasic modulation of tau LLPS by gallic acid (GA), showcasing its ability to expedite the liquid-to-gel transition in tau condensates and effectively impede the formation of deleterious fibrillar aggregates.

Polycatechols inhibit ferroptosis and modulate tau liquid-liquid phase separation to mitigate Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder that affects learning, memory, and cognition. Current treatments targeting amyloid-ß (Aß) and tau have shown limited effectiveness, necessitating further research on the aggregation and toxicity mechanisms. One of these mechanisms involves the liquid-liquid phase separation (LLPS) of tau, contributing to the formation of pathogenic tau aggregates, although their conformational details remain elusive. Another mechanism is ferroptosis, a type of iron-dependent lipid peroxidation-mediated cell death, which has been implicated in AD. There is a lack of therapeutic strategies that simultaneously target amyloid toxicity and ferroptosis. This study aims to explore the potential of polycatechols, PDP and PLDP, consisting of dopamine and L-Dopa, respectively, as multifunctional agents to modulate the pathological nexus between ferroptosis and AD. Polycatechols were found to sequester the labile iron pool (LIP), inhibit Aß and tau aggregation, scavenge free radicals, protect mitochondria, and prevent ferroptosis, thereby rescuing neuronal cell death. Interestingly, PLDP promotes tau LLPS, and modulates their intermolecular interactions to inhibit the formation of toxic tau aggregates, offering a conceptually innovative approach to tackle tauopathies. This is a first-of-its-kind polymer-based integrative approach that inhibits ferroptosis, counteracts amyloid toxicity, and modulates tau LLPS to mitigate the multifaceted toxicity of AD.

Hybrid molecules synergistically mitigate ferroptosis and amyloid-associated toxicities in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the build-up of extracellular amyloid ß (Aß) plaques and intracellular neurofibrillary tangles (NFTs). Ferroptosis, an iron (Fe)-dependent form of cell death plays a significant role in the multifaceted AD pathogenesis through generation of reactive oxygen species (ROS), mitochondrial damage, lipid peroxidation, and reduction in glutathione peroxidase 4 (GPX4) enzyme activity and levels. Aberrant liquid-liquid phase separation (LLPS) of tau drives the growth and maturation of NFTs contributing to AD pathogenesis. In this study, we strategically combined the structural and functional properties of gallic acid (GA) and cyclic dipeptides (CDPs) to synthesize hybrid molecules that effectively target both ferroptosis and amyloid toxicity in AD. This innovative approach marks a paradigm shift from conventional therapeutic strategies. This is the first report of a synthetic small molecule (GCTR) that effectively combats ferroptosis, simultaneously restoring enzymatic activity and enhancing cellular levels of its master regulator, GPX4. Further, GCTR disrupts Fe3+-induced LLPS of tau, and aids in attenuation of abnormal tau fibrillization. The synergistic action of GCTR in combating both ferroptosis and amyloid toxicity, bolstered by GPX4 enhancement and modulation of Fe3+-induced tau LLPS, holds promise for the development of small molecule-based novel therapeutics for AD.

Nano-quercetin mitigates triazophos-induced testicular toxicity in rats by suppressing oxidative stress and apoptosis.

The present study was designed to evaluate the testicular toxicity of triazophos in rats and to check the ameliorative effect of nano-quercetin against triazophos-induced toxicity. Nano-quercetin was synthesized from quercetin and characterized. Male Wistar rats were divided into seven groups. The control group received olive oil as a vehicle orally. The high-dose triazophos group and the low-dose triazophos group received 1/10th LD50 of triazophos (7.6 mg/kg) and 1/20th LD50 of triazophos (3.8 mg/kg), respectively. Two groups of animals were dosed with quercetin and nano-quercetin, both at 50 mg/kg body weight orally. The final two groups received high-dose triazophos with co-administration of quercetin and nano-quercetin, respectively. Triazophos disrupted the male endocrine axis by reducing the levels of steroidogenic enzymes 3-ß-HSD and 17-ß-HSD in testicular cells, further reducing FSH and testosterone. Also, triazophos increased the reactive oxygen species, induced lipid peroxidation, decreased the mitochondrial membrane potential, and elevated the number of apoptotic cells in rat testes. Nano-quercetin ameliorated the testicular oxidative stress and apoptotic and endocrine parameters more efficiently than quercetin. Besides, nano-quercetin alleviated the histopathological and biochemical alterations of triazophos. It is concluded that nano-quercetin has higher anti-oxidant efficacy than quercetin in protecting rats against triazophos-induced testicular toxicity.

A natural polyphenol activates and enhances GPX4 to mitigate amyloid-ß induced ferroptosis in Alzheimer's disease.

Ferroptosis, an iron-dependent cell death, plays a crucial role in the pathology of Alzheimer's disease (AD). Several characteristics of AD, including excessive iron accumulation, elevated lipid peroxide and reactive oxygen species (ROS) levels, and decreased glutathione peroxidase 4 (GPX4) levels, align with the features of ferroptosis. While traditional methods of inhibiting ferroptosis have centered on chelating Fe and trapping radicals, therapeutic strategies that modulate the GPX4 axis to mitigate ferroptosis in AD are yet to be explored. This report introduces naturally occurring polyphenols (PPs) as dual-acting therapeutic agents to synergistically alleviate ferroptosis and AD. The mechanisms of action encompass modulation of amyloid and tau cascade, reduction of oxidative stress, mitochondrial rescue, and inhibition of ferroptosis. For the first time, we show that a single multifunctional molecule, tannic acid (TA) binds at the activator site of GPX4, augmenting both its activity and cellular levels, providing a conceptually innovative and integrated approach for treating AD via the GPX4-ferroptosis axis. The ability of TA to enhance GPX4 levels under conditions of AD pathology opens up newer promising therapeutic avenues for combating the crosstalk between ferroptosis and AD.

Multipronged diagnostic and therapeutic strategies for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and a major contributor to dementia cases worldwide. AD is clinically characterized by learning, memory, and cognitive deficits. The accumulation of extracellular amyloid ß (Aß) plaques and neurofibrillary tangles (NFTs) of tau are the pathological hallmarks of AD and are explored as targets for clinical diagnosis and therapy. AD pathology is poorly understood and there are no fully approved diagnosis and treatments. Notwithstanding the gap, decades of research in understanding disease mechanisms have revealed the multifactorial nature of AD. As a result, multipronged and holistic approaches are pertinent to targeting multiple biomarkers and targets for developing effective diagnosis and therapeutics. In this perspective, recent developments in Aß and tau targeted diagnostic and therapeutic tools are discussed. Novel indirect, combination, and circulating biomarkers as potential diagnostic targets are highlighted. We underline the importance of multiplexing and multimodal detection of multiple biomarkers to generate biomarker fingerprints as a reliable diagnostic strategy. The classical therapeutics targeting Aß and tau aggregation pathways are described with bottlenecks in the strategy. Drug discovery efforts targeting multifaceted toxicity involving protein aggregation, metal toxicity, oxidative stress, mitochondrial damage, and neuroinflammation are highlighted. Recent efforts focused on multipronged strategies to rationally design multifunctional modulators targeting multiple pathological factors are presented as future drug development strategies to discover potential therapeutics for AD.

Rationally Designed Molecules Synergistically Modulate Multifaceted Aß Toxicity, Microglial Activation, and Neuroinflammation.

Synergistic modulation of multifaceted toxicity is the key to tackle multifactorial Alzheimer's disease (AD). The etiology of AD includes amyloid ß (Aß) amyloidosis, metal ion dyshomeostasis, reactive oxygen species (ROS), oxidative stress, mitochondrial damage, and neuroinflammation. We rationally designed multifunctional modulators by integrating pharmacophores for metal chelation, antioxidant and anti-inflammatory properties, and modulation of Aß42 aggregation on the naphthalene monoimide (NMI) scaffold. The in vitro and cellular studies of NMIs revealed that M3 synergistically modulates metal-independent and -dependent amyloid toxicity, scavenges ROS, alleviates oxidative stress, and emulates Nrf2-mediated stress response in neuronal cells. M3 effectively reduced structural and functional damage of mitochondria, reduced Cyt c levels, and rescued cells from apoptosis. The biological atomic force microscopy and Western blot analysis revealed the ability of M3 to suppress microglial activation and neuroinflammation through inhibition of the NF-κß pathway. The synergistic action of M3 is in agreement with our design strategy to develop a multifunctional therapeutic candidate by integrating multiple pharmacophores with distinct structural and functional elements to ameliorate the multifaceted toxicity of AD.

Multifunctional molecules with a bipyridyl core ameliorate multifaceted amyloid toxicity.

A series of 2,2'-bipyridine derivatives appended with structurally unique biomolecular auxiliaries were synthesized and investigated for their ability to ameliorate multifaceted amyloid toxicity. Our results highlight the roles of the metal-chelating bipyridine core and functional auxiliaries in effectively modulating metal-independent and -dependent amyloid toxicity, and oxidative stress observed in Alzheimer's disease (AD).