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.

Site-Specific Hyperphosphorylation Inhibits, Rather than Promotes, Tau Fibrillization, Seeding Capacity, and Its Microtubule Binding.

The consistent observation of phosphorylated tau in the pathology of Alzheimer's disease has contributed to the emergence of a model where hyperphosphorylation triggers both tau disassociation from microtubules and its subsequent aggregation. Herein, we applied a total chemical synthetic approach to site-specifically phosphorylate the microtubule binding repeat domain of tau (K18) at single (pS356) or multiple (pS356/pS262 and pS356/pS262/pS258) residues. We show that hyperphosphorylation of K18 inhibits 1) its aggregation in vitro, 2) its seeding activity in cells, 3) its binding to microtubules, and 4) its ability to promote microtubule polymerization. The inhibition increased with increasing the number of phosphorylated sites, with phosphorylation at S262 having the strongest effect. Our results argue against the hyperphosphorylation hypothesis and underscore the importance of revisiting the role of site-specific hyperphosphorylation in regulating tau functions in health and disease.

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.

Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation.

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington's disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.

Amyloid-ß-Derived Peptidomimetics Inhibits Tau Aggregation.

The aggregation of tau protein is one of the hallmarks for Alzheimer's disease, resulting in neurodegeneration. The peptidomimetics strategy to prevent tau aggregation is more specific over other small molecules. In the present study, we analyzed the effect of amyloid-ß-derived peptidomimetics for inhibiting heparin-induced tau aggregation in vitro. These peptides and their derivatives were known to prevent aggregation of amyloid-ß. KLVFF is a hydrophobic sequence of the pentapeptide that prevented tau aggregation as observed by thioflavin S fluorescence, transmission electron microscopy, and circular dichroism spectroscopy. P4 and P5 also prevented assembly of tau into aggregates and formed short fibrils. The ß-sheet breaker LPFFD was however ineffective in preventing tau aggregation. The peptides further demonstrated reversal of tau-induced cytotoxicity in a dose-dependent manner. Our results suggested that these peptides can also be used to inhibit tau aggregation and also, toxicity induced by tau could be considered as potential molecules that have an effect on tau as well as amyloid-ß.

Antioxidant Berberine-Derivative Inhibits Multifaceted Amyloid Toxicity.

Multiple lines of evidence indicate that amyloid beta (Aß) peptide is responsible for the pathological devastation caused in Alzheimer's disease (AD). Aß aggregation species predominantly contribute to multifaceted toxicity observed in neuronal cells including generation of reactive oxygen species (ROS), mitochondrial dysfunction, interfering with synaptic signaling, and activation of premature apoptosis. Herein, we report a natural product berberine-derived (Ber-D) multifunctional inhibitor to ameliorate in cellulo multifaceted toxicity of AD. The structural attributes of polyphenolic Ber-D have contributed to its efficient Cu chelation and arresting the redox cycle to prevent the generation of ROS and rescue biomacromolecules from oxidative damage. Ber-D inhibits metal-dependent and -independent Aß aggregation, which is supported by in silico studies. Ber-D treatment averts mitochondrial dysfunction and corresponding neuronal toxicity contributing to premature apoptosis. These key multifunctional attributes make Ber-D a potential therapeutic candidate to ameliorate multifaceted Aß toxicity in AD.

Molecular Architectonics of Cyclic Dipeptide Amphiphiles and Their Application in Drug Delivery.

Assembly and co-assemblies of peptide amphiphiles through specific noncovalent forces expand the space of molecular architectonics-driven construction of diverse nanoarchitectures with potential biological applications. In this work, cyclic dipeptide amphiphiles (CDPAs) of cyclo(Gly-Asp) with varying lengths of alkyl chains (C8-C18) were synthesized, and their molecular organization was studied. The noncovalent interactions of the components, CDP and alkyl chain, drive the molecular self-assembly of CDPAs into well-defined and diverse nanoarchitectures such as nanotubes, nanospheres, nano/microsheets, and flowers. The co-assembly of CDPAs with biological molecules such as nucleosides was studied to ascertain their utility as potential drug delivery vehicles. Mechanical properties of these nanoarchitectures in nanoindentation study established them as robust in nature. A temperature-dependent NMR study confirmed the formation of stable co-assembly of CDPAs, primarily driven by the intermolecular hydrogen bonding interactions. Computational modeling of oligomers of CDPAs and their co-assembly with nucleosides/nucleotides reveal the molecular level interactions and driving force behind such assemblies. CDPAs exhibit good biocompatibility and cytocompatibility, as revealed by the cellular studies which substantiated their suitability for drug delivery applications. The co-assembly of CDPA with an anticancer drug 5-bromo-2'-deoxyuridine (BrdU) was studied as a drug delivery platform and cytotoxicity was successfully assessed in HeLa cells. Computational modeling of the oligomers of CDPAs and their co-assembly with the drug molecule was performed to understand the molecular level interactions and driving force behind the assemblies. Our findings reveal the design strategy to construct diverse structural architectures using CDP as the modular building unit and specific molecular interactions driven co-assembly for potential application as drug delivery carrier.

Small Molecule Inhibits Metal-Dependent and -Independent Multifaceted Toxicity of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most devastating forms of dementia, without reliable treatments to cure, delay the onset, or prevent the disease progression. The proposed toxic mechanisms of AD include amyloidogenesis of amyloid ß (Aß), metal ion dyshomeostasis, redox active metal-Aß inclusion complex formation, and generation of excessive reactive oxygen and nitrogen species (ROS and RNS). The imbalance in redox homeostasis causes oxidative stress, DNA damage, mitochondrial dysfunction, and inflammation, which collectively become a major hurdle in the development of effective therapeutic agents for multifactorial AD. This necessitates a multifunctional strategy to develop effective therapeutic agents to inhibit multifaceted toxicity. In this context, we report a rational design, synthesis, and detailed study to identify a small molecule multifunctional modulator (MFM) inspired by the human origin tripeptide. The lead, MFM 4, chelates and sequesters metal ions, disrupts their redox cycles, prevents excessive ROS production and oxidative stress, ameliorates oxidative DNA damage and mitochondrial dysfunction, and modulates Nrf2 protein signaling under oxidative stress conditions by eliminating the toxic stress elements. The MFM 4 was found to inhibit metal-dependent and -independent Aß aggregation and qualified as a suitable candidate to inhibit Aß-induced neuronal toxicity. The NMR spectroscopy study revealed molecular-level interactions of 4 with Aß42, which explain the mechanism of aggregation inhibition. Furthermore, 4 effectively inhibited inflammation as revealed by reduction in nitric oxide (NO) production in LPS-activated glial cells. These key features make 4 a potential MFM platform to develop therapeutic agents for metal (Cu, Zn and Fe)-dependent and -independent multifaceted Aß toxicity of AD.

Synthesis of Hybrid Cyclic Peptoids and Identification of Autophagy Enhancer.

Cyclic peptoids are potential candidates for diverse biological activities. However, applications of cyclic peptoids are limited by the synthetic difficulties, conformational flexibility of large cyclic peptoids, and lack of secondary amide in the backbone. Herein, an elegant methodology for the synthesis of small and medium-size cyclic hybrid peptoids is developed. α N-Alkyl and α N-acyl substituents in N-(2-aminoethyl)glycine monomers enforce intra- and intermolecular cyclization to form stable six- and 12-membered cyclic products, respectively. NMR studies show inter- and intramolecular hydrogen bonding in six- and 12-membered cyclic peptoids, respectively. Screening of a cyclic peptoid library resulted in the identification of a potential candidate that enhanced autophagic degradation of cargo in a live cell model. Such upregulation of autophagy using small molecules is a promising approach for elimination of intracellular pathogens and neurodegenerative protein aggregates.