Protein aggregation: Consequences, mechanism, characterization and inhibitory strategies.

Proteins play a major role in the regulation of various cellular functions including the synthesis of structural components. But proteins are stable under physiological conditions only. A slight variation in environmental conditions can cost them huge in terms of conformational stability ultimately leading to aggregation. Under normal conditions, aggregated proteins are degraded or removed from the cell by a quality control system including ubiquitin-proteasomal machinery and autophagy. But they are burdened under diseased conditions or are impaired by the aggregated proteins leading to the generation of toxicity. The misfolding and aggregation of protein such as amyloid-ß, α-synuclein, human lysozyme etc., are responsible for certain diseases including Alzheimer, Parkinson, and non- neuropathic systemic amyloidosis respectively. Extensive research has been done to find the therapeutics for such diseases but till now we have got only symptomatic treatment that will reduce the disease severity but will not target the initial formation of nucleus responsible for disease progression and propagation. Hence there is an urgent need to develop the drugs targeting the cause of the disease. For this, a wide knowledge related to misfolding and aggregation under the same heading is required as described in this review alongwith the strategies hypothesized and implemented till now. This will contribute a lot to the work of researchers in the field of neuroscience.

Inhibition of primary and secondary nucleation alongwith disruption of amyloid fibrils and alleviation of associated cytotoxicity: A biophysical insight of a novel property of Chlorpropamide (an anti-diabetic drug).

Aggregation of physiologically synthesized soluble proteins to insoluble, cytotoxic fibrils is a pre-requisite for pathogenesis of amyloid associated disorders including Alzheimer's disease, non-systemic amyloidosis, Parkinson's disease, etc. Considerable advancement has been made to understand the mechanism behind aggregation process but till date we have no efficient cure and preventive therapy for associated diseases. Strategies to prevent protein aggregation are nevertheless many which have been proved promisingly successful in vitro. One of those is repurposing already approved drugs that saves time and money too and has been employed in this study. Here, for the first time we are reporting the effectiveness of an anti-diabetic drug chlorpropamide (CHL) under dosage conditions, a novel property to inhibit aggregation in human lysozyme (HL) in vitro. Spectroscopic (Turbidity, RLS, ThT, DLS, ANS) and microscopic (CLSM) results demonstrates that CHL has the potency to suppress aggregation in HL up to 70 %. CHL is shown to affect the elongation of fibrils with IC50 value of 88.5 µM as clear from the kinetics results, may be by interacting near/with aggregation prone regions of HL. Hemolytic assay also revealed the reduced cytotoxicity in the presence of CHL. Disruption of amyloid fibrils and inhibition of secondary nucleation in the presence of CHL was also evidenced by ThT, CD and CLSM results with reduced cytotoxicity as confirmed by hemolytic assay. We also performed preliminary studies on α-synuclein fibrillation inhibition and surprisingly found that CHL is not just inhibiting the fibrillation but also stabilizing the protein in its native state. These findings insinuate that CHL (anti-diabetic) possess multiple roles and can be a promising drug for developing therapeutic against non-systemic amyloidosis, Parkinson's disease and other amyloid associated disorders.

Biophysical insight into the anti-fibrillation potential of Glyburide for its possible implication in therapeutic intervention of amyloid associated diseases.

Protein aggregation is an underlying cause of many neurodegenerative diseases. Also, the overlapping pathological disturbances between neurodegenerative diseases and type-2 diabetes mellitus have urged the scientific community to explore potential of already available anti-diabetic medications in impeding amyloid formation too. Recent study brief out promising potential of an anti-diabetic drug Glyburide(GLY) as an inhibitor of amyloid fibrillation utilizing several biophysical techniques, computational methods and imaging tools. The mechanism of interaction was elucidated and the structural alterations in human serum albumin(HSA) as well as the microenvironment changes of its fluorophores(tryptophan, tyrosine) upon interacting with GLY were studied by spectroscopic techniques like Circular dichroism and synchronous fluorescence. Binding studies detailing about the GLY-HSA complex distance and the energy transfer efficiency was obtained by Fluorescence resonance energy transfer. For aggregation inhibition studies, the existence and size of aggregates formed in HSA and their inhibition by GLY was determined by Turbidity assay, Dynamic light scattering and Rayleigh light scattering along with dye binding assays. The ThT kinetics measurements analysis suggested that GLY deaccelerates fibrillation by decrement of apparent rate(Kapp) constant. The inhibitory effect of GLY might be attributed to native structure stabilization of HSA by obstruction into ß-sheet conversion as confirmed by CD spectroscopy results. Amyloid inhibition and suppression of amyloid-induced hemolysis by GLY was further delineated by TEM and SEM analysis respectively. All these findings for the first time report the new facet of the anti-amyloidogenic potential of GLY, making it a promising candidate to treat neurodegenerative diseases too in the near future.

Cholic acid inhibits amyloid fibrillation: Interplay of protonation and deprotonation.

Amyloidopathies are the consequence of misfolding with subsequent aggregation affecting people worldwide. Irrespective of speedy advancement in the field of therapeutics no agent for treating amyloidopathies has been discovered and thus targeting amyloid fibrillation process via repositioning of small molecules can be fruitful. According to previous reports potential amyloid inhibitors possess unique features like, hydrophobicity, aromaticity, charge etc. Herein, we have explored the effect of Cholic acid (CA) on amyloid fibrillation irrespective of the charge (determined by Zetasizer) using four proteins Human Serum Albumin, Bovine Serum Albumin, Human Insulin and Beta-lactoglobulin (HSA, BSA, HI and BLG) employing biophysical, imaging and computational techniques. ThT results revealed that CA in both protonated and deprotonated form is potent to curb HSA, BSA, BLG aggregation ~50% and HI aggregation ~96% in a dose dependent manner (in accord with CD, ANS and Congo red assay). Interestingly, CA treated samples displayed reduced cytotoxicity (Hemolytic assay) with altered morphology (TEM) and mechanism behind inhibition may be the interaction of CA with proteins via hydrophobic interactions and hydrogen bonding (supported by molecular docking results). This study proved CA (irrespective of the pH) a potential inhibitor of amyloidosis thus can be helpful in generalizing and repurposing the related drugs/compounds for their anti-aggregation behavior as an implication towards treating amyloidopathies.

Mechanistic insight into inhibition of amyloid fibrillation of human serum albumin by Vildagliptin.

Protein aggregation leads to several human pathologies such as Alzheimer's disease (AD), type 2 diabetes (T2D), Parkinson's disease (PD), etc. Due to the overlap in the mechanisms of type 2 diabetes and brain disorders, common effective pharmacological interventions to treat both T2D and AD is under extensive research. Therefore, major aim of research is to repurpose already established treatment of diabetes to cure AD as well. This study evaluates mechanistic insight into anti-amyloidogenic potential of anti-diabetic drug Vildagliptin (VLD) on human serum albumin fibrillation (HSA) by using biophysical, calorimetric, imaging techniques along with hemolytic assay. Dynamic light scattering (DLS) and Rayleigh light scattering (RLS) results showed presence of few small-sized aggregates in the presence of VLD which are formed by deaccelerating the amyloidogenesis as shown by thioflavin T (ThT) fluorescence and Congo red (CR) binding assay. Further, Isothermal titration calorimetry (ITC), steady state fluorescence quenching, molecular docking results revealed that VLD form complex with amyloid facilitating state of HSA and consequently mask the hydrophobic residues involved in amyloidogenesis as evident from decrease in ANS fluorescence. Differential scanning calorimetry (DSC) results confirm that VLD stabilizes the amyloid facilitating state of HSA. In addition, SEM images demonstrated that VLD alleviates the hemolytic effect induced by fibrils of HSA. This study reports VLD as a potential inhibitor of amyloid fibrillation and provides promising results to repurpose VLD as a drug candidate for the cure of Alzheimer's diseases along with diabetes.

Discovery of a tetracyclic indole alkaloid that postpones fibrillation of hen egg white lysozyme protein.

Protein aggregation, such as amyloid fibril formation, is molecular hallmark of many neurodegenerative disorders including Alzheimer's, Parkinson's, and Prion disease. Indole alkaloids are well-known as the compounds having the ability to inhibit protein fibrillation. In this study, we experimentally and computationally have investigated the anti-amyloid property of a derivative of a synthesized tetracyclic indole alkaloid (TCIA), possessing capable functional groups. The fibrillation reaction of Hen White Egg Lysozyme (HEWL) was performed in absence and presence of the indole alkaloid. For quantitative analysis, we used Thioflovin T binding assay which showed ~50% reduction in fibril formation in the presence of 20 µM TCIA. Using TEM imaging, we observed a significant morphological change in our model protein in the presence of TCIA. In addition, we exploited FT-IR assay by which Amide I peak's shifting toward lower wavenumber was clearly observed. Using Molecular Docking, the interaction of the inhibitor (TCIA) with the protein's amyloidogenic region was modeled. Also, different biophysical parameters were calculated by Molecular Dynamics (MD) simulation. Various biochemical assays, conformational change, and hydrophobicity exposure of the protein during amyloid formation indicated that the compound assists HEWL to keep its native structure via destabilizing ß-sheet structure.

Biophysical insight into the interaction of levocabastine with human serum albumin: spectroscopy and molecular docking approach.

Interaction of levocabastine with human serum albumin (HSA) is investigated by applying fluorescence spectroscopy, circular dichroism spectroscopy and molecular docking methods. Levocabastine is an important drug in treatment of allergy and currently a target drug for drug repurposing to treat other diseases like vernal keratoconjuctivitis. Fluorescence quenching data revealed that levocabastine bind weakly to protein with binding constant in the order of 103 M-1. Förster resonance energy transfer results indicated the binding distance of 2.28 nm for levocabastine. Synchronous fluorescence result suggest slight blue shift for tryptophan upon levocabastine binding, binding of levocabastine impelled rise in α-helical structure in protein, while there are minimal changes in tertiary structure in protein. Moreover, docking results indicate levocabastine binds to pocket near to the drug site-I in HSA via hydrogen bonding and hydrophobic interactions. Understanding the interaction of levocabastine with HSA is significant for the advancement of therapeutic and diagnostic strategies for optimal treatment results.Communicated by Ramaswamy H. Sarma.

Both beta sheet breaker and alpha helix forming pentapeptide inhibits protein fibrillation: Implication for the treatment of amyloid disorders.

For the first time, the effect of two novel designed pentapeptides on amyloid growth of human insulin using combined biophysical, microscopic, cell viability and computational approaches. Collective experimental data from ThT, ANS, and TEM demonstrate that in spite of having contrasting features, both peptides can effectively inhibit amyloid formation by prolonging lag phase, slowing down aggregation rate, and reducing final fibril formation (up to 84.26% and 85.24% by P1 and P7 respectively). Although pure amyloid caused profound cellular toxicity in SH-SY5Y neuronal cells, amyloid formed in the presence of peptides showed much reduced cellular toxicity. Such an inhibitory effect can be attributed to interference with the structural transition of insulin toward ß-sheet structure by peptides. Furthermore, molecular dynamic simulations confirm that peptide preferentially binds to nearby region which is more prone to form aggregates that consequently disrupts self-assembly into amyloid fibrils (P1 and P7 possess inhibition constant value of 0.000183 and 0.000216 nm, respectively), supporting our experimental observations. This study underscores the information about the sequence based inhibition mechanism of peptides that might dictate their inhibition or modulation capacity, which might be helpful in designing anti-amyloid therapeutics.

Unravelling the inhibitory and cytoprotective potential of diuretics towards amyloid fibrillation.

Protein misfolding and deposition of aggregated proteins inside as well as outside of the cells have been associated with several neurotoxic and neurodegenerative disorders like Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. that could be controlled by anti-aggregation methodologies employing either inhibition or disaggregation of toxic aggregates. Also, the Alzheimer's disease develops in later life is somehow related to the high mid-life blood pressure. Therefore the present work targets the amyloid inhibiting potential of diuretics (a class of antihypertensive drugs) - Indapamide (INDP) and Hydrochlorothiazide (HCTZ) against human serum albumin (HSA) and human lysozyme (HL) fibrillogenesis. The effect of both INDP and HCTZ on the kinetics of amyloid formation of HSA and HL was illustrated and various biophysical techniques like Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, Congo red measurements and circular dichroism (CD) measurements depicted the inhibitory action of both INDP and HCTZ in a dose dependent manner. Transmission Electronic Microscopy (TEM) confirmed the absence of fibrillar structures when HSA and HL were co-incubated with INDP and HCTZ. In addition, molecular docking results revealed that both the drugs interacts with HSA and HL through hydrophobic interactions as well as hydrogen bonding, and also showed non-hemolytic activity on human RBCs demonstrated by the Hemolytic assay. Thus, both INDP and HCTZ could be propitious as a therapeutic agent and aid in the cure of amyloid related diseases.

Biophysical Elucidation of Amyloid Fibrillation Inhibition and Prevention of Secondary Nucleation by Cholic Acid: An Unexplored Function of Cholic Acid.

Protein misfolding and its deviant self-assembly to converge into amyloid fibrils is associated with the perturbation of cellular functions and thus with debilitating neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, etc. A great deal of research has already been carried out to discover a potential amyloid inhibitor that can slow down, prevent, or remodel toxic amyloids. In the present study with the help of a combination of biophysical, imaging, and computational techniques, we investigated the mechanism of interaction of cholic acid (CA), a primary bile acid, with human insulin and Aß-42 and found CA to be effective in inhibiting amyloid formation. From ThT data, we inferred that CA encumbers amyloid fibrillation up to 90% chiefly by targeting elongation of fibrils with an insignificant effect on lag time, while in the case of Aß-42, CA stabilizes the peptide in its native state preventing its fibrillation. Strikingly upon adding initially at the secondary nucleation stage, CA also detained the progression/growth of insulin fibrils. CA is unable to prevent the conformational changes completely during fibrillation but tends to resist and maintain an α helical structure up to a significant extent at a primary nucleation stage while reducing the ß sheet rich content at the secondary nucleation stage. Moreover, CA treated samples exhibited reduced cytotoxicity and different morphology. Furthermore, the results obtained after molecular docking indicated that CA is interacting with insulin via hydrogen bonds. For future research, this study can be considered as preliminary research for the development of CA, a metabolite of our body, as a potential therapeutic agent against Alzheimer's disease without even stimulating the immunological responses.

Cytotoxic species in amyloid-associated diseases: Oligomers or mature fibrils.

Amyloid diseases especially, Alzheimer's disease (AD), is characterized by an imbalance between the production and clearance of amyloid-ß (Aß) species. Amyloidogenic proteins or peptides can transform structurally from monomers into ß-stranded fibrils via multiple oligomeric states. Among various amyloid species, structured oligomers are proposed to be more toxic than fibrils; however, the identification of amyloid oligomers has been challenging due to their heterogeneous and metastable nature. Multiple techniques have recently helped in better understanding of oligomer's assembly details and structural properties. Moreover, some progress on elucidating the mechanisms of oligomer-triggered toxicity has been made. Based on the collection of current findings, there is growing consensus that control of toxic amyloid oligomers could be a valid approach to regulate amyloid-associated toxicity, which could advance development of new diagnostics and therapeutics for amyloid-related diseases. In this review, we have described the recent scenario of amyloid diseases with a great deal of information about the recent understanding of oligomers' assembly, structural properties, and toxicity. Also comprehensive details have been provided to differentiate the degree of toxicity associated with prefibrillar aggregates.

Amyloid Oligomers, Protofibrils and Fibrils.

Amyloid diseases are of major concern all over the world due to a number of factors including: (i) aging population, (ii) increasing life span and (iii) lack of effective pharmacotherapy options. The past decade has seen intense research in discovering disease-modifying multi-targeting small molecules as therapeutic options. In recent years, targeting the amyloid cascade has emerged as an attractive strategy to discover novel neurotherapeutics. Formation of amyloid species, with different degrees of solubility and neurotoxicity is associated with the gradual decline in cognition leading to dementia/cell dysfunction. Here, in this chapter, we have described the recent scenario of amyloid diseases with a great deal of information about the structural features of oligomers, protofibrils and fibrils. Also, comprehensive details have been provided to differentiate the degree of toxicity associated with prefibrillar aggregates. Moreover, a review of the technologies that aid characterisation of oligomer, protofibrils and fibrils as well as various inhibition strategies to overcome protein fibrillation are also discussed.

Stabilizing proteins to prevent conformational changes required for amyloid fibril formation.

Amyloid fibrillation is associated with several human maladies, such as Alzheimer's, Parkinson's, Huntington's diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti-inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in a linear fashion by influencing the nucleation phase as assayed by thioflavin T fluorescence, transmission electron microscopy, and dynamic light scattering. Furthermore, circular dichroism analysis supported the DSC results, showing that IBFN binds to the native state of proteins and almost completely prevents their tendency to lose secondary and tertiary structures. Cell toxicity assay confirms that species formed in the presence of IBFN are less toxic to neuronal cells (SH-SY5Y). These results demonstrate the feasibility of using a small molecule to stabilize the native state of proteins, thereby preventing the amyloidogenic conformational changes, which appear to be the common link in several human amyloid diseases.

DARK Classics in Chemical Neuroscience: Opium, a Friend or Foe.

Opium has found great use medicinally for its analgesic properties and has been witnessed as one of the most popular medications used in psychiatry. Opium derivatives have been shown as efficacious for relieving pain and the treatment of epileptic seizures, but progressive research toward their use in the treatment of neurodegenerative diseases remain elusive. To gain more insight into the other properties of opium such as anti-inflammatory properties, herein we discuss basic information regarding opium, opium content and mechanism of action, pharmacology of opium derivatives, the role of opium in the prevention of neurodegeneration, and adverse effects of opium derivatives on neuronal health.

Elucidating the Inhibitory Potential of Designed Peptides Against Amyloid Fibrillation and Amyloid Associated Cytotoxicity.

Inhibition of fibrillation process and disaggregation of mature fibrils using small peptide are the promising remedial strategies to combat neurodegenerative diseases. However, designing peptide-based drugs to target ß-sheet-rich amyloid has been a major challenge. The current work describes, for the first time, the amyloid inhibitory potential of the two short peptides (selected on the basis of predisposition of their amino acid residues toward ß-sheet formation) using combination of biophysical, imaging methods, and docking approaches. Results showed that peptides employed different mechanisms to inhibit the amyloid fibrillation. Furthermore, they were also effective in blocking the amyloid fibrillation pathway. In contrary to the insulin fibrillar mesh, significantly less fibrillar species appeared in the presence of peptides, as confirmed by transmission electron microscopy. Circular dichroism analysis indicated that although peptides did not stabilize the native state of insulin, they inhibited amyloid aggregation by reducing the formation of ß-sheet rich structures. Hemolytic assay revealed the non-hemolytic nature of the species formed when insulin was co-incubated with the peptides. Therefore, despite the inherent potential to form ß-sheet structure, these peptides inhibited the amyloid formation and potentially can be used as therapeutics for the treatment of amyloid-related diseases.

Biophysical insight reveals tannic acid as amyloid inducer and conformation transformer from amorphous to amyloid aggregates in Concanavalin A (ConA).

The aggregation phenomenon (amyloid and amorphous) is associated with several pathological complications in human, such as Alzheimer's, Parkinson's, Huntington, Cataract diseases, and Diabetes mellitus type 2. In the present study we are offering evidence and breaking the general belief with regard to the polyphenols action as protein aggregate inhibitors. Herein we confirm that tannic acid (TA) is not only an amyloid inducer, but also it switches one type of conformation, ultimately morphology, into another. We ascertain based on our findings that aggregates are not rigid structures and the stability can be challenged under certain conditions. This study also confirms that unfolded and amorphous aggregates can serve as precursors of amyloids and TA interactions with unordered aggregates (amorphous) bringing orderliness in the conformation via amyloidosis. The shifting of unordered conformation toward orderliness is governed by the modulation in surface hydrophobic patches in Concanavalin A (ConA). Hence, a degree of exposed hydrophobic cluster can be claimed as a strong parameter to detect and distinguish the native, amorphous and both types of amyloids. Turbidity and Rayleigh light scattering measurements followed similar pattern while Thioflavin T and 1-anilino-8-naphthalene sulfonate fluorescence assays of the binding with amorphous and amyloid followed an inverse relation. Electron microscopic studies revealed the morphological variation in the ConA at 65°C as amorphous while the ConA treated with TA followed by heat treatment at 65°C was defined as amyloid in nature. Interestingly for the first time we are reporting the slight agglutination activity by the ConA amyloids.