Mohammad Khursheed Siddiqi

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Comparative insight into surfactants mediated amyloidogenesis of lysozyme

Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation.

Anti-amyloidogenic behavior and interaction of Diallylsulfide with Human Serum Albumin

In this work, binding of garlic component-Diallysulfide (DAS) with major human blood transport protein, Human Serum Albumin (HSA) and its anti- amyloidogenic behavior has been studied by utilizing various spectroscopic and molecular docking strategies. The HSA exhibit significant reduction in fluorescence intensity upon interaction with DAS. DAS quenches the fluorescence of HSA in concentration dependent manner with binding affinity of 1.14×103M-1. UV-visible spectroscopy results confirm the formation of DAS-HSA complex and secondary structure of HSA get stabilized upon complexation with DAS as observed by far UV CD spectroscopy and Differential Scanning Calorimetry. The topology of HSA in absence and presence of DAS was monitored through Dynamic Light Scattering (DLS) technique, inferred that protein becomes more compact in presence of DAS. Further, molecular docking study shows that DAS bind to the nearby site II in subdomain III of HSA. Moreover, effect of DAS was studied on HSA fibrillation process. ThT binding, ANS fluorescence assay, CD measurement, DLS and Transmission Electron Microscopy (TEM) results altogether confirm the anti-amyloidogenic property of DAS. This work will provide biophysical insight into the interaction of DAS with HSA and will help in designing more potential therapeutic strategies against protein aggregation by exploiting other related compounds.

Ascorbic acid inhibits human insulin aggregation and protects against amyloid induced cytotoxicity

Protein aggregation into oligomers and fibrils are associated with many human pathophysiologies. Compounds that modulate protein aggregation and interact with preformed fibrils and convert them to less toxic species, expect to serve as promising drug candidates and aid to the drug development efforts against aggregation diseases. In present study, the kinetics of amyloid fibril formation by human insulin (HI) and the anti-amyloidogenic activity of ascorbic acid (AA) were investigated by employing various spectroscopic, imaging and computational approaches. We demonstrate that ascorbic acid significantly inhibits the fibrillation of HI in a dose-dependent manner. Interestingly ascorbic acid destabilise the preformed amyloid fibrils and protects human neuroblastoma cell line (SH- SY5Y) against amyloid induced cytotoxicity. The present data signifies the role of ascorbic acid that can serve as potential molecule in preventing human insulin aggregation and associated pathophysiologies.

Attenuation of amyloid fibrillation in presence of Warfarin: A biophysical investigation

Protein misfolding and aggregation are associated with more than twenty diseases, such as neurodegenerative diseases. The amyloid oligomers and fibrils may induce cell membrane disruption and lead to cell apoptosis. A great number of studies have focused on discovery of amyloid inhibitors which may prevent or treat amyloidosis. In this study, we used human serum albumin (HSA) as an amyloid model to test the anti-amyloid effects of warfarin (WFN), a very well-known drug for treatment of thrombosis and also used by biophysicists to characterize the specific binding site on HSA (site I of subdomain IIA). We have used a combination of different biophysical, spectroscopic and imaging techniques to prove the anti-amyloidogenic behavior of WFN. Our results demonstrated that WFN is capable enough to inhibit the HSA fibrillation. Exposed HSA surface hydrophobicity was decreased by 50% as judged by ANS analysis. Moreover, anti-amyloidegenic behavior of WFN was found to be concentration dependent as supported by decreased ThT fluorescence by 22.4% and 46% at WFN concentrations of 500 and 1000μM, respectively. Circular dichroism technique showed the change in secondary structure of native HSA as well as in presence of WFN. These results suggests that WFN is capable of inhibiting amyloid aggregation, hence, WFN related compounds may thus be further explored for designing effective anti-amyloidosis compounds.

Vitamin B12 offers neuronal cell protection by inhibiting Aβ-42 amyloid fibrillation

Protein misfolding and aggregation has been implicated as the cause of more than 20 diseases in humans such as Alzheimers and Parkinsons and systemic amyloidosis. Retardation of Aβ- 42 aggregation is considered as a promising and challenging strategy for developing effective therapeutics against Alzheimers disease. Herein, we demonstrated the effect of vitamin B12 (VB) on inhibiting amyloid formation by employing ThT fluorescence assay, circular dichroism, ANS fluorescence assay, dynamic light scattering measurements and transmission electron microscopy and cell viability assay. Our results demonstrate that vitamin B12 (VB), inhibits Aβ- 42 aggregation in a concentration dependent manner. Further VB also provide protection against amyloid induced cytotoxicity in human neuronal cell line. This study points towards a promising strategy to combat Aβ- 42 aggregation and may have broader implication for targeting other neurological disorders whose distinct hallmark is also amyloid formation.

Biophysical insights into the interaction of hen egg white lysozyme with therapeutic dye clofazimine: modulation of activity and SDS induced aggregation of model protein.

The present study details the binding process of clofazimine to hen egg white lysozyme (HEWL) using spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), and molecular docking techniques. Clofazimine binds to the protein with binding constant (Kb) in the order of 1.57 × 104 at 298 K. Binding process is spontaneous and exothermic. Molecular docking results suggested the involvement of hydrogen bonding and hydrophobic interactions in the binding process. Bacterial cell lytic activity in the presence of clofazimine increased to more than 40% of the value obtained with HEWL only. Interaction of the drug with HEWL induced ordered secondary structure in the protein and molecular compaction. Clofazimine also effectively inhibited the sodium dodecyl sulfate (SDS) induced amyloid formation in HEWL and caused disaggregation of preformed fibrils, reinforcing the notion that there is involvement of hydrophobic interactions and hydrogen bonding in the binding process of clofazimine with HEWL and clofazimine destabilizes the mature fibrils. Further, TEM images confirmed that fibrillar species were absent in the samples where amyloid induction was performed in the presence of clofazimine. As clofazimine is a drug less explored for the inhibition of fibril formation of the proteins, this study reports the inhibition of SDS-induced amyloid formation of HEWL by clofazimine, which will help in the development of clofazimine-related molecules for the treatment of amyloidosis.

A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins

Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra.

Probing the interaction of cephalosporin antibiotic–ceftazidime with human serum albumin: A biophysical investigation

The fate of drug administered to a living organism depends on drugs pharmacokinetics as well as pharmacological behavior. Serum albumins (proteins in blood plasma of human) act as a carrier molecule to deliver the drug at specific site. In the present study, we have explored the mechanism of interaction between cephalosporin antibiotic-ceftazidime (CFD) and human serum albumin (HSA) by spectroscopic and molecular docking studies. Quenching of HSA fluorescence by CFD inferred that it binds to HSA through static quenching mechanism; with binding affinity in order of 104M-1. Fluorescence resonance energy transfer (FRET) results shows that donor and acceptor molecule are at 2.08nm apart and also reflects the high probability of energy transfer between HSA and CFD. Change in secondary structure as well as microenvironment around both tryptophan and tyrosine residue, were monitored by Circular Dichroism (CD) and Synchronous fluorescence spectroscopy respectively; confirms that CFD increases the alpha helical secondary structure as well as altered the environment around tryptophan and tyrosine. The specific binding site of CFD on HSA was determined by site-specific markers and molecular docking methods. CFD preferably bind to subdomain IIIA (Sudlow site II) on HSA.

Fibrillogenesis of human serum albumin in the presence of levodopa - spectroscopic, calorimetric and microscopic studies.

Studying amyloid associated neurodegenerative diseases is an active area of research. Cure for these diseases are still to be discovered. In the present study we have performed comprehensive biophysical and computational experiments showing levodopa not only significantly inhibits heat induced fibrillization of human serum albumin but also disaggregates preformed fibrils. Thioflavin T (ThT) binding assay was used to monitor the fibrillation process of human serum albumin (HSA) at 65°C in the presence and absence of levodopa. Binding of levodopa was studied using isothermal titration calorimetry (ITC), binding constant was found to be 3.6×103M-1. Thermal stabilization effect of levodopa on HSA was studied using differential scanning calorimetry (DSC). Microscopic imaging techniques were employed to analyze the morphology of aggregates and effect of levodopa on aggregation. Further, molecular docking study was also utilized to decipher the amino acid residues involved in the binding interaction of levodopa with HSA. Levodopa interferes in the Fibrillogenesis of HSA by interacting with the amino acid residues near to drug binding site II on the HSA with the binding constant of the order of 103 and stabilizes the protein. The results are indicative of the potential use of levodopa as a therapeutic agent for the treatment of amyloid diseases.