Unraveling the Interaction of Diflunisal with Cyclodextrin and Lysozyme by Fluorescence Spectroscopy.

Understanding the interaction between the drug:carrier complex and protein is essential for the development of a new drug-delivery system. However, the majority of reports are based on an understanding of interactions between the drug and protein. Here, we present our findings on the interaction of the anti-inflammatory drug diflunisal with the drug carrier cyclodextrin (CD) and the protein lysozyme, utilizing steady-state and time-resolved fluorescence spectroscopy. Our findings reveal a different pattern of molecular interaction between the inclusion complex of ß-CD (ß-CD) or hydroxypropyl-ß-CD (HP-ß-CD) (as the host) and diflunisal (as the guest) in the presence of protein lysozyme. The quantum yield for the 1:2 guest:host complex is twice that of the 1:1 guest:host complex, indicating a more stable hydrophobic microenvironment created in the 1:2 complex. Consequently, the nonradiative decay pathway is significantly reduced. The interaction is characterized by ultrafast solvation dynamics and time-resolved fluorescence resonance energy transfer. The solvation dynamics of the lysozyme becomes 10% faster under the condition of binding with the drug, indicating a negligible change in the polar environment after binding. In addition, the fluorescence lifetime of diflunisal (acceptor) is increased by 50% in the presence of the lysozyme (donor), which indicates that the drug molecule is bound to the binding pocket on the surface of the protein, and the average distance between active tryptophan in the hydrophobic region and diflunisal is calculated to be approximately 50 Å. Excitation and emission matrix spectroscopy reveals that the tryptophan emission increases 3-5 times in the presence of both diflunisal and CD. This indicates that the tryptophan of lysozyme may be present in a more hydrophobic environment in the presence of both diflunisal and CD. Our observations on the interaction of diflunisal with ß-CD and lysozyme are well supported by molecular dynamics simulation. Results from this study may have an impact on the development of a better drug-delivery system in the future. It also reveals a fundamental molecular mechanism of interaction of the drug-carrier complex with the protein.

An Innate Host Defense Protein ß2-Microglobulin Keeps a Check on α-Synuclein amyloid Assembly: Implications in Parkinson's Disease.

Amyloid formation due to protein misfolding has gained significant attention due to its association with neurodegenerative diseases. α-Synuclein (α-syn) is one such protein that undergoes a profound conformational switch to form higher order cross-ß-sheet structures, resulting in amyloid formation, which is linked to the pathophysiology of Parkinson's disease (PD). The present status of research on α-syn aggregation and PD reveals that the disease progression may be linked with many other diseases, such as kidney-related disorders. Unraveling the link between PD and non-neurological diseases may help in early detection and a better understanding of PD progression. Herein, we investigated the modulation of α-syn in the presence of ß2-microglobulin (ß2m), a structural protein associated with dialysis-related amyloidosis. We took a multi-disciplinary approach to establish that ß2m mitigates amyloid formation by α-syn. Our fluorescence, microscopy and toxicity data demonstrated that sub-stoichiometric ratio of ß2m drives α-syn into off-pathway non-toxic aggregates incompetent of transforming into amyloids. Using AlphaFold2 and all-atom MD simulation, we showed that the ß-strand segments (ß1 and ß2) of α-synuclein, which frequently engage in interactions within amyloid fibrils, interact with the last ß-strand at the C-terminal of ß2m. The outcome of this study will unravel the yet unknown potential linkage of PD with kidney-related disorders. Insights from the cross-talk between two amyloidogenic proteins will lead to early diagnosis and new therapeutic approaches for treating Parkinson's disease. Finally, disruption of the nucleation process of α-syn amyloids by targeting the ß1-ß2 region will constitute a potential therapeutic approach for inhibiting amyloid formation.

Interaction of Ibuprofen with Partially Unfolded Bovine Serum Albumin in the Presence of Ionic Micelles and Oligosaccharides at Different λex and pH: A Spectroscopic Analysis.

The interaction between the plasma protein bovine serum albumin (BSA) and the drug ibuprofen (IBU) has been investigated at three different pH values (7.4, 6.5, and 8.0) in the presence of oligosaccharides and surfactants. The interaction analysis of BSA with oligosaccharides and surfactants has also been studied in the absence of the drug ibuprofen. The results obtained give convenient and efficient access to understand the mechanism of binding of ibuprofen to BSA, and the major forces involved are found to be hydrophobic forces, hydrogen bonding and ionic interactions. In addition to that, the formation of inclusion complexes of ibuprofen with oligosaccharides (ß-CD and 2-HP-ß-CD) has been observed, which has depicted that due to the hydrophobic nature of ibuprofen, it becomes more soluble in the presence of oligosaccharides, but due to the larger size of the inclusion complexes, these could not be able to access the hydrophobic pocket of BSA where tryptophan-212 (Trp-212) resides. The binding interaction between BSA and ibuprofen is observed in the presence of surfactants (SDS and CTAB), which partially unfold the protein. Non-radiative fluorescence resonance energy transfer (FRET) from Trp and Tyr residues of BSA in the presence of an anionic surfactant SDS to ibuprofen has depicted that there is a possibility of drug binding even in the partially unfolded state of BSA protein. Furthermore, the distance between the protein and the drug has been calculated from the FRET efficiency, which gives a comprehensive overview of ibuprofen binding to BSA even in its partially denatured state. The hydrophobic drug binding to the partially unfolded serum albumin protein (BSA) supports the "necklace and bead structures" model and opens up a new direction of drug loading and delivery system, which will have critical therapeutic applications in the efficient delivery of pharmacologically prominent drugs.

Molecular Mechanism of Interaction of Curcumin with BSA, Surfactants and Live E. Coli Cell Membrane Revealed by Fluorescence Spectroscopy and Confocal Microscopy.

Clinical trials on the therapeutic effect of curcumin have proven to be highly effective against many diseases including cancer and Alzheimer's. However, the molecular mechanism of interaction of curcumin with protein and live cell membrane is poorly understood. Here, we report the mechanism of interaction of curcumin with bovine serum albumin (BSA) and live E. coli cell membrane in the presence of organized assemblies of sodium dodecyl sulfate (SDS) and cetrimonium bromide (CTAB) by fluorescence spectroscopy, laser-scanning confocal microscopy, and computation. Enhanced binding constant, blue-shifted emission spectra, and imaging of heterogeneous FRET on live bacteria cell membrane strongly indicate the complex formation of curcumin with strong hydrophobic interaction, which is further validated by computation. Finally, our results may shed light on the efficient strategy of applications of curcumin as a natural therapeutic lead in clinical trials against many life-threatening diseases.