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Rifampicin Induced Aggregation of Ovalbumin: Malicious Behaviour of Antibiotics
Naveed A Fazili, Gufran A Siddiqui, Sheraz A Bhat
1Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh 202 002, India. aabgeenanaim@gmail.com.
Abstract:
Molecular modeling deciphered the site of interaction of rifampicin in the structure of ovalbumin at a site which is surrounded by residues Glu-214, Asp-98, Pro-85, Asp-91 and Asp-47. Isothermal calorimetric analysis determined the thermodynamic parameters i.e. ΔH and ΔS which came out be -8.086 cal/mol and -131 cal/mol/deg. respectively. Ovalbumin is a secretory protein of hen oviduct, present in the human blood serum and interacts with the drug rifampicin in vivo, when administered. Simulating these conditions in vitro revealed that rifampicin induced the aggregated state at 6 µM concentration which was featured by a decrease in the ANS fluorescence intensity relative to the native state while as the pre-molten and molten globule state were obtained at 3 µM and 5 µM concentration of rifampicin respectively displaying a hike in the ANS fluorescence intensity. Far-UV CD analysis suggested β-sheet rich structure with negative ellipticity peak at 217 nm for native ovalbumin incubated with 6 µM rifampicin. Increase in absorbance at 450 nm, red shift of 50 nm in the congo red binding assay and a hike of 10 fold in the ThT fluorescence intensity compared to the native state further confirmed aggregate formation. Moreover, TEM images displayed aggregates to be spherical morphologically. Aggregates formed at 6 µM rifampicin concentration were found to be cytotoxic as there was a reduction of cell viability to 28%. Thus, protein-drug interaction primarily facilitates a structural alteration in the native structure of proteins leading to their aggregation which were proved to be cytotoxic in nature.
Insights
Rifampicin binding to ovalbumin protein induces structural changes, leading to cytotoxic aggregate formation. This protein aggregation, characterized by specific molecular interactions, has implications for drug safety and protein behavior in vivo.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Ovalbumin, a secretory protein found in hen oviduct and human blood serum, can interact with administered drugs like rifampicin.
- Understanding protein-drug interactions is crucial for predicting drug efficacy and potential adverse effects.
- Rifampicin is a widely used antibiotic with known interactions with various biological molecules.
Purpose of the Study:
- To investigate the molecular interactions between ovalbumin and rifampicin.
- To characterize the structural changes and aggregation propensity of ovalbumin upon rifampicin binding.
- To assess the cytotoxicity of rifampicin-induced ovalbumin aggregates.
Main Methods:
- Molecular modeling to identify the drug-protein binding site.
- Isothermal titration calorimetry (ITC) to determine thermodynamic parameters.
- Spectroscopic techniques (ANS fluorescence, Far-UV CD) to monitor protein structural changes.
- Congo red and Thioflavin T (ThT) assays to detect amyloid-like aggregate formation.
- Transmission electron microscopy (TEM) for morphological analysis of aggregates.
- Cell viability assays to evaluate cytotoxicity.
Main Results:
- Molecular modeling pinpointed a specific binding site for rifampicin on ovalbumin.
- Rifampicin induced concentration-dependent structural transitions, including pre-molten and molten globule states, and ultimately aggregation at 6 µM.
- Spectroscopic and binding assays confirmed the formation of β-sheet rich, spherical aggregates.
- The formed aggregates exhibited significant cytotoxicity, reducing cell viability by 72%.
Conclusions:
- Protein-drug interactions, exemplified by rifampicin and ovalbumin, can lead to significant structural alterations and the formation of cytotoxic aggregates.
- The study highlights the potential for drug-induced protein aggregation with implications for therapeutic safety.
- Further research into drug-protein interactions is warranted to mitigate potential toxicities associated with protein aggregation.
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