Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A.

Protein aggregation is related to numerous pathological conditions like Alzheimer's and Parkinson's disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg2Cl2 (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen ß-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native ß-barrel conformation to ß-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg2Cl2 treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the ß-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.

Protective role of chlorogenic acid in preserving cytochrome-c stability against HFIP-induced molten globule state at physiological pH.

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation.