Phloroglucinol inhibited glycation via entrapping carbonyl intermediates

Abstract

Advanced glycation end products (AGEs) play an important role in the pathogenesis of age-linked disorders and diabetes mellitus. The aim of this study was to assess the repurposing potential of Phloroglucinol (PHL the antispasmodic drug), as an anti-glycation agent using Fructose-BSA model. The ability of PHL to inhibit AGE formation was evaluated using AGEs formation (Intrinsic fluorescence), fructosamine adduct (NBT) and free lysine availability (TNBSA) assays. The BSA protein conformation was assessed through Thioflavin-T, Congo-Red and Circular Dichroism assays. The lysine blockade and carbonyl entrapment were explored as possible mode of action. Our data showed that PHL significantly decreased the formation of AGEs with an IC50 value of 0.3mM. The fructosamine adducts and free lysine load was found to be reduced. Additionally, the BSA conformation was preserved by PHL. Mechanistic assays did not reveal involvement of lysine blockade as underlying reason for reduction in AGEs load. This was also supported by computational data whereby PHL failed to engage any catalytic residue involved in early fructose-BSA interaction. However, it was found to entrap the carbonyl moieties. In conclusion, the PHL demonstrated anti-glycation potential, which can be attributed to its ability to entrap carbonyl intermediates. Hence, the clinically available antispasmodic drug, presents itself as a promising candidate to be repurposed as anti-glycation agent.

Fig 1. Structure of Phloroglucinol.

Fig 2. Effect of Phloroglucinol on AGES formation.

The figure shows the formation of advance glycation end products (as intrinsic fluorescence) in the presence of AG and PHL. The gBSA group exhibit enhanced fluorescence as compared to nBSA. The AG and PHL has significantly reduced the formation of AGEs in dose dependent manner. The hash (###) represents the significant (p<0.005) difference as compared to nBSA while asterisks [* (p<0.05), ** (p<0.01) and ***(p<0.005)] represents the statistical comparison with the gBSA. All values are expressed as mean ± SEM of intrinsic AGEs fluorescence intensity (n = 3).

Fig 3. Effect of Phloroglucinol on fructosamine adduct assay.

The figure shows the formation of fructosamine adducts as absorbance in the presence of AG and PHL. The gBSA group exhibit enhanced abosrbance as compared to nBSA. The AG and PHL has significantly reduced the formation of adducts in dose dependent manner. The hash (###) represents the significant (p<0.005) difference as compared to nBSA while asterisks [* (p<0.05), ** (p<0.01) and ***(p<0.005)] represents the statistical comparison with the gBSA. All values are expressed as mean ± SEM of absorbance (n = 3).

Fig 4. Effect of Phloroglucinol on TNBSA assay.

The figure depicts mean ± SEM of absorbance indicative of free lysine in the presence of AG and PHL (n = 3). The gBSA group exhibit reduction in the absorbance as compared to nBSA. The AG and PHL shows significant increase in the absorbance in comparison with gBSA. The hash (###) represents the significant (p<0.005) difference as compared to nBSA while asterisks [* (p<0.05), ** (p<0.01) and ***(p<0.005)] represents the statistical comparison with the gBSA.

Fig 5. Effect of Phloroglucinol on Thioflavin-T assay.

The figure shows the mean ± SEM of ThT fluorescence in the presence of AG and PHL (n = 3). The gBSA group exhibit enhanced fluorescence as compared to nBSA. The AG and PHL treatment groups exhibit significant decrease in fluorescence as compared to gBSA. The hash (###) represents the significant (p<0.005) difference as compared to nBSA while asterisks [* (p<0.05), ** (p<0.01) and ***(p<0.005)] represents the statistical comparison with the gBSA.

Fig 6. Effect of Phloroglucinol on Congo Red assay.

The figure shows the formation of amyloid-like aggregates as absorbance in the presence of AG and PHL. The gBSA group exhibit enhanced absorbance as compared to nBSA. The AG (5mM) and PHL (0.25, 0.5 and 1 mM) demonstrated significant reduction in the absorbance as compared to gBSA. The hash (###) represents the significant (p<0.005) difference as compared to nBSA while asterisks [* (p<0.05), ** (p<0.01) and ***(p<0.005)] represents the statistical comparison with the gBSA. All values are expressed as mean ± SEM of absorbance (n = 3).

Fig 7. Effect of Phloroglucinol on CD spectra of BSA.

The figure shows the CD spectra of BSA underwent glycation reaction with fructose in the presence and absence of AG and PHL. The nBSA showed its characteristic alpha helical spectra with two lambda max at 208 and 222 nm, while the spectra of gBSA exhibited lambda max only at 218 nm, which is indicative of loss of alpha helicity. The AG and PHL exposure gave the BSA spectra similar to that of nBSA, which suggests the preservation of secondary structure of BSA in the presence of fructose.

Fig 8. Effect of Phloroglucinol on lysine blockage assay.

The figure shows mean ± SEM of fluorescence intensity as lysine blockade in the presence of AG and PHL (n = 3). The data shows the insignificant alterations in fluorescence intensity in both standard as well as test drug.

Fig 9. Binding interaction of Phloroglucinol with different residues of BSA.

The computational demonstrated interaction of PHL with the GLY, HIS, ILE, LYS, PHE, TRP and VAL residues of BSA.

Fig 10. Effect of Phloroglucinol on carbonyl entrapping assay using HPLC.

The figure depicts the representative chromatograms of carbonyl entrapping assay in the presence of AG and PHL. The 2-MQ peak was found in the negative control group, while it was significantly reduced following AG and PHL treatments. The percent change following AG treatment was found to 52% whereas in case of PHL, the dose dependent increase in percent inhibitions were [(0.5mM (92%), 1mM (94%), 2mM (97%)] respectively.