From the earlier inhibition study, we observed effective inhibition of hIAPP amyloid formation while co-incubating hIAPP with BSBHps (2C and 2D). The hIAPP-induced cytotoxicity may arise due to the presence of the oligomers and amyloid of the aggregating peptide. Hence, to be an effective therapeutic agent, the peptidomimetics must disrupt the preformed amyloid into a non-oligomeric state. Therefore, we analysed the potential of the BSBHps in disrupting the preformed hIAPP amyloid in vitro. From the initial ThT fluorescence assay (black, Figure 2.16 (a)), we witnessed that hIAPP fibrillization attained its maximum at ~45-50h. Hence, to study the effects of the BSBHps on preformed hIAPP amyloid, we sketched a study where the BSBHps were added to the preformed mature fibrils of hIAPP. The hIAPPwas incubated exclusively at the physiological condition in PBS for 48h to allow to form matured amyloid fibrils. Different molar ratios (2-, 5- and 10-fold) of
BSBHps were added to it. Fibrillogenesis was monitored by various biophysical techniques.
The samples for the different studies were prepared similarly as described above in section 2.6. Since BSBHp 2E was inefficient in inhibiting the fibrillization of hIAPP, we excluded it from the disruption study.
2.7.1. Monitoring conformational transition by CD and FTIR studies:
Incubating for 7 (2+5) days, it was observed from the CD analysis that hIAPP alone showed a negative band centered at ~ 220 nm and a simultaneous positive band centered at ~ 200 nm, confirming β-sheet conformation of hIAPP (Figure 2.19 (a)). However, when hIAPP was co- incubated with 10-fold molar ratios of 2C and 2D (Figure 2.19 (a)), such β-sheet conformation was not observed, suggesting appreciable disruption of hIAPP amyloid. The dose-dependent study revealed that in the presence of the BSBHps, β-sheet content reduced gradually (Figure 2.18) as the doses were increased, giving mixtures of conformation. Similar results were obtained in the presence of the control peptide 2B (Figure 2.19 (a) and 2.20), which also indicated the conversion of β-sheet conformation into a mixture of conformations.
From the FTIR spectra, we noticed a sharp band of amide I at 1630 cm-1 when only hIAPP was incubated at physiological condition, confirming characteristic β-sheet conformation (Figure 2.19 (b)). However, the band shifted to 1650 cm-1 and 1652 cm-1 in co-incubation with 10-fold molar ratios of 2C and 2D respectively indicating conversion of β-sheet conformation. In contrast, in the presence of 2-fold and 5-fold molar ratios of BSBHps with hIAPP, similar results with less efficiency were observed (Figure 2.21). Meanwhile, in the presence of the control peptidomimetics, 2B with various doses (Figure 2.19 (b) and 2.21), reduction of β-sheet conformation of hIAPP was also achieved.
Figure 2.19: (a) CD and (b) FTIR spectra of hIAPP alone (black) and in co-incubation with 10 fold molar ratios of peptidomimetics 2B (red), 2C (blue) and 2D (orange). Spectra were recorded after seven days of incubation of the peptides in PBS (50 mM) at pH 7.4 and 37 °C.
Figure 2.20: CD spectra of hIAPP alone (black) and in co-incubation with (a) 2-fold and (b) 5-fold molar ratios of peptidomimetics 2B (red), 2C (blue) and 2D (orange). Spectra were recorded after seven days of incubation of the peptides in PBS (50 mM) at pH 7.4 and 37 °C.
Figure 2.21: FTIR spectra of hIAPP alone (black) and in co-incubation with of (a) 2-fold and (b) 5- fold molar ratios of peptidomimetics 2B (red), 2C (blue) and 2D (orange). Spectra were recorded after seven days of incubation of the peptides in PBS (50 mM) at pH 7.4 and 37 °C.
2.7.2. Monitoring the kinetics of amyloid disruption by ThT fluorescence assay:
The kinetics of the disruption of preformed amyloid was monitored by Thioflavin T (ThT) fluorescence experiments, where the BSBHps were added to the aggregating hIAPP after incubating 48h in PBS (50 mM) at pH 7.4 and 37 °C. The ThT fluorescence analysis distinctly showed that fluorescence intensity exhibited increment with time when hIAPP was incubated exclusively and reached a maximum after which it becomes saturated (black, Figure 2.22 (a); however, in co-incubation with 10-fold molar ratio of control peptidomimetic 2B we noticed ~ 23% disruption (red, Figure 2.22) of preformed amyloid. On the contrary, when peptidomimetic 2C (blue, Figure 2.22) was added to the solution of hIAPP, we witnessed a sharp decrease in fluorescence intensity up to ~ 40%, indicating disruption of preformed hIAPP amyloid. While in the presence of peptidomimetic 2D (orange, Figure 2.22), we noted disruption of ~ 33% of the preformed amyloid fibril.
Figure 2.22: (a) Time dependent ThT fluorescence spectra of hIAPP (40 µM) alone (black) and in co- incubation with 10-fold molar ratios of 2B (red), 2C (blue) and 2D (orange). (b) Dose dependent ThT fluorescence spectra at 168h of hIAPP (40 µM) alone (black) and in co-incubation with different molar ratios of 2B (red), 2C (blue) and 2D (orange).
Figure 2.23: Time dependent ThT fluorescence spectra of hIAPP (40 µM) alone (black) and in co- incubation with (a) 2-fold and (b) 5-fold molar ratios of peptidomimetics 2B (red), 2C (blue) and 2D (orange).
The dose-dependent disruption of preformed amyloid with 2- and 5-fold molar ratios (Figure 2.23) of BSBHps was also performed. Better results were achieved by increasing the doses from 2- to 10-fold molar excess.
2.7.3. Monitoring disruption of preformed amyloid by TEM and Congo red stained birefringence assay:
Incubating for 7 (2+5) days in PBS at physiological conditions, hIAPP alone showed distinct fibrillar morphology under TEM (Figure 2.24 (a) (i)), supporting the formation of amyloid.
However, hIAPP in co-incubation with 10-fold molar ratio of peptidomimetic 2B (Figure 2.24 (a) (ii)), significant fibrillar morphology was observed, indicating less efficiency of 2B in disrupting the amyloid. On the other hand, in the presence of BSBHps 2C (Figure 2.24 (a) (iii)) and 2D (Figure 2.24 (a) (iv)) with the same molar ratio, no such fibrillar assembly was detected, suggesting sufficient disruption of preformed hIAPP amyloid.
Again, the disruption of amyloid was further monitored by Congo red stained birefringence assay. hIAPP alone displayed green-gold birefringence under cross-polarized light upon staining with Congo red (Figure 2.24 (b) (i)), indicating the development of hIAPP amyloid.
On the contrary, hIAPP in co-incubation with 10-fold molar ratio of 2B with hIAPP, sufficient green-gold birefringence persisted (Figure 2.24 (b) (ii)), signifying inefficient disruption. However, when BSBHps 2C (Figure 2.24 (b) (iii)) and 2D (Figure 2.24 (b) (iv)) were present with hIAPP in the same molar ratio, no characteristic birefringence was displayed, supporting sufficient disruption of hIAPP amyloid by BSBHps.
Figure 2.24: (a) TEM and (b) Congo red birefringence images of hIAPP alone (i) and in co- incubation with 10-fold molar ratios of peptidomimetics, 2B (ii), 2C (iii) and 2D (iv). Images were captured after seven days of incubation of the peptides in PBS (50 mM) at pH 7.4 and 37 °C.