reveal that both hydrogen bonding and hydrophobic interaction are the major contributing forces in the PYZ-HEWL complex formation; however DCS interact mainly through hydrophobic interaction. Hence, molecular docking results support the potent anti-amyloid action of PYZ over DCS. 3.10. Cell viability assay The aggregated/amyloid fibrils are toxic to neurons; though pre-amyloid fibrils or oligomers are believed to be more cytotoxic. Thus, the cytotoxicity of 120 h aged HEWL fibrils were examined on two cell lines i.e., PC-12 and SH-SY5Y by MTT reduction assay at 24 h. MTT undergoes reduction by mitochondrial dehydrogenases to soluble formazan that serves as the indicator of metabolically active cells and inhibition of this reaction is indicative of cytotoxicity. As shown in Fig 10A and 10B, the formation of amyloid fibrils with high prevalence of the cross–sheet conformation in 120h leads to cytotoxicity and reduced PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729642 the percent cell viability to around 30 and 35 in PC-12 and SH-SY5Y cell lines, respectively.However, when HEWL fibrils were allowed to form in presence of PYZ and DCS, cell viability increased significantly as compare to that of HEWL fibrils with increase in concentration of both drugs. It may be due to the decrease in cross–sheet conformation in HEWL fibrils in presence of both 
drugs. In both PC-12 and SH-SY5Y cell lines the percent cell viability increased to 78%, 66% and 66%, 58% with exposure to lysozyme fibrils in presence of 500 M PYZ and DCS respectively. Since cross–sheet conformation in HEWL is lesser in presence of PYZ as compare to DCS with respect to HEWL fibrils, consequently cell cytotoxicity PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19731547 was HC-067047 chemical information affected much by PYZ. Moreover, PYZ and DCS alone in buffer were not showing any significant effect on cell viability. To further exclude the other protective effect of drugs on cell viability; prior to expose with HEWL fibrils, cells were pre-incubated with either PYZ or DCS and no protective effect against HEWL fibrils was found. This confirmed that 14 / 21 Anti-Amyloid Behavior of Pyrazinamide and D-Cycloserine Fig 9. Detailed view of the docking poses of drug -HEWL interaction. Anti-Amyloid Behavior of Pyrazinamide and D-Cycloserine Fig 10. MTT reduction assay for cell cytotoxicity of 120 h aged HEWL amyloid fibrils in SH-SY5Y and PC-12 cell lines in absence and presence of different concentration of DCS and PYZ. Control represents cell lines without prior exposure to HEWL fibrils. Statistically different from the control group, p 0.05 and statistically different from the HEWL, p 0.05 for DCS. Statistically different from the control group, p 0.01 and statistically different from the HEWL, p 0.01 for PYZ. doi:10.1371/journal.pone.0136528.g010 fluorescence whereas incubation of A-42 in presence of 500 M PYZ and DCS reduced the fluorescence to 70.17 and 36.80% of untreated A-42 respectively as shown in Fig 11B. Anti-Amyloid Behavior of Pyrazinamide and D-Cycloserine Similarly, Fig 11C shows an increase in Congo red absorption upon binding with A-42 with absorption maxima at 520 nm along with red shift of 30 nm which indicates the presence of A-42 matured fibril. However, presence of 500 M PYZ and DCS leads to reduction in absorbance with absorption maxima at around 20 and 25 nm respectively. The reduction in absorbance indicates the inhibitory action of drugs against A-42 fibrillation; though more pronounced effect is of PYZ. These findings are further confirm by TEM micrographs of A42, 70 h aged A-42, 70 h aged A
