An insight into the effects of point mutations on stability and catalytic mechanism of Pseudomonas aeruginosa Exotoxin A for immunotoxin therapy

Recombinant immunotoxins are a class of medicines which consist of an antibody or fragments of it fused to a toxic moiety. The most famous toxic moieties are based on Pseudomonas exotoxin A (PE), Diphtheria toxin (DT) and ricin. Denileukin diftitox is the first immunotoxin which was initially approved in US in 1999 and targets interleukin 2 (IL2) receptors and redirects its cytotoxic effects by part of DT. Today‟s studies are focused mainly on PE toxins. Truncated PE has shown a significant decrease on tumor sizes. However, one of the major complications of immunotoxins in cancer therapy is immunogenicity of the toxic moiety. Different tactics have been deployed to reduce immunogenicity of PE. These tactics mainly aim to reduce the number of B-cell epitopes in the 3D structure of domain III. For this purpose, different point mutations have been designed and studies to reduce immunogenicity of the toxin. The successfully modified PE toxin had 7point mutations, R505A, R427A, R490A, R467A, D463A, R538A and R456A (the numbering is based on the residual number in secreted PE with 613 amino acids) and the toxin containing these mutations was named PEX7. Here in this study, we have applied a molecular dynamics approach to investigate the effects of point mutations on overall structure of shortened PE. Also, we have compared the results of MD simulations with CD spectroscopy and cell culture assay to get a deep insight into the catalytic mechanism of PE. For this purpose, we ran a 100ns MD simulations in Gromacs® platform and compared the catalytic activities of PE and PEX7 in an interaction with their natural target, NAD. Protein stability and secondary structure analysis were done using CD spectroscopy and MD simulations. Toxic effects (antitumor activities) on 2 cancerous cell lines (SKOV3 and MCF-7) were also examined and finally the results were confirmed by flowcytometry.The results showed that protein stability increases a bit in PEX7 rather than PE. It shows that as a medicine expressing PEX7 will be more beneficial. When NAD attaches the toxin active sites, a dramatic increase of stability happens in the active sites of both proteins. Cell culture studies showed that both toxins have maintained their catalytic activity well with a little increased IC50 for PEX7. So, PE and PEX7 both showed great toxic effects on turmeric cell lines and can have anticancer effects; but, PE is more potent because its active site is intact. On the other side, because of reduced immunogenicity of PEX7 in comparison to PE, PEX7 can have a longer time of exposure to cancerous cells. So, applying point mutations on PE will not affect its antitumor activities because PEX7 show an increased half life in comparison to intact PE.