Abstract
BackgroundEntolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins.MethodsWe designed and constructed cyclic entolimod using split Nostoc punctiforme DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Both of linear and circular entolimod were first purified by Ni-chelating affinity chromatography, and then the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively.ResultsThe circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice.ConclusionOur data indicates that circular entolimod might be a good candidate for further clinical investigation.
Highlights
Entolimod is a truncated derivative of the Salmonella flagellin protein
Nostoc punctiforme (Npu) DnaE split intein-mediated entolimod cyclization According to the principle of protein trans-splicing (PTS), the two fragments of intein, IN and IC, interact with each other to form an active intein that splices to cyclize the proteins or peptides placed in between
The entire entolimod gene was sandwiched between the IC and IN of Synechocystis sp. PCC6803 (Ssp) or Npu intein with CFN residues at the C terminus from the native C-terminal extein sequence of DnaE intein, and HM and GS residues at its C terminus and N terminus due to cloning (Figs. 1A–1C)
Summary
Entolimod (previously called CBLB502) is a truncated derivative of the Salmonella flagellin protein. Entolimod has prospective clinical applications as a radioprotective and anticancer agent; being a flagellin variant, it can still cause a “flu-like” syndrome after injection, indicating some type of immunogenicity and toxicity (Ding et al, 2012; Burdelya et al, 2013; Hossain et al, 2014; Kojouharov et al, 2014; Yang et al, 2016; Brackett et al, 2016). Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation It caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Conclusion: Our data indicates that circular entolimod might be a good candidate for further clinical investigation
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