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TLR5 binding and activation by KMRC011, a flagellin-derived radiation countermeasure

Song, Wan Seok, Kim, Jee-Hyeon, Choi, Chi-Min, Lee, Woo-Jong, Yoon, Sung-il
Biochemical and biophysical research communications 2019 v.508 no.2 pp. 570-575
Toll-like receptor 5, adverse effects, alanine, clinical trials, dissociation, drug design, drugs, flagellin, humans, mutagenesis, radioprotective effect, toxicity
Entolimod (CBLB502) is a flagellin-derived radiation countermeasure currently under clinical trial. Entolimod exerts radioprotective activity by directly interacting with TLR5, an innate immune receptor, using the conserved domains of flagellin. Entolimod was designed to contain an artificially introduced N-terminal region that is not related to drug effects and might trigger unexpected toxic immunogenic reactions in humans. To refine the entolimod drug design, we engineered entolimod into KMRC011 by removing its ancillary region. The TLR5 binding and activating capacities of KMRC011 were assessed through biophysical and cellular analyses. KMRC011 forms an exceptionally stable complex with TLR5 at a 1:1 molar ratio with an equilibrium dissociation constant of ∼100 pM and potently activates TLR5. Moreover, alanine scanning mutagenesis identified the R90 and E114 residues of KMRC011 as a TLR5 activation hotspot. Further comparative analysis demonstrated that KMRC011 binds and activates TLR5 in a mode similar to that of entolimod. Thus, we propose that KMRC011 can be used in place of entolimod as a second-generation radiation countermeasure that shows none of the immunogenic side effects derived from the entolimod ancillary region.