caa a22 4500 606236252 CHVBK 20210128101246.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s11010-015-2447-6 doi (NATIONALLICENCE)springer-10.1007/s11010-015-2447-6 Effects of metalloprotease anthrax lethal factor on its peptide-based inhibitor R9LF-1 [Elektronische Daten] [Qingsheng Kong, Yuezhang Song, Minlei Mu, Xiaolin Han, Chuanping Si, Feng Li] The metalloprotease lethal factor (LF) from Bacillus anthracis plays a vital role in anthrax toxin action, and thus becomes a target for anti-anthrax therapy. Following the guidelines based on existing metalloprotease inhibitors, we designed a ‘first-generation' LF inhibitor R9LF-1. This inhibitor was shown to be very stable by itself in a wide range of pH and temperature and able to inhibit LF activity in vitro. However, as we reported previously in the presence of LF, this inhibitor was degraded to a small molecular weight species, resulting in a significantly decreased ability to protect MAPKK from cleavage by LF as well as to protect murine macrophages from lethal toxin. In order to elucidate this unusual phenomenon to build solid basis for high-efficiency LF inhibitor development, we performed extensive research to study the effect of LF on its peptide-based inhibitor. Effects of temperature and incubation period of time on generation of the smaller peptide (short version R9LF-1) by LF as well as its catalytic domain were analyzed. We found that LF degraded R9LF-1 with maximum efficiency in the pH range of 7.0-8.5, which correlates well with the range of LF enzymatic activity with its native substrate. The degradation showed a deviation from normal hyperbolic kinetics but a similarity to the kinetics profile of an enzyme-catalyzed reaction with positive cooperativity. The short version R9LF-1 had decreased inhibitory activity toward LF; surprisingly, BIAcore results suggested a better affinity for its binding to LF. In addition, R9LF-1 was not hydrolyzed by other common proteases, such as chymotrypsin and pepsin, suggesting hydrolysis of the bond between amino acid and hydroxamate groups is unique to LF. This study calls for caution when designing peptide-based LF inhibitors and when interpreting effects of these types of inhibitors. Springer Science+Business Media New York, 2015 Anthrax lethal factor nationallicence Inhibitors nationallicence Metalloprotease nationallicence LF : Lethal factor nationallicence LT : Lethal toxin nationallicence LF-N : N-terminal domain of wild type LF nationallicence MAPKK : Mitogen-activated protein kinase kinase nationallicence MALDI-TOF : Matrix-assisted laser desorption/ionization time of flight nationallicence MW : Molecular weight nationallicence PA : Protective antigen nationallicence SPR : Surface plasmon resonance nationallicence WT : Wild type nationallicence Kong Qingsheng Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut Song Yuezhang Laiwu Municipal Center for Disease Control and Prevention, 271199, Laiwu, Shandong, People's Republic of China aut Mu Minlei Rizhao Municipal Chinese Medicine Hospital, 276800, Rizhao, Shandong, People's Republic of China aut Han Xiaolin Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut Si Chuanping Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut Li Feng Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 406/1-2(2015-08-01), 293-299 0300-8177 406:1-2<293 2015 406 11010 https://doi.org/10.1007/s11010-015-2447-6 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s11010-015-2447-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kong Qingsheng Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut NATIONALLICENCE 700 1- Song Yuezhang Laiwu Municipal Center for Disease Control and Prevention, 271199, Laiwu, Shandong, People's Republic of China aut NATIONALLICENCE 700 1- Mu Minlei Rizhao Municipal Chinese Medicine Hospital, 276800, Rizhao, Shandong, People's Republic of China aut NATIONALLICENCE 700 1- Han Xiaolin Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut NATIONALLICENCE 700 1- Si Chuanping Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut NATIONALLICENCE 700 1- Li Feng Jining Medical University, 272067, Jining, Shandong, People's Republic of China aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 406/1-2(2015-08-01), 293-299 0300-8177 406:1-2<293 2015 406 11010