Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group

The metalloprotease activity of lethal factor (LF) from Bacillus anthracis (B. anthracis) is a main source of toxicity in the lethality of anthrax infection. Thus, the understanding of the enzymatic activity and inhibition of B. anthracis LF is of scientific and clinical interests. We have designed, synthesized, and studied a peptide inhibitor of LF, R9LF-1, with the structure NH(2)-(D: -Arg)(9)-Val-Leu-Arg-CO-NHOH in which the C-terminal hydroxamic acid is commonly used in the inhibitors of metalloproteases to chelate the active-site zinc. This inhibitor was shown to be very stable in solution and effectively inhibited LF in kinetic assays. However, its protection on murine macrophages against lethal toxin's lysis activity was relatively weak in longer assays. We further observed that the hydroxamic acid group in R9LF-1 was hydrolyzed by LF, and the hydrolytic product of this inhibitor is considerably weaker in inhibition of potency. To resist this unique hydrolytic activity of LF, we further designed a new inhibitor R9LF-2 which contained the same structure as R9LF-1 except replacing the hydroxamic acid group with N,O-dimethyl hydroxamic acid (DMHA), -N(CH(3))-O-CH(3). R9LF-2 was not hydrolyzed by LF in long-term incubation. It has a high inhibitory potency vs. LF with an inhibition constant of 6.4 nM had a better protection of macrophages against LF toxicity than R9LF-1. These results suggest that in the development of new LF inhibitors, the stability of the chelating group should be carefully examined and that DMHA is a potentially useful moiety to be used in new LF inhibitors.