Dirofilaria immitis: effect of fluoromethyl ketone cysteine protease inhibitors on the third- to fourth-stage molt.
D. immitis third-stage larvae (L3) were cultured with fluoromethyl ketone cysteine protease inhibitors. By Day 5 in culture, none of the larvae cultured with 0.1, 0.2, 0.6, or 1.0 mM benzyloxycarbonyl-Phe-Ala-CH2F (Z-Phe-Ala-CH2F) has molted, while 63.2% of larvae in media without inhibitor had molted. At the two lower concentrations of inhibitor more larvae had initiated, but not completed, the molt. In addition to Z-Phe-Ala-CH2F, four other fluoromethyl ketone derivatives, Z-Phe-Arg-CH2F, amorpholine urea-(Mu)-Leu-Phe-CH2F, Mu-Tyr-Phe-CH2F, and Mu-Phe-Phe-CH2F, were tested to determine their effects on L3 in culture. All fluoromethyl ketones tested except Z-Phe-Arg-CH2F inhibited molting. Larvae cultured in inhibitors were determined to be alive as judged qualitatively by motility and quantitatively by reduction of 3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium. Electron microscopy demonstrated that L3 which were unable to molt after being cultured in a fluoromethyl ketone derivative had synthesized the new fourth-stage (L4) cuticle but had not shed the L3 cuticle. The same fluoromethyl ketone derivative that did not inhibit molting, Z-Phe-Arg-CH2F, was a slightly less effective inhibitor of larval extract-initiated hydrolysis of the synthetic peptide substrate, Z-Val-Leu-Arg-7-amino-4-methyl coumarin. L3 were also cultured through the molt in media containing the synthetic peptide substrate Z-Val-Leu-Arg-4- methoxy-B-naphthylamide to examine cysteine protease activity in situ. Fluorescence as seen on Days 0-4 during the molting process was first observed on the anterior tip of the larvae, and subsequently in the pharynx, with progression down the L4 as it shed the L3 cuticle.
Richer
J K
JK
Department of Pathology, Colorado State University, Fort Collins 80523.
Hunt
W G
WG
Sakanari
J A
JA
Grieve
R B
RB
D000704:Analysis of Variance; D000818:Animals; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D004183:Dirofilaria immitis / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure; D007659:Ketones / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure; D008854:Microscopy, Electron
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007659:Ketones; C057383:MDL 201053