Carried out as described previously (27, 34, 35). LmCPB2.eight was recombinantly expressed as described previously (36). CL and CB had been bought from Calbiochem (Schwalbach, Germany). The fluorimetric substrate Cbz-Phe-Arg-AMC was bought from Bachem (Bubendorf, Switzerland). The assay buffer for CL and CB contained 50 mM Tris (pH 6.5), 5 mM EDTA, 200 mM NaCl, and 0.005 polyoxyethyleneglycol dodecyl ether (Brij 35). The enzyme buffer for CL and CB contained 50 mM Tris (pH six.five), 5 mM EDTA, 200 mM NaCl, and 2 mM DTT. For LmCPB2.8 and LmaCatB, the assay buffer consisted of 50 mM phosphate buffer (pH six.five) and five mM EDTA. The enzyme buffer for LmCPB2.eight and LmaCatB consisted of 50 mM phosphate buffer (pH six.five), five mM EDTA, and 5 mM DTT. Substrates and inhibitor stock solutions had been ready in dimethyl sulfoxide (DMSO) and diluted with assay buffer (to a final DMSO concentration of 7.5 ). A Varian Cary Eclipse fluorescence spectrophotometer (Varian,February 2016 Volume 60 NumberAntimicrobial Agents and Chemotherapyaac.asm.orgSchad et al.TABLE 1 Inhibition of CL, CB, LmCPB2.8, and LmaCatB, antileishmanial activity against L. major promastigotes, and cytotoxicity by trans-aziridine-2,3-dicarboxylates 13b, 13e, and s1 to s38cKi ( M) (reference) Compound 13b 13e s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12 s13 s14 s15 s16 s17 s18 s19 s20 s21 s22 s23 s24 s25 s26 s27 s28 s29 s30 s31 s32 s33 s34 s35 s36 s37 saIC50 ( M) LmCPB2.8 1.7 2.1 NI NI NI NI NI NI NI NI 3.eight three.4 4.three 4.1 four.0 4.six 8.1 two.6 two.9 3.1 four.six NI NI NI 25.four four.3 NI NI 2.eight NI NI ten.three 1.7 1.five 2.7 ND ND 1.five 1.eight 0.six 0.two 0.4 LmaCatB ND ND NI NI NI NI 15.2 NI NI 10.3 18.2 NI 0.69 0.82 0.72 1.5 4.eight 17.9 NI NI 17.three 7.0 six.two NI NI NI 8.7 NI NI NI NI 20.six NI NI NI 1.5 1.1 NI NI 67.0 L. major promastigotes 33.4 2.five 47.0 11.eight ND ND ND ND ND ND ND ND 37.four 95 one hundred 100 one hundred one hundred 34.IL-18 Protein Gene ID 2 32.6 40.7 47.two 41.0 one hundred one hundred 100 46.1 34.eight 9.eight NDp NDp 31.three NDp one hundred 48.0 one hundred one hundred one hundred one hundred 100 one hundred one hundred Cytotoxicity on J774.1 cells 100 100 ND ND ND ND ND ND ND ND 68 one hundred 87 100 89 one hundred 100 100 250 100 one hundred 100 100 100 one hundred 250 250 100 100 100 one hundred 100 100 one hundred one hundred 42.five one hundred one hundred 100R2 Boc-(S)-Leu-(R)-Pro Boc-(R)-Leu-(S)-Pro Boc-(S)-Leu-(R)-Pro Boc-(R)-Leu-(S)-Pro Boc-(S)-Leu-(R)-Pro Boc-(R)-Leu-(S)-Pro Boc-(S)-Pro-(S)-Leu Boc-(R)-Pro-(S)-Leu Boc-(S)-Pro-(R)-Leu Boc-(R)-Pro-(R)-Leu Boc-(S)-Leu-(R)-Pro Boc-(R)-Leu-(S)-Pro Boc-(S)-Pro-(S)-Leu Boc-(R)-Pro-(S)-Leu Boc-(S)-Pro-(R)-Leu Boc-(R)-Pro-(R)-Leu Boc-Gly-(S)-Leu-(R)-Pro Boc-(S)-Pro-(S)-Leu Boc-(R)-Pro-(S)-Leu Boc-(S)-Pro-(R)-Leu Boc-(R)-Pro-(R)-Leu Boc-Gly-(R)-Pro Boc- -Ala-(R)-Pro Boc-Aib-(R)-Pro Boc-(S)-Ala-(R)-Pro Boc-(S)-Val-(R)-Pro Boc-(S)-Ile-(R)-Pro Boc-(S)-Nva-(R)-Pro Boc-(S)-Nle-(R)-Pro Boc-(S)-Chg-(R)-Pro Boc-(S)-Cha-(R)-Pro Boc-(S)-Phg-(R)-Proa Boc-(S)-Phe-(R)-Pro Boc-(S)-hPhe-(R)-Pro Boc-(S)-Trp-(R)-Pro Boc-(S)-Leu-(R)-Orn(Boc) Boc-(S)-Leu-(R)-Arg(NO2) Boc-(S/R)-Nipa Boc-(S/R)-Nipb Boc-(S)-Leu-(S/R)-NipR1 Bn Bn Et Et Et Et Et Et Et Et Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn Bn pBr-Bn1a, 1b S,S S,S S,S S,S R,R R,R S,S S,S S,S S,S R,R R,R S,S S,S S,S S,S R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R R,R S,S R,R S,SCL six.IL-6 Protein Source 0 0.PMID:28739548 eight (14) four.0 0.two (14) NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI 14.two 0.1 19.7 four.1 NI 70.9 0.3 53.1 eight.7 NI NI NI 51.five 1.6 NI NI NI NI NI NI NI NI NI NI NI NI 75.9 8.7 NICB NI (14) NI (14) NI NI NI NI NI NI NI NI NI NI NI NI NI NI 26.4 4.9 29.2 1.1 44.0 two.1 24.six 7.9 NI NI NI NI NI 54.six 1.five N.
