Hosn Kalid N, Jefferson Mary Margaret, Leding Carlton, Shokouh-Amiri Solomon, Thomas Edwin L
Periodontology Department University of Tennessee Health Science Center Memphis Tennessee USA.
Bioscience Research Department, College of Dentistry University of Tennessee Health Science Center Memphis Tennessee USA.
Clin Exp Dent Res. 2015 Oct 27;1(1):18-25. doi: 10.1002/cre2.4. eCollection 2015 Oct.
Locally applied therapeutic agents have become established in the treatment of periodontal disease. Inhibition of human metalloproteases by metal-chelating antibiotics contributes to the utility of local therapy. Adding inhibitors of bacterial proteases might extend and improve local therapy. The periodontal pathogen (Pg) produces two extracellular cysteine proteases (gingipains Rgp and Kgp) that are virulence factors and contribute to destruction of oral tissues. Our aims were to compare efficacy of protease inhibitors against gingipains and evaluate bactericidal activity of the inhibitors. Protease activity was measured in fluorescent assays with specific Rgp and Kgp substrates. Bacterial viability was measured with Light™ (Invitrogen, Inc., Carlsbad, CA) reagents. Pairs of inhibitors of Rgp and Kgp, respectively, were leupeptin and cathepsin B inhibitor II, KYT-1 and KYT-36, and PPACK and Z-FK-ck. The cysteine-protease inhibitor E64 was also tested. Rgp activity was higher than Kgp activity, and activity was higher in Pg 33277 and 49417 cell suspensions than in media. Concentrations required for 50% inhibition of Rgp in cell suspensions were 2 × 10, 2 × 10, 2 × 10, and 5 × 10 M for KYT-1, PPACK, leupeptin, and E64, respectively. Concentrations required for 50% Kgp inhibition were 5 × 10, 1 × 10, and 5 × 10 M for Z-FK-ck, KYT-36, and cathepsin B inhibitor II. E64 did not inhibit Kgp. Inhibition of Rgp could be accounted for by competition for binding between the arginine residue of the substrate and the guanidinobutane portion of E64. PPACK was the least selective, with a 10-fold difference in concentrations that inhibited Rgp and Kgp. KYT-1 and Z-FK-ck inhibited both Rgp and Kgp, but inhibitory concentrations differed by 10,000-fold. At up to 1 × 10 M, only Z-FK-ck was bactericidal. KYT-1 and KYT-36 were remarkably effective even when used in cell suspensions in which bacterial proteins could bind inhibitors or compete for binding to gingipains. These inhibitors might prove useful as an addition to locally applied therapeutic agents.
局部应用的治疗药物已在牙周病治疗中确立了地位。金属螯合抗生素对人金属蛋白酶的抑制作用有助于局部治疗的效用。添加细菌蛋白酶抑制剂可能会扩展和改善局部治疗。牙周病原体（Pg）产生两种细胞外半胱氨酸蛋白酶（牙龈蛋白酶Rgp和Kgp），它们是毒力因子，会导致口腔组织破坏。我们的目的是比较蛋白酶抑制剂对牙龈蛋白酶的疗效，并评估抑制剂的杀菌活性。在使用特定Rgp和Kgp底物的荧光测定中测量蛋白酶活性。用Light™（英杰生命技术有限公司，加利福尼亚州卡尔斯巴德）试剂测量细菌活力。分别用于Rgp和Kgp的抑制剂对为亮抑蛋白酶肽和组织蛋白酶B抑制剂II、KYT-1和KYT-36，以及PPACK和Z-FK-ck。还测试了半胱氨酸蛋白酶抑制剂E64。Rgp活性高于Kgp活性，并且在Pg 33277和49417细胞悬液中的活性高于培养基中的活性。在细胞悬液中对Rgp产生50%抑制所需的浓度，对于KYT-1、PPACK、亮抑蛋白酶肽和E64分别为2×10、2×10、2×10和5×10 M。对Kgp产生50%抑制所需的浓度，对于Z-FK-ck、KYT-36和组织蛋白酶B抑制剂II分别为5×10、1×10和5×10 M。E64不抑制Kgp。Rgp的抑制作用可能是由于底物的精氨酸残基与E64的胍丁烷部分之间的结合竞争。PPACK的选择性最低，抑制Rgp和Kgp的浓度相差10倍。KYT-1和Z-FK-ck同时抑制Rgp和Kgp，但抑制浓度相差10000倍。在高达1×10 M时，只有Z-FK-ck具有杀菌作用。即使在细菌蛋白可结合抑制剂或竞争与牙龈蛋白酶结合的细胞悬液中使用，KYT-1和KYT-36也非常有效。这些抑制剂可能作为局部应用治疗药物的补充证明是有用的。
