Lin Gang, Tsu Christopher, Dick Lawrence, Zhou Xi K, Nathan Carl
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10065, USA.
J Biol Chem. 2008 Dec 5;283(49):34423-31. doi: 10.1074/jbc.M805324200. Epub 2008 Oct 1.
The proteasome of Mycobacterium tuberculosis (Mtb) is a validated and drug-treatable target for therapeutics. To lay ground-work for developing peptide-based inhibitors with a useful degree of selectivity for the Mtb proteasome over those of the host, we used a library of 5,920 N-acetyl tripeptide-aminomethylcoumarins to contrast the substrate preferences of the recombinant Mtb proteasome wild type and open gate mutant, the Rhodococcus erythropolis proteasome, and the bovine proteasome with activator PA28. The Mtb proteasome was distinctive in strictly preferring P1 = tryptophan, particularly in combination with P3 = glycine, proline, lysine or arginine. Screening results were validated with Michalis-Menten kinetic analyses of 21 oligopeptide aminomethyl-coumarin substrates. Bortezomib, a proteasome inhibitor in clinical use, and 17 analogs varying only at P1 were used to examine the differential impact of inhibitors on human and Mtb proteasomes. The results with the inhibitor panel confirmed those with the substrate panel in demonstrating differential preferences of Mtb and mammalian proteasomes at the P1 amino acid. Changing P1 in bortezomib from Leu to m-CF(3)-Phe led to a 220-fold increase in IC(50) against the human proteasome, whereas changing a P1 Ala to m-F-Phe decreased the IC(50) 400-fold against the Mtb proteasome. The change of a P1 Ala to m-Cl-Phe led to an 8000-fold shift in inhibitory potency in favor of the Mtb proteasome, resulting in 8-fold selectivity. Combinations of preferred amino acids at different sites may thus improve the species selectivity of peptide-based inhibitors that target the Mtb proteasome.
结核分枝杆菌（Mtb）的蛋白酶体是经过验证的、可通过药物治疗的治疗靶点。为了开发对Mtb蛋白酶体具有比宿主蛋白酶体更高选择性的基于肽的抑制剂奠定基础，我们使用了一个包含5920种N - 乙酰三肽 - 氨基甲基香豆素的文库，来对比重组Mtb蛋白酶体野生型和开放门突变体、红平红球菌蛋白酶体以及具有激活剂PA28的牛蛋白酶体的底物偏好。Mtb蛋白酶体的独特之处在于它严格偏好P1 = 色氨酸，特别是与P3 = 甘氨酸、脯氨酸、赖氨酸或精氨酸结合时。通过对21种寡肽氨基甲基香豆素底物进行米氏动力学分析验证了筛选结果。临床使用的蛋白酶体抑制剂硼替佐米以及仅在P1处不同的17种类似物，用于研究抑制剂对人和Mtb蛋白酶体的不同影响。抑制剂组的结果证实了底物组的结果，表明Mtb和哺乳动物蛋白酶体在P1氨基酸上存在不同偏好。将硼替佐米中的P1从亮氨酸改为间三氟甲基苯丙氨酸，导致对人蛋白酶体的IC50增加220倍，而将P1丙氨酸改为间氟苯丙氨酸则使对Mtb蛋白酶体的IC50降低400倍。将P1丙氨酸改为间氯苯丙氨酸导致抑制效力向有利于Mtb蛋白酶体的方向转变8000倍，产生8倍选择性。因此，不同位点的优选氨基酸组合可能会提高靶向Mtb蛋白酶体的基于肽的抑制剂的物种选择性。
