Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry, Universität Leipzig, 04109 Leipzig, Germany.
Center for Biotechnology and Biomedicine, Universität Leipzig, 04109 Leipzig, Germany.
Int J Mol Sci. 2022 Mar 15;23(6):3150. doi: 10.3390/ijms23063150.
Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of , a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif DKPRPYLPRP with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using BW25113 and the corresponding knockout mutants. The dissociation constant (K) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and Δ and Δ knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.
富含脯氨酸的抗菌肽 (PrAMPs) 是治疗细菌感染的有前途的候选药物。设计肽 ARV-1502 对体外和体内均表现出强烈的抗菌作用。由于 ARV-1502 对 70 kDa 热休克蛋白 DnaK 的抑制作用不能完全解释其 176 个取代类似物的抗菌活性，因此我们进一步研究了它们对细菌 70S 核糖体的影响，这是 PrAMPs 的已知靶标。用 5(6)-羧基荧光素标记的 (Cf-)ARV-1502 和从 BW25113 中分离的 70S 核糖体进行竞争性荧光偏振 (FP) 结合筛选试验，测试了在结合基序 DKPRPYLPRP 中位置 3、4 和 8 至 12 (下划线) 用天冬氨酸、赖氨酸、丝氨酸、苯丙氨酸或亮氨酸取代的 ARV-1502 类似物。在无细胞表达系统 (体外翻译) 中用绿色荧光蛋白 (GFP) 研究了它们对核糖体蛋白表达的影响，并用 BW25113 和相应的敲除突变体研究了已知 PrAMP 转运蛋白 SbmA 和 MdtM 的重要性。对于 Cf-ARV-1502，获得的解离常数 (K) 为 201±16 nmol/L，表明与 70S 核糖体的强结合。抑制结合测定表明，结合位点与其他 PrAMPs 重叠，包括 Onc112 和吡咯菌素以及非肽类抗生素红霉素和氯霉素。所有这些药物和药物候选物都与 70S 核糖体的出口隧道结合。结合基序 C 末端片段的取代降低了结合。同时，GFP 表达的抑制作用随着净肽电荷的增加而增加。有趣的是，野生型和 Δ和 Δ 敲除突变体的 MIC 值表明，核糖体结合基序的取代也改变了细菌摄取，这通常通过掺入疏水性残基得到改善。总之，大多数取代的 ARV-1502 类似物与 ARV-1502 相比，与 70S 核糖体的结合较弱，这强调了 YLPRP 结合基序的重要性。核糖体结合较弱与体外抗菌活性降低密切相关。具有更高疏水性或正净电荷的取代 ARV-1502 类似物改善了核糖体结合、翻译抑制和细菌摄取。
