Faculty of Chemistry and Biochemistry, Inorganic Chemistry I - Bioinorganic Chemistry, Ruhr University Bochum, Bochum, Germany.
UMR7242, ESBS, University of Strasbourg, 67413, Illkirch, Strasbourg, France.
Chemistry. 2023 Sep 6;29(50):e202300364. doi: 10.1002/chem.202300364. Epub 2023 Aug 4.
A series of new conjugates comprised from a small synthetic antimicrobial peptide (AMP) and a siderophore-type vector component was designed and tested for activity on P. aeruginosa PAO1 and several genetically modified strains. As AMP, the well-established arginine-tryptophane combination K(RW) (P1) was chosen with an added lysine for siderophore attachment. This peptide is easy to prepare, modify, and possesses good anti-bacterial activity. On the vector part, we examined several moieties: (i) the natural siderophore deferoxamine (DFO); (ii) bidentate iron chelators based on the hydroxamate building block (4 a-c) ; (iii) the non-siderophore chelators deferasirox (DFX) and deferiprone-carboxylate (DFP-COOH). All conjugates were prepared by solid phase synthesis techniques and fully characterized by HPLC and mass spectrometry (including HR-MS). Fe uptake assays indicate a receptor-mediated uptake for 4 a-c, DFP-COOH and DFO, which is dependent on the outer membrane transporter FoxA in the case of DFO. All conjugates showed increased antibacterial activity against P. aeruginosa compared to the parent peptide P1 alone when investigated in iron-depleted medium. MIC values were as low as 2 μM (for P1-DFP) on wild type P. aeruginosa. The activity of P1-DFO and P1-DFP was even better on genetically mutated strains unable to produce siderophores (down to 0.5 μM). Although the DFX vector on its own was not able to transport iron inside the bacterial cell as shown by Fe uptake studies, the P1-DFX conjugate had excellent antibacterial activity compared to P1 (2 μM, and as low as 0.25 μM on a receptor-deficient strain unable to produce siderophores), suggesting that the conjugates were indeed recognized and internalized by an (unknown) transporter. Control experiments with an equimolar mixture of P1 and DFX confirm that the observed activity is intrinsic to vectorization. This work thus demonstrates the power of linking small AMPs covalently to siderophores for a new class of Trojan Horse antibiotics, with P1-DFP and P1-DFX being the most potent conjugates.
设计并测试了一系列由小的合成抗菌肽（AMP）和铁载体型载体组成的新缀合物，以评估其对铜绿假单胞菌 PAO1 和几种遗传修饰株的活性。选择了经过充分验证的精氨酸-色氨酸组合 K(RW)（P1）作为 AMP，其添加了一个赖氨酸以用于铁载体附着。该肽易于制备、修饰，并具有良好的抗菌活性。在载体部分，我们研究了几种部分：（i）天然铁载体去铁胺（DFO）；（ii）基于羟肟酸构建块的双齿铁螯合剂（4a-c）；（iii）非铁载体螯合剂地拉罗司（DFX）和去铁酮羧酸酯（DFP-COOH）。所有缀合物均通过固相合成技术制备，并通过 HPLC 和质谱（包括高分辨率质谱）进行了完全表征。Fe 摄取实验表明，4a-c、DFP-COOH 和 DFO 是通过外膜转运蛋白 FoxA 进行受体介导摄取的，对于 DFO 则是如此。与单独的母体肽 P1 相比，在缺铁培养基中研究时，所有缀合物对铜绿假单胞菌的抗菌活性均有所提高。MIC 值低至 2 μM（对于 P1-DFP）在野生型铜绿假单胞菌上。对于无法产生铁载体的遗传突变株（低至 0.5 μM），P1-DFO 和 P1-DFP 的活性甚至更好。尽管 DFX 载体本身不能像 Fe 摄取研究所示那样将铁运入细菌细胞内，但与 P1 相比，P1-DFX 缀合物具有极好的抗菌活性（2 μM，对于无法产生铁载体的受体缺陷株低至 0.25 μM），表明缀合物确实被（未知）转运蛋白识别和内化。用 P1 和 DFX 的等摩尔混合物进行的对照实验证实，观察到的活性是载体化的固有性质。这项工作因此证明了将小的 AMP 共价连接到铁载体上以形成新一类木马抗生素的强大功能，其中 P1-DFP 和 P1-DFX 是最有效的缀合物。
