Jacob Binu, Rajasekaran Ganesan, Kim Eun Young, Park Il-Seon, Bang Jeong-Kyu, Shin Song Yub
Department of Medical Science, Graduate School, Chosun University, Gwangju, 501-759, Republic of Korea.
Department of Cellular and Molecular Medicine, School of Medicine, Chosun University, Gwangju, 501-759, Republic of Korea.
Amino Acids. 2016 May;48(5):1241-51. doi: 10.1007/s00726-016-2170-y. Epub 2016 Jan 21.
Sheep myeloid antimicrobial peptide-29 (SMAP-29) is a cathelicidin-related antimicrobial peptide derived from sheep myeloid cells. In order to investigate the effects of L-to-D-amino acid substitution in SMAP-29 on bacterial selectivity, membrane interaction and anti-inflammatory activity, we synthesized its two D-enantiomeric peptides (SMAP-29-E1 and SMAP-29-E2 containing D-Ile and D-allo-Ile, respectively) and two diastereomeric peptides (SMAP-29-D1 and SMAP-29-D2). Additionally, in order to address the effect of L-to-D-amino acid substitution in the N-terminal helical peptide of SMAP-29 (named SMAP-18) on antimicrobial activity, we synthesized its two D-enantiomeric peptides (SMAP-18-E1 and SMAP-18-E2), which are composed of D-amino acids entirely. L-to-D-amino acid substitution in membrane-targeting AMP, SMAP-29 did not affect its antimicrobial activity. However, D-allo-Ile containing-SMAP-29-E2 and SMAP-29-D2 exhibited less hemolytic activity compared to D-Ile containing-SMAP-29-E1 and SMAP-29-D1, respectively. L-to-D-amino acid substitution in intracellular targeting-AMPs, SMAP-18 and buforin-2 improved antimicrobial activity by 2- to eightfold. The improved antimicrobial activity of the D-isomers of SMAP-18 and buforin-2 seems to be due to the stability against proteases inside bacterial cells. Membrane depolarization and dye leakage suggested that the membrane-disruptive mode of SMAP-29-D1 and SMAP-29-D2 is different from that of SMAP-29, SMAP-29-E1, and SMAP-29-E2. L-to-D-amino acid substitution in SMAP-29 improved anti-inflammatory activity in LPS-stimulated RAW 264.7 cells. In summary, we propose here that D-allo-Ile substitution is a more powerful strategy for increasing bacterial selectivity than D-Ile substitution in the design of D-enantiomeric and diastereomeric AMPs. SMAP-29-D1, and SMAP-29-D2 with improved bacterial selectivity and anti-inflammatory activity can serve as promising candidates for the development of anti-inflammatory and antimicrobial agents.
绵羊髓系抗菌肽-29（SMAP-29）是一种源自绵羊髓系细胞的与cathelicidin相关的抗菌肽。为了研究SMAP-29中L型氨基酸向D型氨基酸取代对细菌选择性、膜相互作用和抗炎活性的影响，我们合成了其两种D型对映体肽（分别含有D-异亮氨酸和D-别异亮氨酸的SMAP-29-E1和SMAP-29-E2）以及两种非对映体肽（SMAP-29-D1和SMAP-29-D2）。此外，为了研究SMAP-29的N端螺旋肽（命名为SMAP-18）中L型氨基酸向D型氨基酸取代对抗菌活性的影响，我们合成了其两种完全由D型氨基酸组成的D型对映体肽（SMAP-18-E1和SMAP-18-E2）。在靶向膜的抗菌肽SMAP-29中进行L型氨基酸向D型氨基酸的取代，并未影响其抗菌活性。然而，与分别含有D-异亮氨酸的SMAP-29-E1和SMAP-29-D1相比，含有D-别异亮氨酸的SMAP-29-E2和SMAP-29-D2表现出较低的溶血活性。在靶向细胞内的抗菌肽SMAP-18和蟾蜍灵-2中进行L型氨基酸向D型氨基酸的取代，使抗菌活性提高了2至8倍。SMAP-18和蟾蜍灵-2的D型异构体抗菌活性的提高似乎归因于其对细菌细胞内蛋白酶的稳定性。膜去极化和染料泄漏表明，SMAP-29-D1和SMAP-29-D2的膜破坏模式与SMAP-29、SMAP-29-E1和SMAP-29-E2不同。在SMAP-29中进行L型氨基酸向D型氨基酸的取代，提高了脂多糖刺激的RAW 264.7细胞中的抗炎活性。总之，我们在此提出，在设计D型对映体和非对映体抗菌肽时，D-别异亮氨酸取代是一种比D-异亮氨酸取代更有效的提高细菌选择性的策略。具有提高的细菌选择性和抗炎活性的SMAP-29-D1和SMAP-29-D2可作为开发抗炎和抗菌药物的有前景的候选物。
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