De Novo Design and Synthesis of Ultra-Short Peptidomimetic Antibiotics Having Dual Antimicrobial and Anti-Inflammatory Activities

Ravichandran N. Murugan, Mija Ahn, Eunha Hwang, Ji-Hyung Seo, Chaejoon Cheong, Jeong Kyu Bang, Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk, Republic of KoreaBinu Jacob, Song Yub Shin, Department of Bio-Materials, Graduate School and Department of Cellular and Molecular Medicine, School of Medicine, Chosun University, Gwangju, Republic of KoreaHoik Sohn, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, United States of AmericaHyo-Nam Park, Jae-Kyung Hyun, Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon, Republic of KoreaEunjung Lee, Ki-Woong Jeong, Yangmee Kim, Department of Bioscience and Biotechnology, Institute of SMART Biotechnology, Konkuk University, Seoul, Republic of KoreaKy-Youb Nam, Bioinformatics and Molecular Design Research Center, Yonsei University Research Complex, Seoul, Republic of KoreaBackground: Much attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability.-- Methodology/Principal Findings: In an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti–methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes. -- Conclusion/Significance: The combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics.This work was supported in part by the Korea Basic Science Institute's research program grants T33418 (J.K.B) and T33518 (J-k.H.), and the Korea Research Foundation, funded by the Korean Government (KRF-2011-0009039 to S.Y.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.ChemistryBiochemistryEmail: [email protected] (JKB)Email: [email protected] (SYS

Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization

Photograph of a scene at the Philbrook Museum of Art

Exploring the binding nature of pyrrolidine pocket-dependent interactions in the polo-box domain of polo-like kinase 1.

BACKGROUND: Over the years, a great deal of effort has been focused on the design and synthesis of potent, linear peptide inhibitors targeting the polo-like kinase 1 (Plk1), which is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain, and inhibiting the Plk1 polo-box domain has been considered as an approach to circumvent the specificity problems associated with inhibiting the conserved adenosine triphosphate-binding pocket. The polo-box domain consists of two different binding regions, such as the unique, broader pyrrolidine-binding pocket and the conserved, narrow, Tyr-rich hydrophobic channel, among the three Plk polo-box domains (Plks 1-3), respectively. Therefore, the studies that provide insights into the binding nature of the unique, broader pyrrolidine-binding pocket might lead to the development of selective Plk1-inhibitory compounds. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to retain the monospecificity by targeting the unique, broader pyrrolidine-binding pocket, here, for the first time, a systematic approach was undertaken to examine the structure-activity relationship of N-terminal-truncated PLHSpTM derivatives, to apply a site-directed ligand approach using bulky aromatic and non-aromatic systems, and to characterize the binding nature of these analogues using X-ray crystallographic studies. We have identified a new mode of binding interactions, having improved binding affinity and retaining the Plk1 polo-box domain specificity, at the pyrrolidine-binding pocket. Furthermore, our data revealed that the pyrrolidine-binding pocket was very specific to recognize a short and bulky hydrophobic ligand like adamantane, whereas the Tyr-rich hydrophobic channel was specific with lengthy and small hydrophobic groups. CONCLUSION/SIGNIFICANCE: The progress made using our site-directed ligands validated this approach to specifically direct the ligand into the unique pyrrolidine-binding region, and it extends the applicability of the strategy for discovering selective protein-protein interaction inhibitors

Design and Synthesis of a Cell-Permeable, Drug-Like Small Molecule Inhibitor Targeting the Polo-Box Domain of Polo-Like Kinase 1

<div><p>Background</p><p>Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.</p><p>Methodology/Principal Findings</p><p>In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.</p><p>Conclusion/Significance</p><p>We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.</p></div

Structures of PPG, poloxin, thymoquinone, PLHSpT, and 4j.

<p>Structures of PPG, poloxin, thymoquinone, PLHSpT, and 4j.</p

Crystal structures of Plk1 PBD in complex with PL-74.

<p>The overlay of the crystal structures of the Plk1 PBD-bound PL-74 (white) over the previously reported unligated Plk1 PBD (cyan) revealed that the <i>N-</i>terminal cyclohexyl moiety of PL-74 bound deeply into the Tyr-rich hydrophobic channel. </p

Selected <i>N-</i>terminal peptide derivatives synthesized from the second-phase diversification and quantification of their inhibitory activities against Plk1 PBD.

<p>(A) Second-phase N-terminal-truncated PLHSpTM peptide derivatives generated using solid-phase peptide synthesis (SPPS) to study the binding nature of the pyrrolidine-binding region. (B) ELISA-based Plk1 PBD-binding IC₅₀ graph (O.D., optical density). A representative graph from three independent experiments.</p

The crystal structure of the previously reported unligated Plk1 PBD, which shows the presence of both the unique pyrrolidine-binding pocket and conserved, Tyr-rich hydrophobic channel.

<p>The crystal structure of the previously reported unligated Plk1 PBD, which shows the presence of both the unique pyrrolidine-binding pocket and conserved, Tyr-rich hydrophobic channel.</p

Crystal structures of Plk1 PBD in complex with PL-42 or PL-49.

<p>(A) The crystal structure of the Plk1 PBD-PL-42 complex showed that the <i>N-</i>terminal moiety of PL-42 interacts with the pyrrolidine hydrophobic region. (B) The overlay of the two distinct crystal structures of the Plk1 PBD-PL-49 complex revealed that the <i>N-</i>terminal cyclohexyl moiety of PL-49 possesses the dual-binding ability and interacts with either the Tyr-rich hydrophobic channel or the pyrrolidine hydrophobic region. Note that since the <i>N-</i>terminal arm of PL-49 (gray backbone) that points to the Tyr-rich hydrophobic channel could not be localized in the density maps because of its high flexibility, we were not able to model the cyclohexylmethyl segment using PyMOL [19].</p

First-phase diversification of peptides, and quantification of their inhibitory activities against Plk1 PBD.

<p>(A) First-phase <i>N-</i>terminal-truncated peptides generated using solid-phase peptide synthesis (SPPS). i) R-COOH, HBTU, HOBt, DIPEA, DMF, 2 h; ii) TFA/TIS/H₂O (90:5:5), 2.5 h; (B) ELISA-based Plk1 PBD-binding assay result using first-phase <i>N-</i>terminal-truncated peptide derivatives. Representative graphs from three independent experiments are shown (O.D., optical density).</p