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

Bilateral inhibition of HAUSP deubiquitinase by a viral interferon regulatory factor protein

Herpesvirus-associated ubiquitin specific protease (HAUSP) regulates the stability of p53 and MDM2, implicating HAUSP as a therapeutic target for tuning p53-mediated anti-tumor activity. Here, we report the structural analysis of HAUSP with Kaposi’s sarcoma-associated herpesvirus vIRF4 and the discovery of two vIRF4-derived peptides, vif1 and vif2, as potent and selective HAUSP antagonists. This analysis reveals a bilateral belt-type interaction resulting in inhibition of HAUSP. The vif1 peptide binds the HAUSP TRAF domain, competitively blocking substrate binding, while the vif2 peptide binds both the HAUSP TRAF and catalytic domains, robustly suppressing its deubiquitination activity. Consequently, peptide treatments comprehensively blocked HAUSP, leading to p53-dependent cell cycle arrest and apoptosis in culture and tumor regression in xenograft mouse model. Thus, the virus has developed a unique molecular strategy to target the HAUSP-MDM2-p53 pathway, and these virus-derived short peptides represent biologically active HAUSP antagonists

Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography

Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For structural studies, RNA purification requires a single nucleotide resolution. Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective. To overcome these problems in high-throughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography. A tag sequence is added at the 3′ end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 3′ end tag is then removed by sequence-specific cleavage using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number. This purification method is simpler and faster than the conventional method

Characterization of the Antimicrobial Activities of <i>Trichoplusia ni</i> Cecropin A as a High-Potency Therapeutic against Colistin-Resistant <i>Escherichia coli</i>

The spread of colistin-resistant bacteria is a serious threat to public health. As an alternative to traditional antibiotics, antimicrobial peptides (AMPs) show promise against multidrug resistance. In this study, we investigated the activity of the insect AMP Tricoplusia ni cecropin A (T. ni cecropin) against colistin-resistant bacteria. T. ni cecropin exhibited significant antibacterial and antibiofilm activities against colistin-resistant Escherichia coli (ColREC) with low cytotoxicity against mammalian cells in vitro. Results of permeabilization of the ColREC outer membrane as monitored through 1-N-phenylnaphthylamine uptake, scanning electron microscopy, lipopolysaccharide (LPS) neutralization, and LPS-binding interaction revealed that T. ni cecropin manifested antibacterial activity by targeting the outer membrane of E. coli with strong interaction with LPS. T. ni cecropin specifically targeted toll-like receptor 4 (TLR4) and showed anti-inflammatory activities with a significant reduction of inflammatory cytokines in macrophages stimulated with either LPS or ColREC via blockade of TLR4-mediated inflammatory signaling. Moreover, T. ni cecropin exhibited anti-septic effects in an LPS-induced endotoxemia mouse model, confirming its LPS-neutralizing activity, immunosuppressive effect, and recovery of organ damage in vivo. These findings demonstrate that T. ni cecropin exerts strong antimicrobial activities against ColREC and could serve as a foundation for the development of AMP therapeutics

1H, 15N and 13C resonance assignment of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii

Acinetobacter baumannii is a multiple drug resistant pathogen that causes pneumonia, bacteremia, wound infections and urinary tract infections in immunocompromised patients. Outer membrane protein A of Acinetobacter baumannii (AbOmpA) is a major surface protein and induces host cell death. AbOmpA is supposed to be composed of N-terminal β-barrel transmembrane domain and C-terminal periplasmic domain. The periplasmic domain of AbOmpA forms an OmpA-like fold, which is involved in the non-covalent interaction with peptidoglycan. However, its three dimensional structure is not determined yet. Here, we report the cloning, expression, purification and NMR spectroscopy study of recombinant AbOmpA periplasmic domain (AbOmpA-PD). The NMR spectrum of AbOmpA-PD is well dispered and almost signals exhibit homogeneous, which allow us to assign the backbone signals thoroughly. This study provides a clue for structural study of AbOmpA-PD

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