Directed Molecular Engineering of Mig6 Peptide Selectivity between Proto-oncogene ErbB Family Receptor Tyrosine Kinases

Zhijun Qiao,Shuai Wang 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.2

The ErbB signaling pathway plays important roles in normal physiology and cancer, which consists of four proto-oncogene receptor tyrosine kinases ErbB1/EGFR, ErbB2/Her2, ErbB3/Her3, and ErbB4/Her4. Selective targeting of different ErbB kinases would result in distinct therapeutic effects, but traditional small-molecule inhibitors generally exhibit a strong cross-reactivity over these kinases due to the very high conservation in kinase’s active site. Instead of developing small-molecule drugs to selectively target the conserved active site of ErbB kinases, we herein attempt to design peptide agents for selectively disrupting the dimerization event of these kinases at their asymmetric dimer interfaces that have a relatively low homology. Three hotspot peptides S1P1, S1P2, and S1P3 are split from the functional segment 1 (Seg1) of mitogen-inducible gene 6 (Mig6), a natural EGFR-inhibitory protein that has been observed to inactivate the kinase by disrupting its dimerization. We demonstrate that the Mig6 peptides not only inhibit EGFR, but also bind Her2, Her3, and Her4, although the peptide affinities to the four ErbB kinases are different considerably, exhibiting a typical selectivity. The S1P2 peptide locates in the core binding region of Mig6 Seg1 and contributes significantly to the segment interaction with kinases. An iteration algorithm is employed to guide the directed molecular engineering of S1P2 peptide selectivity towards each of the four kinases. Hundreds of parallel evolution running yield a series of peptide candidates with potential selectivity, which are then substantiated by fluorescence-based assays. The designed EGFR-selective peptide S1P2-p1EGFR is determined to have a moderate affinity to EGFR (Kd = 56 μM) and a high selectivity for EGFR over Her2, Her3, and H4 (FEGFR = 10.1-fold), which is improved considerably relative to wild-type S1P2 peptide (FEGFR = 2.7-fold). Structural examination observes different noncovalent interaction modes at the complex interfaces of S1P2-p1EGFR with EGFR and other three kinases, revealing a molecular origin of the peptide selectivity.

Design of potent 9-mer antimicrobial peptide analogs of protaetiamycine and investigation of mechanism of antimicrobial action

Shin, Soyoung,Kim, Jin-Kyoung,Lee, Jee-Young,Jung, Ki-Woong,Hwang, Jae-Sam,Lee, Juneyoung,Lee, Dong Gun,Kim, Iksoo,Shin, Song Yub,Kim, Yangmee John Wiley Sons, Ltd. 2009 Journal of Peptide Science Vol.15 No.9

<P>Protaetiamycine is an insect defensin, a naturally occurring 43-amino-acid-residue antimicrobial peptide derived from the larvae of the beetle Protaetia brevitarsis. In a previous work that aimed at developing short antibiotic peptides, we designed 9-mer peptide analogs of protaetiamycine. Among them, RLWLAIGRG-NH<SUB>2</SUB> showed good antifungal activity against Candida albicans. In this study, we designed four 9-mer peptide analogs based on the sequence of RLWLAIGRG-NH<SUB>2</SUB>, in which Gly or Ile was substituted with Arg, Lys, or Trp to optimize the balance between the hydrophobicity and cationicity of the peptides and to increase bacterial cell selectivity. We measured their toxicity to bacteria and mammalian cells as well as their ability to permeabilize model phospholipid membranes. Substitution of Arg for Gly9 at the C-terminus (9Pbw1) resulted in two- to fourfold improvement in antibacterial activity. Further substitution of Gly7 with Lys (9Pbw2 and 9Pbw4) caused four- to eightfold improvement in the antibacterial activity without increase in cytotoxocity, while substitution of Gly7 with Trp (9Pbw3) increased cytotoxicity as well as antibacterial activity. The peptides 9Pbw2 and 9Pbw4 with the highest bacterial cell selectivity were not effective in depolarizing the membrane of Staphylococcus aureus cytoplasmic membranes and showed almost no leakage of a fluorescent dye entrapped within the vesicles. Gel-retardation experiments indicated that 9Pbw2 and 9Pbw4 inhibited the migration of DNA at concentrations >20 µM. Three positively charged residues at the C-terminus in 9Pbw2 and 9Pbw4 may facilitate effective penetration into the negatively charged phospholipid membrane of bacteria. The results obtained in this study suggest that the bactericidal action of our potent antibacterial peptides, namely 9Pbw2 and 9Pbw4, may be attributed to the inhibition of the functions of intracellular components after penetration of the bacterial cell membrane. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.</P>

Composition Variation of Papain-catalyzed Esterification of a Fibroin Peptide Mixture

정재호,이신영,허원 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.4

Composition variation of a complex peptide mixture under enzymatic transformation can be tracked by mass spectrometry (MS). In this report, papain-catalyzed esterification of fibroin peptides was investigated at the individual peptide level using liquid chromatography-mass spectrometry with selected ion monitoring. Optimal conditions for maximizing ester formation were obtained using a water-to-pentanol ratio of 1:9 at pH 2.8 and 40°C;however, the optimum conditions varied for individual peptides. The optimum pH levels were 2.5 and 2.8 for the tetrapeptides with a tyrosine or a valine residue and those with alanine or serine residues, respectively. The optimum pH shifted to 3.4 for dipeptide esters with a tyrosine residue. Tetrapeptides had a relatively higher rate of esterification above 50°C. Alhough, the profiles of peptides and their esters in the esterification reaction were significantly affected by the reaction conditions, alanyl-glycine ester represented the largest fraction in the mixture under most reaction conditions. As demonstrated here, MS analysis of peptide mixtures can be used to elucidate specific reaction conditions for the enrichment of particular peptide products.

Structure-Activity Relationships of Peptide Antibiotics with Improved Bacterial Cell Selectivity of Pseudin

Lee, Yeongjoon,Jeon, Dasom,Kim, Jin-Kyoung,Kim, Yangmee Korean Magnetic Resonance Society 2017 Journal of the Korean Magnetic Resonance Society Vol.21 No.3

Pseudin is a naturally occurring 24 amino-acid-residue antimicrobial peptide derived from the skin of paradoxical frog Pseud's paradoxa. It shows potency against the bacteria and antibiotic-resistant bacteria strain, but has high cytotoxicity against mammalian cell. In our previous study, substitution of $Pro^{11}$ for Gly (Ps-P) increased bacterial cell selectivity but decreased the antibacterial activity of pseudin. In this study, we designed pseudin analogue, Ps-4K-P with increased cationicity up to +7 in Ps-P by substituting Glu14, Gln10, Gln24, and Leu18 with Lys. Ps-4K-P showed improved potent antibacterial activity with high bacterial cell selectivity. We determined the tertiary structure of Ps-4K-P in the presence of DPC micelles by NMR spectroscopy and it has a hinge structure at $Pro^{11}$ followed by three turn helices from $Pro^{11}$ to $Val^{23}$ at the C-terminus. Amphipathicity with increased cationicity as well as helix-hinge-helix structural motif provided by introduction of a Pro at position $Gly^{11}$ are the crucial factors which confer antibacterial activity with bacterial cell selectivity to Ps-4K-P.

Structure-Activity Relationships of Peptide Antibiotics with Improved Bacterial Cell Selectivity of Pseudin

이영준,전다솜,김진경,김양미 한국자기공명학회 2017 Journal of the Korean Magnetic Resonance Society Vol.21 No.3

Pseudin is a naturally occurring 24 amino-acid-residue antimicrobial peptide derived from the skin of paradoxical frog Pseud’s paradoxa. It shows potency against the bacteria and antibiotic-resistant bacteria strain, but has high cytotoxicity against mammalian cell. In our previous study, substitution of Pro11 for Gly (Ps-P) increased bacterial cell selectivity but decreased the antibacterial activity of pseudin. In this study, we designed pseudin analogue, Ps-4K-P with increased cationicity up to +7 in Ps-P by substituting Glu14, Gln10, Gln24, and Leu18 with Lys. Ps-4K-P showed improved potent antibacterial activity with high bacterial cell selectivity. We determined the tertiary structure of Ps-4K-P in the presence of DPC micelles by NMR spectroscopy and it has a hinge structure at Pro11 followed by three turn helices from Pro11 to Val23 at the C-terminus. Amphipathicity with increased cationicity as well as helix-hinge-helix structural motif provided by introduction of a Pro at position Gly11 are the crucial factors which confer antibacterial activity with bacterial cell selectivity to Ps-4K-P.

Improvement of Cell Selectivity of a Bovine Antimicrobial Peptide Indolicidin by Pro → D-Pro or Nala Substitution

Shin, Song Yub 朝鮮大學校 附設 醫學硏究所 2007 The Medical Journal of Chosun University Vol.32 No.2

In this study, we investigated the effect of Pro -> D-Pro or Nala (Ala peptoid residue) substitution on cell selectivity of a Trp/Pro-rich antimicrobial peptide, indolicidin (IN) with poor cell selectivity. We synthesized two IN analogues, IN-D-Pro and IN-Nala, in which all proline residues of IN were replaced by D-Pro and Nala, respectively, Compared to a parent antimicrobial peptide IN, IN-D-Pro and IN-Nala displayed lower activity in hemolysis against human red blood cells and cytotoxicity against mouse fibroblastic NIH 3T3 cells while retaining strong antibacterial activity against Gram-positive and Gram-negative bacterial cells. Furthermore, tryptophan fluorescence spectroscopy suggested that the improvement in therapeutic index/cell selectivity of IN-D-Pro and IN-Nala is correlated with their low permeability to zwitterionic membranes.

Design of Pro-rich Model Antimicrobial Peptides with Improved Bacterial Selectivity

Nan, Yong-Hai,Shin, Song-Yub 朝鮮大學校 附設 醫學硏究所 2009 The Medical Journal of Chosun University Vol.33 No.S

To develop novel Pro-rich model antimicrobial peptides with shorter length and higher bacterial selectivity/therapeutic index than a natural Trp/Pro-rich antimicrobial peptide, indolicidin, we synthesized three Pro-rich model antimicrobial peptides composed of Trp, Lys, Leu and Pro resides. Compared to natural indolicidin, the designed Pro-rich model peptides had approximate 6- to 11-fold higher bacterial selectivity/therapeutic index. These designed peptides selectively bind to negatively charged liposomes [L-α-phosphatidylethanolamine (EYPE) / L-α-phosphatidyl-DL-glycerol (EYPG) (7:3, w/w)], mimicking bacterial membranes. This result indicated that the high bacterial selectivity of designed Pro-rich model peptides may due to their selective interaction with negatively charged phospholipids. Taken together, our designed peptides appear to be excellent candidates for future development as a novel antimicrobial agent.

Sequence-selective Peptide-binding Metallomacrocycles

Kum Hee Lee,Seung Soo Yoon* 대한화학회 2006 Bulletin of the Korean Chemical Society Vol.27 No.1

Synthesis and Peptide-binding Properties of C3-symmetric Metallomacrocycles

Eun Ho Song,Seung Kyu Lee,Seung Soo Yoon* 대한화학회 2007 Bulletin of the Korean Chemical Society Vol.28 No.9

The design of a cell-selective fowlicidin-1-derived peptide with both antimicrobial and anti-inflammatory activities

Rajasekaran, Ganesan,Kumar, S. Dinesh,Yang, Sungtae,Shin, Song Yub Elsevier 2019 European journal of medicinal chemistry Vol.182 No.-

<P><B>Abstract</B></P> <P>Fowlicidin-1 (Fowl-1), a cathelicidin expressed in chicken intestine, is known to have both antimicrobial and anti-inflammatory properties. However, its pharmaceutical development has been ultimately compromised by its high host cytotoxicity. In this study, a series of N- and C-terminal-truncated 19-meric Fowl-1 peptides were synthesized. Among these truncated peptides, Fowl-1 (8–26) exhibited broad-spectrum antimicrobial activity without human erythrocyte cytotoxicity while reducing anti-inflammatory activity. Further, Fowl-1 (8–26)-WRK was designed via Thr<SUP>5</SUP>→Trp, Ile<SUP>7</SUP>→Arg, and Asn<SUP>11</SUP>→Lys substitutions in Fowl-1 (8–26) to exhibit more amphipathicity. The results revealed that it exhibited both antimicrobial and anti-inflammatory properties. This study also demonstrated that the inhibitory activity of Fowl-1 (8–26)-WRK against LPS-induced inflammation was mainly due to the binding of LPS to the peptide. Interestingly, compared with human cathelicidin LL-37 and melittin, Fowl-1 (8–26)-WRK showed more potent activity against drug-resistant bacteria. It was also resistant to physiological salts and human serum and acted synergistically in combination with conventional antibiotics, such as chloramphenicol, ciprofloxacin, and oxacillin, suggesting that combined with conventional antibiotics, it is a promising adjuvant. Furthermore, membrane depolarization, SYTOX Green uptake, and flow cytometry revealed that it kills bacteria by damaging their membrane integrity. Therefore, this study suggests that Fowl-1 (8–26)-WRK has considerable potential for future development as an antimicrobial and anti-inflammatory agent for treating antibiotic-resistant infections.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Fowl-1 (8–26)-WRK exerts dual antimicrobial and anti-inflammatory activities. </LI> <LI> Compared to LL-37, Fowl-1 (8–26)-WRK showed more potent activity against drug-resistant bacteria. </LI> <LI> Fowl-1 (8–26)-WRK acted synergistically when combined with conventional antibiotics. </LI> <LI> Fowl-1 (8–26)-WRK has considerable potential for future development as an antimicrobial and anti-inflammatory agent. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>