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1. Lung SPLUNC1 Peptide Derivatives in the Lipid Membrane Headgroup Kill Gram-Negative Planktonic and Biofilm Bacteria

   Jakkampudi, Tanvi; Lin, Qiao; Mitra, Saheli; Qin, Weiheng; Kang, Ann; Chen, Jespar; Ryan, Emma; Wang, Runwuan; Gong, Yuqi; Heinrich, Frank; et al (May 2023, Biomacromolecules)

   Lu, Hua (Ed.)

   SPLUNC1 (short palate lung and nasal epithelial clone 1) is a multifunctional host defense protein found in human respiratory tract with antimicrobial properties. In this work we compare the biological activities of four SPLUNC1 antimicrobial peptide (AMP) derivatives using paired clinical isolates of the Gram-negative (G(-)) bacteria Klebsiella pneumoniae, obtained from eleven patients with/without colistin resistance. Secondary structural studies were carried out to study interactions between the AMPs and lipid model membranes (LMMs) utilizing circular dichroism (CD). Two peptides were further characterized using x-ray diffuse scattering (XDS) and neutron reflectivity (NR). A4-153 displayed superior antibacterial activity in both G(-) planktonic cultures and biofilms. NR and XDS revealed that A4-153 (highest activity) is located primarily in membrane headgroups, while A4-198 (lowest activity) is located in hydrophobic region. CD revealed that A4-153 is helical while A4-198 has little helical character, demonstrating that helicity and efficacy are correlated in these SPLUNC1 AMPs. 

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2. Arsinothricin, an arsenic-containing non-proteinogenic amino acid analog of glutamate, is a broad-spectrum antibiotic

   https://doi.org/10.1038/s42003-019-0365-y  

   Nadar, Venkadesh Sarkarai; Chen, Jian; Dheeman, Dharmendra S.; Galván, Adriana Emilce; Yoshinaga-Sakurai, Kunie; Kandavelu, Palani; Sankaran, Banumathi; Kuramata, Masato; Ishikawa, Satoru; Rosen, Barry P.; et al (April 2019, Communications Biology)

   Abstract The emergence and spread of antimicrobial resistance highlights the urgent need for new antibiotics. Organoarsenicals have been used as antimicrobials since Paul Ehrlich’s salvarsan. Recently a soil bacterium was shown to produce the organoarsenical arsinothricin. We demonstrate that arsinothricin, a non-proteinogenic analog of glutamate that inhibits glutamine synthetase, is an effective broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, suggesting that bacteria have evolved the ability to utilize the pervasive environmental toxic metalloid arsenic to produce a potent antimicrobial. With every new antibiotic, resistance inevitably arises. ThearsN1gene, widely distributed in bacterial arsenic resistance (ars) operons, selectively confers resistance to arsinothricin by acetylation of the α-amino group. Crystal structures of ArsN1N-acetyltransferase, with or without arsinothricin, shed light on the mechanism of its substrate selectivity. These findings have the potential for development of a new class of organoarsenical antimicrobials and ArsN1 inhibitors. 

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3. Novel Helical Trp‐ and Arg‐Rich Antimicrobial Peptides Locate Near Membrane Surfaces and Rigidify Lipid Model Membranes

   https://doi.org/10.1002/anbr.202300013  

   Mitra, Saheli; Coopershlyak, Mark; Li, Yunshu; Chandersekhar, Bhairavi; Koenig, Rachel; Chen, Mei-Tung; Evans, Brandt; Heinrich, Frank; Deslouches, Berthony; Tristram-Nagle, Stephanie (April 2023, Advanced NanoBiomed Research)

   Antibiotics are losing effectiveness as bacteria become resistant to conventional drugs. To find new alternatives, antimicrobial peptides (AMPs) are rationally designed with different lengths, charges, hydrophobicities (H), and hydrophobic moments (μH), containing only three types of amino acids: arginine, tryptophan, and valine. Six AMPs with low minimum inhibitory concentrations (MICs) and <25% toxicity to mammalian cells are selected for biophysical studies. Their secondary structures are determined using circular dichroism (CD), which finds that the % α‐helicity of AMPs depends on composition of the lipid model membranes (LMMs): gram‐negative (G(−)) inner membrane (IM) >gram‐positive (G(+))> Euk33 (eukaryotic with 33 mol% cholesterol). The two most effective peptides, E2‐35 (16 amino acid [AA] residues) and E2‐05 (22 AAs), are predominantly helical in G(–) IM and G(+) LMMs. AMP/membrane interactions such as membrane elasticity, chain order parameter, and location of the peptides in the membrane are investigated by low‐angle and wide‐angle X‐ray diffuse scattering (XDS). It is found that headgroup location correlates with efficacy and toxicity. The membrane bending modulusK_Cdisplays nonmonotonic changes due to increasing concentrations of E2‐35 and E2‐05 in G(–) and G(+) LMMs, suggesting a bacterial killing mechanism where domain formation causes ion and water leakage. 

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4. Modification of narrow‐spectrum peptidomimetic polyurethanes with fatty acid chains confers broad‐spectrum antibacterial activity

   https://doi.org/10.1002/pi.5773  

   Peng, Chao; Zhang, Tian; Ortiz‐Ortiz, Deliris_N; Vishwakarma, Apoorva; Barton, Hazel_A; Joy, Abraham (February 2019, Polymer International)

   Abstract Each year, thousands of patients die from antimicrobial‐resistant bacterial infections that fail to respond to conventional antibiotic treatment. Antimicrobial polymers are a promising new method of combating antibiotic‐resistant bacterial infections. We have previously reported the synthesis of a series of narrow‐spectrum peptidomimetic antimicrobial polyurethanes that are effective against Gram‐negative bacteria, such asEscherichia coli; however, these polymers are not effective against Gram‐positive bacteria, such asStaphylococcus aureus. With the aim of understanding the correlation between chemical structure and antibacterial activity, we have subsequently developed three structural variants of these antimicrobial polyurethanes using post‐polymerization modification with decanoic acid and oleic acid. Our results show that such modifications converted the narrow‐spectrum antibacterial activity of these polymers into broad‐spectrum activity against Gram‐positive species such asS. aureus, however, also increasing their toxicity to mammalian cells. Mechanistic studies of bacterial membrane disruption illustrate the differences in antibacterial action between the various polymers. The results demonstrate the challenge of balancing antimicrobial activity and mammalian cell compatibility in the design of antimicrobial polymer compositions. © 2019 Society of Chemical Industry 

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5. Rational Framework for the Design of Trp- and Arg-Rich Peptide Antibiotics Against Multidrug-Resistant Bacteria

   https://doi.org/10.3389/fmicb.2022.889791  

   Xiang, Wenyu; Clemenza, Patrice; Klousnitzer, Jessie; Chen, Jespar; Qin, Weiheng; Tristram-Nagle, Stephanie; Doi, Yohei; Di, Y. Peter; Deslouches, Berthony (May 2022, Frontiers in Microbiology)

   The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2–4 μM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application. 

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