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13 pages, 3205 KiB

Open AccessArticle

From In Vitro Promise to In Vivo Reality: An Instructive Account of Infection Model Evaluation of Antimicrobial Peptides

by Adam Carrera-Aubesart, Jiarui Li, Estefanía Contreras, Roberto Bello-Madruga, Marc Torrent and David Andreu

Int. J. Mol. Sci. 2024, 25(18), 9773; https://doi.org/10.3390/ijms25189773 - 10 Sep 2024

Cited by 1 | Viewed by 1709

Abstract

Antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics in the face of ever-increasing resistance. However, many AMPs fail to progress into clinics due to unexpected difficulties found in preclinical in vivo phases. Our research has focused on crotalicidin (Ctn), an AMP from snake venom, and a fragment thereof, Ctn[15-34], with improved in vitro antimicrobial and anticancer activities and remarkable serum stability. As the retroenantio versions of both AMPs maintained favorable profiles, in this work, we evaluate the in vivo efficacy of both the native-sequence AMPs and their retroenantio counterparts in a murine infection model with Acinetobacter baumannii. A significant reduction in bacterial levels is found in the mice treated with Ctn[15-34]. However, contrary to expectations, the retroenantio analogs either exhibit toxicity or lack efficacy when administered to mice. Our findings underscore the critical importance of in vivo infection model evaluation to fully calibrate the therapeutic potential of AMPs. Full article

(This article belongs to the Topic Peptoids and Peptide Based Drugs)

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28 pages, 7654 KiB

Open AccessEditor’s ChoiceReview

Topoisomeric Membrane-Active Peptides: A Review of the Last Two Decades

by Adam Carrera-Aubesart, Maria Gallo, Sira Defaus, Toni Todorovski and David Andreu

Pharmaceutics 2023, 15(10), 2451; https://doi.org/10.3390/pharmaceutics15102451 - 12 Oct 2023

Cited by 7 | Viewed by 2221

Abstract

In recent decades, bioactive peptides have been gaining recognition in various biomedical areas, such as intracellular drug delivery (cell-penetrating peptides, CPPs) or anti-infective action (antimicrobial peptides, AMPs), closely associated to their distinct mode of interaction with biological membranes. Exploiting the interaction of membrane-active peptides with diverse targets (healthy, tumoral, bacterial or parasitic cell membranes) is opening encouraging prospects for peptides in therapeutics. However, ordinary peptides formed by L-amino acids are easily decomposed by proteases in biological fluids. One way to sidestep this limitation is to use topoisomers, namely versions of the peptide made up of D-amino acids in either canonic (enantio) or inverted (retroenantio) sequence. Rearranging peptide sequences in this fashion provides a certain degree of native structure mimicry that, in appropriate contexts, may deliver desirable biological activity while avoiding protease degradation. In this review, we will focus on recent accounts of membrane-active topoisomeric peptides with therapeutic applications as CPP drug delivery vectors, or as antimicrobial and anticancer candidates. We will also discuss the most common modes of interaction of these peptides with their membrane targets. Full article

(This article belongs to the Special Issue State of the Art of Membrane Active Peptides)

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13 pages, 8804 KiB

Open AccessArticle

Examining Topoisomers of a Snake-Venom-Derived Peptide for Improved Antimicrobial and Antitumoral Properties

by Adam Carrera-Aubesart, Sira Defaus, Clara Pérez-Peinado, Daniel Sandín, Marc Torrent, Maria Ángeles Jiménez and David Andreu

Biomedicines 2022, 10(9), 2110; https://doi.org/10.3390/biomedicines10092110 - 29 Aug 2022

Cited by 8 | Viewed by 2466

Abstract

Ctn[15-34], the C-terminal section of crotalicidin (Ctn), a cathelicidin from a South American pit viper, is an antimicrobial and antitumoral peptide with remarkably longer stability in human serum than the parent Ctn. In this work, a set of topoisomers of both Ctn and Ctn[15-34], including the retro, enantio, and retroenantio versions, were synthesized and tested to investigate the structural requirements for activity. All topoisomers were as active as the cognate sequences against Gram-negative bacteria and tumor cells while slightly more toxic towards normal cells. More importantly, the enhanced serum stability of the D-amino-acid-containing versions suggests that such topoisomers must be preferentially considered as future antimicrobial and anticancer peptide leads. Full article

(This article belongs to the Special Issue Peptides of Natural Origin as Leads for Drug Discovery: From Native Structures to Peptidomimetics, Peptide-Conjugates, and Peptide-Nanoparticles 2.0)

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20 pages, 1094 KiB

Open AccessArticle

Identification of Somatic Mitochondrial DNA Mutations, Heteroplasmy, and Increased Levels of Catenanes in Tumor Specimens Obtained from Three Endometrial Cancer Patients

by Matthew J. Young, Ravi Sachidanandam, Dale B. Hales, Laurent Brard, Kathy Robinson, Md. Mostafijur Rahman, Pabitra Khadka, Kathleen Groesch and Carolyn K. J. Young

Life 2022, 12(4), 562; https://doi.org/10.3390/life12040562 - 9 Apr 2022

Cited by 7 | Viewed by 4961

Abstract

Endometrial carcinoma (EC) is the most common type of gynecologic malignant epithelial tumor, with the death rate from this disease doubling over the past 20 years. Mitochondria provide cancer cells with necessary anabolic building blocks such as amino acids, lipids, and nucleotides, and EC samples have been shown to increase mitochondrial biogenesis. In cancer, mitochondrial DNA (mtDNA) heteroplasmy studies suggest that heteroplasmic variants encode predicted pathogenic proteins. We investigated the mtDNA genotypes within peri-normal and tumor specimens obtained from three individuals diagnosed with EC. DNA extracts from peri-normal and tumor tissues were used for mtDNA-specific next-generation sequencing and analyses of mtDNA content and topoisomers. The three tumors harbor heteroplasmic somatic mutations, and at least one mutation in each carcinoma is predicted to deleteriously alter a mtDNA-encoded protein. Somatic heteroplasmy linked to two mtDNA tRNA genes was found in separate tumors, and two heteroplasmic non-coding variants were identified in a single EC tumor. While two tumors had altered mtDNA content, all three displayed increased mtDNA catenanes. Our findings support that EC cells require wild-type mtDNA, but heteroplasmic mutations may alter mitochondrial metabolism to help promote cancer cell growth and proliferation. Full article

(This article belongs to the Special Issue Mitochondria: From Physiology to Pathology)

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16 pages, 1216 KiB

Open AccessArticle

Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy

by Olusegun Folarin, Darren Nesbeth, John M. Ward and Eli Keshavarz-Moore

Bioengineering 2019, 6(2), 54; https://doi.org/10.3390/bioengineering6020054 - 19 Jun 2019

Cited by 5 | Viewed by 9542

Abstract

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing. Full article

(This article belongs to the Special Issue Genetic and Process Engineering Tools for Next Generation Cell Factories)

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11 pages, 2120 KiB

Open AccessArticle

Two-Holder Strategy for Efficient and Selective Synthesis of Lk 1 ssDNA Catenane

by Qi Li, Jing Li, Yixiao Cui, Sheng Liu, Ran An, Xingguo Liang and Makoto Komiyama

Molecules 2018, 23(9), 2270; https://doi.org/10.3390/molecules23092270 - 5 Sep 2018

Cited by 5 | Viewed by 4253

Abstract

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it governs the feasibility of dynamic motions. However, there has been no established way to synthesize catenanes in which Lk is controlled to a predetermined value. This paper reports a new methodology to selectively synthesize Lk 1 catenanes composed of single-stranded DNA rings, in which these rings can most freely rotate each other due to minimal inter-ring interactions. To the mixture for the synthesis, two holder strands (oligonucleotides of 18–46 nt) were added, and the structure of the quasi-catenane intermediate was interlocked through Watson–Crick base pairings into a favorable conformation for Lk 1 catenation. The length of the complementary part between the two quasi-rings was kept at 10 bp or shorter. Under these steric constraints, two quasi-rings were cyclized with the use of T4 DNA ligase. By this simple procedure, the formation of undesired topoisomers (Lk ≥ 2) was almost completely inhibited, and Lk 1 catenane was selectively prepared in high yield up to 70 mole%. These Lk 1 catenanes have high potentials as dynamic parts for versatile DNA architectures. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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10 pages, 1607 KiB

Open AccessArticle

Transformation of a Thermostable G-Quadruplex Structure into DNA Duplex Driven by Reverse Gyrase

by Dawei Li, Qiang Wang, Yun Liu, Kun Liu, Qiang Zhuge and Bei Lv

Molecules 2017, 22(11), 2021; https://doi.org/10.3390/molecules22112021 - 22 Nov 2017

Cited by 1 | Viewed by 6190

Abstract

Reverse gyrase is a topoisomerase that can introduce positive supercoils to its substrate DNA. It is demonstrated in our studies that a highly thermal stable G-quadruplex structure in a mini-plasmid DNA was transformed into its duplex conformation after a treatment with reverse gyrase. The structural difference of the topoisomers were verified and analyzed by gel electrophoresis, atomic force microscopy examination, and endonuclease digestion assays. All evidence suggested that the overwinding structure of positive supercoil could provide a driven force to disintegrate G-quadruplex and reform duplex. The results of our studies could suggest that hyperthermophiles might use reverse gyrase to manipulate the disintegration of non-B DNA structures and safekeep their genomic information. Full article

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