There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality.

Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

I and i

Over the past decade, peptide drug discovery has experienced a revival of interest and scientific momentum, as the pharmaceutical industry has come to appreciate the role that peptide therapeutics can play in addressing unmet medical needs and how this class of compounds can be an excellent complement or even preferable alternative to small molecule and biological therapeutics. In this Perspective, we give a concise description of the recent progress in peptide drug discovery in a holistic manner, highlighting enabling technological advances affecting nearly every aspect of this field: from lead discovery, to synthesis and optimization, to peptide drug delivery. An emphasis is placed on describing research efforts to overcome the inherent weaknesses of peptide drugs, in particular their poor pharmacokinetic properties, and how these efforts have been critical to the discovery, design, and subsequent development of novel therapeutics.

The Current State of Peptide Drug Discovery: Back to the Future?

There is great need for stand-alone luminescence-based chemosensors that exemplify selectivity, sensitivity, and applicability and that overcome the challenges that arise from complex, real-world media. Discussed herein are recent developments toward these goals in the field of supramolecular luminescent chemosensors, including macrocycles, polymers, and nanomaterials. Specific focus is placed on the development of new macrocycle hosts since 2010, coupled with considerations of the underlying principles of supramolecular chemistry as well as analytes of interest and common luminophores. State-of-the-art developments in the fields of polymer and nanomaterial sensors are also examined, and some remaining unsolved challenges in the area of chemosensors are discussed.

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Supramolecular Luminescent Sensors

The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where al...

Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules

Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.

CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

Antimicrobial peptides (AMPs), essential elements in host innate immune defenses against numerous pathogens, have received considerable attention as potential alternatives to conventional antibiotics. Most AMPs exert broad-spectrum antimicrobial activity through depolarization and permeabilization of the bacterial cytoplasmic membrane. Here, we introduce a new approach for enhancing the antibiotic activity of AMPs by conjugation of a cationic cell-penetrating peptide (CPP). Interestingly, CPP-conjugated AMPs elicited only a 2- to 4-fold increase in antimicrobial activity against Gram-positive bacteria, but showed a 4- to 16-fold increase in antimicrobial activity against Gram-negative bacteria. Although CPP-AMP conjugates did not significantly increase membrane permeability, they efficiently translocated across a lipid bilayer. Indeed, confocal microscopy showed that, while AMPs were localized mainly in the membrane of Escherichia coli, the conjugates readily penetrated bacterial cells. In addition, the conjugates exhibited a higher affinity for DNA than unconjugated AMPs. Collectively, we demonstrate that CPP-AMP conjugates possess multiple functional properties, including membrane permeabilization, membrane translocation, and DNA binding, which are involved in their enhanced antibacterial activity against Gram-negative bacteria. We propose that conjugation of CPPs to AMPs may present an effective approach for the development of novel antimicrobials against Gram-negative bacteria.

Conjugation of Cell-Penetrating Peptides to Antimicrobial Peptides Enhances Antibacterial Activity.

Site-specific fatty acid-conjugation to prolong protein half-life in vivo.

Self-assembled protein nanocarrier for intracellular delivery of antibody

Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo.

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.

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Sung In Lim

Papers

Conjugation of Cell-Penetrating Peptides to Antimicrobial Peptides Enhances Antibacterial Activity.

Site-specific fatty acid-conjugation to prolong protein half-life in vivo.

Self-assembled protein nanocarrier for intracellular delivery of antibody

Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo.

Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activity.