Marcos Pires

The effect of preorganized versus undefined charge display on the cellular uptake of cationic cell-penetrating peptides (CPPs) was investigated by comparing conformationally well-defined guanidinylated oligoprolines with flexible oligoarginines. Flow cytometry and confocal microscopy studies with different cancer cell lines (HeLa, MCF-7, and HT-29) showed that preorganization of cationic charges in lateral distances of ≈9 Å enhanced the cellular uptake of CPPs. Binding affinity measurements revealed tighter binding of analogues of cell-surface glycans to the guanidinylated octaproline with localized charges compared to flexible octaarginine, a finding that was further correlated to the cellular uptake by studies with CHO cells deficient in glycans on the outer plasma membrane.

Matching charges: Cationic oligoprolines with charges at lateral distances of 9 Å penetrate into cancer cells at submicromolar concentrations. The cellular uptake correlates with binding affinity to the cell-surface glycan analogue heparin with comparable distances between anionic sulfate moieties.

Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49–2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α’-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL⁻¹) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

In for the kill: C-terminal proline-rich antimicrobial peptide (PrAMP) hydrazidation together with its rational tetramerization are shown to be effective means for increasing both diversity and potency of PrAMP action against nosocomial Gram-negative bacteria.Wade et al: C-Terminal Modification & Multimerization Increase Efficacy #Proline-Rich #Antimicrobial #Peptide

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of sequence-selective DNA minor-groove binding agents that form a covalent aminal bond between their C11-position and the C2-NH₂ groups of guanine bases. The first example of a PBD monomer, the natural product anthramycin, was discovered in the 1960s, and the best known PBD dimer, SJG-136 (also known as SG2000, NSC 694501 or BN2629), was synthesized in the 1990s and has recently completed Phase II clinical trials in patients with leukaemia and ovarian cancer. More recently, PBD dimer analogues are being attached to tumor-targeting antibodies to create antibody–drug conjugates (ADCs), a number of which are now in clinical trials, with many others in pre-clinical development. This Review maps the development from anthramycin to the first PBD dimers, and then to PBD-containing ADCs, and explores both structure–activity relationships (SARs) and the biology of PBDs, and the strategies for their use as payloads for ADCs.

PBDs as payloads for ADCs: The pyrrolobenzodiazepines (PBDs) are a family of DNA-interactive antitumor agents that bind to guanine bases in the DNA minor groove in a sequence-selective manner. They have potent cytotoxicity, and are being used as payloads for antibody–drug conjugates (ADCs). This Review outlines their development from the discovery of the natural product anthramycin through to the use of PBD dimer payloads in ADCs.

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence-based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high-throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high-throughput MraY assays.

Total, not semi: Park's nucleotide is an intermediate in bacterial peptidoglycan biosynthesis acting as the substrate of the enzyme MraY. An efficient total synthesis of dansylated Park's nucleotide is reported. The thus obtained amount of the target compound vastly exceeded the amount accessible by semi-synthesis on conventional scale, therefore enabling high-throughput assays for the identification of MraY inhibitors as potential antimicrobial agents.

Aberrant canonical NF-κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many non-canonical NF-κB functions. Here we show that a selective peptide-based inhibitor of canonical NF-κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non-labile bond, shows an about 10-fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NF-κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many protein–protein interactions mediated by such structures.

A peptide-based inhibitor of canonical NF-κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non-labile bond, shows an about 10-fold increased potency relative to the original inhibitor. The success of the synthetic replacement of a peptide loop suggests that the general strategy could be broadly useful for discovering modulators of many protein–protein interactions mediated by such structures.

Gram-negative bacteria represent a challenging task for antibacterial drug discovery owing to their impermeable cell membrane and restricted uptake of small molecules. We herein describe the synthesis of natural-product-derived epoxycyclohexenones and explore their antibiotic activity against several pathogenic bacteria. A compound with activity against Salmonella Typhimurium was identified, and the target enzymes were unraveled by quantitative chemical proteomics. Importantly, two protein hits were linked to bacterial stress response, and corresponding assays revealed an elevated susceptibility to reactive oxygen species upon compound treatment. The consolidated inhibition of these targets provides a rationale for antibacterial activity and highlights epoxycyclohexenones as natural product scaffolds with suitable properties for killing Gram-negative Salmonella.

Aminoepoxycyclohexenones inhibit the growth of Gram-negative Salmonella in the low micromolar range. The bacterial targets of these antibiotics were analyzed by quantitative ABPP, which revealed a link to bacterial stress response that was validated by various assays. Aminoepoxybenzoquinones were thus validated as natural product scaffolds with suitable properties for the growth inhibition of Salmonella.