Synthesis, Structure-Activity Relationship Study, Bioactivity, and Nephrotoxicity Evaluation of the Proposed Structure of the Cyclic Lipodepsipeptide Brevicidine B.

The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.

Towards a High-Affinity Peptidomimetic Targeting Proliferating Cell Nuclear Antigen from Aspergillus fumigatus.

Invasive fungal infections (IFIs) are prevalent in immunocompromised patients. Due to alarming levels of increasing resistance in clinical settings, new drugs targeting the major fungal pathogen Aspergillus fumigatus are required. Attractive drug targets are those involved in essential processes like DNA replication, such as proliferating cell nuclear antigens (PCNAs). PCNA has been previously studied in cancer research and presents a viable target for antifungals. Human PCNA interacts with the p21 protein, outcompeting binding proteins to halt DNA replication. The affinity of p21 for hPCNA has been shown to outcompete other associating proteins, presenting an attractive scaffold for peptidomimetic design. p21 has no A. fumigatus homolog to our knowledge, yet our group has previously demonstrated that human p21 can interact with A. fumigatus PCNA (afumPCNA). This suggests that a p21-based inhibitor could be designed to outcompete the native binding partners of afumPCNA to inhibit fungal growth. Here, we present an investigation of extensive structure-activity relationships between designed p21-based peptides and afumPCNA and the first crystal structure of a p21 peptide bound to afumPCNA, demonstrating that the A. fumigatus replication model uses a PIP-box sequence as the method for binding to afumPCNA. These results inform the new optimized secondary structure design of a potential peptidomimetic inhibitor of afumPCNA.

Designing Fluorescent Nuclear Permeable Peptidomimetics to Target Proliferating Cell Nuclear Antigen.

Human proliferating cell nuclear antigen (PCNA) is a critical mediator of DNA replication and repair, acting as a docking platform for replication proteins. Disrupting these interactions with a peptidomimetic agent presents as a promising avenue to limit proliferation of cancerous cells. Here, a p21-derived peptide was employed as a starting scaffold to design a modular peptidomimetic that interacts with PCNA and is cellular and nuclear permeable. Ultimately, a peptidomimetic was produced which met these criteria, consisting of a fluorescein tag and SV40 nuclear localization signal conjugated to the N-terminus of a p21 macrocycle derivative. Attachment of the fluorescein tag was found to directly affect cellular uptake of the peptidomimetic, with fluorescein being requisite for nuclear permeability. This work provides an important step forward in the development of PCNA targeting peptidomimetics for use as anti-cancer agents or as cancer diagnostics.

PPARγ Corepression Involves Alternate Ligand Conformation and Inflation of H12 Ensembles.

Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.

Factors influencing on-resin depsipeptide bond formation: case studies on daptomycin- and brevicidine-derived sequences.

Depsipeptides are an important class of bioactive natural products, where a growing number of genome-mined structures that display anti-microbial activity are macrocyclic depsipeptides. Chemically, peptide ester (depsipeptide) bond formation often displays low yields, and thereby hampers efforts to access these structures for structure-activity studies. Herein, we present a systematic study of the variables that influence depsipeptide bond formation on-resin, using simplified sequences derived from antibiotic peptides, daptomycin and brevicidine, prepared via Fmoc-based solid-phase synthesis. Our study highlights reaction solvent as the key determinant, where switching the solvent from DMF to DCM in almost all cases increased the amount of depsipeptide product. Limiting the number of amino-acids N-terminal to the reactive alcohol was also noted to significantly improve the acylation efficiency. The impact of different N-terminal and side-chain protecting groups, as well as stereochemistry, was also investigated. Additives to the reaction, such as inclusion of surfactants for esterification of long hydrophobic sequences, did not improve conversion. 6-ClHOBt, often added to improve acylation efficiency, notably decreased the amount of depsipeptide observed. Lastly, no significant difference between polystyrene and Tentagel® (PEG-decorated) resins were found for these sequences.

Discovery of an ʟ-amino acid ligase implicated in Staphylococcal sulfur amino acid metabolism.

Enzymes involved in Staphylococcus aureus amino acid metabolism have recently gained traction as promising targets for the development of new antibiotics, however, not all aspects of this process are understood. The ATP-grasp superfamily includes enzymes that predominantly catalyze the ATP-dependent ligation of various carboxylate and amine substrates. One subset, ʟ-amino acid ligases (LALs), primarily catalyze the formation of dipeptide products in Gram-positive bacteria, however, their involvement in S. aureus amino acid metabolism has not been investigated. Here, we present the characterization of the putative ATP-grasp enzyme (SAOUHSC_02373) from S. aureus NCTC 8325 and its identification as a novel LAL. First, we interrogated the activity of SAOUHSC_02373 against a panel of ʟ-amino acid substrates. As a result, we identified SAOUHSC_02373 as an LAL with high selectivity for ʟ-aspartate and ʟ-methionine substrates, specifically forming an ʟ-aspartyl-ʟ-methionine dipeptide. Thus, we propose that SAOUHSC_02373 be assigned as ʟ-aspartate-ʟ-methionine ligase (LdmS). To further understand this unique activity, we investigated the mechanism of LdmS by X-ray crystallography, molecular modeling, and site-directed mutagenesis. Our results suggest that LdmS shares a similar mechanism to other ATP-grasp enzymes but possesses a distinctive active site architecture that confers selectivity for the ʟ-Asp and ʟ-Met substrates. Phylogenetic analysis revealed LdmS homologs are highly conserved in Staphylococcus and closely related Gram-positive Firmicutes. Subsequent genetic analysis upstream of the ldmS operon revealed several trans-acting regulatory elements associated with control of Met and Cys metabolism. Together, these findings support a role for LdmS in Staphylococcal sulfur amino acid metabolism.

A cell permeable bimane-constrained PCNA-interacting peptide.

The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 310-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143-151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with K D values ranging from 570 nM to 3.86 µM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 310-helical binding conformation. This suggests the 310-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.

Approaches to Introduce Helical Structure in Cysteine-Containing Peptides with a Bimane Group.

An i-i+4 or i-i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i-i+4 constrained peptide adopts a 310 -helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.

Unlocking the PIP-box: A peptide library reveals interactions that drive high-affinity binding to human PCNA.

The human sliding clamp, Proliferating Cell Nuclear Antigen (hPCNA), interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes to the features of a PIP-box modulate protein binding and thus how they fine-tune downstream processes. Here, we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterized as KD values determined by surface plasmon resonance, spanned a 4000-fold range, from 1.83 nM to 7.59 µM. The hPCNA-bound peptide structures determined by X-ray crystallography and modeled computationally revealed intermolecular and intramolecular interaction networks that correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA-binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA affinity to regulate DNA replication and repair processes. In addition, these insights will be useful to future design of hPCNA inhibitors.

A turn-on fluorescent PCNA sensor.

The solvatochromic amino-acids 4-DMNA or 4-DAPA, were separately introduced at position 147, 150 or 151 of a short p21 peptide (141-155) known to bind sliding clamp protein PCNA. The ability of these peptides, 1a-3a and 1b-3b, to act as a turn-on fluorescent sensor for PCNA was then investigated. The 4-DMNA-containing peptides (1a-3a) displayed up to a 40-fold difference in fluorescence between a polar (Tris buffer) and a hydrophobic solvent (dioxane with 5 mM 18-crown-6), while the 4-DAPA-containing peptides (1b-3b) displayed a significantly enhanced (300-fold) increase in fluorescence from Tris buffer to dioxane with 18-crown-6. SPR analysis of the peptides against PCNA revealed that the 151-substituted peptides 3a and 3b interacted specifically with PCNA, with KD values of 921 nM and 1.28 µM, respectively. Analysis of the fluorescence of these peptides in the presence of increasing concentrations of PCNA revealed a 10-fold change in fluorescence for 3a at 2.5 equivalents of PCNA, compared to only a 3.5-fold change in fluorescence for 3b. Peptide 3a is an important lead for development of a PCNA-selective turn-on fluorescent sensor for application as a cell proliferation sensor to investigate diseases such as cancer.

Protein detection enabled using functionalised silk-binding peptides on a silk-coated optical fibre.

We present a new coating procedure to prepare optical fibre sensors suitable for use with protein analytes. We demonstrate this through the detection of AlexaFluor-532 tagged streptavidin by its binding to D-biotin that is functionalised onto an optical fibre, via incorporation in a silk fibroin fibre coating. The D-biotin was covalently attached to a silk-binding peptide to provide SBP-biotin, which adheres the D-biotin to the silk-coated fibre tip. These optical fibre probes were prepared by two methods. The first involves dip-coating the fibre tip into a mixture of silk fibroin and SBP-biotin, which distributes the SBP-biotin throughout the silk coating (method A). The second method uses two steps, where the fibre is first dip-coated in silk only, then SBP-biotin added in a second dip-coating step. This isolates SBP-biotin to the outer surface of the silk layer (method B). A series of fluorescence measurements revealed that only the surface bound SBP-biotin detects streptavidin with a detection limit of 15 µg mL-1. The fibre coatings are stable to repeated washing and long-term exposure to water. Formation of silk coatings on fibres using commercial aqueous silk fibroin was found to be inhibited by a lithium concentration of 200 ppm, as determined by atomic absorption spectroscopy. This was reduced to less than 20 ppm by dialysis against water, and was found to successfully form a coating on optical fibres.

A Bimane-Based Peptide Staple for Combined Helical Induction and Fluorescent Imaging.

The thiol-selective fluorescent imaging agent, dibromobimane, has been repurposed to crosslink cysteine- and homocysteine-containing peptides, with the resulting bimane linker acting as both a structural constraint and a fluorescent tag. Macrocyclisation was conducted on nine short peptides containing two cysteines and/or homocysteines, both on-resin and in buffered aqueous solution, to give macrocycles ranging in size from 16 (i,i+2) to 31 (i,i+7) atoms. The structures were defined by CD, NMR structure calculations by using Xplor-NIH, NMR secondary shift and JHαNH analyses to reveal helical structure in the i,i+4 (1, 2), and i,i+3 (5) constrained peptides. Cellular-uptake studies were conducted with three of the macrocycles. Subsequent confocal imaging revealed punctate fluorescence within the cytosol indicative of peptides trapped in endocytic vesicles. These studies demonstrate that dibromobimane is an effective tool for defining secondary structure within short peptides, whilst simultaneously introducing a fluorescent tag suitable for common cell-based experiments.

Targeting PCNA with Peptide Mimetics for Therapeutic Purposes.

Proliferating cell nuclear antigen (PCNA) is an excellent inhibition target to shut down highly proliferative cells and thereby develop a broad-spectrum cancer therapeutic. It interacts with a wide variety of proteins through a conserved motif referred to as the PCNA-interacting protein (PIP) box. There is large sequence diversity between high-affinity PCNA binding partners, but with conservation of the binding structure-a well-defined 310 -helix. Herein, all current PIP-box peptides crystallised with human PCNA are collated to reveal common trends between binding structure and affinity. Key intra- and intermolecular hydrogen-bonding networks that stabilise the 310 -helix of PIP-box partners are highlighted and related back to the canonical PIP-box motif. High correlation with the canonical PIP-box sequence does not directly afford high affinity. Instead, we summarise key interactions that stabilise the binding structure that leads to enhanced PCNA binding affinity. These interactions also implicate the "non-conserved" residues within the PIP-box that have previously been overlooked. Such insights will allow a more directed approach to develop therapeutic PCNA inhibitors.