Recombinant production of the cyclotide kalata B1 by conditional split inteins.

This study describes the design, production, and characterization of a novel conditional intein system for the recombinant production of cyclic peptides. The system is based on two key features: (1) a promiscuous extein recognition site allowing cyclization of virtually any peptide, and (2) a secondary split site within the intein itself enabling triggered splicing at will. Two intein precursors were recombinantly expressed, purified, and then self-assembled in vitro to cyclize the model peptide kalata B1 (kB1). Cyclized kB1 was successfully purified, refolded and characterized by mass spectrometry and NMR, demonstrating correct disulfide bond formation and identical structure to synthetic kB1. Importantly, the intein-derived kB1 retained full biological activity as evidenced by insect cell toxicity assays. This work establishes a versatile and efficient approach for intein-mediated protein cyclization with potential applications in bioengineering and peptide discovery.

Hijacking of N-fixing legume albumin-1 genes enables the cyclization and stabilization of defense peptides.

The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides.

The prospect of substrate-based kinase inhibitors to improve target selectivity and overcome drug resistance.

Human kinases are recognized as one of the most important drug targets associated with cancer. There are >80 FDA-approved kinase inhibitors to date, most of which work by inhibiting ATP binding to the kinase. However, the frequent development of single-point mutations within the kinase domain has made overcoming drug resistance a major challenge in drug discovery today. Targeting the substrate site of kinases can offer a more selective and resistance-resilient solution compared to ATP inhibition but has traditionally been challenging. However, emerging technologies for the discovery of drug leads using recombinant display and stabilization of lead compounds have increased interest in targeting the substrate site of kinases. This review discusses recent advances in the substrate-based inhibition of protein kinases and the potential of such approaches for overcoming the emergence of resistance.

Peptide PaDBS1R6 has potent antibacterial activity on clinical bacterial isolates and integrates an immunomodulatory peptide fragment within its sequence.

BACKGROUND: Resistant infectious diseases caused by gram-negative bacteria are among the most serious worldwide health problems. Antimicrobial peptides (AMPs) have been explored as promising antibacterial, antibiofilm, and anti-infective candidates to address these health challenges. MAJOR CONCLUSIONS: Here we report the potent antibacterial effect of the peptide PaDBS1R6 on clinical bacterial isolates and identify an immunomodulatory peptide fragment incorporated within it. PaDBS1R6 was evaluated against Acinetobacter baumannii and Escherichia coli clinical isolates and had minimal inhibitory concentration (MIC) values from 8 to 32 µmol L-1. It had a rapid bactericidal effect, with eradication showing within 3 min of incubation, depending on the bacterial strain tested. In addition, PaDBS1R6 inhibited biofilm formation for A. baumannii and E. coli and was non-toxic toward healthy mammalian cells. These findings are explained by the preference of PaDBS1R6 for anionic membranes over neutral membranes, as assessed by surface plasmon resonance assays and molecular dynamics simulations. Considering its potent antibacterial activity, PaDBS1R6 was used as a template for sliding-window fr agmentation studies (window size = 10 residues). Among the sliding-window fragments, PaDBS1R6F8, PaDBS1R6F9, and PaDBS1R6F10 were ineffective against any of the bacterial strains tested. Additional biological assays were conducted, including nitric oxide (NO) modulation and wound scratch assays, and the R6F8 peptide fragment was found to be active in modulating NO levels, as well as having strong wound healing properties. GENERAL SIGNIFICANCE: This study proposes a new concept whereby peptides with different biological properties can be derived by the screening of fragments from within potent AMPs.

Enhancing the Intrinsic Antiplasmodial Activity and Improving the Stability and Selectivity of a Tunable Peptide Scaffold Derived from Human Platelet Factor 4.

The control of malaria, a disease caused by Plasmodium parasites that kills over half a million people every year, is threatened by the continual emergence and spread of drug resistance. Therefore, new molecules with different mechanisms of action are needed in the antimalarial drug development pipeline. Peptides developed from host defense molecules are gaining traction as anti-infectives due to theood of inducing drug resistance. Human platelet factor 4 (PF4) has intrinsic activity against P. falciparum, and a macrocyclic helix-loop-helix peptide derived from its active domain recapitulates this activity. In this study, we used a stepwise approach to optimize first-generation PF4-derived internalization peptides (PDIPs) by producing analogues with substitutions to charged and hydrophobic amino acid residues or with modifications to terminal residues including backbone cyclization. We evaluated the in vitro activity of PDIP analogues against P. falciparum compared to their overall helical structure, resistance to breakdown by serum proteases, selective binding to negatively charged membranes, and hemolytic activity. Next, we combined antiplasmodial potency-enhancing substitutions that retained favorable membrane and cell-selective properties onto the most stable scaffold to produce a backbone cyclic PDIP analogue with four-fold improved activity against P. falciparum compared to first-generation peptides. These studies demonstrate the ability to modify PDIP to select for and combine desirable properties and further validate the suitability of this unique peptide scaffold for developing a new molecule class that is distinct from existing antimalarial drugs.

Gene-guided identifications of a structure-chimeric cyclotide viphi I from Viola philippica: Potential functions against cadmium and nematodes.

The cyclic peptides, cyclotides, are identified mostly with 29-31-aa (amino acid residues) but rarely with ≥ 34-aa in plants. Viola philippica is a well-known medicinal plant but a rare metallophyte with cyclotides. A hypothesis was hence raised that the potential novel 34-aa cyclotide of Viola philippica would clearly broaden the structural and functional diversities of plant cyclotides. After homology-cloning the cyclotide precursor gene of VpCP5, a 34-aa cyclotide (viphi I) was identified to be larger than 22 other known cyclotides in V. philippica. It had a chimeric primary structure, due to its unusual loop structures (8 residues in loop 2 and 6 residues in loop 5) and aa composition (3 E and 5 R), by using phylogenetic analyses and an in-house cyclotide analysis tool, CyExcel_V1. A plasmid pCYC-viphi_I and a lab-used recombinant process were specially constructed for preparing viphi I. Typically, 0.12 or 0.25 mg ml-1 co-exposed viphi I could significantly remain cell activities with elevating Cd2+-exposed doses from 10-8 to 10-6 mol l-1 in MCF7 cells. In the model nematode Caenorhabditis elegans, IC50 values of viphi I to inhibit adult ratios and to induce death ratios, were 184.7 and 585.9 µg ml-1, respectively; the median lifespan of adult worms decreased from 14 to 2 d at viphi I doses ranging from 0.05 to 2 mg ml-1. Taken together, the newly identified viphi I exhibits functional potentials against cadmium and nematodes, providing new insights into structural and functional diversity of chimeric cyclotides in plants.

Cyclic tachyplesin I kills proliferative, non-proliferative and drug-resistant melanoma cells without inducing resistance.

Acquired drug resistance is the major cause for disease recurrence in cancer patients, and this is particularly true for patients with metastatic melanoma that carry a BRAF V600E mutation. To address this problem, we investigated cyclic membrane-active peptides as an alternative therapeutic modality to kill drug-tolerant and resistant melanoma cells to avoid acquired drug resistance. We selected two stable cyclic peptides (cTI and cGm), previously shown to have anti-melanoma properties, and compared them with dabrafenib, a drug used to treat cancer patients with the BRAF V600E mutation. The peptides act via a fast membrane-permeabilizing mechanism and kill metastatic melanoma cells that are sensitive, tolerant, or resistant to dabrafenib. Melanoma cells do not become resistant to long-term treatment with cTI, nor do they evolve their lipid membrane composition, as measured by lipidomic and proteomic studies. In vivo studies in mice demonstrated that the combination treatment of cTI and dabrafenib resulted in fewer metastases and improved overall survival. Such cyclic membrane-active peptides are thus well suited as templates to design new anticancer therapeutic strategies.

Chemical synthesis of grafted cyclotides using a "plug and play" approach.

Cyclotides are a diverse class of plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology. Their remarkable structural stability and resistance to proteolytic degradation can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. Thus, cyclotides have emerged as powerful scaffold molecules for designing peptide-based therapeutics. The chemical engineering of cyclotides has generated novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. However key challenges potentially limit the widespread use of cyclotides in molecular grafting applications. Folding of cyclotides containing bioactive epitopes remains a major bottleneck in cyclotide synthesis. Here we present a modular 'plug and play' approach that effectively bypasses problems associated with the oxidative folding of cyclotides. By grafting onto a pre-formed acyclic cyclotide-like scaffold we show that difficult-to-graft sequences can be easily obtained and can target GPCRs with nanomolar affinities and potencies. We further show the suitability of this new method to graft other complex epitopes including structures with additional disulfide bonds that are not readily available via currently employed chemical methods, thus fully unlocking cyclotides to be used in drug design applications.

Conotoxins Targeting Voltage-Gated Sodium Ion Channels.

Voltage-gated sodium (NaV) channels are intimately involved in the generation and transmission of action potentials, and dysfunction of these channels may contribute to nervous system diseases, such as epilepsy, neuropathic pain, psychosis, autism, and cardiac arrhythmia. Many venom peptides selectively act on NaV channels. These include conotoxins, which are neurotoxins secreted by cone snails for prey capture or self-defense but which are also valuable pharmacological tools for the identification and/or treatment of human diseases. Typically, conotoxins contain two or three disulfide bonds, and these internal crossbraces contribute to conotoxins having compact, well defined structures and high stability. Of the conotoxins containing three disulfide bonds, some selectively target mammalian NaV channels and can block, stimulate, or modulate these channels. Such conotoxins have great potential to serve as pharmacological tools for studying the functions and characteristics of NaV channels or as drug leads for neurologic diseases related to NaV channels. Accordingly, discovering or designing conotoxins targeting NaV channels with high potency and selectivity is important. The amino acid sequences, disulfide bond connectivity, and three-dimensional structures are key factors that affect the biological activity of conotoxins, and targeted synthetic modifications of conotoxins can greatly improve their activity and selectivity. This review examines NaV channel-targeted conotoxins, focusing on their structures, activities, and designed modifications, with a view toward expanding their applications. SIGNIFICANCE STATEMENT: NaV channels are crucial in various neurologic diseases. Some conotoxins selectively target NaV channels, causing either blockade or activation, thus enabling their use as pharmacological tools for studying the channels' characteristics and functions. Conotoxins also have promising potential to be developed as drug leads. The disulfide bonds in these peptides are important for stabilizing their structures, thus leading to enhanced specificity and potency. Together, conotoxins targeting NaV channels have both immediate research value and promising future application prospects.