Stitched peptides as potential cell permeable inhibitors of oncogenic DAXX protein.

DAXX (Death Domain Associated Protein 6) is frequently upregulated in various common cancers, and its suppression has been linked to reduced tumor progression. Consequently, DAXX has gained significant interest as a therapeutic target in such cancers. DAXX is known to function in several critical biological pathways including chromatin remodelling, transcription regulation, and DNA repair. Leveraging structural information, we have designed and developed a novel set of stapled/stitched peptides that specifically target a surface on the N-terminal helical bundle domain of DAXX. This surface serves as the anchor point for binding to multiple interaction partners, such as Rassf1C, p53, Mdm2, and ATRX, as well as for the auto-regulation of the DAXX N-terminal SUMO interaction motif (SIM). Our experiments demonstrate that these peptides effectively bind to and inhibit DAXX with a higher affinity than the known interaction partners. Furthermore, these peptides release the auto-inhibited SIM, enabling it to interact with SUMO-1. Importantly, we have developed stitched peptides that can enter cells, maintaining their intracellular concentrations at nanomolar levels even after 24 hours, without causing any membrane perturbation. Collectively, our findings suggest that these stitched peptides not only serve as valuable tools for probing the molecular interactions of DAXX but also hold potential as precursors to the development of therapeutic interventions.

Development of teixobactin analogues containing hydrophobic, non-proteogenic amino acids that are highly potent against multidrug-resistant bacteria and biofilms.

Teixobactin is a cyclic undecadepsipeptide that has shown excellent potency against multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). In this article, we present the design, synthesis, and antibacterial evaluations of 16 different teixobactin analogues. These simplified analogues contain commercially available hydrophobic, non-proteogenic amino acid residues instead of synthetically challenging expensive L-allo-enduracididine amino acid residue at position 10 together with different combinations of arginines at positions 3, 4 and 9. The new teixobactin analogues showed potent antibacterial activity against a broad panel of Gram-positive bacteria, including MRSA and VRE strains. Our work also presents the first demonstration of the potent antibiofilm activity of teixobactin analogoues against Staphylococcus species associated with serious chronic infections. Our results suggest that the use of hydrophobic, non-proteogenic amino acids at position 10 in combination with arginine at positions 3, 4 and 9 holds the key to synthesising a new generation of highly potent teixobactin analogues to tackle resistant bacterial infections and biofilms.

Chemoselective N-Acylation of Amines with Acylsilanes under Aqueous Acidic Conditions.

We report a facile method for forming amide bonds between acylsilanes and a wide range of amines in the presence of a mild chlorinating agent under aqueous acidic conditions. The reaction is highly chemoselective, as exemplified by the late-stage modification of a panel of approved drugs and natural products containing reactive functionalities.

Depleted hexokinase1 and lack of AMPKα activation favor OXPHOS-dependent energetics in retinoblastoma tumors.

Lack of retinoblastoma (Rb) protein causes aggressive intraocular retinal tumors in children. Recently, Rb tumors have been shown to have a distinctly altered metabolic phenotype, such as reduced expression of glycolytic pathway proteins alongside altered pyruvate and fatty acid levels. In this study, we demonstrate that loss of hexokinase 1(HK1) in tumor cells rewires their metabolism allowing enhanced oxidative phosphorylation-dependent energy production. We show that rescuing HK1 or retinoblastoma protein 1 (RB1) in these Rb cells reduced cancer hallmarks such as proliferation, invasion, and spheroid formation and increased their sensitivity to chemotherapy drugs. Induction of HK1 was accompanied by a metabolic shift of the cells to glycolysis and a reduction in mitochondrial mass. Cytoplasmic HK1 bound Liver Kinase B1 and phosphorylated AMP-activated kinase-α (AMPKα Thr172), thereby reducing mitochondria-dependent energy production. We validated these findings in tumor samples from Rb patients compared to age-matched healthy retinae. HK1 or RB1 expression in Rb-/- cells led to a reduction in their respiratory capacity and glycolytic proton flux. HK1 overexpression reduced tumor burden in an intraocular tumor xenograft model. AMPKα activation by AICAR also enhanced the tumoricidal effects of the chemotherapeutic drug topotecan in vivo. Therefore, enhancing HK1 or AMPKα activity can reprogram cancer metabolism and sensitize Rb tumors to lower doses of existing treatments, a potential therapeutic modality for Rb.

Propranolol Ameliorates the Antifungal Activity of Azoles in Invasive Candidiasis.

The effectiveness of current antifungal therapies is hampered by the emergence of drug resistance strains, highlighting an urgent need for new alternatives such as adjuvant antifungal treatments. This study aims to examine the synergism between propranolol and antifungal drugs, based on the premise that propranolol is known to inhibit fungal hyphae. In vitro studies demonstrate that propranolol potentiates the antifungal activity of azoles and that the effect is more pronounced for propranolol-itraconazole combination. Using an in vivo murine systemic candidemia model, we show that propranolol-itraconazole combination treatment resulted in a lower rate of body weight loss, decreased kidney fungal bioburden and renal inflammation when compared to propranolol and azole treatment alone or untreated control. Altogether, our findings suggest that propranolol increases the efficacy of azoles against C. albicans, offering a new therapeutic strategy against invasive fungal infections.

Correction: Peguda et al. The Activity of Polyhomoarginine against Acanthamoeba castellanii. Biology 2022, 11, 1726.

In the original publication [...].

The Activity of Polyhomoarginine against Acanthamoeba castellanii.

Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the negatively charged microbial cell membranes. Acanthamoeba is a free-living protozoan known to cause a rare corneal infection which is difficult to diagnose and treat. This study evaluated the activity of the polyhomoarginines against Acanthamoeba castellanii. Acanthamoeba amoebicidal, amoebistatic, encystation and excystment assays were performed using protocols described in the literature. The activity of polyhomoarginines (PHAs) of different lengths (10 to 400 residues) was measured against the trophozoites and cysts of Acanthamoeba castellanii ATCC30868 in concentrations ranging from 0.93 µM to 15 µM. Data were represented as mean ± SE and analysed using one-way ANOVA. Overall, PHAs demonstrated good anti-acanthamoeba activity against both trophozoites and cysts. PHA 30 reduced the number of viable trophozoites by 99%, inhibited the formation of cysts by 96% and the emergence of trophozoites from cysts by 67% at 3.75 µM. PHA 10 was similarly active, but at a slightly higher concentration of 15 µM, reducing the numbers of viable trophozoites by 98%, inhibiting cyst formation by 84% and preventing the emergence of trophozoites from cysts by 99%. At their greatest anti-amoeba concentrations, PHA 10 gave only 8% haemolysis at 15 µM while PHA 30 gave <40 % haemolysis at 3.75 µM. Polyhomoarginine 10 showed excellent anti-amoebic activity against both forms of Acanthamoeba castellanii and was non-toxic at its most active concentrations. This implies that polyhomoarginines can be developed into a potential therapeutic agent for Acanthamoeba keratitis. However, there is a need to carry out further pre-clinical and then in vivo experiments in the AK animal model.

Selective Induction of Intrinsic Apoptosis in Retinoblastoma Cells by Novel Cationic Antimicrobial Dodecapeptides.

Host defense peptides represent an important component of innate immunity. In this work, we report the anticancer properties of a panel of hyper-charged wholly cationic antimicrobial dodecapeptides (CAPs) containing multiple canonical forms of lysine and arginine residues. These CAPs displayed excellent bactericidal activities against a broad range of pathogenic bacteria by dissipating the cytoplasmic membrane potential. Specifically, we identified two CAPs, named HC3 and HC5, that effectively killed a significant number of retinoblastoma (WERI-Rb1) cells (p ≤ 0.01). These two CAPs caused the shrinkage of WERI-Rb1 tumor spheroids (p ≤ 0.01), induced intrinsic apoptosis in WERI-Rb1 cells via activation of caspase 9 and caspase 3, cleaved the PARP protein, and triggered off the phosphorylation of p53 and γH2A.X. Combining HC3 or HC5 with the standard chemotherapeutic drug topotecan showed synergistic anti-cancer activities. Overall, these results suggest that HC3 and HC5 can be exploited as potential therapeutic agents in retinoblastoma as monotherapy or as adjunctive therapy to enhance the effectiveness of currently used treatment modalities.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Nanobiotics against antimicrobial resistance: harnessing the power of nanoscale materials and technologies.

Given the spasmodic increment in antimicrobial resistance (AMR), world is on the verge of "post-antibiotic era". It is anticipated that current SARS-CoV2 pandemic would worsen the situation in future, mainly due to the lack of new/next generation of antimicrobials. In this context, nanoscale materials with antimicrobial potential have a great promise to treat deadly pathogens. These functional materials are uniquely positioned to effectively interfere with the bacterial systems and augment biofilm penetration. Most importantly, the core substance, surface chemistry, shape, and size of nanomaterials define their efficacy while avoiding the development of AMR. Here, we review the mechanisms of AMR and emerging applications of nanoscale functional materials as an excellent substitute for conventional antibiotics. We discuss the potential, promises, challenges and prospects of nanobiotics to combat AMR.

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Recent advances in copper and copper-derived materials for antimicrobial resistance and infection control.

Antibacterial properties of copper have been known for ages. With the rise of antimicrobial resistance (AMR), hospital-acquired infections, and the current SARS-CoV-2 pandemic, copper and copper-derived materials are being widely researched for healthcare ranging from therapeutics to advanced wound dressing to medical devices. We cover current research that highlights the potential uses of metallic and ionic copper, copper alloys, copper nanostructures, and copper composites as antibacterial, antifungal, and antiviral agents, including those against the SARS-CoV-2 virus. The applications of copper-enabled engineered materials in medical devices, wound dressings, personal protective equipment, and self-cleaning surfaces are discussed. We emphasize the potential of copper and copper-derived materials in combating AMR and efficiently reducing infections in clinical settings.

Host Defense Peptides at the Ocular Surface: Roles in Health and Major Diseases, and Therapeutic Potentials.

Sight is arguably the most important sense in human. Being constantly exposed to the environmental stress, irritants and pathogens, the ocular surface - a specialized functional and anatomical unit composed of tear film, conjunctival and corneal epithelium, lacrimal glands, meibomian glands, and nasolacrimal drainage apparatus - serves as a crucial front-line defense of the eye. Host defense peptides (HDPs), also known as antimicrobial peptides, are evolutionarily conserved molecular components of innate immunity that are found in all classes of life. Since the first discovery of lysozyme in 1922, a wide range of HDPs have been identified at the ocular surface. In addition to their antimicrobial activity, HDPs are increasingly recognized for their wide array of biological functions, including anti-biofilm, immunomodulation, wound healing, and anti-cancer properties. In this review, we provide an updated review on: (1) spectrum and expression of HDPs at the ocular surface; (2) participation of HDPs in ocular surface diseases/conditions such as infectious keratitis, conjunctivitis, dry eye disease, keratoconus, allergic eye disease, rosacea keratitis, and post-ocular surgery; (3) HDPs that are currently in the development pipeline for treatment of ocular diseases and infections; and (4) future potential of HDP-based clinical pharmacotherapy for ocular diseases.

Structural insights into the interaction of antifungal peptides and ergosterol containing fungal membrane.

The treatment of invasive drug-resistant and potentially life-threatening fungal infections is limited to few therapeutic options that are usually associated with severe side effects. The development of new effective antimycotics with a more tolerable side effect profile is therefore of utmost clinical importance. Here, we used a combination of complementary in vitro assays and structural analytical methods to analyze the interaction of the de novo antimicrobial peptide VG16KRKP with the sterol moieties of biological cell membranes. We demonstrate that VG16KRKP disturbs the structural integrity of fungal membranes both invitro and in model membrane system containing ergosterol along with phosphatidylethanolamine lipid and exhibits broad-spectrum antifungal activity. As revealed by systematic structure-function analysis of mutated VG16KRKP analogs, a specific pattern of basic and hydrophobic amino acid side chains in the primary peptide sequence determines the selectivity of VG16KRKP for fungal specific membranes.

Integrated Analysis of Cancer Tissue and Vitreous Humor from Retinoblastoma Eyes Reveals Unique Tumor-Specific Metabolic and Cellular Pathways in Advanced and Non-Advanced Tumors.

Retinoblastoma (Rb) is a pediatric intraocular malignancy that is proposed to originate from maturing cone cell precursors in the developing retina. The molecular mechanisms underlying the biological and clinical behaviors are important to understand in order to improve the management of advanced-stage tumors. While the genetic causes of Rb are known, an integrated understanding of the gene expression and metabolic processes in tumors of human eyes is deficient. By integrating transcriptomic profiling from tumor tissues and metabolomics from tumorous eye vitreous humor samples (with healthy, age-matched pediatric retinae and vitreous samples as controls), we uncover unique functional associations between genes and metabolites. We found distinct gene expression patterns between clinically advanced and non-advanced Rb. Global metabolomic analysis of the vitreous humor of the same Rb eyes revealed distinctly altered metabolites, indicating how tumor metabolism has diverged from healthy pediatric retina. Several key enzymes that are related to cellular energy production, such as hexokinase 1, were found to be reduced in a manner corresponding to altered metabolites; notably, a reduction in pyruvate levels. Similarly, E2F2 was the most significantly elevated E2F family member in our cohort that is part of the cell cycle regulatory circuit. Ectopic expression of the wild-type RB1 gene in the Rb-null Y79 and WERI-Rb1 cells rescued hexokinase 1 expression, while E2F2 levels were repressed. In an additional set of Rb tumor samples and pediatric healthy controls, we further validated differences in the expression of HK1 and E2F2. Through an integrated omics analysis of the transcriptomics and metabolomics of Rb, we uncovered a significantly altered tumor-specific metabolic circuit that reduces its dependence on glycolytic pathways and is governed by Rb1 and HK1.

Rationalisation of Antifungal Properties of α-Helical Pore-Forming Peptide, Mastoparan B.

The high mortality associated with invasive fungal infections, narrow spectrum of available antifungals, and increasing evolution of antifungal resistance necessitate the development of alternative therapies. Host defense peptides are regarded as the first line of defense against microbial invasion in both vertebrates and invertebrates. In this work, we investigated the effectiveness of four naturally occurring pore-forming antimicrobial peptides (melittin, magainin 2, cecropin A, and mastoparan B) against a panel of clinically relevant pathogens, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida glabrata. We present data on the antifungal activities of the four pore-forming peptides, assessed with descriptive statistics, and their cytocompatibility with cultured human cells. Among the four peptides, mastoparan B (MB) displayed potent antifungal activity, whereas cecropin A was the least potent. We show that MB susceptibility of phylogenetically distant non-candida albicans can vary and be described by different intrinsic physicochemical parameters of pore-forming α-helical peptides. These findings have potential therapeutic implications for the design and development of safe antifungal peptide-based drugs.

Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights From Experimental and Molecular Dynamics Simulations Studies.

Background/Aim: Host defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and the secondary structure and underlying mechanism of action of CaD23. Methods: Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard and time-kill assays. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FIC<0.5), additive (FIC between 0.5-1.0), indifferent (FIC between >1.0 and ≤4), or antagonistic (FIC>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes. Circular dichroism (CD) spectroscopy was also performed to examine the secondary structures of CaD23. Results: CaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI = 0.60-0.69) and MRSA43300 (FICI = 0.56-0.60) but an indifferent effect against P. aeruginosa (FIC = 1.03-1.15). CaD23 (at 25 µg/ml; 2xMIC) completely killed S. aureus within 30 min. When used at sub-MIC concentration (3.1 µg/ml; 0.25xMIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23. The secondary structures of CaD23 observed in MD simulations were validated by CD spectroscopy. Conclusion: CaD23 is a novel alpha-helical, membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulations serves as a powerful tool in revealing the peptide secondary structure, dissecting the mechanism of action, and guiding the design and optimisation of HDPs.

Ocular Delivery of Predatory Bacteria with Cryomicroneedles Against Eye Infection.

The development of potent antibiotic alternatives with rapid bactericidal properties is of great importance in addressing the current antibiotic crisis. One representative example is the topical delivery of predatory bacteria to treat ocular bacterial infections. However, there is a lack of suitable methods for the delivery of predatory bacteria into ocular tissue. This work introduces cryomicroneedles (cryoMN) for the ocular delivery of predatory Bdellovibrio bacteriovorus (B. bacteriovorus) bacteria. The cryoMN patches are prepared by freezing B. bacteriovorus containing a cryoprotectant medium in a microneedle template. The viability of B. bacteriovorus in cryoMNs remains above 80% as found in long-term storage studies, and they successfully impede the growth of gram-negative bacteria in vitro or in a rodent eye infection model. The infection is significantly relieved by nearly six times through 2.5 days of treatment without substantial effects on the cornea thickness and morphology. This approach represents the safe and efficient delivery of new class of antimicrobial armamentarium to otherwise impermeable ocular surface and opens up new avenues for the treatment of ocular surface disorders.

Hybrid derivative of cathelicidin and human beta defensin-2 against Gram-positive bacteria: A novel approach for the treatment of bacterial keratitis.

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5-25.0 µg/ml (5.2-10.4 µM)] and S. epidermidis [MIC = 12.5 µg/ml (5.2 µM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 µg/ml (10.4-20.8 µM)]. CaD23 (at 25 µg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 µg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.