Enhancing the stability and therapeutic potential of the antimicrobial peptide Feleucin-K3 against Multidrug-Resistant a. Baumannii through rational utilization of a D-amino acid substitution strategy.

Antimicrobial peptides (AMPs), which have a low probability of developing resistance, are considered the most promising antimicrobial agents for combating antibiotic resistance. Feleucin-K3 is an amphiphilic cationic AMP that exhibits broad-spectrum antimicrobial activity. In our previous research, the first phenylalanine residue was identified as the critical position affecting its biological activity. Here, a series of Feleucin-K3 analogs containing hydrophobic D-amino acids were developed, leveraging the low sensitivity of proteases to unnatural amino acids and the regulatory effect of hydrophobicity on antimicrobial activity. Among them, K-1dF, which replaced the phenylalanine of Feleucin-K3 with its enantiomer (D-phenylalanine), exhibited potent antimicrobial activity with a therapeutic index of 46.97 and MICs between 4 to 8 µg/ml against both sensitive and multidrug-resistant Acinetobacter baumannii. The introduction of D-phenylalanine increased the salt tolerance and serum stability of Feleucin-K3. Moreover, K-1dF displayed a rapid bactericidal effect, a low propensity to develop resistance, and a synergistic effect when combined with antibiotics. More importantly, it exhibited considerable or superior efficacy to imipenem against pneumonia and skin abscess infection. In brief, the K-1dF obtained by simple and effective modification strategy has emerged as a promising candidate antimicrobial agent for tackling multidrug-resistant Acinetobacter baumannii infections.

Novel Peptide DR3penA as a Low-Toxicity Antirenal Fibrosis Agent by Suppressing the TGF-ß1/miR-212-5p/Low-Density Lipoprotein Receptor Class a Domain Containing 4/Smad Axis.

Renal fibrosis is a complex pathological process that contributes to the development of chronic kidney disease due to various risk factors. Conservative treatment to curb progression without dialysis or renal transplantation is widely applicable, but its effectiveness is limited. Here, the inhibitory effect of the novel peptide DR3penA (DHα-(4-pentenyl)-AlaNPQIR-NH2), which was developed by our group, on renal fibrosis was assessed in cells and mice with established fibrosis and fibrosis triggered by transforming growth factor-ß1 (TGF-ß1), unilateral ureteral obstruction, and repeated low-dose cisplatin. DR3penA preserved renal function and ameliorated renal fibrosis at a dose approximately 100 times lower than that of captopril, which is currently used in the clinic. DR3penA also significantly reduced existing fibrosis and showed similar efficacy after subcutaneous or intraperitoneal injection. Mechanistically, DR3penA repressed TGF-ß1 signaling via miR-212-5p targeting of low-density lipoprotein receptor class a domain containing 4, which interacts with Smad2/3. In addition to having good pharmacological effects, DR3penA could preferentially target injured kidneys and exhibited low toxicity in acute and chronic toxicity experiments. These results unveil the advantages of DR3penA regarding efficacy and toxicity, making it a potential candidate compound for renal fibrosis therapy.

Novel antimicrobial peptides modified with fluorinated sulfono-γ-AA having high stability and targeting multidrug-resistant bacteria infections.

The emergence and increasing prevalence of multidrug-resistant (MDR) bacteria have posed an urgent demand for novel antibacterial drugs. Currently, antimicrobial peptides (AMPs), potential novel antimicrobial agents with rare antimicrobial resistance, represent an available strategy to combat MDR bacterial infections but suffer the limitation of protease degradation. In this study, we developed a highly effective method for optimizing the stability of AMPs by introducing fluorinated sulfono-γ-AApeptides, and successfully synthesized novel Feleucin-K3-analogs. The results demonstrated that the incorporation of fluorinated sulfono-γ-AA into Feleucin-K3 effectively improved stability and afforded optimal peptides, such as CF3-K11, which exhibited 8-9 times longer half-lives than Feleucin-K3. Moreover, CF3-K11 displayed potent antimicrobial activity against clinically isolated Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), excellent biosafety, low resistance propensity, and possessed powerful antimicrobial efficacy for both local skin infection and pneumonia infection. The optimal CF3-K11 exhibited strong therapeutic potential and offered a superior approach for treating MDR bacterial infections.

The Peptide DHα-(4-pentenyl)-ANPQIR-NH2 Exhibits Antifibrotic Activity in Multiple Pulmonary Fibrosis Models Induced by Particulate and Soluble Chemical Fibrogenic Agents.

Interstitial lung diseases (ILDs) are a group of restrictive lung diseases characterized by interstitial inflammation and pulmonary fibrosis. The incidence of ILDs associated with exposure to multiple hazards such as inhaled particles, fibers, and ingested soluble chemicals is increasing yearly, and there are no ideal drugs currently available. Our previous research showed that the novel and low-toxicity peptide DHα-(4-pentenyl)-ANPQIR-NH2 (DR3penA) had a strong antifibrotic effect on a bleomycin-induced murine model. Based on the druggability of DR3penA, we sought to investigate its effects on respirable particulate silicon dioxide (SiO2)- and soluble chemical paraquat (PQ)-induced pulmonary fibrosis in this study by using western blot, quantitative reverse-transcription polymerase chain reaction (RT-qPCR), immunofluorescence, H&E and Masson staining, immunohistochemistry, and serum biochemical assays. The results showed that DR3penA alleviated the extent of fibrosis by inhibiting the expression of fibronectin and collagen I and suppressed oxidative stress and epithelial-mesenchymal transition (EMT) in vitro and in vivo. Further study revealed that DR3penA may mitigate pulmonary fibrosis by negatively regulating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway and mitogen-activated protein kinase (MAPK) pathway. Unexpectedly, through the conversion of drug bioavailability under different routes of administration, DR3penA exerted antifibrotic effects equivalent to those of the positive control drug pirfenidone (PFD) at lower doses. In summary, DR3penA may be a promising lead compound for various fibrotic ILDs. SIGNIFICANCE STATEMENT: Our study verified that DHα-(4-pentenyl)-ANPQIR-NH2 (DR3penA) exhibited positive antifibrotic activity in pulmonary fibrosis induced by silicon dioxide (SiO2) particles and soluble chemical paraquat (PQ) and demonstrated a low-dose advantage compared to the small-molecule drug pirfenidone (PFD). The peptide DR3penA can be further developed for the treatment of multiple fibrotic lung diseases.

The novel peptide DR4penA attenuates the bleomycin- and paraquat-induced pulmonary fibrosis by suppressing the TGF-ß/Smad signaling pathway.

Pulmonary fibrosis (PF), which is caused by continuous alveolar epithelial cell injury and abnormal repair, is referred to as a difficult disease of the lung system by the World Health Organization due to its rapid progression, poor prognosis, and high mortality rate. However, there is still a lack of ideal therapeutic strategies. The peptide DR8 (DHNNPQIR-NH2 ), which is derived from rapeseed, exerted antifibrotic activity in the lung, liver, and kidney in our previous studies. By studying the structure-activity relationship and rational design, we introduced an unnatural hydrophobic amino acid (α-(4-pentenyl)-Ala) into DR8 and screened the novel peptide DR4penA (DHNα-(4-pentenyl)-APQIR-NH2 ), which had higher anti-PF activity, higher antioxidant activity and a longer half-life than DR8. Notably, DR4penA attenuated bleomycin- and paraquat-induced PF, and the anti-PF activity of DR4penA was equivalent to that of pirfenidone. Additionally, DR4penA suppressed the TGF-ß/Smad pathway in TGF-ß1-induced A549 cells and paraquat-induced rats. This study demonstrates that the novel peptide DR4penA is a potential candidate compound for PF therapy, and its antifibrotic activity in different preclinical models of PF provides a theoretical basis for further study.

The CaMKII Inhibitory Peptide AIP Alleviates Renal Fibrosis Through the TGF-ß/Smad and RAF/ERK Pathways.

Renal fibrosis is characterized by the excessive deposition of extracellular matrix that destroys and replaces the functional renal parenchyma, ultimately leading to organ failure. It is a common pathway by which chronic kidney disease can develop into end-stage renal disease, which has high global morbidity and mortality, and there are currently no good therapeutic agents available. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been indicated to be closely related to the occurrence of renal fibrosis, and its specific inhibitory peptide, autocamtide-2-related inhibitory peptide (AIP), was shown to directly bind the active site of CaMKII. In this study, we examined the effect of AIP on the progression of renal fibrosis and its possible mechanism. The results showed that AIP could inhibit the expression of the fibrosis markers fibronectin, collagen I, matrix metalloproteinase 2, and α-smooth muscle actin in vivo and in vitro. Further analysis revealed that AIP could inhibit the expression of various epithelial-to-mesenchymal transformation-related markers, such as vimentin and Snail 1, in vivo and in vitro. Mechanistically, AIP could significantly inhibit the activation of CaMKII, Smad 2, Raf, and extracellular regulated protein kinases (ERK) in vitro and in vivo and reduce the expression of transforming growth factor-ß (TGF-ß) in vivo. These results suggested that AIP could alleviate renal fibrosis by inhibiting CaMKII and blocking activation of the TGF-ß/Smad2 and RAF/ERK pathways. Our study provides a possible drug candidate and demonstrates that CaMKII is a potential pharmacological target for the treatment of renal fibrosis. SIGNIFICANCE STATEMENT: We have demonstrated that AIP significantly attenuated transforming growth factor-ß-1-induced fibrogenesis and ameliorated unilateral ureteral obstruction-induced renal fibrosis through the CaMKII/TGF-ß/Smad and CaMKII/RAF/ERK signaling pathways in vitro and in vivo. Our study provides a possible drug candidate and demonstrates that CaMKII can be a potential pharmacological target for the treatment of renal fibrosis.

A novel and low-toxic peptide DR3penA alleviates pulmonary fibrosis by regulating the MAPK/miR-23b-5p/AQP5 signaling axis.

Pulmonary fibrosis (PF) is a pathological change caused by repeated injuries and repair dysfunction of the alveolar epithelium. Our previous study revealed that the residues Asn3 and Asn4 of peptide DR8 (DHNNPQIR-NH2) could be modified to improve stability and antifibrotic activity, and the unnatural hydrophobic amino acids α-(4-pentenyl)-Ala and d-Ala were considered in this study. DR3penA (DHα-(4-pentenyl)-ANPQIR-NH2) was verified to have a longer half-life in serum and to significantly inhibit oxidative damage, epithelial-mesenchymal transition (EMT) and fibrogenesis in vitro and in vivo. Moreover, DR3penA has a dosage advantage over pirfenidone through the conversion of drug bioavailability under different routes of administration. A mechanistic study revealed that DR3penA increased the expression of aquaporin 5 (AQP5) by inhibiting the upregulation of miR-23b-5p and the mitogen-activated protein kinase (MAPK) pathway, indicating that DR3penA may alleviate PF by regulating MAPK/miR-23b-5p/AQP5. Safety evaluation showed that DR3penA is a peptide drug without obvious toxicity or acute side effects and has significantly improved safety compared to DR8. Thus, our findings suggest that DR3penA, as a novel and low-toxic peptide, has the potential to be a leading compound for PF therapy, which provides a foundation for the development of peptide drugs for fibrosis-related diseases.

DR7dA, a Novel Antioxidant Peptide Analog, Demonstrates Antifibrotic Activity in Pulmonary Fibrosis In Vivo and In Vitro.

Pulmonary fibrosis (PF), which is characterized by enhanced extracellular matrix (ECM) deposition, is an interstitial lung disease that lacks an ideal clinical treatment strategy. It has an extremely poor prognosis, with an average survival of 3-5 years after diagnosis. Our previous studies have shown that the antioxidant peptide DR8 (DHNNPQIR-NH2), which is extracted and purified from rapeseed, can alleviate PF and renal fibrosis. However, natural peptides are easily degraded by proteases in vivo, which limits their potency. We have since synthesized a series of DR8 analogs based on amino acid scanning substitution. DR7dA [DHNNPQ (D-alanine) R-NH2] is an analog of DR8 in which L-isoleucine (L-Ile) is replaced with D-alanine (D-Ala), and its half-life is better than that of DR8. In the current study, we verified that DR7dA ameliorated tumor growth factor (TGF)-ß1-induced fibrogenesis and bleomycin-induced PF. The results indicated that DR7dA reduced the protein and mRNA levels of TGF-ß1 target genes in TGF-ß1-induced models. Surprisingly, DR7dA blocked fibrosis in a lower concentration range than DR8 in cells. In addition, DR7dA ameliorated tissue pathologic changes and ECM accumulation in mice. BLM caused severe oxidative damage, but administration of DR7dA reduced oxidative stress and restored antioxidant defense. Mechanistic studies suggested that DR7dA inhibits ERK, P38, and JNK phosphorylation in vivo and in vitro All results indicated that DR7dA attenuated PF by inhibiting ECM deposition and oxidative stress via blockade of the mitogen-activated protein kinase (MAPK) pathway. Hence, compared with its parent peptide, DR7dA has higher druggability and could be a candidate compound for PF treatment in the future. SIGNIFICANCE STATEMENT: In order to improve druggability of DR8, we investigated the structure-activity relationship of it and replaced the L-isoleucine with D-alanine. We found that the stability and antifibrotic activity of DR7dA were significantly improved than DR8, as well as DR7dA significantly attenuated tumor growth factor (TGF)-ß1-induced fibrogenesis and ameliorated bleomycin-induced fibrosis by inhibiting extracellular matrix deposition and oxidative stress via blockade of the MAPK pathway, suggesting DR7dA may be a promising candidate compound for the treatment of PF.

Peptide DR8 analogs alleviate pulmonary fibrosis via suppressing TGF-ß1 mediated epithelial-mesenchymal transition and ERK1/2 pathway in vivo and in vitro.

Pulmonary fibrosis is a chronic progressive lung disease that lacks effective treatments in clinic. It is characterized by repair disorder of epithelial cells, formation of fibroblast foci as well as destruction of alveolar structure. Previously we first determined that parent peptide DR8 (DHNNPQIR-NH2) has anti-fibrotic activity in bleomycin-induced mice. In order to further improve the druggability of DR8, including anti-fibrotic activity, stability and security, the structure-activity relationship was investigated using a series of D-amino acid and alanine scanning analogs of DR8. The results indicated that peptides DR8-3D and DR8-8A exhibited potent anti-fibrotic activity and better stability. Further mechanism research revealed that DR8-3D and DR8-8A ameliorated lung fibrosis by inhibiting TGF-ß1 mediated epithelial-mesenchymal transition process and ERK1/2 signaling pathway in vitro and in vivo. Moreover, we found that anti-fibrotic activity of DR8 was closely related to the residues aspartic acid (Asp)1, histidine (His)2, proline (Pro)5 and glutamine (Gln)6, which suggested that the position of residues asparagine (Asn)3, asparagine (Asn)4, isoleucine (Ile)7 and arginine (Arg)8 could be further modified to optimized its anti-fibrotic effect. Therefore, we consider that DR8-3D and DR8-8A not only could be used as a potential leading compound for the treatment of bleomycin-induced lung fibrosis but also laid a foundation for the development of new anti-fibrotic drugs.

The antimicrobial peptide YD attenuates inflammation via miR-155 targeting CASP12 during liver fibrosis.

The antimicrobial peptide APKGVQGPNG (named YD), a natural peptide originating from Bacillus amyloliquefaciens CBSYD1, exhibited excellent antibacterial and antioxidant properties in vitro. These characteristics are closely related to inflammatory responses which is the central trigger for liver fibrosis. However, the therapeutic effects of YD against hepatic fibrosis and the underlying mechanisms are rarely studied. In this study, we show that YD improved liver function and inhibited the progression of liver fibrosis by measuring the serum transaminase activity and the expression of α-smooth muscle actin and collagen I in carbon tetrachloride-induced mice. Then we found that YD inhibited the level of miR-155, which plays an important role in inflammation and liver fibrosis. Bioinformatics analysis and luciferase reporter assay indicate that Casp12 is a new target of miR-155. We demonstrate that YD significantly decreases the contents of inflammatory cytokines and suppresses the NF-κB signaling pathway. Further studies show that transfection of the miR-155 mimic in RAW264.7 cells partially reversed the YD-mediated CASP12 upregulation, the downregulated levels of inflammatory cytokines, and the inactivation of the NF-κB pathways. Collectively, our study indicates that YD reduces inflammation through the miR-155-Casp12-NF-κB axis during liver fibrosis and provides a promising therapeutic candidate for hepatic fibrosis.

Peptide DR8 suppresses epithelial-to-mesenchymal transition via the TGF-ß/MAPK signaling pathway in renal fibrosis.

AIMS: Renal fibrosis is a progressive disease that leads to renal dysfunction and end-stage renal failure, and there is currently no specific treatment. Our previous study showed that the 8-residue peptide DR8 (DHNNPQIR) exhibits potent antioxidant and antifibrotic properties, and accumulating evidence suggests that oxidative stress contributes greatly to fibrosis. The effects and mechanisms of DR8 on renal fibrosis remain unknown. MATERIALS AND METHODS: The effects of DR8 were assessed in a unilateral ureteral obstruction mouse model that received a daily, single-dose subcutaneous injection of 500 µg/kg DR8 for 14 days and in cultured cells (HK-2 and NIH-3T3 cells) treated with 5 ng/mL TGF-ß1 and 80 µM DR8. Western blotting, immunohistochemical staining, real-time qPCR and other tools were conducted to study the molecular mechanisms underlying antifibrotic effects. KEY FINDINGS: DR8 improved renal function and reduced injury and extracellular matrix (ECM) deposition. Inflammation and oxidative stress were alleviated by DR8 in vivo. DR8 also inhibited the activation of fibroblasts and ECM deposition in HK-2 and NIH-3T3 cells induced by TGF-ß1. In addition, epithelial-to-mesenchymal transition (EMT) was inhibited by DR8 both in vivo and in vitro. Mechanistic studies supported that DR8 inhibited ERK and p38 mitogen-activated protein kinase (MAPK) activation. These results indicate that DR8 attenuates renal fibrosis via suppression of EMT by antagonizing the MAPK pathway. SIGNIFICANCE: We provide mechanistic details for a potential therapeutic agent and establish a foundation for peptide therapeutics.

Protective effect of peptide DR8 on bleomycin-induced pulmonary fibrosis by regulating the TGF-ß/MAPK signaling pathway and oxidative stress.

Pulmonary fibrosis (PF) is a fatal and irreversible lung disease that eventually causes respiratory failure, lung dysfunction and death. The peptide DHNNPQIR-NH2 (DR8) has been reported to possess potent antioxidant activity, and an imbalance of oxidation/antioxidation is a crucial mechanism that causes PF. Here, we studied the ability of DR8 to improve PF and further explored the pathway in which DR8 plays a critical role. We found that after prophylactic or therapeutic treatment with DR8, fibrosis-associated indices, including marker proteins, proinflammatory cytokines and profibrogenic cytokines, were significantly downregulated. Importantly, DR8 could reduce bleomycin-induced pathological changes and collagen deposition, especially collagen I content. Furthermore, DR8 prominently upregulated nonenzymatic antioxidants and enzymatic antioxidants. Consistent with the in vivo results, we observed that DR8 significantly inhibited the proliferation and reactive oxygen species (ROS) generation of A549 cells and NIH3T3 cells stimulated with transforming growth factor-ß1 (TGF-ß1), as well as decreased NADPH oxidase 4 (NOX4) levels under the same conditions. Moreover, DR8 reversed the TGF-ß1-induced upregulation of phosphorylated ERK1/2 and p38 MAPK in cells and the bleomycin-induced upregulation of these indices in mice. Our results indicate that DR8 could prevent and treat PF by reducing oxidative damage and suppressing the TGF-ß/MAPK pathway. Because of the high efficiency and low toxicity of DR8, we consider that DR8 could be a candidate drug for PF, and our studies establish a foundation for the development of a lead compound to be used as a therapy for fibrosis-related diseases.

Rapeseed protein-derived antioxidant peptide RAP alleviates renal fibrosis through MAPK/NF-κB signaling pathways in diabetic nephropathy.

INTRODUCTION: Kidney fibrosis is the main pathologic change in diabetic nephropathy (DN), which is the major cause of end-stage renal disease. Current therapeutic strategies slow down but cannot reverse the progression of renal dysfunction in DN. Plant-derived bioactive peptides in foodstuffs are widely used in many fields because of their potential pharmaceutical and nutraceutical benefits. However, this type of peptide has not yet been studied in renal fibrosis of DN. Previous studies have indicated that the peptide YWDHNNPQIR (named RAP), a natural peptide derived from rapeseed protein, has an antioxidative stress effect. The oxidative stress is believed to be associated with DN. The aim of this study was to evaluate the pharmacologic effects of RAP against renal fibrosis of DN and high glucose (HG)-induced mesangial dysfunction. MATERIALS AND METHODS: Diabetes was induced by streptozotocin and high-fat diet in C57BL/6 mice and these mice were treated by subcutaneous injection of different doses of RAP (0.1 mg/kg and 0.5 mg/kg, every other day) or PBS for 12 weeks. Later, functional and histopathologic analyses were performed. Parallel experiments verifying the molecular mechanism by which RAP alleviates DN were carried out in HG-induced mesangial cells (MCs). RESULTS: RAP improved the renal function indices, including 24-h albuminuria, triglyceride, serum creatinine, and blood urea nitrogen levels, but did not lower blood glucose levels in DN mice. RAP also simultaneously attenuated extracellular matrix accumulation in DN mice and HG-induced MCs. Furthermore, RAP reduced HG-induced cell proliferation, but it showed no toxicity in MCs. Additionally, RAP inhibited the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways. CONCLUSION: RAP can attenuate fibrosis in vivo and in vitro by antagonizing the MAPK and NF-κB pathways.