DUCNP@Mn-MOF/FOE as a Highly Selective and Bioavailable Drug Delivery System for Synergistic Combination Cancer Therapy.

Heterostructures comprising lanthanide-doped upconversion nanoparticles (DUCNPs) and metal-organic frameworks (MOFs) are emerging as promising nanosystems for integrating medical diagnosis and treatment. Here, the DUCNP@Mn-MOF nanocarrier was developed, which showed good efficiency for loading and delivering a cytotoxic antitumor agent (3-F-10-OH-evodiamine, FOE). The combined advantages of the pH-responsive and peroxidase-like properties of Mn-MOF and the unique optical features of DUCNPs granted the DUCNP@Mn-MOF/FOE system synergistic chemodynamic and chemotherapeutic effects. The DUCNP@Mn-MOF nanocarrier effectively overcame the intrinsic limitations of FOE, such as its unfavorable physicochemical properties and limited in vivo potency. This complexed nanosystem was responsive to the tumor microenvironment and showed excellent tumor targeting capability. Thus, DUCNP@Mn-MOF/FOE exhibited highly selective and bioavailable drug delivery properties and is promising for cancer therapy. In a mouse breast cancer model, DUCNP@Mn-MOF/FOE inhibited tumor growth without significant toxicity. Therefore, the proposed nanosystem represents a promising theragnostic platform for multimodal combination diagnosis and therapy of tumors.

Configuration-Specific Antibody for Bacterial Heptosylation: An Antiadhesion Therapeutic Strategy.

Alternative antibacterial therapies refractory to existing mechanisms of antibiotic resistance are urgently needed. One such attractive therapy is to inhibit bacterial adhesion and colonization. Ser O-heptosylation (Ser O-Hep) on autotransporters of Gram-negative bacteria is a novel glycosylation and has been proven to be essential for bacterial colonization. Herein, we chemically synthesized glycopeptides containing this atypical glycan structure and an absolute C6 configuration through the assembly of Ser O-Hep building blocks. Using glycopeptides as haptens, we generated first-in-class poly- and monoclonal antibodies, termed Anti-SerHep1a and Anti-SerHep1b, that stereoselectively recognize Ser O-heptosylation (d/l-glycero) with high specificity in vitro and in vivo. Importantly, these antibodies effectively blocked diffusely adhering Escherichia coli 2787 adhesion to HeLa cells and in mice in a dose- and Ser O-Hep-dependent manner. Together, these antibodies represent not only useful tools for the discovery of unknown serine O-heptosylated proteins bearing various C6 chiral centers but also a novel class of antiadhesion therapeutic agents for the treatment of bacterial infection.

Benzyl stapled modification and anticancer activity of antimicrobial peptide A4K14-Citropin 1.1.

A4K14-Citropin 1.1 (GLFAVIKKVASVIKGL-NH2) is a derived antimicrobial peptide (AMP) with a more stable α-helical structure at the C-terminal compared to prototype Citropin 1.1 which was obtained from glandular skin secretions of Australian freetail lizards. In a previous report, A4K14-Citropin 1.1 has been considered as an anti-cancer lead compound. However, linear peptides are difficult to maintain stable secondary structure, resulted in poor pharmacokinetic properties. In this study, we designed and synthesized a series of benzyl-stapled derivatives of A4K14-Citropin 1.1. And their physical and chemical properties, as well as biological activity, were both explored. The result showed that AC-CCSP-2-o and AC-CCSP-3-o exhibited a higher degree of helicity and greater anti-cancer activity compared with the prototype peptide. Besides, there was no significant difference in the hemolytic effect between the stapled peptides and the prototype peptide. AC-CCSP-2-o and AC-CCSP-3-o could serve as promising anti-cancer lead compounds for the novel anti-cancer drug development.

Stapled Helical Peptides Bearing Different Anchoring Residues.

A large proportion of protein-protein interactions (PPIs) occur between a short peptide and a globular protein domain; the peptides involved in surface interactions play important roles, and there is great promise for using peptide motifs to interfere with protein interactions. Peptide inhibitors show more promise in blocking large surface protein interactions compared to small molecule inhibitors. However, peptides have drawbacks including poor stability against circulating proteolytic enzymes and an intrinsic inability to penetrate cell membranes. Stapled helical peptides, by adopting a preformed, stable α-helical conformation, exhibit improved proteolytic stability and membrane permeability compared to linear bioactive peptides. In this review, we summarize the broad aspects of peptide stapling for chemistry, biophysics, and biological applications and specifically highlight the methodology by providing an inventory of different anchoring residues categorized into two natural amino acids, two nonnatural amino acids, or a combination of natural and nonnatural amino acids. Additional advantages of specific peptide stapling techniques, including but not limited to reversibility, bio-orthogonal reactivity, and photoisomerization, are also discussed individually. This review is expected to provide a broad reference for the rational design of druggable stapled peptides targeting therapeutic proteins, particularly those involved in PPIs, by considering the impact of anchoring residues, functional cross-linkers, physical staple length, staple components, and the staple motif on the biophysical properties of the peptides.

Design, synthesis, and anti-tumor activities of novel Brevinin-1BYa peptidomimetics.

Brevinin-1BYa is an amphibian skin-derived peptide that exhibits promising anti-microbial activity against gram-positive and -negative bacteria. However, the anti-tumor activity of Brevinin-1BYa remains unclear, and, more importantly, its therapeutic application is limited owing to its poor protease and reduction stability. In this study, a series of novel Brevinin-1BYa derivatives, including O-linked N-acetyl-glucosamine glyclopeptides and disulfide bond mimetics, were designed and synthesized. Additionally, their anti-tumor activity against human prostate cancer cell line C4-2B, human NSCLC cell line A549 (adenocarcinoma), and human hepatoma cells line HuH-7 was investigated. Among these, the thioether bridge substituted peptidomimetic Brevinin-1BYa-3 displayed improved reduction stability, more stable secondary structure, greater protease stability, and increased anti-tumor activity compared with the original peptide, rendering it a promising leading compound for drug development, particularly for applications against malignant tumors.

Design, synthesis, and biological evaluation of stapled ascaphin-8 peptides.

Ascaphin-8 is an α-helical anti-tumor and antimicrobial peptide containing 19 residues, which was isolated from norepinephrine-stimulated skin secretions of the North American tailed frog Ascaphus truei. To improve both its stability and biological activities, a series of hydrocarbon-stapled analogs of Ascaphin-8 were synthesized and investigated for their potential antiproliferative activities. The activity studies were evaluated using the CCK-8 method and colony formation assay on human cancer cell lines. Ascaphin-8-3, as the most active peptide, showed a stronger inhibition effect when compared with the parent peptide for the tested cell lines. In addition, the effect of Ascaphin-8-3 on inhibiting the metastatic capabilities of A549 cells was more powerful than that of the parent peptide. This peptide derivative showed potentiality for further optimization in antitumor drugs.

Total Synthesis and Antibacterial Study of Cyclohexapeptides Desotamide B, Wollamide B and Their Analogs.

As natural-product-derived antibiotics, desotamides A - D and wollamides exhibit growth inhibitory activity against Gram-posivite bacteria (IC50 0.6 - 7 µm) and are noncytotoxic to mammalian cells (IC50  > 30 µm). Herein we firstly report the total synthesis of above two cyclohexapeptides as well as a series of structural variants through solid phase peptide synthesis, of which 3 displayed a 2-fold increase of antibacterial activity when compared with the original peptide 1. This strategy may offer good improvements for the synthesis of other cyclic peptides.

A novel matrine derivate inhibits differentiated human hepatoma cells and hepatic cancer stem-like cells by suppressing PI3K/AKT signaling pathways.

Matrine is an alkaloid extracted from a Chinese herb Sophora flavescens Ait, which has shown chemopreventive potential against various cancers. In this study, we evaluated the anticancer efficacy of a novel derivative of matrine, (6aS, 10S, 11aR, 11bR, 11cS)-10- methylamino-dodecahydro- 3a,7a-diazabenzo (de) (MASM), against human hepatocellular carcinoma (HCC) cells and their corresponding sphere cells in vitro and in vivo. Human HCC cell lines (Hep3B and Huh7) were treated with MASM. Cell proliferation was assessed using CCK8 and colony assays; cell apoptosis and cell cycle distributions were examined with flow cytometry. The expression of cell markers and signaling molecules was detected using Western blot and qRT-PCR analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Hep3B and Huh7 cells. The in vivo antitumor efficacy of MASM was evaluated in Huh7 cell xenograft model in BALB/c nude mice, which were administered MASM (10 mg·kg-1·d-1, ig) for 3 weeks. After the treatment was completed, tumor were excised and weighed. A portion of tumor tissue was enzymatically dissociated to obtain a single cell suspension for the spheroid formation assays. MASM (2, 10, 20 µmol/L) dose-dependently inhibited the proliferation of HCC cells, and induced apoptosis, which correlated with a reduction in Bcl-2 expression and an increase in PARP cleavage. MASM also induced cell cycle arrest in G0/G1 phase, which was accompanied by increased p27 and decreased Cyclin D1 expression. Interestingly, MASM (2, 10, and 20 µmol/L) drastically reduced the EpCAM+/CD133+ cell numbers, suppressed the sphere formation, inhibited the expression of stem cell marker genes and promoted the expression of mature hepatocyte markers in the Hep3B and Huh7 spheroids. Additionally, MASM dose-dependently suppressed the PI3K/AKT/mTOR and AKT/GSK3ß/ß-catenin signaling pathways in Hep3B and Huh7 cells. In Huh7 xenograft bearing nude mice, MASM administration significantly inhibited Huh7 xenograft tumor growth and markedly reduced the number of surviving cancer stem-like cells in the tumors. MASM administration also reduced the expression of stem cell markers while increasing the expression of mature hepatocyte markers in the tumor tissues. The novel derivative of matrine, MASM, markedly suppresses HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the treatment of hepatocellular carcinoma.

The Matrine Derivate MASM Prolongs Survival, Attenuates Inflammation, and Reduces Organ Injury in Murine Established Lethal Sepsis.

BACKGROUND: MASM, a novel derivative of matrine, has inhibitory effects on activation of macrophages, dendritic cells, and hepatic stellate cells and binds to ribosomal protein S5 (RPS5). This study was designed to evaluate the effect of MASM on murine-established lethal sepsis and its mechanisms. METHODS: Mouse peritoneal macrophages and RAW264.7 cells that were infected with recombinant lentiviruses encoding shRPS5 were incubated with lipopolysaccharide (LPS) in the absence or presence of MASM in vitro. Endotoxemia induced by LPS injection and sepsis induced by cecal ligation and puncture was followed by MASM treatment. RESULTS: MASM markedly attenuated LPS-induced release and messenger RNA expression of tumor necrosis factor α, interleukin 6, and NO/inducible NO synthase in murine peritoneal macrophages and RAW264.7 cells. Meanwhile, MASM inhibited LPS-induced activation of nuclear factor κB and MAPK pathways. Consistently, RPS5 suppressed LPS-induced inflammatory responses and at least in part mediated the antiinflammatory effect of MASM in vitro. Remarkably, delayed administration of MASM could significantly reduce mortality in mouse sepsis models, which was associated with the reduction in the inflammatory response, the attenuation in multiple organ injury, and the enhanced bacterial clearance. CONCLUSIONS: MASM could be further explored for the treatments of sepsis, especially for administration later after the onset of sepsis.

Bioactive compound reveals a novel function for ribosomal protein S5 in hepatic stellate cell activation and hepatic fibrosis.

UNLABELLED: Liver fibrosis and its endstage, cirrhosis, represent a major public health problem worldwide. Activation of hepatic stellate cells (HSCs) is a central event in hepatic fibrosis. However, the proteins that control HSC activation are incompletely understood. Here we show that (6aS, 10S, 11aR, 11bR, 11cS)-10-methylamino-dodecahydro-3a, 7a-diaza-benzo [de]anthracene-8-thione (MASM) exhibits potent inhibitory activity against liver fibrosis in vitro and in vivo associated with the reduction of Akt phosphorylation. Furthermore, ribosomal protein S5 (RPS5) was identified as a direct target of MASM, which stabilized RPS5 in cultured HSCs and in the liver of experimental animals after dimethylnitrosamine (DMN) or bile duct ligation (BDL). Functional studies revealed that RPS5 could prevent HSC activation. RPS5 overexpression in HSCs resulted in Akt dephosphorylation at both Ser473 and Thr308, and led to subsequent dephosphorylation of GSK3ß or P70S6K. Progression of DMN- and BDL-induced hepatic fibrosis was aggravated by Rps5 knockdown and alleviated by RPS5 overexpression, which correlated with the modulation of Akt phosphorylation and HSC number in the fibrotic livers. Moreover, RPS5 was substantially reduced in the transdifferentiated HSCs, experimental fibrotic livers, and human cirrhosis samples. CONCLUSION: These results demonstrate that RPS5 is implicated in hepatic fibrogenesis and may represent a promising target for potential therapeutic intervention in liver fibrotic diseases.

Discover, Develop & Validate--Advance and Prospect of Tumor Biomarkers.

Tumor biomarkers can serve as tools for early detection, diagnosis, prognosis, therapeutic target, response predicting, therapy monitoring of tumors or can be used as surrogate endpoints. Their abilities to assist various clinical decisions make them indispensable tools for current oncotherapies. Therefore, in the past decades, significant effort has been put into finding sensitive, specific, noninvasive, inexpensive, and clinically feasible tumor biomarkers. This review will summarize the recent progress made in different kinds of tumor biomarkers, introduce a promising and versatile novel tumor biomarker called DEK oncoprotein, and lastly, discuss the prospects of tumor biomarkers.

Cadmium contamination of rice from various polluted areas of China and its potential risks to human health.

A total of 484 rice samples were collected from five polluted areas in China to investigate the cadmium (Cd) contamination of rice and its potential health risks. The mean Cd contents of analyzed rice samples obtained from different areas ranged from 0.149 to 0.189 mg·kg(-1). Cd concentrations in more than 18% of rice samples exceeded the maximum allowable Cd concentration, and the highest level of 41.1% was observed in samples from Hezhang, Guizhou, which was characterized by serious Cd pollution. Target hazard quotient (THQ) values of 1.5 to 7.8 from rice intake indicated a significant non-carcinogenic health risk for humans, particularly for highly exposed consumers. Children are more at risk than adults, as indicated by the higher THQs. Moreover, carcinogenic risks of Cd from rice intake for average and high consumers in the selected areas were two to three and four to eight greater, respectively, than the threshold value recommended by the International Commission on Radiological Protection.

Synthesis of and specific antibody generation for glycopeptides with arginine N-GlcNAcylation.

As a unique and unappreciated protein posttranslational modification, arginine N-glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N-glycosylation, we report the first general strategy for a rapid and cost-effective synthesis of glycopeptides carrying single or multiple arginine N-GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N-GlcNAcylated peptides or proteins in a sequence-independent manner.

Suggestion of GLYAT gene underlying variation of bone size and body lean mass as revealed by a bivariate genome-wide association study.

Bone and muscle, two major tissue types of musculoskeletal system, have strong genetic determination. Abnormality in bone and/or muscle may cause musculoskeletal diseases such as osteoporosis and sarcopenia. Bone size phenotypes (BSPs), such as hip bone size (HBS), appendicular bone size (ABS), are genetically correlated with body lean mass (mainly muscle mass). However, the specific genes shared by these phenotypes are largely unknown. In this study, we aimed to identify the specific genes with pleiotropic effects on BSPs and appendicular lean mass (ALM). We performed a bivariate genome-wide association study (GWAS) by analyzing ~690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) followed by a replication study in 2,286 unrelated US Caucasians (558 males and 1,728 females). We identified 14 interesting single nucleotide polymorphisms (SNPs) that may contribute to variation of both BSPs and ALM, with p values <10(-6) in discovery stage. Among them, the association of three SNPs (rs2507838, rs7116722, and rs11826261) in/near GLYAT (glycine-N-acyltransferase) gene was replicated in US Caucasians, with p values ranging from 1.89 × 10(-3) to 3.71 × 10(-4) for ALM-ABS, from 5.14 × 10(-3) to 1.11 × 10(-2) for ALM-HBS, respectively. Meta-analyses yielded stronger association signals for rs2507838, rs7116722, and rs11826261, with pooled p values of 1.68 × 10(-8), 7.94 × 10(-8), 6.80 × 10(-8) for ALB-ABS and 1.22 × 10(-4), 9.85 × 10(-5), 3.96 × 10(-4) for ALM-HBS, respectively. Haplotype allele ATA based on these three SNPs was also associated with ALM-HBS and ALM-ABS in both discovery and replication samples. Interestingly, GLYAT was previously found to be essential to glucose metabolism and energy metabolism, suggesting the gene's dual role in both bone development and muscle growth. Our findings, together with the prior biological evidence, suggest the importance of GLYAT gene in co-regulation of bone phenotypes and body lean mass.

Design, synthesis and antitumor activity of Ascaphin-8 derived stapled peptides based on halogen-sulfhydryl click chemical reactions.

Ascaphin-8 (GFKDLLKGAAKALVKTVLF-NH2), isolated from the norepinephrine-stimulated skin secretion of the North American-tailed frog Ascaphus truei, is a C-terminal α-helical antimicrobial peptide with potential antitumor activity. However, linear peptides are difficult to be applied directly as drugs because of their inherent defects, such as low hydrolytic enzyme tolerance and poor structural stability. In this study, we designed and synthesized a series of stapled peptides based on Ascaphin-8 via thiol-halogen click chemistry. Most of the stapled peptide derivatives showed enhanced antitumor activity. Among them, A8-2-o and A8-4-Dp had the most improved structural stability, stronger hydrolytic enzyme tolerance and highest biological activity. This research may provide a reference for the stapled modification of other similar natural antimicrobial peptides.

Highly reliable and efficient encoding systems for hexadecimal polypeptide-based data storage.

Polypeptides consisting of amino acid (AA) sequences are suitable for high-density information storage. However, the lack of suitable encoding systems, which accommodate the characteristics of polypeptide synthesis, storage and sequencing, impedes the application of polypeptides for large-scale digital data storage. To address this, two reliable and highly efficient encoding systems, i.e. RaptorQ-Arithmetic-Base64-Shuffle-RS (RABSR) and RaptorQ-Arithmetic-Huffman-Rotary-Shuffle-RS (RAHRSR) systems, are developed for polypeptide data storage. The two encoding systems realized the advantages of compressing data, correcting errors of AA chain loss, correcting errors within AA chains, eliminating homopolymers, and pseudo-randomized encrypting. The coding efficiency without arithmetic compression and error correction of audios, pictures and texts by the RABSR system was 3.20, 3.12 and 3.53 Bits/AA, respectively. While that using the RAHRSR system reached 4.89, 4.80 and 6.84 Bits/AA, respectively. When implemented with redundancy for error correction and arithmetic compression to reduce redundancy, the coding efficiency of audios, pictures and texts by the RABSR system was 4.43, 4.36 and 5.22 Bits/AA, respectively. This efficiency further increased to 7.24, 7.11 and 9.82 Bits/AA by the RAHRSR system, respectively. Therefore, the developed hexadecimal polypeptide-based systems may provide a new scenario for highly reliable and highly efficient data storage.

Cyclobutane-bearing restricted anchoring residues enabled geometry-specific hydrocarbon peptide stapling.

Stapled peptides are regarded as the promising next-generation therapeutics because of their improved secondary structure, membrane permeability and metabolic stability as compared with the prototype linear peptides. Usually, stapled peptides are obtained by a hydrocarbon stapling technique, anchoring from paired olefin-terminated unnatural amino acids and the consequent ring-closing metathesis (RCM). To investigate the adaptability of the rigid cyclobutane structure in RCM and expand the chemical diversity of hydrocarbon peptide stapling, we herein described the rational design and efficient synthesis of cyclobutane-based conformationally constrained amino acids, termed (E)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (E7) and (Z)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (Z7). All four combinations including E7-E7, E7-Z7, Z7-Z7 and Z7-E7 were proven to be applicable in RCM-mediated peptide stapling to afford the corresponding geometry-specific stapled peptides. With the aid of the combined quantum and molecular mechanics, the E7-E7 combination was proven to be optimal in both the RCM reaction and helical stabilization. With the spike protein of SARS-CoV-2 as the target, a series of cyclobutane-bearing stapled peptides were obtained. Among them, E7-E7 geometry-specific stapled peptides indeed exhibit higher α-helicity and thus stronger biological activity than canonical hydrocarbon stapled peptides. We believe that this methodology possesses great potential to expand the scope of the existing peptide stapling strategy. These cyclobutane-bearing restricted anchoring residues served as effective supplements for the existing olefin-terminated unnatural amino acids and the resultant geometry-specific hydrocarbon peptide stapling provided more potential for peptide therapeutics.

Methionine restriction promotes cGAS activation and chromatin untethering through demethylation to enhance antitumor immunity.

Cyclic GMP-AMP synthase (cGAS) is the major sensor for cytosolic DNA and activates type I interferon signaling and plays an essential role in antitumor immunity. However, it remains unclear whether the cGAS-mediated antitumor activity is affected by nutrient status. Here, our study reports that methionine deprivation enhances cGAS activity by blocking its methylation, which is catalyzed by methyltransferase SUV39H1. We further show that methylation enhances the chromatin sequestration of cGAS in a UHRF1-dependent manner. Blocking cGAS methylation enhances cGAS-mediated antitumor immunity and suppresses colorectal tumorigenesis. Clinically, cGAS methylation in human cancers correlates with poor prognosis. Thus, our results indicate that nutrient stress promotes cGAS activation via reversible methylation, and suggest a potential therapeutic strategy for targeting cGAS methylation in cancer treatment.

Design of stapled peptide-based PROTACs for MDM2/MDMX atypical degradation and tumor suppression.

Rationale: Although stapled peptides offer a powerful solution to overcome the susceptibility of linear peptides to proteolytic degradation and improve their ability to cross membranes, an efficient and durable disease treatment strategy has not yet been developed due to the inevitable elimination of peptide inhibitors and rapid accumulation of target proteins. Methods: Herein we developed stapled peptide-based proteolysis-targeting chimeras (SP-PROTACs), that simultaneously exhibited improved cellular uptake and proteolytic stability attributed to the stapled peptides, and efficient target protein degradation promoted by the PROTACs. Based on the PMI peptide with dual specificity for both MDM2 and MDMX, a series of SP-PROTACs were designed. Results: Among them, the optimized SPMI-HIF2-1 exhibited similar binding affinity with MDM2 and MDMX but obviously higher helical contents, improved proteolytic stability, better cellular permeability, and a better pharmacokinetic profile compared with its linear counterpart. Importantly, SPMI-HIF2-1 could effectively kill cancer cells and inhibit tumor progression in subcutaneous and orthotopic colorectal cancer xenograft models through simultaneously promoting the atypical degradation of both MDM2 and MDMX and durable p53 activation. An FP-based binding assay and structural modeling analysis of the ternary complex suggested that SPMI-HIF2-1 simultaneously bound with the target protein and E3 ligase. Conclusion: Our findings not only provide a new class of anticancer drug candidates, but also bridge the gap and reduce the physical distance between peptides and PROTACs.

On-column refolding purification of DT389-hIL13 recombinant protein expressed in Escherichia coli.

Protein refolding is a bottleneck in the production of therapeutic proteins from inclusion bodies. In recent years, several studies have described on-column refolding of recombinant proteins. DT389-hIL13 is a recombinant protein that targets the glioma. In our study, the recombinant protein DT389-hIL13 was expressed in Escherichia coli (E. coli). The isolated inclusion bodies were refolded using size exclusion chromatography (SEC) and further purified using anion exchange chromatography. Three different methods of SEC on-column refolding were studied. In vitro tests on U251 cells showed that the recombinant protein could effectively inhibit the proliferation of U251 cells, especially the protein refolded by urea and pH gradient method. The half-maximal inhibitory concentration (IC50) of 0.887 nM was achieved with this new method, unlike an IC50 of 11.4 nM achieved in the non-gradient method.