Thyclotides, tetrahydrofuran-modified peptide nucleic acids that efficiently penetrate cells and inhibit microRNA-21.

Peptide nucleic acids (PNAs) are promising therapeutic molecules for gene modulation; however, they suffer from poor cell uptake. Delivery of PNAs into cells requires conjugation of the PNA to another large molecule, typically a cell-penetrating peptide or nanoparticle. In this study, we describe a new PNA-based molecule with cyclic tetrahydrofuran (THF) backbone modifications that in some cases considerably improve cell uptake. We refer to these THF-PNA oligomers as thyclotides. With THF groups at every position of the oligomer, the cell uptake of thyclotides targeted to miR-21 is enhanced compared with the corresponding unmodified PNA based on an aminoethylglycine backbone. An optimized thyclotide can efficiently enter cells without the use of cell-penetrating peptides, bind miR-21, its designated microRNA target, decrease expression of miR-21 and increase expression of three downstream targets (PTEN, Cdc25a and KRIT1). Using a plasmid with the PTEN-3'UTR coupled with luciferase, we further confirmed that a miR-21-targeted thyclotide prevents miR-21 from binding to its target RNA. Additionally, the thyclotide shows no cytotoxicity when administered at 200 times its active concentration. We propose that thyclotides be further explored as therapeutic candidates to modulate miRNA levels.

Conformational constraints of cyclopentane peptide nucleic acids facilitate tunable binding to DNA.

We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (Tm) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the Tm of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a Tm around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9-DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.

Allosteric modulation of ß-cell M3 muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice.

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic ß cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured ß cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by ß-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in ß cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.

Proteomics analysis reveals a potential new target protein for the lipid-lowering effect of Berberine8998.

Berberine8998 is a newly synthesized berberine derivative with better lipid-lowering activity and improved absorption. The objective of this study was to investigate the effects of berberine8998 on serum cholesterol and lipid levels in vivo and to examine the mechanisms involved. Hamsters on high-fat diet (HFD) were administered berberine or berberine8998 (50 mg·kg-1·d-1, ig) for 3 weeks. Berberine8998 administration significantly lowered the total cholesterol, triglycerides and LDL-C levels in HFD hamsters. Bioinformatics revealed that berberine and berberine8998 shared similar metabolic pathways and fatty acid metabolism was the predominant pathway. Western blot validation results showed that peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and long-chain fatty acid-CoA ligase 1 (ACSL1), two proteins involved in fatty acid metabolism, were expressed differently in the berberine8998 group than in the untreated group and the berberine treatment group. Biochemistry results showed that berberine8998 significantly lowered the non-esterified fatty acid (NEFA) levels, which may lead to a reduction in TG levels in the berberine8998 treatment group and the differences observed in proteomics analyses. Pharmacokinetic analysis conducted in rats. After administration of berberine or berberine8998 (50 mg/kg, ig), berberine8998 exhibited a remarkably improved absorption with increasing bioavailability by 6.7 times compared with berberine. These findings suggest that berberine8998 lowers cholesterol and lipid levels via different mechanisms than berberine, and its improved absorption makes it a promising therapeutic candidate for the treatment of hypercholesterolemia and obesity.

Atorvastatin attenuates homocysteine-induced migration of smooth muscle cells through mevalonate pathway involving reactive oxygen species and p38 MAPK.

Statins have been reported to have an antioxidant effect against homocysteine (Hcy)-induced endothelial dysfunction. It is unknown whether they have the same effect against migration of vascular smooth muscle cells (VSMCs) induced by Hcy. In this study, it was investigated whether and how atorvastatin could inhibit the Hcy-induced migration in cultured VSMCs and revealed the possible redox mechanism. VSMCs were isolated from the thoracic aortas of Sprague-Dawley rats. The migration of VSMCs was examined using a transwell technique and cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay. Reactive oxygen species (ROS) were measured using the fluoroprobe 2'7'-dichlorodihydrofluorescein diacetate. The activity of NADPH oxidase was assessed by lucigenin enhanced chemiluminescence. Expressions of Nox1 mRNA and p-p38MAPK protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The results showed that atorvastatin inhibited the migration of VSMCs induced by Hcy, which was reversed by the mevalonate. In addition, pretreatment with the NADPH oxidase inhibitor DPI, the free radical scavenger NAC and the p38 MAPK inhibitor SB203580 blocked Hcy-induced VSMCs migration. Furthermore, atorvastatin suppressed Hcy-induced activation of NADPH oxidase and ROS, attenuated Hcy-induced overexpression of Nox1mRNA. Similar effects occurred with VSMCs transfected with Nox1 siRNA. Moreover, atorvastatin other than DPI, NAC, SB203580 and Nox1 siRNA transfection blocked Hcy-induced p38 MAPK phosphorylation, which was also reversed by the mevalonate. The data demonstrates that atorvastatin inhibits Hcy-induced VSMCs migration in a mevalonate pathway. Furthermore, a part of the biological effect of atorvastatin involves a decrease in the levels of Nox1-dependent ROS generation and p38 MAPK activation.

Gold-catalyzed diastereoselective cycloisomerization of alkylidene-cyclopropane-bearing 1,6-diynes.

An unprecedented gold-catalyzed diastereoselective cycloisomerization of 1,6-diynes bearing an alkylidene cyclopropane moiety has been developed. This methodology enables rapid access to a variety of 1,2-trimethylenenorbornanes, which are important building blocks in the preparations of abiotic and sesquiterpene core structures.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Exploration of the Potential Biomarker FNDC5 for Discriminating Heart Failure in Patients with Coronary Atherosclerosis.

Coronary atherosclerosis leading to ischemic artery disease is one of the etiological factors to develop heart failure (HF). This study aimed to investigate potential biomarkers for discriminating HF in atherosclerotic patients. This study included 40 consecutive atherosclerotic patients who underwent angiography. Concentrations of B-type natriuretic peptide (BNP), fibronectin type III domain containing 5 (FNDC5), and Phosphodiesterase 9A (PDE9A) were measured in 20 atherosclerotic patients with HF symptoms/signs and 20 without HF symptoms/signs. Circulating BNP levels were elevated, while FNDC5 levels were reduced in atherosclerotic patients with HF symptoms/signs compared to those without HF symptoms/signs. Pearson correlation analysis showed a significant correlation between FNDC5 and BNP. Receiver Operating Characteristics analysis indicated that both FNDC5 and BNP were able to discriminate HF in atherosclerotic patients. Our findings suggest that FNDC5, along with BNP, has independent value as a biomarker for discriminating HF in patients with coronary atherosclerosis.

Detecting the FLJ22447 lncRNA in Ovarian Cancer with Cyclopentane-Modified FIT-PNAs (cpFIT-PNAs).

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.

Discovery of Novel Macrocyclic MERTK/AXL Dual Inhibitors.

MERTK and AXL are members of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases that are aberrantly expressed and have been implicated as therapeutic targets in a wide variety of human tumors. Dual MERTK and AXL inhibition could provide antitumor action mediated by both direct tumor cell killing and modulation of the innate immune response in some tumors such as nonsmall cell lung cancer. We utilized our knowledge of MERTK inhibitors and a structure-based drug design approach to discover a novel class of macrocyclic dual MERTK/AXL inhibitors. The lead compound 43 had low-nanomolar activity against both MERTK and AXL and good selectivity over TYRO3 and FLT3. Its target engagement and selectivity were also confirmed by NanoBRET and cell-based MERTK and AXL phosphorylation assays. Compound 43 had excellent pharmacokinetic properties (large AUC and long half-life) and mediated antitumor activity against lung cancer cell lines, indicating its potential as a therapeutic agent.

GTF2H4 regulates partial EndMT via NF-κB activation through NCOA3 phosphorylation in ischemic diseases.

Partial endothelial-to-mesenchymal transition (EndMT) is an intermediate phenotype observed in endothelial cells (ECs) undergoing a transition toward a mesenchymal state to support neovascularization during (patho)physiological angiogenesis. Here, we investigated the occurrence of partial EndMT in ECs under hypoxic/ischemic conditions and identified general transcription factor IIH subunit 4 (GTF2H4) as a positive regulator of this process. In addition, we discovered that GTF2H4 collaborates with its target protein excision repair cross-complementation group 3 (ERCC3) to co-regulate partial EndMT. Furthermore, by using phosphorylation proteomics and site-directed mutagenesis, we demonstrated that GTF2H4 was involved in the phosphorylation of receptor coactivator 3 (NCOA3) at serine 1330, which promoted the interaction between NCOA3 and p65, resulting in the transcriptional activation of NF-κB and the NF-κB/Snail signaling axis during partial EndMT. In vivo experiments confirmed that GTF2H4 significantly promoted partial EndMT and angiogenesis after ischemic injury. Collectively, our findings reveal that targeting GTF2H4 is promising for tissue repair and offers potential opportunities for treating hypoxic/ischemic diseases.

Correction: Cyclopentane FIT-PNAs: bright RNA sensors.

Correction for 'Cyclopentane FIT-PNAs: bright RNA sensors' by Odelia Tepper et al., Chem. Commun., 2021, 57, 540-543, https://doi.org/10.1039/D0CC07400D.

Variation of Tetrahydrofurans in Thyclotides Enhances Oligonucleotide Binding and Cellular Uptake of Peptide Nucleic Acids.

Selective incorporation of conformational constraints into thyclotides can be used to modulate their binding to complementary oligonucleotides, increase polarity, and optimize uptake into HCT116 cells without assistance from moieties known to promote cell uptake. The X-ray structure and biophysical studies of a thyclotide-DNA duplex reveal that incorporation of tetrahydrofurans into an aegPNA backbone promotes a helical conformation that enhances binding to complementary DNA and RNA. Selective incorporation of tetrahydrofurans into the aegPNA backbone allows polarity to be increased incrementally so that uptake into HCT116 cells can be optimized. The enhanced binding, polarity, and cellular uptake properties of thyclotides were used to demonstrate effective inhibition of microRNA-21 in HCT116 cells.

Foam Cells in Atherosclerosis: Novel Insights Into Its Origins, Consequences, and Molecular Mechanisms.

Foam cells play a vital role in the initiation and development of atherosclerosis. This review aims to summarize the novel insights into the origins, consequences, and molecular mechanisms of foam cells in atherosclerotic plaques. Foam cells are originated from monocytes as well as from vascular smooth muscle cells (VSMC), stem/progenitor cells, and endothelium cells. Novel technologies including lineage tracing and single-cell RNA sequencing (scRNA-seq) have revolutionized our understanding of subtypes of monocyte- and VSMC-derived foam cells. By using scRNA-seq, three main clusters including resident-like, inflammatory, and triggering receptor expressed on myeloid cells-2 (Trem2 hi ) are identified as the major subtypes of monocyte-derived foam cells in atherosclerotic plaques. Foam cells undergo diverse pathways of programmed cell death including apoptosis, autophagy, necroptosis, and pyroptosis, contributing to the necrotic cores of atherosclerotic plaques. The formation of foam cells is affected by cholesterol uptake, efflux, and esterification. Novel mechanisms including nuclear receptors, non-coding RNAs, and gut microbiota have been discovered and investigated. Although the heterogeneity of monocytes and the complexity of non-coding RNAs make obstacles for targeting foam cells, further in-depth research and therapeutic exploration are needed for the better management of atherosclerosis.

Low-density lipoprotein receptor and apolipoprotein A 5, myocardial infarction biomarkers in plasma-derived exosomes.

OBJECTIVES: Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. Previously, rare mutations in low-density lipoprotein receptor (LDLR) genes and apolipoprotein A V (APOA5) have been shown to contribute to MI risk in individual families. Exosomes provide a potential source of biomarkers for MI. This study is to determine the role of LDLR and APOA5 as biomarkers for early diagnosis of MI. METHODS: In this study, we detected the levels of LDLR, APOA5, and cardiac troponin T in plasma-derived exosomes in MI patients and age-matched healthy people by enzyme linked immunosorbent assay and observed the morphology and number of exosomes using transmission electron microscope and nanoparticle tracking analysis. Oxygen-glucose deprivation (OGD) method was used to induce MI in H9C2 cardiomyocytes to explore the effect of exosomes. RESULTS: We found that the levels of LDLR and APOA5 in plasma-derived exosomes in MI patients were significantly decreased. Furthermore, exosomes of MI patients were significantly larger in size and the concentration of exosomes was higher than that of age-matched non-MI people. In vitro experiments showed that OGD treatment induced apoptosis of myocardial cells and decreased the expression of LDLR and APOA5, while addition of exosomes isolated from healthy people rescued these phenotypes. CONCLUSION: Exosomal APOA5 and LDLR are intimately associated with MI, and thereby have the potential to function as diagnostic markers of MI.

Alkyl-substituted N-methylaryl-N'-aryl-4-aminobenzamides: A new series of small molecule inhibitors for Wip1 phosphatase.

The wild-type p53 induced phosphatase 1 (Wip1), a member of the serine/threonine-specific PP2C family, is overexpressed in numerous human cancers. Wip1 dephosphorylates p53 as well as several kinases (such as p38 MAPK, ATM, Chk1, and Chk2) in the DNA damage response pathway that are responsible for maintaining genomic stability and preventing oncogenic transformation. As a result, Wip1 is an attractive target for synthetic inhibitors that could be further developed into therapeutics to treat some cancers. In this study, we report a series of alkyl-substituted N-methylaryl-N'-aryl-4-aminobenzamides and their inhibitory activity of the Wip1 phosphatase. A straightforward synthetic route was developed to synthesize the target compounds from commercially available starting materials. Three different portions (R1, R2, R3) of the core scaffold were extensively modified to examine structure-activity relationships. This study revealed interesting trends about a new molecular scaffold to inhibit Wip1.

Cyclopentane FIT-PNAs: bright RNA sensors.

Cyclopentane modified FIT-PNA (cpFIT-PNA) probes are reported as highly emissive RNA sensors with the highest reported brightness for FIT-PNAs. Compared to FIT-PNAs, cpFIT-PNAs have improved mismatch discrimination for several pyrimidine-pyrimidine single nucleotide variants (SNVs).

miR-33a-5p Suppresses ox-LDL-Stimulated Calcification of Vascular Smooth Muscle Cells by Targeting METTL3.

Oxidized low-density lipoprotein (ox-LDL) accumulation in the vascular wall plays a pivotal role in the development of atherosclerosis and vascular calcification. However, few studies focus on the regulatory roles of microRNAs in ox-LDL stimulated vascular calcification. The aim of the present study was to investigate how miR-33a-5p regulated vascular calcification stimulated by ox-LDL. In the present study, miR-33a-5p was downregulated during vascular smooth muscle cells (VSMCs) calcification and upon ox-LDL treatment. ox-LDL significantly stimulated VSMCs calcification, while miR-33a-5p overexpression by its mimics transfection inhibited alkaline phosphatase (ALP) activity, mineralization and marker genes associated with VSMCs calcification even in the presence of ox-LDL. Methyltransferase like 3 (METTL3) was the target gene of miR-33a-5p. METTL3 was upregulated during VSMCs calcification and upon ox-LDL treatment. When VSMCs were transfected with miR-33a-5p mimics, METTL3 was downregulated. METTL3 downregulation by siRNA method decreased VSMCs calcification even in the presence of ox-LDL. Taken together, these results suggest miR-33a-5p suppresses VSMCs calcification stimulated by ox-LDL via targeting METTL3, highlighting the critical role of miR-33a-5p/METTL3 in vascular calcification.

The Effective Role of Natural Product Berberine in Modulating Oxidative Stress and Inflammation Related Atherosclerosis: Novel Insights Into the Gut-Heart Axis Evidenced by Genetic Sequencing Analysis.

The exacerbation of oxidative and inflammatory reactions has been involved in atherosclerotic cardiovascular diseases leading to morbidity and mortality worldwide. Discovering the underlying mechanisms and finding optimized curative approaches to control the global prevalence of cardiovascular diseases is needed. Growing evidence has demonstrated that gut microbiota is associated with the development of atherosclerosis, while berberine, a natural product exhibits antiatherogenic effects in clinical and pre-clinical studies, which implies a potential link between berberine and gut microbiota. In light of these novel discoveries, evidence of the role of berberine in modulating atherosclerosis with a specific focus on its interaction with gut microbiota is collected. This review synthesizes and summarizes antioxidant and anti-inflammatory effects of berberine on combating atherosclerosis experimentally and clinically, explores the interaction between berberine and intestinal microbiota comprehensively, and provides novel insights of berberine in managing atherosclerotic cardiovascular diseases via targeting the gut-heart axis mechanistically. The phenomenon of how berberine overcomes its weakness of poor bioavailability to conduct its antiatherogenic properties is also discussed and interpreted in this article. An in-depth understanding of this emerging area may contribute to identifying therapeutic potentials of medicinal plant and natural product derived pharmaceuticals for the prevention and treatment of atherosclerotic cardiovascular diseases in the future.

The potential value of Copeptin and Pentraxin3 for evaluating the severity of coronary stenosis in patients with coronary artery disease.

INTRODUCTION: Coronary artery disease (CAD) is an ischemic heart disease due to the narrowing of the coronary arteries resulting from atherosclerosis. Blood biomarkers have been well utilized for the diagnosis and prognosis of CAD. However, the value of biomarkers for evaluating coronary atherosclerosis remains to be clarified. This clinical investigation aimed to explore the potential value of biomarkers for evaluating the severity of coronary stenosis in CAD patients. METHODS: The extent of coronary atherosclerosis was accessed by the angiography-based quantitative measurement Gensini score (GS). Blood levels of Brain natriuretic peptide, Copeptin (CPP), Phosphodiesterase 9A, and Pentraxin3 (PTX3) were measured in 56 patients divided into three levels as low GS (n = 17), intermediate GS (n = 19) and high GS (n = 20) based on GS tertiles. RESULTS: We found that plasma concentrations of CPP and PTX3 were significantly elevated in patients with high GS compared with the low GS group. In addition, Pearson correlation analysis showed that CPP and PTX3 were positively correlated with the GS. Furthermore, Receiver operating characteristics analysis demonstrated that both CPP and PTX3 exhibited discriminative capacities for evaluating the extent of coronary stenosis. CONCLUSIONS: Laboratory tests of CPP and PTX3 via non-invasive means may provide novel information for risk stratification and disease management in CAD patients before invasive angiographic approaches. This study opens the door for enormous opportunities to explore new biomarkers with better efficiency, sensitivity and specificity as alternative/additional methods for evaluating the severity of coronary atherosclerosis in CAD patients in future research.