Neuropilin-1 is a novel host factor modulating the entry of hepatitis B virus.

BACKGROUND & AIMS: Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for hepatitis B virus (HBV). However, hepatocytes expressing NTCP exhibit varying susceptibilities to HBV infection. This study aimed to investigate whether other host factors modulate the process of HBV infection. METHODS: Liver biopsy samples obtained from children with hepatitis B were used for single-cell sequencing and susceptibility analysis. Primary human hepatocytes, HepG2-NTCP cells, and human liver chimeric mice were used to analyze the effect of candidate host factors on HBV infection. RESULTS: Single-cell sequencing and susceptibility analysis revealed a positive correlation between neuropilin-1 (NRP1) expression and HBV infection. In the HBV-infected cell model, NRP1 overexpression before HBV inoculation significantly enhanced viral attachment and internalization, and promoted viral infection in the presence of NTCP. Mechanistic studies indicated that NRP1 formed a complex with LHBs and NTCP. The NRP1 b domain mediated its interaction with conserved arginine residues at positions 88 and 92 in the preS1 domain of the HBV envelope protein LHBs. This NRP1-preS1 interaction subsequently promoted the binding of preS1 to NTCP, facilitating viral infection. Moreover, disruption of the NRP1-preS1 interaction by the NRP1 antagonist EG00229 significantly attenuated the binding affinity between NTCP and preS1, thereby inhibiting HBV infection both in vitro and in vivo. CONCLUSIONS: Our findings indicate that NRP1 is a novel host factor for HBV infection, which interacts with preS1 and NTCP to modulate HBV entry into hepatocytes. IMPACT AND IMPLICATIONS: HBV infection is a global public health problem, but the understanding of the early infection process of HBV remains limited. Through single-cell sequencing, we identified a novel host factor, NRP1, which modulates HBV entry by interacting with HBV preS1 and NTCP. Moreover, antagonists targeting NRP1 can inhibit HBV infection both in vitro and in vivo. This study could further advance our comprehension of the early infection process of HBV.

A rapid and simple non-radioactive assay for measuring uptake by solute carrier transporters.

Introduction: Solute carrier (SLC) transport proteins play a crucial role in maintaining cellular nutrient and metabolite homeostasis and are implicated in various human diseases, making them potential targets for therapeutic interventions. However, the study of SLCs has been limited due to the lack of suitable tools, particularly cell-based substrate uptake assays, necessary for understanding their biological functions and for drug discovery purposes. Methods: In this study, a cell-based uptake assay was developed using a stable isotope-labeled compound as the substrate for SLCs, with detection facilitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay aimed to address the limitations of existing assays, such as reliance on hazardous radiolabeled substrates and limited availability of fluorescent biosensors. Results: The developed assay was successfully applied to detect substrate uptakes by two specific SLCs: L-type amino acid transporter 1 (LAT1) and sodium taurocholate co-transporting polypeptide (NTCP). Importantly, the assay demonstrated comparable results to the radioactive method, indicating its reliability and accuracy. Furthermore, the assay was utilized to screen for novel inhibitors of NTCP, leading to the identification of a potential NTCP inhibitor compound. Discussion: The findings highlight the utility of the developed cell-based uptake assay as a rapid, simple, and environmentally friendly tool for investigating SLCs' biological roles and for drug discovery purposes. This assay offers a safer alternative to traditional methods and has the potential to contribute significantly to advancing our understanding of SLC function and identifying therapeutic agents targeting SLC-mediated pathways.

Corrigendum: Prostaglandin reductase 1 as a potential therapeutic target for cancer therapy.

[This corrects the article DOI: 10.3389/fphar.2021.717730.].

Rapid access to C2-quaternary 3-methyleneindolines via base-mediated post-Ugi Conia-ene cyclization.

Highly efficient synthesis of diverse 2,2-disubstituted 3-methyleneindoline derivatives through a one-pot base-promoted post-Ugi 5-exo-dig "Conia-ene"-type cyclization has been disclosed. The mechanism study indicates that an intramolecular hydrogen bond may play a vital role in this process. The antiproliferative evaluation of cancer cell lines reveals that this protocol provides practical use in the green synthesis of bioactive compound libraries.

Signaling Pathway and Small-Molecule Drug Discovery of FGFR: A Comprehensive Review.

Targeted therapy is a groundbreaking innovation for cancer treatment. Among the receptor tyrosine kinases, the fibroblast growth factor receptors (FGFRs) garnered substantial attention as promising therapeutic targets due to their fundamental biological functions and frequently observed abnormality in tumors. In the past 2 decades, several generations of FGFR kinase inhibitors have been developed. This review starts by introducing the biological basis of FGF/FGFR signaling. It then gives a detailed description of different types of small-molecule FGFR inhibitors according to modes of action, followed by a systematic overview of small-molecule-based therapies of different modalities. It ends with our perspectives for the development of novel FGFR inhibitors.

Influence of stereochemistry on the activity of rapadocin, an isoform-specific inhibitor of the nucleoside transporter ENT1.

Rapadocin is a novel rapamycin-inspired polyketide-tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1). Rapadocin contains an epimerizable chiral center in phenylglycine and an olefin group, and can thus exist as a mixture of four stereoisomers. Herein, we report the first total synthesis of the four stereoisomers of rapadocin using two different synthetic strategies and the assignment of their structures. The inhibitory activity of each of the four synthetic isomers on both hENT1 and hENT2 was determined. It was found that the stereochemistry of phenylglycine played a more dominant role than the configuration of the olefin in the activity of rapadocin. These findings will guide the future design and development of rapadocin analogs as new modulators of adenosine signaling.

Prostaglandin Reductase 1 as a Potential Therapeutic Target for Cancer Therapy.

Altered tumor metabolism is a hallmark of cancer and targeting tumor metabolism has been considered as an attractive strategy for cancer therapy. Prostaglandin Reductase 1 (PTGR1) is a rate-limiting enzyme involved in the arachidonic acid metabolism pathway and mainly responsible for the deactivation of some eicosanoids, including prostaglandins and leukotriene B4. A growing evidence suggested that PTGR1 plays a significant role in cancer and has emerged as a novel target for cancer therapeutics. In this review, we summarize the progress made in recent years toward the understanding of PTGR1 function and structure, highlight the roles of PTGR1 in cancer, and describe potential inhibitors of PTGR1. Finally, we provide some thoughts on future directions that might facilitate the PTGR1 research and therapeutics development.

Evolved resistance to partial GAPDH inhibition results in loss of the Warburg effect and in a different state of glycolysis.

Aerobic glycolysis or the Warburg effect (WE) is characterized by increased glucose uptake and incomplete oxidation to lactate. Although the WE is ubiquitous, its biological role remains controversial, and whether glucose metabolism is functionally different during fully oxidative glycolysis or during the WE is unknown. To investigate this question, here we evolved resistance to koningic acid (KA), a natural product that specifically inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-controlling glycolytic enzyme, during the WE. We found that KA-resistant cells lose the WE but continue to conduct glycolysis and surprisingly remain dependent on glucose as a carbon source and also on central carbon metabolism. Consequently, this altered state of glycolysis led to differential metabolic activity and requirements, including emergent activities in and dependences on fatty acid metabolism. These findings reveal that aerobic glycolysis is a process functionally distinct from conventional glucose metabolism and leads to distinct metabolic requirements and biological functions.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays.

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell-cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

Activation of BMP Signaling by FKBP12 Ligands Synergizes with Inhibition of CXCR4 to Accelerate Wound Healing.

The combination of AMD3100 and low-dose FK506 has been shown to accelerate wound healing in vivo. Although AMD3100 is known to work by releasing hematopoietic stem cells into circulation, the mechanism of FK506 in this setting has remained unknown. In this study, we investigated the activities of FK506 in human cells and a diabetic-rat wound model using a non-immunosuppressive FK506 analog named FKVP. While FKVP was incapable of inhibiting calcineurin, wound-healing enhancement with AMD3100 was unaffected. Further study showed that both FK506 and FKVP activate BMP signaling in multiple cell types through FKBP12 antagonism. Furthermore, selective inhibition of BMP signaling abolished stem cell recruitment and wound-healing enhancement by combination treatment. These results shed new light on the mechanism of action of FK506 in acceleration of wound healing, and raise the possibility that less toxic FKBP ligands such as FKVP can replace FK506 for the treatment of chronic wounds.

Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation.

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib.IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors.

Inhibition of Eukaryotic Translation by the Antitumor Natural Product Agelastatin A.

Protein synthesis plays an essential role in cell proliferation, differentiation, and survival. Inhibitors of eukaryotic translation have entered the clinic, establishing the translation machinery as a promising target for chemotherapy. A recently discovered, structurally unique marine sponge-derived brominated alkaloid, (-)-agelastatin A (AglA), possesses potent antitumor activity. Its underlying mechanism of action, however, has remained unknown. Using a systematic top-down approach, we show that AglA selectively inhibits protein synthesis. Using a high-throughput chemical footprinting method, we mapped the AglA-binding site to the ribosomal A site. A 3.5 Å crystal structure of the 80S eukaryotic ribosome from S. cerevisiae in complex with AglA was obtained, revealing multiple conformational changes of the nucleotide bases in the ribosome accompanying the binding of AglA. Together, these results have unraveled the mechanism of inhibition of eukaryotic translation by AglA at atomic level, paving the way for future structural modifications to develop AglA analogs into novel anticancer agents.

Enzyme-instructed molecular self-assembly confers nanofibers and a supramolecular hydrogel of taxol derivative.

By covalently connecting taxol with a motif that is prone to self-assemble, we successfully generate the precursor (5a), the hydrogelator (5b), and hydrogel of a taxol derivative without compromising the cytotoxic activity of the taxol. This approach promises a general method to create nanofibers of therapeutic molecules that have a dual role, as both the delivery vehicle and the drug itself.

Amino terminus of the SARS coronavirus protein 3a elicits strong, potentially protective humoral responses in infected patients.

The 3a protein of severe acute respiratory syndrome (SARS)-associated coronavirus is expressed and transported to the plasma membrane in tissue cells of infected patients. Its short N-terminal ectodomain was found to elicit strong humoral responses in half of the patients who had recovered from SARS. The ectodomain-specific antibodies from the convalescent-phase plasma readily recognized and induced destruction of 3a-expressing cells in the presence of the human complement system, demonstrating their potential ability to provide immune protection by recognizing and eliminating SARS coronavirus-infected cells that express the target protein. In addition, when coupled to a carrier protein, the ectodomain peptide elicited 3a-specific antibodies in mice and rabbit at high titres. These results showed that the N terminus of the 3a protein is highly immunogenic and elicits potentially protective humoral responses in infected patients. Therefore, the short extracellular domain may be a valuable immunogen in the development of a vaccine for infectious SARS.

B-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the S2 domain of the surface spike glycoprotein.

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel strain of coronavirus. Examination of the immune responses of patients who have recovered from SARS should provide important information for design of a safe and effective vaccine. We determined the continuous viral epitopes targeted by antibodies in plasma samples from convalescent SARS patients through biopanning with a vast M13 phage display dodecapeptide library. These epitopes converged to very short peptide fragments, one on each of the structural proteins spike and nucleocapsid and the nonstructural proteins 3a, 9b, and nsp 3. Immunoassays found that most of the patients who had recovered from SARS developed complementary antibodies to the epitope-rich region on the spike S2 protein, indicating that this is an immunodominant site on the viral envelope comprising the spike, matrix, and small envelope glycoproteins. These S2-targeting antibodies were shown to effectively neutralize the coronavirus, indicating that they provided protective immunity to help the patients recover from the viral infection. These results suggest that the SARS coronavirus might have an antigenic profile distinct from those of other human or animal coronaviruses. Due to the tested safety and protective effects of the convalescent-phase serological antibodies, identification of their complementary antigens may enable the design of an epitope-based vaccine to prevent potential antibody-mediated immunopathology.

[Study of direct fluorophotometric and flow-injection fluorophotometric methods based on the inhibitory effect for the determination of trace formaldehyde].

Sulfite reacts with o-phthalaldehyde in the presence of ammonium forming the highly fluorescing isoindole-1-sulfonate in neutral or weakly acid solution, and formaldehyde has inhibitory effect on it. Based on this principle, the authors developed the direct fluorophotometric and flow-injection fluorophotometric methods for the determination of trace formaldehyde. The maximum excitation wavelength and the maximum emission wavelength are 320 and 390 nm respectively. With the direct fluorophotometric method, formaldehyde in the concentration range of 0.10-1.60 microg x mL(-1) can be determined with a detection limit of 0.046 microg x mL(-1). With the flow-injection fluorophotometric method, formaldehyde in the concentration range of 0.10-2.00 microg x mL(-1) can be determined with a detection limit of 0.085 microg x mL(-1). The methods were applied respectively to the analysis of river water with satisfactory results.