DNA G-Quadruplexes as Targets for Natural Product Drug Discovery

DNA guanine(G)-quadruplexes(G4s)are unique secondary structures formed by two or more stacked Gtetrads in G-rich DNA sequences.These structures have been found to play a crucial role in highly transcribed genes,especially in cancer-related oncogenes,making them attractive targets for cancer therapeutics.Significantly,targeting oncogene promoter G4 structures has emerged as a promising strategy to address the challenge of undruggable and drug-resistant proteins,such as MYC,BCL2,KRAS,and EGFR.Natural products have long been an important source of drug discovery,particularly in the fields of cancer and infectious diseases.Noteworthy progress has recently been made in the discovery of naturally occurring DNA G4-targeting drugs.Numerous DNA G4s,such as MYC-G4,BCL2-G4,KRAS-G4,PDGFR-b-G4,VEGF-G4,and telomeric-G4,have been identified as potential targets of natural products,including berberine,telomestatin,quindoline,sanguinarine,isaindigotone,and many others.Herein,we summarize and evaluate recent advancements in natural and nature-derived DNA G4 binders,focusing on understanding the structural recognition of DNA G4s by small molecules derived from nature.We also discuss the challenges and opportunities associated with developing drugs that target DNA G4s.

A proteomic landscape of pharmacologic perturbations for functional relevance

Pharmacological perturbation studies based on protein-level signatures are fundamental for drug discovery. In the present study, we used a mass spectrometry (MS)-based proteomic platform to profile the whole proteome of the breast cancer MCF7 cell line under stress induced by 78 bioactive compounds. The integrated analysis of perturbed signal abundance revealed the connectivity between phenotypic behaviors and molecular features in cancer cells. Our data showed functional relevance in exploring the novel pharmacological activity of phenolic xanthohumol, as well as the noncanonical targets of clinically approved tamoxifen, lovastatin, and their derivatives. Furthermore, the rational design of synergistic inhibition using a combination of histone methyltransferase and topoisomerase was identified based on their complementary drug fingerprints. This study provides rich resources for the proteomic landscape of drug responses for precision therapeutic medicine.

Highly Aromatic Norditerpenoid Heterodimers and Monomers from Trigonostemon fragilis

Four new norditerpenoid heterodimers with different dimerization patterns-namely,trigofragiloids A-C(denoted as compounds 1-3)and(+)-and(-)-trigofragiloid D(compound 4)-and three new phenanthrenone norditerpenoids-namely,trigofragiloids E-G(compounds 5-7)-were isolated from Trigonostemon fragilis.Compounds 1 and 2 feature a novel heterodimeric carbon skeleton formed by the conjugation of a tetra-norditerpenoid and an ennea-norditerpenoid;they have been identified as class 2 atropisomers by means of quantum chemical calculations.Compound 3 is an unprecedented phenylpropanoid-norditerpenoid adduct with a new dimerization pattern.Compounds(+)-and(-)-4 are the first example of S-shaped 1,4-dioxane-fused norditerpenoid dimers.Inspired by the structure elucidation of compound 4,two co-occurring analogues,actephilol A and epiactephilol A,were structurally revised as a pair of geometrical isomers and were identified as two pairs of enantiomers,(+)-and(-)-8 and(+)-and(-)-9,respectively.Their structures were characterized using a combined method.Notably,compound 7 exhibits remarkable adenosine triphosphate-citrate lyase(ACLY)inhibition with a halfmaximal inhibition concentration(IC50)value of(0.46±0.11)lmol·L^(-1),as active as the positive control BMS-303141,and a molecular docking study offers deep insight into the interaction between compound 7 and ACLY.

Transient receptor potential channels and calcium dysregulation: a pathogenic duo in Parkinson's disease

Parkinson's disease(PD) has a complex and multifactorial pathophysiology. Various studies, conducted both in pre-clinical models and PD patients, have reported a link between the disruption of calcium(Ca^(2+)) homeostasis and the subsequent development of PD. Ca^(2+) regulation is crucial for neuronal survival, differentiation,exocytosis at synapses,gene transcription,and proliferation.

A novel saliva-based miRNA profile to diagnose and predict oral cancer

Oral cancer (OC) is the most common form of head and neck cancer. Despite the high incidence and unfavourable patient outcomes, currently, there are no biomarkers for the early detection of OC. This study aims to discover, develop, and validate a novel saliva-based microRNA signature for early diagnosis and prediction of OC risk in oral potentially malignant disorders (OPMD).The Cancer Genome Atlas (TCGA) miRNA sequencing data and small RNA sequencing data of saliva samples were used to discover differentially expressed miRNAs. Identified miRNAs were validated in saliva samples of OC (n=50), OPMD (n=52), and controls(n=60) using quantitative real-time PCR. Eight differentially expressed miRNAs (miR-7-5p, miR-10b-5p, miR-182-5p, miR-215-5p,miR-431-5p, miR-486-3p, miR-3614-5p, and miR-4707-3p) were identified in the discovery phase and were validated. The efficiency of our eight-miRNA signature to discriminate OC and controls was:area under curve (AUC):0.954, sensitivity:86%, specificity:90%,positive predictive value (PPV):87.8%and negative predictive value (NPV):88.5%whereas between OC and OPMD was:AUC:0.911,sensitivity:90%, specificity:82.7%, PPV:74.2%and NPV:89.6%. We have developed a risk probability score to predict the presence or risk of OC in OPMD patients. We established a salivary miRNA signature that can aid in diagnosing and predicting OC,revolutionising the management of patients with OPMD. Together, our results shed new light on the management of OC by salivary miRNAs to the clinical utility of using miRNAs derived from saliva samples.

Indoleamine 2,3-dioxygenase: As a potential prognostic marker and immunotherapeutic target for hepatocellular carcinoma

Tumor cells induce an immunosuppressive microen-vironment which leads towards tumor immune escape. Understanding the intricacy of immunomodulation by tumor cells is essential for immunotherapy. Indoleamine 2,3-dioxygenase(IDO) is an immunosuppressive enzyme which mediates tumor immune escape in various cancers including hepatocellular carcinoma(HCC). IDO up-regulation in HCC may lead to recruitment of regulatory T-cells into tumor microenvironment and therefore inhibit local immune responses and promote metastasis. HCC associated fibroblasts stimulate natural killer cells dysfunction through prostaglandin E2 and subsequently IDO promotes favorable condition for tumor metastasis. IDO up-regulation induces immuno-suppression and may enhance the risk of hepatitis C virus and hepatitis B virus induced HCC. Therefore, IDO inhibitors as adjuvant therapeutic agents may have clinical implications in HCC. This review proposes future prospects of IDO not only as a therapeutic target but also as a prognostic marker for HCC.

Reliable cell purification and determination of cell purity:crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair

Transplantation of olfactory ensheathing cells,the glia of the primary olfactory nervous system,has been trialed for spinal cord injury repair with promising but variable outcomes in animals and humans.Olfactory ensheathing cells can be harvested either from the lamina propria beneath the neuroepithelium in the nasal cavity,or from the olfactory bulb in the brain.As these areas contain several other cell types,isolating and purifying olfactory ensheathing cells is a critical part of the process.It is largely unknown how contaminating cells such as fibroblasts,other glial cell types and supporting cells affect olfactory ensheathing cell function post-transplantation;these cells may also cause unwanted side-effects.It is also,however,possible that the presence of some of the contaminant cells can improve outcomes.Here,we reviewed the last decade of olfactory ensheathing cell transplantation studies in rodents,with a focus on olfactory ensheathing cell purity.We analyzed how purification methods and resultant cell purity differed between olfactory mucosa-and olfactory bulb-derived cell preparations.We analyzed how the studies reported on olfactory ensheathing cell purity and which criteria were used to define cells as olfactory ensheathing cells.Finally,we analyzed the correlation between cell purity and transplantation outcomes.We found that olfactory bulb-derived olfactory ensheathing cell preparations are typically purer than mucosa-derived preparations.We concluded that there is an association between high olfactory ensheathing cell purity and favourable outcomes,but the lack of olfactory ensheathing cell-specific markers severely hampers the field.

Olfactory ensheathing cells for spinal cord repair: crucial differences between subpopulations of the glia

OECs for spinal cord repair： Is repairing the iniured spinal cord by olfactory ensheathing cell （OEC） transplantation pos- sible？ A recent human trial in which a paralysed man regained some function after transplantation of partially purified OECs suggests that this therapy may be a successful approach （Ta- bakow et al., 2014）. In another human trial in which olfactory mucosa lamina propria was transplanted, patients recovered some motor and sensory function （Wang et al., 2015）. While these results show promise, it is clear that improvements are needed to provide patients with increased functional output. Strategies to improve the therapeutic use of OECs may include improving the purification of the OECs used for transplantation, using them in combination with growth factors to combat the inhibitory environment and improve anon growth, the use of nerve bridges, advanced physiotherapy and the use of exo- skeleton robotics to reinforce functional connections. Of all these approaches, it is probably is primarily addressed to ensure crucial that the purity of OECs consistency in outcomes.

Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosorbent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were successfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias%ranged from -12.2% to -5.2%,precision ranged from -12.4% to -1.4%,and the relative standard deviation(RSD)was less than 6.6% and 8.7%,respectively.The total error was less than 20.4%.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Exploration of Activated Pathways for Improving Antifungal Agent FR901469 Productivity in Fungal Species No.11243 Using Comprehensive Pathway Model

Secondary metabolites are important for various industrial applications. The production of secondary metabolites is often improved by the activation of substrate supply pathways for biosynthesis. However, many important pathways have remained unclear. In this study, we explored possible pathways related to substrate supply for the biosynthesis of the antifungal agent FR901469 which is a nonribosomal peptide and a fungal secondary metabolite. To clarify the unknown activated pathways, we utilized the Comprehensive Pathway Model (CPM) which was developed in our previous study. We verified that the overexpression of the hypothetical beta-alanine-aminotransferase (BAL-AT), which was included in the explored pathways, improved the FR901469 productivity. The genes encoding the BAL metabolic enzymes are considered to be important for improving the FR901469 productivity.

Development of an Algorithm for Reconstructing a Comprehensive Pathway Model: Application to <i>Saccharomyces cerevisiae</i>

The generation of bioactive products by microbial bioprocesses is important for drug discovery, functional food development, and other beneficial purposes. Many pathways contribute to the production of these bioactive compounds, but important knowledge for improving productivity still remains in hidden pathways. Recently, an abundance of knowledge about metabolic pathways has been accumulated in metabolic pathway databases, such as BioCyc and KEGG. Many by-products are chemically transformed and actually used in other enzymatic reactions. In this work, we developed an algorithm for the reconstruction of a comprehensive genetic pathway model from a known metabolic pathway database. This model considers the interactions of the by-products, in addition to the main products. Furthermore, we developed a method for the construction of a comprehensive pathway model in a specific organism. In this study, we reconstructed a Saccharomyces cerevisiae model. From this model, the pathways among enzymes that contributed to galactose metabolism were explored. Using S. cerevisiae DNA microarray data, the activated pathways were found among the explored pathways.

Structure-based drug discovery of novel fusedpyrazolone carboxamide derivatives as potent and selective AXL inhibitors

a novel and promising antitumor target,AXL plays an important role in tumor growth,metastasis,immunosuppression and drug resistance of various malignancies,which has attracted extensive research interest in recent years.In this study,by employing the structure-based drug design and bioisosterism strategies,we designed and synthesized in total 54 novel AXL inhibitors featuring a fusedpyrazolone carboxamide scaffold,of which up to 20 compounds exhibited excellent AXL kinase and BaF3/TEL-AXL cell viability inhibitions.Notably,compound 59 showed a desirable AXL kinase inhibitory activity(IC_(50):3.5 nmol/L)as well as good kinase selectivity,and it effectively blocked the cellular AXL signaling.In turn,compound 59 could potently inhibit BaF3/TEL-AXL cell viability(IC_(50):1.5 nmol/L)and significantly suppress GAS6/AXL-mediated cancer cell invasion,migration and wound healing at the nanomolar level.More importantly,compound 59 oral administration showed good pharmacokinetic profile and in vivo antitumor efficiency,in which we observed significant AXL phosphorylation suppression,and its antitumor efficacy at 20 mg/kg(qd)was comparable to that of BGB324 at 50 mg/kg(bid),the most advanced AXL inhibitor.Taken together,this work provided a valuable lead compound as a potential AXL inhibitor for the further antitumor drug development.

Development of SV2A Ligands for Epilepsy Treatment:A Review of Levetiracetam,Brivaracetam,and Padsevonil

Epilepsy is a common neurological disorder that is primarily treated with antiseizure medications(ASMs).Although dozens of ASMs are available in the clinic,approximately 30%of epileptic patients have medically refractory seizures;other limitations in most traditional ASMs include poor tolerability and drug-drug interactions.Therefore,there is an urgent need to develop alternative ASMs.Levetiracetam(LEV)is a first-line ASM that is well tolerated,has promising efficacy,and has little drug-drug interaction.Although it is widely accepted that LEV acts through a unique therapeutic target synaptic vesicle protein(SV)2A,the molecular basis of its action remains unknown.Even so,the next-generation SV2A ligands against epilepsy based on the structure of LEV have achieved clinical success.This review highlights the research and development(R&D)process of LEV and its analogs,brivaracetam and padsevonil,to provide ideas and experience for the R&D of novel ASMs.

A novel strategy for treating oncogene-mutated tumors by targeting tumor microenvironment and synergistically enhancing anti-PD-1 immunotherapy

Oncogenes are critical factors in tumorigenesis of diverse cancer types and play essential roles in tumor immune escape.Mutations in Kirsten rat sarcoma viral oncogene homolog(KRAS)and epidermal growth factor receptor(EGFR)are among the most frequent gain-of-function alterations[1].After many years of in-depth research,inhibitors targeting EGFR or KRAS mutations have been successfully developed,however,their clinical benefit is relatively limited,and they will inevitably encounter the challenge of drug resistance.The emergence of resistance is attributed to secondary mutations in driver genes and other complicated factors.

Composition analysis of Compound Shenhua Tablet,a seven-herb Chinese medicine for IgA nephropathy:evaluation of analyte-capacity of the assays

Compound Shenhua Tablet,a medicine comprising seven herbs,is employed in treating IgA nephropathy.This study aimed to meticulously analyze its chemical composition.Based on a list of candidate compounds,identified through extensive literature review pertinent to the tablet’s herbal components,the composition analysis entailed the systematic identification,characterization,and quantification of the constituents.The analyte-capacity of LC/ESI-MS-based and GC/EI-MS-based assays was evaluated.The identified and characterized constituents were quantified to determine their content levels and were ranked based on the constituents’daily doses.A total of 283 constituents,classified into 12 distinct categories,were identified and characterized in the Compound Shenhua Tablet.These constituents exhibited content levels of 1−10982μg·g^(−1),with daily doses of 0.01−395μmol·d^(−1).The predominant constituents,with daily doses of≥10μmol·d^(−1),include nine organic acids(citric acid,quinic acid,chlorogenic acid,cryptochlorogenic acid,gallic acid,neochlorogenic acid,isochlorogenic acid C,isochlorogenic acid B,and linoleic acid),five iridoids(specnuezhenide,nuezhenoside G13,nuezhenidic acid,secoxyloganin,and secologanoside),two monoterpene glycosides(paeoniflorin and albiflorin),a sesquiterpenoid(curzerenone),a triterpenoid(oleanolic acid),and a phenylethanoid(salidroside).Additionally,there were 83,126,and 55 constituents detected in the medicine with daily doses of 1–10,0.1–1,and 0.01–0.1μmol·d^(−1),respectively.The combination of the LC/ESI-MS-based and GC/EI-MS-based assays demonstrated a complementary relationship in their analyte-capacity for detecting the constituents present in the medicine.This comprehensive composition analysis establishes a solid foundation for further pharmacological research on Compound Shenhua Tablet and facilitates the quality evaluation of this complex herbal medicine.

Discovery of a potent and dual-selective bisubstrate inhibitor for protein arginine methyltransferase 4/5

Protein arginine methyltransferases(PRMTs)have been implicated in the progression of many diseases.Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery.Herein,we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine(SAM)analogue moiety and a tripeptide through an alkyl substituted guanidino group.Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration(IC_(50)) of 2.8 and0.42 nmol/L,respectively.Computational studies provided a plausible explanation for the high potency and selectivity of AH237 for PRMT4/5 over other 40 methyltransferases.This proof-of-principle study outlines an applicable strategy to develop potent and selective bisubstrate inhibitors for PRMTs,providing valuable probes for future structural studies.

P2X7 receptor signaling during adult hippocampal neurogenesis

Neurogenesis is a persistent and essential feature of the adult mammalian hippocampus.Granular neurons generated from resident pools of stem or progenitor cells provide a mechanism for the formation and consolidation of new memories.Regulation of hippocampal neurogenesis is complex and multifaceted,and numerous signaling pathways converge to modulate cell proliferation,apoptosis,and clearance of cellular debris,as well as synaptic integration of newborn immature neurons.The expression of functional P2X7 receptors in the central nervous system has attracted much interest and the regulatory role of this purinergic receptor during adult neurogenesis has only recently begun to be explored.P2X7 receptors are exceptionally versatile:in their canonical role they act as adenosine triphosphate-gated calcium channels and facilitate calcium-signaling cascades exerting control over the cell via calcium-encoded sensory proteins and transcription factor activation.P2X7 also mediates transmembrane pore formation to regulate cytokine release and facilitate extracellular communication,and when persistently stimulated by high extracellular adenosine triphosphate levels large P2X7 pores form,which induce apoptotic cell death through cytosolic ion dysregulation.Lastly,as a scavenger receptor P2X7 directly facilitates phagocytosis of the cellular debris that arises during neurogenesis,as well as during some disease states.Understanding how P2X7 receptors regulate the physiology of stem and progenitor cells in the adult hippocampus is an important step towards developing useful therapeutic models for regenerative medicine.This review considers the relevant aspects of adult hippocampal neurogenesis and explores how P2X7 receptor activity may influence the molecular physiology of the hippocampus,and neural stem and progenitor cells.

Combined VEGF/PDGF improves olfactory regeneration after unilateral bulbectomy in mice

The olfactory receptor neurons lining the nasal cavity have a remarkable capacity to regenerate throughout life. They are replenished continuously and their axons make new connections within the olfactory bulb. However, some factors such as head trauma and skull base surgery damage the olfactory nerve which lead to olfactory dysfunction. Losing the sense of smell has considerable effects on quality of life and life-expectancy. Therefore, there is a clear need to find a treatment for olfactory dysfunction. One such potential treatment is growth factor therapy which showed promising results in the spinal cord and brain injuries. The aim of the present study was to investigate whether combined delivery of two growth factors, vascular endothelial growth factor and platelet-derived growth factor treatment can improve the olfactory neurons regeneration in mice. The degeneration of the olfactory neurons was induced by unilateral bulbectomy. The treatment group received 1.5 μg of the combined growth factors intranasally, while the control injured group received saline. Growth factor treatment significantly increased the number of immature neurons at 5 and 7 days post injury and also the number of mature olfactory neurons at 10 and 14 days post bulbectomy. Regenerating axons extended over a larger volume in the operated cavity in the treatment group compared to control group at 14 days post bulbectomy. The growth factor treatment also significantly reduced astrocytic glia scar in the operated cavity. The results indicate that the combined delivery of the growth factors has the potential to improve olfactory dysfunction.

Factors that modulate olfactory dysfunction

The olfactory system is one of a few areas in the nervous system which is capable of regeneration throughout the life.Olfactory sensory neurons reside in the nasal cavity are continuously replenished with new neurons arising from stem cells.Some factors such as aging,neurodegenerative diseases,head trauma,brain tumor extraction and infection cause olfactory dysfunction which significantly influences physical wellbeing,quality of life,mental health,nutritional status,memory processes,identifying danger and is associated with increased mortality.Therefore,finding a treatment to improve olfactory dysfunction is needed.Recent research efforts in the field have shown some very promising new approaches to treat olfactory dysfunction.This review explores the current studies that have addressed therapeutic approaches to improve olfactory neuron regeneration based on cell transplantation therapy,modulation of physiological olfactory dysfunction and drug treatments.

Review of 10 years of research on breast cancer patients:Focus on indoleamine 2,3-dioxygenase

Therapeutic manipulation of the immune system in cancer has been an extensive area of research in the field of oncoimmunology.Immunosuppression regulates antitumour immune responses.An immunosuppressive enzyme,indoleamine 2,3-dioxygenase(IDO)mediates tumour immune escape in various malignancies including breast cancer.IDO upregulation in breast cancer cells may lead to the recruitment of regulatory T(T-regs)cells into the tumour microenvironment,thus inhibiting local immune responses and promoting metastasis.Immunosuppression induced by myeloid derived suppressor cells activated in an IDOdependent manner may enhance the possibility of immune evasion in breast cancer.IDO overexpression has independent prognostic significance in a subtype of breast cancer of emerging interest,basal-like breast carcinoma.IDO inhibitors as adjuvant therapeutic agents may have clinical implications in breast cancer.This review proposes future prospects of IDO not only as a therapeutic target but also as a valuable prognostic marker for breast cancer.