Differential Scanning Fluorimetry Research Articles

Erianin inhibits the progression of triple-negative breast cancer by suppressing SRC-mediated cholesterol metabolism

Triple-negative breast cancer (TNBC) is highly malignant and lacks effective biotherapeutic targets. The development of efficient anticancer drugs with low toxicity and few side effects is a hotspot in TNBC treatment research. Although erianin is known to have potent antitumor activity, its regulatory mechanism and target in TNBC have not been fully elucidated, hampering further drug development. This study showed that erianin can significantly inhibit TNBC cell proliferation and migration, promote cell apoptosis, and inhibit the growth of transplanted tumors in mice. Mechanistically, through network pharmacology analysis, molecular docking and cellular thermal shift assays, we preliminarily identified SRC as the cellular target of erianin. Erianin potently inhibited the expression of SRC, which mediated the anticancer effect of erianin in TNBC. Moreover, erianin can downregulate the expression of genes related to cholesterol synthesis and uptake by targeting SRC, interfering with cholesterol levels in TNBC, thereby inhibiting the progression of TNBC in vivo and in vitro. Taken together, our results suggest that erianin may inhibit the progression of TNBC by suppressing SRC-mediated cholesterol metabolism, and erianin has the great potential to be an effective treatment for TNBC patients.

Naringin as a natural candidate for anti-autoimmune hepatitis: Inhibitory potency and hepatoprotective mechanism

BackgroundAutoimmune hepatitis (AIH), primarily mediated by T cells, is characterized by liver inflammation. Despite the advancements in understanding its pathogenesis, effective therapeutic options are limited. Naringin, a flavonoid abundant in citrus fruits, is recognized for its anti-inflammatory properties and ability to protect against various inflammatory diseases, including drug-induced liver injury. However, the exact effects of naringin on AIH and the mechanisms involved remain poorly understood. PurposeWe aim to determine the role of naringin in AIH, exploring its targets and actions in this disease. MethodsNetwork pharmacology, molecular docking, and molecular dynamics simulations were utilized to predict the HUB targets connecting naringin, T cell-mediated autoimmune disorders, and AIH. Cellular thermal shift assays were used to determine the binding abilities of naringin with the HUB targets. An in vivo experiment confirmed the impact of naringin treatment on AIH development and underlying mechanisms. ResultsNaringin demonstrated therapeutic effects on ConA-induced AIH. There were 455 shared targets between naringin, T cell-mediated autoimmune diseases, and AIH. Ten HUB genes (AKT1, ALB, IL-6, IL-1β, CTNNB1, TNF, TP53, MAPK3, VEGFA, and JUN) were identified through the PPI network. Gene ontology analysis revealed involvement in gene expression regulation, lipopolysaccharide-mediated signaling, and I-kappa kinase/NFκB signaling. Pathway analysis suggested TNF, Th1/Th2 cell differentiation, and Toll-like receptor pathways, with favorable naringin-HUB gene binding. Molecular docking confirmed albumin (ALB), IL-1β, IL-6, and TNF as primary targets for naringin. Molecular dynamics simulations showed stable binding in ALB-naringin, TNF-naringin, and IL-1β-naringin complexes. Naringin's hepatoprotective effect on AIH was supported by increased serum ALB and decreased hepatic inflammatory cytokines including IL-1β, IL-6, and TNF-α. ConclusionOur data underscore the potential of naringin as a preventive or therapeutical agent in T cell-mediated autoimmune diseases including AIH.

Targeted blocking of EGFR and GLUT1 by compound H reveals a new strategy for treatment of triple-negative breast cancer and nasopharyngeal carcinoma

BackgroundCytoplasmic epidermal growth factor receptor (EGFR) is overexpressed in both nasopharyngeal carcinoma (NPC) and triple-negative breast cancer (TNBC), while clinical outcome and prognosis vary greatly among patients treated with gefitinib, and all patients eventually develop resistance to this agent. Therefore, we propose a new concept for synthesizing multitarget compounds and reveal new therapeutic strategies for NPC and TNBC expressing EGFR. MethodsCompound H was synthesized in our previous study. Molecular docking, and cell thermal shift assays (CETSAs) and drug affinity responsive target stability(DARTS) were used to confirm the binding of compound H to EGFR and GLUT1. Methylthiazolyldiphenyl-tetrazolium bromide(MTT), annexin V-PE assays, mitochondrial membrane potential (MMP) assays, and animal models were used to evaluate the inhibitory effect of compound H on TNBC cell lines. Energy metabolism tests, Western blotting, and immunofluorescence staining were performed to evaluate the synergistic effects on EGFR- and glucose transporter type 1(GLUT1)-mediated energy metabolism. ResultsCompound H can simultaneously act on the EGFR tyrosine kinase ATP-binding site and inhibit GLUT1-mediated energy metabolism, resulting in reductions in ATP, MMP, intra-cellular lactic acid, and EGFR nuclear transfer. The anti-tumor activity of compound H is significantly superior to the combination of GLUT1 inhibitor BAY876 and EGFR inhibitor gefitinib. Compound H has remarkable anti-proliferative effects on TNBC MDA-MB231 cells, and importantly, no obvious toxicity effects of compound H were found in vivo. ConclusionsSynergistic effects of inhibition of EGFR- and GLUT1-mediated energy metabolism by compound H may present a new strategy for the treatment of TNBC and NPC.

Abstract PO3-26-04: Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells

Abstract KAT6A is a histone acetyltransferase (HAT) which directs chromatin structural rearrangement and subsequent changes in gene transcription through acetylation of histones, primarily specific lysine residues on histone H3. KAT6A is modified in cancer, including genomic amplification, overexpression, mutation, as well as genetic rearrangement resulting in the production of fusion proteins. Dysregulation of this MYST family member and its acetyl transferase activity is known to drive gene expression and tumorigenic progression in cancers. An inhibitor of KAT6A and related MYST family member KAT6B has entered phase 1 clinical trials with durable partial responses seen in a variety of tumor types, including estrogen-receptor positive (ER+) breast cancer previously treated with a CDK4/6 inhibitor (Sommerhalder, ASCO2023). Evidence of dose dependent pharmacodynamics (H3K23Ac inhibition) was observed in peripheral blood mononuclear cells and paired tumor biopsies. Isosterix has developed 2 distinct series of KAT6A selective inhibitors which engage in the active site of KAT6A in the acetyl CoA substrate binding pocket and inhibit histone acetylation activity. The kinetics of target enzyme binding and inhibition will be discussed for both series of inhibitors. These compounds have been characterized for potency and cellular activity in a variety of assays. Both series of compounds inhibit KAT6A-mediated histone acetylation with single digit nM potency in a biochemical assay as well as significant potency in the breast cancer tumor cell line ZR-75-1, inhibiting H3K23 acetylation. Using a thermal shift assay (CETSA) and mass spectrometry-based protein quantitation, direct target engagement of KAT6A within cells has been shown. These compounds reduce cell viability of a number of breast cancer cell lines with 10 days of compound administration confirming the dependence of these tumor cell lines on KAT6A acetyl transferase activity for proliferation and cell cycle progression. Compounds are orally bioavailable with excellent pharmacokinetic (PK) properties in mice including half-lives >5 hours with oral administration. Finally, Isosterix inhibitors demonstrated anti-tumor efficacy with oral administration in a ZR-75-1 mouse xenograft tumor model. Inhibitors against this novel epigenetic target demonstrate the potential for efficacy in ER+ breast cancer as well as other tumor indications through suppression of KAT6A-directed transcriptional induction and chromatin remodeling. Table. Compound Kat6A Biochemical IC50 (µM) ZR-75-1 H3K23 Acetylation EC50 (µM) Mouse PK (p.o.) Mouse PK (i.v.) Inhibitor class %F T½ (h) Cl (mL/min/kg) SL-IST-001 0.007 0.01 100 5.2 0.47 Series 1 SL-IST-002 0.007 0.005 100 6 0.48 Series 2 Citation Format: Leslie Holsinger, Roopa RaI, Rahul R. Nagawade, Benoy Pal, Samir Pawar, Pankaj Singh, Krishna Putta, Mahaboobi Jaleel, Tove Bostrom, Jakob Karen, Qianyang Huang, Alison Hannah. Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-04.

Discovery and pharmacological characterization of 1,2,3,4-tetrahydroquinoline derivatives as RORγ inverse agonists against prostate cancer.

The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.

Abstract PO5-24-01: Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models

Abstract Despite improvement of outcomes for HR+, HER2- metastatic breast cancer by cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors, safety concerns and disease progression raise a critical need to identify novel approaches. Narazaciclib (ON123300), a novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens and in endometrial cancer in combination with letrozole (NCT05705505). Our study explored the activity of narazaciclib and its metabolite ON1232580 in comparisons to the FDA approved CDK4/6i and identify additional targets engaged by narazaciclib. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) and Integrative Inferred Kinase Activity (INKA) analysis were performed. Both CETSA and INKA analysis identified additional targets engaged by narazaciclib compared to palbociclib in both MDA-MB-231 lysates and intact cells such as BUB1, CHEK1, AURKA, GSK3α and GSK3β. In TNBC patients with BUB1 overexpression, bioinformatics analysis indicates a low survival correlation. Molecular docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. The differential activity of narazaciclib was explored by using cohorts of human and mouse breast cancer cell lines and cell based assays. Comparison of narazaciclib’s in vitro IC50 profile to other CDK4/6i was studied against a panel of 370 kinases (HotSpot) and Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Narazaciclib and abemaciclib were found to be the most promiscuous in vitro kinase inhibitors and ribociclib the most specific. Abemaciclib and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abemaciclib (0.08 nM), narazaciclib (0.18 nM), palbociclib (0.75 nM) and ribociclib (1.3 nM). Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. A stronger inhibition of cell proliferation, as well as induction of apoptosis and senescence were detected in narazaciclib and its metabolite ON1232580 treated PYMT cells, a murine mammary carcinoma model which recapitulates luminal BC subtype, compared to the other CDK4/6i. Both narazaciclib and ON1232580 enhanced CCL5 and CXCL10 mRNA levels, while a higher reduction of PD-L1 and pRb protein levels and a promoting effect on the H2D1 and B2M mRNA levels was shown in narazaciclib and its metabolite treated PYMT cells. Narazaciclib showed significant synergy with anti-PD1 in the EMT-6 syngeneic breast cancer model. A differential effect of narazaciclib on the expression levels of 62 cytokines was detected compared to abemaciclib and palbociclib. Lastly, inhibition of autophagy sensitizes cancer cells to both ON123300 and ON1232580, and induces irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our findings identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement. Understanding the role of the differential targets engaged by narazaciclib, the potential enhanced antitumor immunity and the sensitization by autophagy inhibitors to cell death, will guide future clinical studies. Citation Format: Petros Kechagioglou, Debomita Chakraborty, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Julianne Mooz, Mahil Lamber, Volker Dötsch, Stephen Cosenza, Steven Fruchtman, Krishnaraj Rajalingam. Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-01.

Sesamolin serves as an MYH14 inhibitor to sensitize endometrial cancer to chemotherapy and endocrine therapy via suppressing MYH9/GSK3β/β-catenin signaling

BackgroundEndometrial cancer (EC) is one of the most common gynecological cancers. Herein, we aimed to define the role of specific myosin family members in EC because this protein family is involved in the progression of various cancers.MethodsBioinformatics analyses were performed to reveal EC patients’ prognosis-associated genes in patients with EC. Furthermore, colony formation, immunofluorescence, cell counting kit 8, wound healing, and transwell assays as well as coimmunoprecipitation, cycloheximide chase, luciferase reporter, and cellular thermal shift assays were performed to functionally and mechanistically analyze human EC samples, cell lines, and a mouse model, respectively.ResultsMachine learning techniques identified MYH14, a member of the myosin family, as the prognosis-associated gene in patients with EC. Furthermore, bioinformatics analyses based on public databases showed that MYH14 was associated with EC chemoresistance. Moreover, immunohistochemistry validated MYH14 upregulation in EC cases compared with that in normal controls and confirmed that MYH14 was an independent and unfavorable prognostic indicator of EC. MYH14 impaired cell sensitivity to carboplatin, paclitaxel, and progesterone, and increased cell proliferation and metastasis in EC. The mechanistic study showed that MYH14 interacted with MYH9 and impaired GSK3β-mediated β-catenin ubiquitination and degradation, thus facilitating the Wnt/β-catenin signaling pathway and epithelial–mesenchymal transition. Sesamolin, a natural compound extracted from Sesamum indicum (L.), directly targeted MYH14 and attenuated EC progression. Additionally, the compound disrupted the interplay between MYH14 and MYH9 and repressed MYH9-regulated Wnt/β-catenin signaling. The in vivo study further verified sesamolin as a therapeutic drug without side effects.ConclusionsHerein, we identified that EC prognosis-associated MYH14 was independently responsible for poor overall survival time of patients, and it augmented EC progression by activating Wnt/β-catenin signaling. Targeting MYH14 by sesamolin, a cytotoxicity-based approach, can be applied synergistically with chemotherapy and endocrine therapy to eventually mitigate EC development. This study emphasizes MYH14 as a potential target and sesamolin as a valuable natural drug for EC therapy.Graphical

Creating a more strategic small molecule biophysical hit characterization workflow

To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.

Discovery of a SUCNR1 antagonist for potential treatment of diabetic nephropathy: In silico and in vitro studies

Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus. Succinate Receptor 1 (SUCNR1), a member of the G-protein-coupled receptor (GPCR) family, represents a potential target for treatment of DN. Here, utilizing multi-strategy in silico virtual screening methods containing AlphaFold2 modelling, molecular dynamics (MD) simulation, ligand-based pharmacophore screening, molecular docking and machine learning-based similarity clustering, we successfully identified a novel antagonist of SUCNR1, AK-968/12117473 (Cpd3). Through extensive in vitro experiments, including dual-luciferase reporter assay, cellular thermal shift assay, immunofluorescence, and western blotting, we substantiated that Cpd3 could specifically target SUCNR1, inhibit the activation of NF-κB pathway, and ameliorate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells (NRK-52E) under high glucose conditions. Further in silico simulations revealed the molecular basis of the SUCNR1-Cpd3 interaction, and the in vitro metabolic stability assay indicated favorable drug-like pharmacokinetic properties of Cpd3. This work not only successfully pinpointed Cpd3 as a specific antagonist of SUCNR1 to serve as a promising candidate in the realm of therapeutic interventions for DN, but also provides a paradigm of dry-wet combined discovery strategies for GPCR-based therapeutics.

Honokiol suppress the PD-L1 expression to improve anti-tumor immunity in lung cancer

Lung cancer is a serious health issue globally, and current treatments have proven to be inadequate. Therefore, immune checkpoint inhibitors (ICIs) that target the PD-1/PD-L1 pathway have become a viable treatment option in lun cancer. Honokiol, a lignan derived from Magnolia officinalis, has been found to possess anti-inflammatory, antioxidant, and antitumor properties. Our research found that honokiol can effectively regulate PD-L1 through network pharmacology and transcriptome analysis. Cell experiments showed that honokiol can significantly reduce PD-L1 expression in cells with high PD-L1 expression. Molecular docking, cellular thermal shift assay (CETSA) and Bio-Layer Interferometry (BLI)indicated that Honokiol can bind to PD-L1. Co-culture experiments on lung cancer cells and T cells demonstrated that honokiol mediates PD-L1 degradation, stimulates T cell activation, and facilitates T cell killing of tumor cells. Moreover, honokiol activates CD4 + and CD8 + T cell infiltration in vivo, thus suppressing tumor growth in C57BL/6 mice. In conclusion, this study has demonstrated that honokiol can inhibit the growth of lung cancer by targeting tumor cell PD-L1, suppressing PD-L1 expression, blocking the PD-1/PD-L1 pathway, and enhancing anti-tumor immunity.

Marine-derived EGFR inhibitors: novel compounds targeting breast cancer growth and drug resistance

Breast cancer (BC) continues to be a major health challenge globally, ranking as the fifth leading cause of cancer mortality among women, despite advancements in cancer detection and treatment. In this study, we identified four novel compounds from marine organisms that effectively target and inhibit the Epidermal Growth Factor Receptor (EGFR), crucial for BC cell growth and proliferation. These compounds not only induced early apoptosis through Caspase-3 activation but also showed significant inhibitory effects on EGFR mutations associated with drug resistance (T790M, L858R, and L858R/T790M), demonstrating high EGFR kinase selectivity. Cell Thermal Shift Assay (CETSA) experiments indicated that Tandyukisin stabilizes EGFR in a concentration-dependent manner. Furthermore, binding competition assays using surface plasmon resonance technology revealed that Tandyukisin and Trichoharzin bound to distinct sites on EGFR and that their combined use enhanced apoptosis in BC cells. This discovery may pave the way for developing new marine-derived EGFR inhibitors, offering a promising avenue for innovative cancer treatment strategies and addressing EGFR-mediated drug resistance.

The anti-non-small cell lung cancer effect of Diosbulbin B: targeting YY1 induced cell cycle arrest and apoptosis

BackgroundToxic components frequently exhibit unique characteristics and activities, offering ample opportunities for the advancement of anti-cancer medications. As the main hepatotoxic component of Dioscorea bulbifera L. (DB), Diosbulbin B (DIOB) has been widely studied for its anti-tumor activity at non-toxic doses. However, the effectiveness and mechanism of DIOB against non-small cell lung cancer (NSCLC) remains unclear. PurposeTo evaluate the anti-NSCLC activity of DIOB and to elucidate the specific mechanism of action. MethodThe effect of DIOB on NSCLCL in vitro was evaluated through CCK8, colony formation, and flow cytometry. The in vivo efficacy and safety of DIOB in treating NSCLC were assessed using various techniques, including HE staining, tunel staining, immunohistochemistry, and biochemical index detection. To understand the underlying mechanism, cell transfection, western blotting, molecular docking, cellular thermal shift assay (CESTA), and surface plasmon resonance (SPR) were employed for investigation. ResultsDIOB effectively hindered the progression of NSCLC both in vitro and in vivo settings at a no-observed-adverse-effect concentration (NOAEC) and a safe dosage. Specifically, DIOB induced significant G0/G1 phase arrest and apoptosis in A549, PC-9, and H1299 cells, while also notably inhibiting the growth of subcutaneous tumors in nude mice. Mechanistically, DIOB could directly interact with oncogene Yin Yang 1 (YY1) and inhibit its expression. The reduction in YY1 resulted in the triggering of the tumor suppressor P53, which induced cell cycle arrest and apoptosis in NSCLC cells by inhibiting the expression of Cyclin A2, B2, CDK1, CDK2, CDK4, BCL-2, and inducing the expression of BAX. In NSCLC cells, the induction of G0/G1 phase arrest and apoptosis by DIOB was effectively reversed when YY1 was overexpressed or P53 was knocked down. Importantly, we observed that DIOB exerted the same effect by directly influencing the expression of YY1-regulated c-Myc and BIM, particularly in the absence of P53. ConclusionFor the inaugural investigation, this research unveiled the anti-NSCLC impact of DIOB, alongside its fundamental mechanism. DIOB has demonstrated potential as a treatment agent for NSCLC due to its impressive efficacy in countering NSCLC.

Inhibition of ALDOA and ENO1‐mediated Glycolysis of Oridonin as a Novel Anti‐tumor Strategy of Non‐small Cell Lung Cancer

AbstractNon‐small cell lung cancer (NSCLC) is the most common type of lung cancer. Oridonin (ORI), the main pharmacodynamic component of the Chinese herb Herba Rabdosiae with anti‐cancer activity has been reported to inhibit various types of cancer including NSCLC. However, its direct target proteins acting on NSCLC still require further investigation. In this study, we analyze the direct protein profile and identify the key targets of ORI by combining activity‐based protein profiling (ABPP) and the cellular thermal shift assay (CETSA). As a result, ORI can significantly promote LLC cell apoptosis, decrease mitochondrial membrane potential (MMP), and increase reactive oxygen species (ROS) accumulation. In addition, both ABPP and CETSA indicate that ORI directly binds to key proteins of the glycolysis pathway, including Aldolase A (ALDOA), Enolase 1 (ENO1), Pyruvate kinase M2 (PKM2) and Lactate dehydrogenase (LDHA). Based on the LLC mouse model, ORI markedly inhibits the growth of lung tumors, increases the serum levels of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH‐Px), and reduces the production of malondialdehyde (MDA). To sum up, ORI can be regarded as a novel anti‐tumor strategy of NSCLC by inhibiting ALDOA and ENO1‐mediated glycolysis.This article is protected by copyright. All rights reserved

Glycoprotein-glycoprotein receptor binding detection using bioluminescence resonance energy transfer.

The glycoprotein receptors, members of the large G protein-coupled receptors (GPCRs) family, are characterized by a large extracellular domains responsible of binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity as these assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer (BRET) in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or luteinizing hormone) fused to the enhanced green fluorescent protein (eGFP). The Gluc-LHCGR, as well as other Gluc-GPCRs such as the somatostatin and the C-X-cytokine 4 receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric eGFP-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, BRET was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR, and show that this bioassay provides a fast and effective, safe and cost efficient tool to assist the molecular characterization of mutations in either the receptor or ligand, and it is compatible with downstream cellular assays to determine receptor activation/function.

Redesigning the kinetics of lysozyme amyloid aggregation by cephalosporin molecules.

In lysozyme amyloidosis, fibrillar aggregates of lysozyme are associated with severe renal, hepatic, and gastrointestinal manifestations, with no definite therapy. Current drugs are now being tested in amyloidosis clinical trials as aggregation inhibitors to mitigate disease progression. The tetracycline group among antimicrobials in use is in phase II of clinical trials, whereas some macrolides and cephalosporins have shown neuroprotection. In the present study, two cephalosporins, ceftazidime (CZD) and cefotaxime (CXM), and a glycopeptide, vancomycin (VNC), are evaluated for inhibition of amyloid aggregation of hen egg white lysozyme (HEWL) under two conditions (i) 4 M guanidine hydrochloride (GuHCl) at pH 6.5 and 37° C, (ii) At pH 1.5 and 65 °C. Fluorescence quench titration and molecular docking methods report that CZD, CXM, and VNC interact more strongly with the partially folded intermediates (PFI) in comparison to the protein's natural state (N). However, only CZD and CXM proficiently inhibit the aggregation. Transmission electron microscopy, tinctorial assessments, and aggregation kinetics all support oligomer-level inhibition. Transition structures in CZD-HEWL and CXM-HEWL aggregation are shown by circular dichroism (CD). On the other hand, kinetic variables and soluble fraction assays point to a localized association of monomers. Intrinsic fluorescence (IF),1-Anilino 8-naphthalene sulphonic acid, and CD demonstrate structural and conformational modifications redesigning the PFI. GuHCl-induced unfolding and differential scanning fluorimetry suggested that the PFI monomers bound to CZD and CXM exhibited partial stability. Our results present two mechanisms that function in both solution conditions, creating a novel avenue for the screening of putative inhibitors for drug repurposing. We extend our proposed mechanisms in the designing of physical inhibitors of amyloid aggregation considering shorter time frames and foolproof methods.Communicated by Ramaswamy H. Sarma.

Competitive Inhibition of Okanin against Plasmodium falciparum Tyrosyl-tRNA Synthetase.

Malaria is a severe disease that presents a significant threat to human health. As resistance to current drugs continues to increase, there is an urgent need for new antimalarial medications. Aminoacyl-tRNA synthetases (aaRSs) represent promising targets for drug development. In this study, we identified Plasmodium falciparum tyrosyl-tRNA synthetase (PfTyrRS) as a potential target for antimalarial drug development through a comparative analysis of the amino acid sequences and three-dimensional structures of human and plasmodium TyrRS, with particular emphasis on differences in key amino acids at the aminoacylation site. A total of 2141 bioactive compounds were screened using a high-throughput thermal shift assay (TSA). Okanin, known as an inhibitor of LPS-induced TLR4 expression, exhibited potent inhibitory activity against PfTyrRS, while showing limited inhibition of human TyrRS. Furthermore, bio-layer interferometry (BLI) confirmed the high affinity of okanin for PfTyrRS. Molecular dynamics (MD) simulations highlighted the stable conformation of okanin within PfTyrRS and its sustained binding to the enzyme. A molecular docking analysis revealed that okanin binds to both the tyrosine and partial ATP binding sites of the enzyme, preventing substrate binding. In addition, the compound inhibited the production of Plasmodium falciparum in the blood stage and had little cytotoxicity. Thus, okanin is a promising lead compound for the treatment of malaria caused by P. falciparum.

Biophysical and structural study of La Crosse virus endonuclease inhibition for the development of new antiviral options.

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors.

Crystal structure of human peptidylarginine deiminase type VI (PAD6) provides insights into its inactivity.

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.

Arjunolic acid ameliorates lipopolysaccharide-induced depressive behavior by inhibiting neuroinflammation via microglial SIRT1/AMPK/Notch1 signaling pathway

Ethnopharmacological relevanceNeuroinflammation is involved in the pathogenesis of depression disorder by activating microglia cells, increasing proinflammatory cytokines, effecting serotonin synthesis and metabolism, and neuronal apoptosis and neurogenesis. Arjunolic acid (ARG) is a triterpenoid derived from the fruits of Akebia trifoliata for treating psychiatric disorders in TCM clinic, which exhibits anti-inflammatory and neuroprotective effects. However, its anti-depressive effect and underlying mechanism are unknown. Aim of the studyThe aim of this study is to explore the effect of arjunolic acid on depression and its possible mechanisms. MethodsIntraperitoneal injection of LPS in mice and LPS stimulated-BV2 microglia were utilized to set up in vivo and in vitro models. Behavioral tests, H&E staining and ELISA were employed to evaluate the effect of arjunolic acid on depression. RT-qPCR, immunofluorescence, molecular docking and Western blot were performed to elucidate the molecular mechanisms. ResultsArjunolic acid dramatically ameliorated depressive behavior in LPS-induced mice. The levels of BDNF and 5-HT in the hippocampus of the mice were increased, while the number of iNOS + IBA1+ cells in the brain were decreased and Arg1+IBA1+ positive cells were increased after arjunolic acid treatment. In addition, arjunolic acid promoted the polarization of BV2 microglia from M1 to M2 type. Notably, drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and molecular docking technologies identified SIRT1 as the target of arjunolic acid. Moreover, after SIRT1 inhibition by using EX-527, the effects of arjunolic acid on ameliorating LPS-induced depressive behavior in mice and promoting M2 Microglia polarization were blocked. In addition, arjunolic acid activated AMPK and decreased Notch1 expression, however, inhibition of AMPK, the effect of arjunolic acid on the downregulation of Notch1 expression were weaken. ConclusionsThis study elucidates that arjunolic acid suppressed neuroinflammation through modulating the SIRT1/AMPK/Notch1 signaling pathway. Our study demonstrates that arjunolic acid might serve as a potiential anti-depressant.

Gambogic acid exhibits promising anticancer activity by inhibiting the pentose phosphate pathway in lung cancer mouse model

BackgroundThe pentose phosphate pathway (PPP) plays a crucial role in the material and energy metabolism in cancer cells. Targeting 6-phosphogluconate dehydrogenase (6PGD), the rate-limiting enzyme in the PPP metabolic process, to inhibit cellular metabolism is an effective anticancer strategy. In our previous study, we have preliminarily demonstrated that gambogic acid (GA) induced cancer cell death by inhibiting 6PGD and suppressing PPP at the cellular level. However, it is unclear whether GA could suppress cancer cell growth by inhibiting PPP pathway in mouse model. PurposeThis study aimed to confirm that GA as a covalent inhibitor of 6PGD protein and to validate that GA suppresses cancer cell growth by inhibiting the PPP pathway in a mouse model. MethodsCell viability was detected by CCK-8 assays as well as flow cytometry. The protein targets of GA were identified using a chemical probe and activity-based protein profiling (ABPP) technology. The target validation was performed by in-gel fluorescence assay, the Cellular Thermal Shift Assay (CETSA). A lung cancer mouse model was constructed to test the anticancer activity of GA. RNA sequencing was performed to analyze the global effect of GA on gene expression. ResultsThe chemical probe of GA exhibited high biological activity in vitro. 6PGD was identified as one of the binding proteins of GA by ABPP. Our findings revealed a direct interaction between GA and 6PGD. We also found that the anti-cancer activity of GA depended on reactive oxygen species (ROS), as evidenced by experiments on cells with 6PGD knocked down. More importantly, GA could effectively reduce the production of the two major metabolites of the PPP in lung tissue and inhibit cancer cell growth in the mouse model. Finally, RNA sequencing data suggested that GA treatment significantly regulated apoptosis and hypoxia-related physiological processes. ConclusionThese results demonstrated that GA was a covalent inhibitor of 6PGD protein. GA effectively suppressed cancer cell growth by inhibiting the PPP pathway without causing significant side effects in the mouse model. Our study provides in vivo evidence that elucidates the anticancer mechanism of GA, which involves the inhibition of 6PGD and modulation of cellular metabolic processes.