Target Binding Sites Research Articles

A non-canonical target-binding site in Munc18-1 domain 3b for assembling the Mint1-Munc18-1-syntaxin-1 complex

As the prototype of Sec1/Munc18 (SM) family proteins, Munc18-1 can manipulate the distinct conformations of syntaxin-1 for controlling intracellular membrane fusion. The Munc18-1-interacting domain of Mint1 (Mint1-MID) binds to Munc18-1 together with syntaxin-1 to form a Mint1-Munc18-1-syntaxin-1 complex, but the mechanism underlying the complex assembly remains unclear. Here, we determine the structure of the Mint1-MID-Munc18-1-syntaxin-1 complex. Unexpectedly, Munc18-1 recognizes Mint1-MID and syntaxin-1 simultaneously via two opposite sites. The canonical central cavity between domains 1 and 3a of Munc18-1 embraces closed syntaxin-1, whereas the non-canonical basic pocket in domain 3b captures the acidic Mint1-MID helix. The domain 3b-mediated recognition of an acidic-helical motif is distinct from other target-recognition modes of Munc18-1. Mutations in the interface between domain 3b and Mint1-MID disrupt the assembly of the Mint1-Munc18-1-syntaxin-1 complex. This work reveals a non-canonical target-binding site in Munc18-1 domain 3b for assembling the Mint1-Munc18-1-syntaxin-1 complex.

Abstract 2567: Selectivity determinants of structurally-conserved loops near the target-binding site of SH2 and SH3 domains using chimeric proteins

Abstract 2567: Selectivity determinants of structurally-conserved loops near the target-binding site of SH2 and SH3 domains using chimeric proteins

Computational Biology and Machine Learning Approaches Identify Rubber Tree (Hevea brasiliensis Muell. Arg.) Genome Encoded MicroRNAs Targeting Rubber Tree Virus 1

Tapping panel dryness (TPD), a complex physiological syndrome associated with the rubber tree (Hevea brasiliensis Muell. Arg.), causes cessation of latex drainage upon tapping and thus threatens rubber production. Rubber tree virus 1 (RTV1) is a novel positive-sense single-stranded RNA virus from the Betaflexiviridae (genus Capillovirus), which has been established to cause TPD. MicroRNAs (miRNAs) play an important role in the interplay between viruses and host cells. In this study, we identified the rubber tree genome-encoded miRNAs and their therapeutic targets against RTV1. We applied computational algorithms to predict target binding sites of rubber tree miRNAs potentially targeting RTV1 RNA genome. Mature rubber-tree miRNAs are retrieved from the miRBase database and are used for hybridization of the RTV1 genome. A total of eleven common rubber-tree miRNAs were identified based on consensus genomic positions. The consensus of four algorithms predicted the hybridization sites of the hbr-miR396a and hbr-miR398 at common genomic loci (6676 and 1840), respectively. A miRNA-regulatory network of rubber tree was constructed with the RTV1— ORFs using Circos, is illustrated to analyze therapeutic targets. Overall, this study provides the first computational evidence of the reliable miRNA–mRNA interaction between specific rubber tree miRNAs and RTV1 genomic RNA transcript. Therefore, the predicted data offer valuable evidence for the development of RTV1-resistant rubber tree in the future. Our work suggests that similar computational host miRNA prediction strategies are warranted for identification of the miRNA targets in the other viral genomes.

MiRBind: A Deep Learning Method for miRNA Binding Classification

The binding of microRNAs (miRNAs) to their target sites is a complex process, mediated by the Argonaute (Ago) family of proteins. The prediction of miRNA:target site binding is an important first step for any miRNA target prediction algorithm. To date, the potential for miRNA:target site binding is evaluated using either co-folding free energy measures or heuristic approaches, based on the identification of binding 'seeds', i.e., continuous stretches of binding corresponding to specific parts of the miRNA. The limitations of both these families of methods have produced generations of miRNA target prediction algorithms that are primarily focused on 'canonical' seed targets, even though unbiased experimental methods have shown that only approximately half of in vivo miRNA targets are 'canonical'. Herein, we present miRBind, a deep learning method and web server that can be used to accurately predict the potential of miRNA:target site binding. We trained our method using seed-agnostic experimental data and show that our method outperforms both seed-based approaches and co-fold free energy approaches. The full code for the development of miRBind and a freely accessible web server are freely available.

14-3-3 Activated Bacterial Exotoxins AexT and ExoT Share Actin and the SH2 Domains of CRK Proteins as Targets for ADP-Ribosylation.

Bacterial exotoxins with ADP-ribosyltransferase activity can be divided into distinct clades based on their domain organization. Exotoxins from several clades are known to modify actin at Arg177; but of the 14-3-3 dependent exotoxins only Aeromonas salmonicida exoenzyme T (AexT) has been reported to ADP-ribosylate actin. Given the extensive similarity among the 14-3-3 dependent exotoxins, we initiated a structural and biochemical comparison of these proteins. Structural modeling of AexT indicated a target binding site that shared homology with Pseudomonas aeruginosa Exoenzyme T (ExoT) but not with Exoenzyme S (ExoS). Biochemical analyses confirmed that the catalytic activities of both exotoxins were stimulated by agmatine, indicating that they ADP-ribosylate arginine residues in their targets. Side-by-side comparison of target protein modification showed that AexT had activity toward the SH2 domain of the Crk-like protein (CRKL), a known target for ExoT. We found that both AexT and ExoT ADP-ribosylated actin and in both cases, the modification compromised actin polymerization. Our results indicate that AexT and ExoT are functional homologs that affect cytoskeletal integrity via actin and signaling pathways to the cytoskeleton.

Ginsenoside Rg1 Inhibits High Glucose-Induced Proliferation, Migration, and Angiogenesis in Retinal Endothelial Cells by Regulating the lncRNA SNHG7/miR-2116-5p/SIRT3 Axis.

Diabetic retinopathy (DR), including retinal angiogenesis and endothelial cell proliferation and migration, is a serious complication in diabetic patients. It has been reported that ginsenoside Rg1 can prevent retinal damage. However, the mechanism by which Rg1 prevents retinal damage is unknown. Therefore, the aim of the present study was to investigate the mechanism by which Rg1 inhibits high glucose-induced complications through the regulation of the lncRNA SNHG7/miR-2116-5p/SIRT3 axis. Under high glucose (HG) conditions, human retinal endothelial cells (HRECs) were cultured to simulate a DR environment, and Rg1 was added after 48 h. Negative control (NC), miR-2116-5p mimic, si-SNHG7, pc-DNA SIRT3, and miR-2116-5p inhibitor were transfected into HRECs, and CCK-8 assay was used to detect the cell viability. Angiogenesis and transwell assays were used to evaluate angiogenesis and cell migration, respectively. qRT-PCR and Western blot were used to detect the expression of related genes and proteins. Luciferase reporter assays and bioinformatics were used to analyze the target binding sites of miR-2116-5p to lncRNA SNHG7 and SIRT3. The proliferation, migration and angiogenesis of HRECs were induced by HG. As expected, HG upregulated miR-2116-5p and VEGF expression but downregulated lncRNA SNHG7 and SIRT3 expression. Importantly, Rg1 inhibited HG-induced HREC proliferation, migration, and angiogenesis by upregulating the lncRNA SNHG7, and miR-2116-5p had a target regulatory relationship with both lncRNA SNHG7 and SIRT3. Rg1 inhibits HG-induced proliferation, migration, angiogenesis, and VEGF expression in retinal endothelial cells through the lncRNA SNG7/miR-2116-5p/SIRT3 axis. This finding provides theoretical evidence for the clinical application of Rg1 in DR.

Investigating the Micrornas as Key Regulator in Diabetic Cardiomyopathy

Diabetic Cardiomyopathy (DCM) is a state in which cardiomyopathy occurs in patients due to diabetes mellitus without valvular and coronary artery disease. The DCM prevalence is about 50% in patients diagnosed with diabetes at either clinical or preclinical stages. DCM is characterized by structural and metabolic myocardial changes established in patients with diabetes mellitus. These changes are triggered by insulin resistance and hyperinsulinemia that lead to the dysregulation of different signaling pathways. Despite the improvement in pharmacological interventions in DCM, still, new reliable strategies are required. MicroRNAs are small non-coding molecules that play a significant role in the regulation of genes at transcriptional and translational levels. We have found the clinical tested genes involved in DCM from gene target registry (GTR), and different miRNAs targeting these genes were identified by using bioinformatics tools NCBI, miRanda, TargetScan, miRBase, and TarBase. From the clinical data of DCM patients, we found that the number of genes are dysregulated which can be the putative target of miRNAs as they directly target these genes by binding with 8mer unit i.e STAT3 (having target binding site for miR-124-3p.1, miR125-5p), MAPK14 (targeted by miR128-3p, miR-124-3p.2, miR-124.3p.1), PRKAA1 (target site for miR-137, miR-19-3p, miR-148-3p), FOXO1 (target binding site for miR-135-3p, miR-96-5p), STK11 (putative target of miR17-5p/20-5p), and SOCS3 can be a potential target of miR-30-5p, miR-19-3p, miR-218-5p. Conclusively, we put forward the different miRNAs which are targeting the genes involved in DCM which may serve as a platform for the development of a miRNA-based therapeutic or diagnostic strategy for the treatment of diabetic cardiomyopathy.

Intertwined Wdr47-NTD dimer recognizes a basic-helical motif in Camsap proteins for proper central-pair microtubule formation

Calmodulin-regulated spectrin-associated proteins (Camsaps) bind to the N-terminal domain of WD40-repeat 47 (Wdr47-NTD; featured with a LisH-CTLH motif) to properly generate axonemal central-pair microtubules (CP-MTs) for the planar beat pattern of mammalian motile multicilia. The underlying molecular mechanism, however, remains unclear. Here, we determine the structures of apo-Wdr47-NTD and Wdr47-NTD in complex with a characteristic Wdr47-binding region (WBR) from Camsap3. Wdr47-NTD forms an intertwined dimer with a special cross-over region (COR) in addition to the canonical LisH and globular α-helical core (GAC). The basic WBR peptide adopts an α-helical conformation and anchors to a tailored acidic pocket embedded in the COR. Mutations in this target-binding pocket disrupt the interaction between Wdr47-NTD and Camsap3. Impairing Wdr47-Camsap interactions markedly reduces rescue effects of Wdr47 on CP-MTs and ciliary beat of Wdr47-deficient ependymal cells. Thus, Wdr47-NTD functions by recognizing a specific basic helical motif in Camsap proteins via its non-canonical COR, a target-binding site in LisH-CTLH-containing domains.

De novo design of anti-tuberculosis agents using a structure-based deep learning method

Mycobacterium tuberculosis (Mtb) is a pathogen of major concern due to its ability to withstand both first- and second-line antibiotics, leading to drug resistance. Thus, there is a critical need for identification of novel anti-tuberculosis agents targeting Mtb-specific proteins. The ceaseless search for novel antimicrobial agents to combat drug-resistant bacteria can be accelerated by the development of advanced deep learning methods, to explore both existing and uncharted regions of the chemical space. The adaptation of deep learning methods to under-explored pathogens such as Mtb is a challenging aspect, as most of the existing methods rely on the availability of sufficient target-specific ligand data to design novel small molecules with optimized bioactivity. In this work, we report the design of novel anti-tuberculosis agents targeting the Mtb chorismate mutase protein using a structure-based drug design algorithm. The structure-based deep learning method relies on the knowledge of the target protein's binding site structure alone for conditional generation of novel small molecules. The method eliminates the need for curation of a high-quality target-specific small molecule dataset, which remains a challenge even for many druggable targets, including Mtb chorismate mutase. Novel molecules are proposed, that show high complementarity to the target binding site. The graph attention model could identify the probable key binding site residues, which influenced the conditional molecule generator to design new molecules with pharmacophoric features similar to the known inhibitors.

Recognition of Aβ oligomer by LilrB2 acceptor: a tetracoordinated zipper mechanism.

Leukocyte immunoglobulin-like receptor B2 (LilrB2) is one of discovered cell surface β-amyloid (Aβ) receptors and taken as a promising therapeutic target for the treatment of Alzheimer's disease (AD). Aβ42 oligomer rather than monomer is toxic to neuronal cells and can directly bind to LilrB2, resulting in synaptic loss and cognitive impairment in the development of AD. Therefore, uncovering the mechanism of interaction between Aβ42 oligomer and LilrB2 becomes the first step to obtain a clear drug target and specific binding sites. Herein, a tetracoordinated mechanism for the Aβ oligomer-LilrB2 binding was first put forward by employing Aβ42 dimer mimic-antiparallel copies of Aβ42 core fragment 16KLVFFA21, to bind LilrB2 as models, in which four key residues (F5/F6/L12/F14) in the Aβ42 mimic are bound strongly with LilrB2 residue(s) or accommodated by four hydrophobic cavities in LilrB2 to generate a stable complex. Bi-dentate binding, however, cannot keep the complex Aβ42 mimic-LilrB2 stable. The inhibitor fluspirilene can disturb the binding of four key residues of Aβ42 to LilrB2, justifying the tetracoordinated zipper mechanism on the other hand.

Long non-coding RNA WAC antisense RNA 1 mediates hepatitis B virus replication in vitro by reinforcing miR-192-5p/ATG7-induced autophagy.

Long non-coding RNA WAC antisense RNA 1 (lncRNA WAC-AS1) is involved in the replication of the hepatitis B virus (HBV). The purpose of this study was to determine its functions and specific mechanism. The levels of lncRNA WAC-AS1, RNA (miR)-192-5p and were examined in serum of HBV-infected patients and in HepG2.2.15 cells using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. Using the database starBase, the target binding sites of lncRNA WAC-AS1 and miR-192-5p were predicted and confirmed by dual-luciferase reporter assay and RNA pull-down assay. The expression of pgRNA and HBV DNA was determined by qRT-PCR, while the levels of HBeAg and HBsAg were measured by enzymelinked immunosorbent assay (ELISA). Using laser scanning confocal microscopy, the light chain 3 (LC3) expression was analyzed. qRT-PCR and Western blotting were used to assess the expression of beclin-1, p62, and LC3I/II. Overexpression of lncRNA WAC-AS1, upregulation of ATG7. and downregulation of miR-192-5p were observed in the serum of HBV-infected patients and the in vitro model. miR-192-5p directly targets lncRNA WAC-AS1. LncRNA WAC-AS1 was downregulated in lncRNA WAC-AS1-shRNA‒transfected cells. miR-192-5p was upregulated in lncRNA WAC-AS1-shRNA-transfected cells and downregulated in cells transfected with a miR-192-5p inhibitor. In HepG2 2.15 cells, the downregulation of lncRNA WAC-AS1 inhibited HBV replication and autophagy. In contrast, the miR-192-5p inhibitor-transfected group exhibited the opposite results, and ATG7 overexpression reversed the effects of miR-192-5p mimic or lncRNA WAC-AS1-shRNA on HBV replication and cell autophagy. Our findings indicate that lncRNA WAC-AS1 regulates HBV replication by reinforcing the autophagy induced by miR-192-5p/ATG7. Consequently, lncRNA WAC-AS1 may serve as a therapeutically- promising target in HBV patients.

Photoperiods induced the circRNA differential expression in the thyroid gland of OVX+E2 ewes.

Circular RNAs (circRNAs) are non-coding RNAs newly identified and play important roles in RNA regulation. However, little is known regarding photoperiods induced circRNAs in the thyroid gland. In this study, we performed a comprehensive analysis of circRNA profiles in the thyroid gland of OVX+E2 ewes at different photoperiods by whole transcriptome sequencing. A total of 37,470 novel circRNAs were detected in different photoperiods (42 days of short photoperiod treatment, SP42; 42 days of long photoperiod treatment, LP42; SP42 transfer to LP42, SPLP42), with a total of 817 circRNAs for SP42-LP42 (down: 132; up: 114), LP42-SPLP42 (down: 136; up: 112) and SP42-SPLP42 (down: 182; up: 141) having differentially expressed. Functional enrichment annotation analysis of DE-circRNAs for GO and KEGG by R package, features that influence photoperiod response in Sunite ewes through the Inositol phosphate metabolism, cGMP-PKG signaling pathway, Calcium signaling pathway, MAPK signaling pathway, and Oocyte meiosis. In addition, competitive endogenous RNA (ceRNA) network analysis revealed target binding sites for identified miRNAs in DE-cirRNAs such as oar-miR-10b, oar-miR-200c, oar-miR-21, oar-miR-370-3p, oar-miR-377-3p, oar-miR-181a, oar-miR-432, and oar-miR-495-3p. These results of this study will provide some new information for understanding circRNA function as well as the changes in the sheep thyroid gland under different photoperiods.

Investigation of anti-microbial activity of imidazol [2, 1-B][1,3,4] thiadiazole by using molecular docking and ADMET studies

This report consists of molecular docking based on series of imidazol [2,1-b], , thiadiazole-benzimidazole derivative. Molecular docking is software which gives information about molecular modeling in which molecule fits into target binding sites and predict structure of intermolecular complex. These molecules were investigated by protein ligand binding score, protein ligand interaction and ADME studies. All the target molecules were analyzed against which is a gram positive bacteria found on skin and upper respiratory tract. The protein molecule selected for the analysis was PDB code 4LAE protein ligand. Basically it is a oxidoreductase inhibitor and its structure is based on 7(benzimidazole-1-yl)-2, 4-diaminoquinazolines. Out of all twenty nine compounds five compounds (5B,5G,5H,5N and 5Q) were estimated as most potent molecules as antibacterial agent.

Predicting target binding sites in plant immune receptors

Predicting target binding sites in plant immune receptors

Extensive Study of DFT-Quantum Calculations Based QSAR Modeling of Fused 1,2,4-Triazine Derivatives Revealed Potent CYP1A1 Inhibitors

In a continuing effort for our research group, we identify new active 1,2,4-triazine derivatives as novel cancer preventive agents that target CYP1A1 activity, through the application of quantitative structure–activity relationship (QSAR) theory. For this purpose, novel fused 1,2,4-triazine derivatives, namely, 4-Amino-6-substituted benzyl-3-(2-substituted benzylidenehydrazinyl)-1,2,4-triazin-5(4H)-ones and acetylated 6-substituted benzyl-7-oxo-3-substituted phenyl-2,3-dihydro-7[Formula: see text]-[1,2,4]triazolo[4,3-[Formula: see text]][1,2,4]triazine analogs were utilized for the prediction of the QSAR model. Furthermore, the application of this QSAR equation served successfully as a rational guide for predicting the activities of newly synthesized analogs. The DFT calculation was applied to recognize the physicochemical parameters at B3LYP/6- 311G* level to gain a clear view of global and local molecular reactivity. Besides, the molecular features of the investigated compounds were identified through HOMO, LUMO and molecular electrostatic potential (MEP) which were plotted to determine the charge transfer within the molecules. Promising two novel 1,2,4-triazine compounds were identified with observed significant inhibitions (83% and 67%) and CYP1A1 activity was predicted. The chemical configuration of the two actives showed good alignment of [Formula: see text]-methoxy benzyl aminotriazinone fragments, hydrazino and carbonyl linker in structure–activity relationship and interaction. Molecular modeling analysis supported with MD simulations was undertaken to investigate different binding interactions with the target binding site. These projects offered a good application of a promising QSAR model for prediction of highly active compounds for further lead optimization.

Structural and Dynamics Studies of the Spcas9 Variant Provide Insights into the Regulatory Role of the REC1 Domain

CRISPR-Cas9 from Streptococcus pyogenes has been harnessed as a powerful biotechnological tool for DNA sequence modification. The gRNA binding the recognition lobe (REC lobe) of Cas9 is the initial step of Cas9-mediated DNA cleavage and is a prerequisite to trigger the subsequent conformational transition of Cas9. The functions of REC1–3 domains have been summarized as “sense” nucleic acids, “regulate” the HNH conformational transition, and finally “lock” the HNH domain at the cleavage site. Therefore, understanding the dynamic mechanism of the REC lobe is important to design and engineer better Cas9 enzymes. Here, we solved the apo form structure of a SpCas9 variant (xCas9 P411T) that harbors seven mutations in the SpCas9 REC region and one mutation in the C-terminal domain. This structure reveals that the mutations in the REC lobe resulted in a distinct conformation of the REC1 domain. Together with the biochemical results, we hypothesized that the dynamic properties of the REC1 domain are associated with Cas9 PAM specificity. Furthermore, using molecular dynamics simulations to model the conformational activation process of xCas9 P411T, we found that REC1 acts as a hub that modulates multiple domain motion during Cas9 activation and target site binding. These results lay the foundation for engineering an improved CRISPR-Cas9 system.

Quantitative structure–activity relationship modeling and docking of some synthesized bioactive oxopyrolidines against Staphylococcus aureus

Quantitative correlations of chemical compounds' biological activity (response variables) with their molecular structures are known as QSARs (independent variables). The molecular descriptors account for a molecule's conformational, constitutional, thermodynamic, steric effects, and electronic characteristics. Molecular docking is one of the most commonly used strategies in structure-based drug design because of its ability to predict the binding-conformation of small-molecule ligands to the proper target binding site. The study of binding behaviour aids in the rational design of therapeutics at the molecular level. A series of novel bioactive oxopyrrolidine derivatives were reported to be synthesized from benzimidazole, pyrrolidinone, pyrrole, 1,2,4-triazole, thiazole, and oxadiazole with 1-(3-(1,3-thiazol-2-yl)phenyl)-5-oxopyrrolidines substituents. Consequently, oxopyrrolidines used possess thiazole ring obtained from reaction of either benzenecarbothioamide, tioureido acid or thiocarbamide with 1-(4-(bromoacetyl)phenyl)-5-oxopyrrolidine-3-carboxylic acid heterocyclic fragments. It was further compared with control Oxytetracycline in which quinoxaline containing fragment showed the more marked effect than of the used control antibiotic. Therefore, this study aimed to build a robust QSAR model to predict the anti-bacterial activity of some oxopyrolidines compounds against Staphylococcus aureus and carry out molecular docking studies between the compounds of the tyrosyltRNAsynthethase receptor. Based on the obtained values of the validation parameters, such as R2 (0.9049), R2adj (0.8789), and Q2cv(0.8365), necessary to evaluate the strength and robustness of the QSAR model, the developed QSAR model was deemed to be statistically significant. Some compounds were chosen and docked with the Staphylococcustyrosyl tRNA synthethase receptor, and the results suggest that C3, C7, C12, C17, C18, and C19 with free binding energies (FBE) −10.0 kcal/mol, −9.6 kcal/mol, −10.2 kcal/mol, −10.1 kcal/mol, −10.6 kcal/mol and −9.7 kcal/mol respectively were better than the standard tyrosyltRNAsynthethase inhibitor (Linezolid, FBE = −8.3 kcal mol−1). Furthermore, these compounds met the drug-likeness criteria and were orally bioavailable. The six compounds' modeling, docking, and drug similarity features broaden the scope of novel derivative design, manufacturing, and testing against Staphylococcus aureus.

Abstract 4259: PT199, a next generation anti-CD73 mAb that inhibits both membrane-bound and soluble CD73 activity to completion without “hook effect”

Abstract CD73, also known as ecto-5’-nucleotidase, is implicated in promoting immunosuppressive tumor microenvironment (TME) through production of adenosine. The first generation of anti-CD73 therapeutic antibodies currently in clinical studies have largely exhibited subdued clinical activities mainly due to several unfavorable characteristics such as incomplete enzyme inhibition and/or “hook effect”. These weaknesses are caused by the unfavorable target binding sites and enzyme inhibition mechanisms, which explains why these molecules do not inhibit soluble CD73 enzyme activity, or why the enzyme inhibition depends on certain stochiometric ratios of CD73 and the antibody and exhibits “hook effect” in dose-response curves. Here we report a next generation humanized monoclonal antibody against human CD73 (hCD73), PT199, which binds to a unique site on human CD73 that confers a different mechanism of enzyme inhibition and consequently does not have the flaws associated with the first generation of anti-CD73 mAbs currently in clinic. By directly binding to an epitope that is in or near the CD73 catalytic domain, PT199 potently inhibits both soluble shed CD73 and membrane-bound CD73 activity to completion without “hook effect”. Enzyme kinetic studies demonstrate that PT199 inhibits CD73 enzyme activity in a noncompetitive manner, allowing complete CD73 inhibition even at high AMP concentrations reported in TME. In a cell-based functional assay, PT199 inhibition of CD73 alleviates the immunosuppression effect of AMP on T cell proliferation. Compared with the first-generation molecules, PT199 completely inhibits CD73 activity in cancer patient serum. Furthermore, when administrated to hCD73 knock-in mice, PT199 greatly elevates endogenous AMP level, demonstrating a robust pharmacodynamic effect in blocking the conversion of AMP to adenosine. Comparative mechanistic studies indicate that PT199 binds to both the “open conformation” (inactive enzyme state) and the “closed conformation” (active enzyme state) with high affinity but with shifted binding curves, suggesting that PT199 may inhibit the CD73 enzyme activity by preventing the transition of the enzyme from the “open conformation” to the “closed conformation”. PT199 did not cause B cell reduction in either acute or repeated dosing studies in NHP. In NHP toxicology studies, PT199 exhibits no clinical observations at up to 300 mg/kg weekly repeated dosing and has an excellent PK profile typical of mAbs. Taken together, our data confirms PT199 as a potential best-in-class anti-hCD73 mAb. Phase 1 clinical trial is planned to start in the first quarter of 2022. Citation Format: Haiqun (John) Jia, Jack Li, Fen Pei, Lorraine Greenwood, Lauren Pejza, Yan Long, Kelan Chen, Jessica Perer, Ming Wang, Hui Zou. PT199, a next generation anti-CD73 mAb that inhibits both membrane-bound and soluble CD73 activity to completion without “hook effect” [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4259.

LncRNA PVT1 Regulates miR-1207-5p to Affect Colon Cancer Proliferation and Migration via the Wnt6/β-catenin2 Pathway.

Background: We aimed to evaluate the effects of lncRNA PTV1 on colon cancer proliferation and migration via the Wnt6/β-catenin2 pathway. Materials and Methods: A total of 117 colon cancer and normal adjacent tissue samples were collected. LncRNA PVT1 and miR-1207-5p expressions in these samples and colon cancer cell lines were detected by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). LncRNA PVT1-silencing cells and miR-1207-5p-overexpressing Caco-2-siPVT1 cells were constructed, respectively. The effects of lncRNA PVT1 silencing on cell proliferation were assessed by MTT and colony formation assays. The effects on invasion and migration were tested by Transwell and scratch assays respectively. The targeting regulatory relationship between miR-1207-5p and Wnt6 was analyzed by a dual-luciferase reporter assay. The relationship between lncRNA PVT1 and miR-1207-5p was studied by RNA-binding protein immunoprecipitation and RNA pull-down assays. The expressions of proteins in the Wnt6/β-catenin2 pathway were detected by Western blotting. Results: The lncRNA PVT1 mRNA expression in colon cancer tissue was significantly higher than that in normal adjacent tissue (p < 0.05). The expression in lncRNA PVT1-silencing cells was significantly down-regulated (p < 0.05). The colonies of Caco-2-siPVT1 cells decreased, accompanied by a reduced number of cells penetrating Matrigel and migration (p < 0.05). Compared with siPVT1 + NC group, the number of colonies and migration of siPVT1 + miR-1207-5p-overexpressing group increased significantly (p < 0.05). There was a targeting relationship between miR-1207-5p and PVT1. MiR-1207-5p had a targeted binding site with Wnt6. The protein expressions of Wnt6/β-catenin2 in Caco-2-siPVT1 group were significantly lower than those of control and Caco-2-siNC groups (p < 0.05). Conclusion: LncRNA PVT1 was highly expressed in colon cancer. It may enhance the proliferation and migration of colon cancer cells by up-regulating miR-1207-5p level and enhancing the Wnt6/β-catenin2 pathway.

Quercetin improves contrast-induced acute kidney injury through the HIF-1α/lncRNA NEAT1/HMGB1 pathway

Context The risk of contrast-induced acute kidney injury (CI-AKI) is increasing and the harm is great. Quercetin is the main active component in Abelmoschus manihot (L.) Medik (Malvaceae) and was reported to reduce the expression of HIF-1α. Objective We investigate whether quercetin improves the CI-AKI through the HIF-1α/lncRNA NEAT1/HMGB1 pathway. Materials and methods HK-2 cells were treated with iohexol (200 mg/mL) for 6 h to establish a CI-AKI model. Quercetin (20 μM) was administered to CI-AKI cells cultured in dishes for 24 h. Cell morphology was observed by a fluorescence microscope. MTT and TUNEL assays were used to detect cell survival rate and apoptosis. Relative mRNA levels were measured by qRT-PCR. Protein levels were detected using western blotting. IL-6 and TNF-α protein levels were tested by Elisa assay. Targeting binding sites of HIF-1α and lncRNA NEAT1 were detected by luciferase assay. Results The IC50 value of quercetin was 163.25 μM. The expression levels of HIF-1α, lncRNA NEAT1 and HMGB1 were upregulated in the CI-AKI cell model. Quercetin diminished cell injury and apoptosis via inhibiting HIF-1α. Silencing of HIF-1α targeting lncRNA MEAT1 diminished cell injury and apoptosis. Silencing lncRNA NEAT1 has the same effect via suppressing HMGB1 expression. Collectively, quercetin diminished cell injury and apoptosis in CI-AKI cell model via the inhibition of HIF-1α on lncRNA NEAT1/HMGB1 signalling pathway. Discussion and conclusions Quercetin diminished cell injury and apoptosis in CI-AKI cell mode via the inhibition of HIF-1α on the lncRNA NEAT1/HMGB1 signalling pathway, offering a potential novel therapeutic target for CI-AKI therapy.