Isothermal Titration Calorimetry Analysis Research Articles

The simultaneous application of fulvic acid and protein hydrolysate biostimulants enhances cucumber responses to Fe deficiency

Iron (Fe) is widely recognized as a critical factor in limiting crop production; however, eco-friendly strategies to address its deficiency are still required. The use of biostimulants has displayed promising results in mitigating Fe deficiency. Our hypothesis was that the combined application of two biostimulants with distinct molecular structures - fulvic acid (FA) and protein hydrolysate (PH) - could be more effective than the use of a single compound. The simultaneous presence of FA and PH (MIX) in a Fe-free nutrient solution led to a redistribution of endogenous Fe, resulting in a higher leaf SPAD index. Furthermore, the addition of FeCl3 as a Fe source (resupply) in MIX-treated plants enhanced the biostimulant effect, as evidenced by increased dry root and shoot weight and a more developed root system. In addition, the expression of Strategy-I-related genes, CsFRO1 and CsIRT1, remained elevated. These effects can be attributed to improved interaction between the roots and biostimulants through the formation of the FA-PH complex, as demonstrated by circular dichroism and isothermal titration calorimetry analyses.

Formononetin Induces Ferroptosis in Activated Hepatic Stellate Cells to Attenuate Liver Fibrosis by Targeting NADPH Oxidase 4.

Ferroptosis is a newly discovered type of cell death that exerts a crucial role in hepatic fibrosis. Formononetin (FMN), a natural isoflavone compound mainly isolated from Spatholobus suberectus Dunn, shows multiple biological activities, including antioxidant, anti-inflammatory, and hepatoprotection. This research aims to explore the regulatory mechanism of FMN in liver fibrosis and the relationship between NADPH oxidase 4 (NOX4) and ferroptosis. The effects of FMN on HSC ferroptosis were evaluated in rat model of CCl4-induced hepatic fibrosis. In vitro, N-acetyl-L-cysteine (NAC) and deferoxamine (DFO) were used to block ferroptosis and then explored the anti-fibrotic effect of FMN. The target protein of FMN was identified by bio-orthogonal click chemistry reaction as well as drug affinity responsive target stability (DARTS), cellular thermal shift (CETSA), surface plasmon resonance (SPR) assays, and isothermal titration calorimetry (ITC) analysis. Here, we found that FMN exerted anti-fibrotic effects via inducing ferroptosis in activated HSCs. NAC and DFO prevented FMN-induced ferroptotic cell death and collagen reduction. Furthermore, FMN bound directly to NOX4 through possible active amino acid residues sites, and increased NOX4-based NADPH oxidase activity to enhance levels of NADP+/NADPH, thus promoting ferroptosis of activated HSCs and relieving liver fibrosis. These results demonstrate that the direct target and mechanism by which FMN improves liver fibrosis, suggesting that FMN may be a natural candidate for further development of liver fibrosis therapy.

The phytopathogen Xanthomonas campestris senses and effluxes salicylic acid via a sensor HepR and an RND family efflux pump to promote virulence in host plants.

Salicylic acid (SA) plays an essential role in plant defense against biotrophic and semi-biotrophic pathogens. Following pathogen recognition, SA biosynthesis dramatically increases at the infection site of the host plant. The manner in which pathogens sense and tolerate the onslaught of SA stress to survive in the plant following infection remains to be understood. The objective of this work was to determine how the model phytopathogen Xanthomonas campestris pv. campestris (Xcc) senses and effluxes SA during infection inside host plants. First, RNA-Seq analysis identified an SA-responsive operon Xcc4167-Xcc4171, encoding a MarR family transcription factor HepR and an RND (resistance-nodulation-cell division) family efflux pump HepABCD in Xcc. Electrophoretic mobility shift assays and DNase I footprint analysis revealed that HepR negatively regulated hepABCD expression by specifically binding to an AT-rich region of the promoter of the hepRABCD operon, Phep. Second, isothermal titration calorimetry and further genetic analysis suggest that HepR is a novel SA sensor. SA binding released HepR from its cognate promoter Phep and then induced the expression of hepABCD. Third, the RND family efflux pump HepABCD was responsible for SA efflux. The hepRABCD cluster was also involved in the regulation of culture pH and quorum sensing signal diffusible signaling factor turnover. Finally, the hepRABCD cluster was transcribed during the XC1 infection of Chinese radish and was required for the full virulence of Xcc in Chinese radish and cabbage. These findings suggest that the ability of Xcc to co-opt the plant defense signal SA to activate the multidrug efflux pump may have evolved to ensure Xcc survival and virulence in susceptible host plants.

Effects of imidazolium-based ionic liquids on the elasticity of DNA revealed by magnetic tweezers

The functions of DNA are related to its mechanical properties, including elasticity and conformational transitions. Ionic liquids, known as green solvents, are used for DNA separation and as a solvent medium for long-term DNA storage. This paper elucidated DNA interaction mechanisms with two imidazolium-based ionic liquids: 1-butyl-3-methylimi-dazolium chloride ([bmim]Cl) and 1-octyl-3-methylimidazolium chloride ([omim]Cl), via magnetic tweezers, bulk techniques and molecular dynamic simulation (MD). By stretching and twisting individual DNA molecules, we measured the elasticity of DNA in the presence of ILs ([bmim]Cl and [omim]Cl). As results, the persistence length, stretch modulus, torsional stiffness and twist-stretch coupling exhibited a significant decrease compared to the DNA without ILs binding. Isothermal titration calorimetry analysis indicated that the ILs in the presence of DNA were thermodynamically favored driven by enthalpy and entropy. DNA underwent size transition and conformational change induced by ILs, and the charges of DNA were neutralized by the added ILs. The binding of ILs induced unwinding and softening of DNA. Circular dichroism spectra and MD results indicated that DNA maintained B-conformation after interacting with ILs. Compared to [bmim]+, [omim]+ showed stronger binding affinity to DNA and tended to aggregate on the DNA surface. ILs binding reduced Na+ and water binding on DNA, which likely prevented the hydrolytic reactions that denatured DNA and helped stabilize DNA for the long term. This research provides a critical theoretical foundation for the utilization of ionic liquids in life sciences.

Structural basis for TNIP1 binding to FIP200 during mitophagy

TNIP1 has been increasingly recognized as a security check to finely adjust the rate of mitophagy by disrupting the recycling of the Unc-51-like kinase (ULK) complex during autophagosome formation. Through tank-binding kinase 1 (TBK1)-mediated phosphorylation of the TNIP1 FIR motif, the binding affinity of TNIP1 for FIP200, a component of the ULK complex, is enhanced, allowing TNIP1 to outcompete autophagy receptors. Consequently, FIP200 is released from the autophagosome, facilitating further autophagosome expansion. However, the molecular basis by which FIP200 utilizes its claw domain to distinguish the phosphorylation status of residues in the TNIP1 FIP200 interacting region (FIR) motif for recognition is not well understood. Here, we elucidated multiple crystal structures of the complex formed by the FIP200 claw domain and various phosphorylated TNIP1 FIR peptides. Structural and isothermal titration calorimetry (ITC) analyses identified the crucial residues in the FIP200 claw domain responsible for the specific recognition of phosphorylated TNIP1 FIR peptides. Additionally, utilizing structural comparison and molecular dynamics (MD) simulation data, we demonstrated that the C-terminal tail of TNIP1 peptide affected its binding to the FIP200 claw domain. Moreover, the phosphorylation of TNIP1 Ser123 enabled the peptide to effectively compete with the peptide p-CCPG1 (the FIR motif of the autophagy receptor CCPG1) for binding with the FIP200 claw domain. Overall, our work provides a comprehensive understanding of the specific recognition of phosphorylated TNIP1 by the FIP200 claw domain, marking an initial step toward fully understanding the molecular mechanism underlying the TNIP1-dependent inhibition of mitophagy.

Tetrandrine targeting SIRT5 exerts anti-melanoma properties via inducing ROS, ER stress, and blocked autophagy

Tetrandrine (TET), a natural bisbenzyl isoquinoline alkaloid extracted from Stephania tetrandra S. Moore, has diverse pharmacological effects. However, its effects on melanoma remain unclear. Cellular proliferation assays, multi-omics analyses, and xenograft models were used to determine the effect of TET on melanoma. The direct target of TET was identified using biotin-TET pull-down liquid chromatograph-mass spectrometry (LC-MS), cellular thermal shift assays, and isothermal titration calorimetry (ITC) analysis. Our findings revealed that TET treatment induced robust cellular autophagy depending on activating transcription factor 6 (ATF6)-mediated endoplasmic reticulum (ER) stress. Simultaneously, it hindered autophagic flux by inducing cytoskeletal protein depolymerization in melanoma cells. TET treatment resulted in excessive accumulation of reactive oxygen species (ROS) and simultaneously triggered mitophagy. Sirtuin 5 (SIRT5) was ultimately found to be a direct target of TET. Mechanistically, TET led to the degradation of SIRT5 via the ubiquitin (Ub)-26S proteasome system. SIRT5 knockdown induced ROS accumulation, whereas SIRT5 overexpression attenuated the TET-induced ROS accumulation and autophagy. Importantly, TET exhibited anti-cancer effects in xenograft models depending on SIRT5 expression. This study highlights the potential of TET as an antimelanoma agent that targets SIRT5. These findings provide a promising avenue for the use of TET in melanoma treatment and underscore its potential as a therapeutic candidate.

Structure-based drug design of DNA minor groove binders and evaluation of their antibacterial and anticancer properties

Antimicrobial and chemotherapy resistance are escalating medical problem of paramount importance. Yet, research for novel antimicrobial and anticancer agents remains lagging behind. With their reported medical applications, DNA minor groove binders (MGBs) are worthy of exploration. In this study, the approach of structure-based drug design was implemented to generate 11 MGB compounds including a novel class of bioactive alkyne-linked MGBs. The NCI screening protocol was utilized to evaluate the antitumor activity of the target MGBs. Furthermore, a variety of bactericidal, cytopathogenicity, MIC90, and cytotoxicity assays were carried out using these MGBs against 6 medically relevant bacteria: Salmonella enterica, Escherichia coli, Serratia marcescens, Bacillus cereus, Streptococcus pneumoniae and Streptococcus pyogenes. Moreover, molecular docking, molecular dynamic simulations, DNA melting, and isothermal titration calorimetry (ITC) analyses were utilized to explore the binding mode and interactions between the most potent MGBs and the DNA duplex d(CGACTAGTCG)2. NCI results showed that alkyne-linked MGBs (26 & 28) displayed the most significant growth inhibition among the NCI-60 panel. In addition, compounds MGB3, MGB4, MGB28, and MGB32 showed significant bactericidal effects, inhibited B. cereus and S. enterica-mediated cytopathogenicity, and exhibited low cytotoxicity. MGB28 and MGB32 demonstrated significant inhibition of S. pyogenes, whereas MGB28 notably inhibited S. marcescens and all four minor groove binders significantly inhibited B. cereus. The ability of these compounds to bind with DNA and distort its groove dimensions provides the molecular basis for the allosteric perturbation of proteins-DNA interactions by MGBs. This study shed light on the mechanism of action of MGBs and revealed the important structural features for their antitumor and antibacterial activities, which are important to guide future development of MGB derivatives as novel antibacterial and anticancer agents.

Structural characterization and mechanisms of macrophage immunomodulatory activity of a novel polysaccharide with a galactose backbone from the processed Polygonati Rhizoma

A purified polysaccharide with a galactose backbone (SPR-1, Mw 3,622 Da) was isolated from processed Polygonati Rhizoma with black beans (PRWB) and characterized its chemical properties. The backbone of SPR-1 consisted of [(4)-β-D-Galp-(1]9 → 4,6)-β-D-Galp-(1 → 4)-α-D-GalpA-(1 → 4)-α-D-GalpA-(1 → 4)-α-D-Glcp-(1 → 4,6)-α-D-Glcp-(1 → 4)-α/β-D-Glcp, with a branch chain of R1: β-D-Galp-(1 → 3)-β-D-Galp-(1→ connected to the →4,6)-β-D-Galp-(1→ via O-6, and a branch chain of R2: α-D-Glcp-(1 → 6)-α-D-Glcp-(1→ connected to the →4,6)-α-D-Glcp-(1→ via O-6. Immunomodulatory assays showed that the SPR-1 significantly activated macrophages, and increased secretion of NO and cytokines (i.e., IL-1β and TNF-α), as well as promoted the phagocytic activities of cells. Furthermore, isothermal titration calorimetry (ITC) analysis and molecular docking results indicated high-affinity binding between SPR-1 and MD2 with the equilibrium dissociation constant (KD) of 18.8 μM. It was suggested that SPR-1 activated the immune response through Toll-like receptor 4 (TLR4) signaling and downstream responses. Our research demonstrated that the SPR-1 has a promising candidate from PRWB for the TLR4 agonist to induce immune response, and also provided an easily accessible way that can be used for PR deep processing.

5-methyl-salicylaldoxime based ionic liquids for non-destructive separation of protein and cadmium

Confronting with explosive cadmium contaminations to environment, non-destructive separation of protein with cadmium for protecting food safety is hardpressed. Ionic liquids (ILs) are benign bio-compatible stabilizer of proteins with considerable metal affinity, while their application in separation of cadmium from protein is limited owing to their comparative metal affinities with protein. Accordingly, we firstly synthesize 1-methyl-3 (salicylaldoxime-5-methyl) imidazole hexafluorophosphate ([Salenmim][PF6]) to enhance cadmium extraction with hydroxyl and oxime groups as ligands of cadmium. Structure of [Salenmim][PF6] is confirmed by 1H NMR, 13C NMR, 31P NMR, 19F NMR, FT-IR, Raman spectra, and theoretical simulations. [Salenmim][PF6] can remove 85.31 % of Cd2+ from bovine serum albumin (BSA) at pH of 2.0. X-ray photoelectron spectroscopy (XPS) of BSA and [Salenmim][PF6] before and after Cd2+ extraction illustrate that Cd2+ generally bonds with C = N and O-H of [Salenmim][PF6] to achieve detachment of Cd2+ from BSA. Molecular docking of BSA and Cd2+ indicates that 4 amino acids residues listed as ASP561, ASP562, LYS563, and GLU564 coordinate with Cd2+ with coordinate number close to 1, further confirmed as 1.48 by isothermal titration calorimetry (ITC) analysis. DFT calculations of [Salenmim][PF6] with Cd2+ demonstrate that energy gap between HOMO orbital localized at benzene ring is 1.6860 eV higher than LUMO orbital, faciliating formation of [Salenmim][PF6]-Cd2+ complex. Finally, there are no changes in secondary and tertiary structure of BSA and no ILs residual observed after [Salenmim][PF6] assisted Cd2+ extraction. This work firstly reports a method for synthesis of [Salenmim][PF6] with high affinity to cadmium and systemically reveals how do structure of [Salenmim][PF6] enhance heavy metal removal from proteins, providing a possible strategy for heavy metal extraction from proteins.

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M−1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M−1. In the plot of ΔH-T*ΔS, the of Enthalpy−Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

In silico identification and molecular mechanism of novel egg white-derived tyrosinase inhibitory peptides

This study aimed to identify novel tyrosinase inhibitory peptides from egg white proteins including ovalbumin, ovotransferrin and ovomucoid. Peptides GDVA and DEK were identified using computer-aided virtual screening and in vitro tyrosinase inhibition experiments. Especially, peptide DEK exhibited the strongest tyrosinase inhibitory capacity with IC50 value of 0.205 ± 0.07 mmol L−1. Molecular docking and isothermal titration calorimetry analysis (ITC) were performed to explore the molecular mechanism. The results revealed that two peptides interacted with key residues of tyrosinase mainly by hydrogen bonding and hydrophobic interaction. The binding reactions of these peptides with tyrosinase were spontaneous, endothermic and entropy driven. Furthermore, the structure of two peptide-tyrosinase complexes was compact and stable during the molecular dynamic simulation. These findings indicated that egg white-derived peptide DEK had potential as tyrosinase inhibitory peptide.

Zwitterionic Polysulfobetaine Inhibits Cancer Cell Migration Owing to Actin Cytoskeleton Dynamics.

Cell migration is an essential dynamic process for most living cells, mainly driven by the reorganization of actin cytoskeleton. To control actin dynamics, a molecular architecture that can serve as a nucleator has been designed by polymerizing sulfobetaine methacrylate. The synthesized zwitterionic polymer, poly(sulfobetaine methacrylate) (PZI), effectively nucleates the polymerization process of G-actin and substantially accelerates the rate of polymerization. Isothermal titration calorimetry (ITC) and bioinformatics analysis indicated binding between PZI and monomeric G-actin. Thus, in vitro actin dynamics was studied by dynamic light scattering (DLS), pyrene-actin polymerization assay, and total internal reflection fluorescence microscopy (TIRFM). Furthermore, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore-containing monomeric unit was incorporated into the sulfobetaine zwitterionic architecture to visualize the effect of polymer in the cellular environment. The BODIPY-containing zwitterionic sulfobetaine polymer (PZI-F) successfully penetrated the cell and remained in the lysosome with minimal cytotoxicity. Confocal microscopy revealed the influence of this polymer on the cellular actin cytoskeleton dynamics. The PZI-F polymer was successfully able to inhibit the collective migration of the human cervical cancer cell line (HeLa cell) and breast cancer cell line (MDA-MB-231 cell), as confirmed by a wound healing assay. Therefore, polyzwitterionic sulfobetaine could be explored as an inhibitor of cancer cell migration.

ITC analysis of polydisperse systems: Unravelling the impact of sample heterogeneity

Binding interactions often involve heterogeneous samples displaying a distribution of binding sites that vary in affinity and binding enthalpy. Examples include biological samples like proteins and chemically produced samples like modified cyclodextrins. Experimental studies often ignore sample heterogeneity and treat the system as an interaction of two homogeneous species, i.e. a chemically well-defined ligand binding to one type of site. The present study explores, by simulations and experiments, the impact of heterogeneity in isothermal titration calorimetry (ITC) setups where one of the binding components is heterogeneous. It is found that the standard single-site model, based on the assumption of two homogeneous binding components, provides excellent fits to simulated ITC data when the binding free energy is normally distributed and all sites have similar binding enthalpies. In such cases, heterogeneity can easily go undetected but leads to underestimated binding constants. Heterogeneity in the binding enthalpy is a bigger problem and may result in enthalpograms of increased complexity that are likely to be misinterpreted as two-site binding or other complex binding models. Finally, it is shown that heterogeneity can account for previously observed experimental anomalies. All simulations are accessible in Google Colab for readers to experiment with the simulation parameters.

Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting main protease and papain-like protease.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50) values ranging from 1.42 to 32.7 μM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half-maximum effective concentration (EC50) values of 21.73 and 31.19 μM, respectively. The anti-inflammatory roles of azadirachtin and withanolide_A when assessed using HEK293T cells, were found to significantly reduce the levels of CXCL10, TNFα, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-α1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2-specific enzymes and also host immune pathways involved in virus-mediated inflammation.

An Analysis in Identifying the Mechanism of Action of the Alkaloids Found in Berberis vulgaris on the Treatment and Prevention of SARS-CoV-2

Introduction Berberis vulgaris extract is a well-known herbal remedy for treating numerous conditions including coronavirus disease 2019 (COVID-19). The HAB4a method of preparing the extract is a common method used in clinical practice and a further aim of this study was to identify the key alkaloid compounds that are present in an alcohol versus a glycerine-based extraction and determine the mechanism by which these prevent and treat COVID-19. Results and Discussion Identified alkaloid compounds included bervulcine, vulracine, berberrubine, lambertine, berbidine, obamegine, berbamine, berberine, and palmatine in the alcohol-based extract. The glycerine-based extract contained all except bervulcine, vulracine, and berbidine. Berberine and palmatine were the most abundant compounds in both extracts but more berberine was present in the alcohol-based extract and more palmatine in the glycerine-based extraction. Databases revealed compound-gene interactions with human targets involved in signalling and gene regulation networks that could synergistically change cell function. The SARS-CoV-2 spike 1 protein purification yielded about 5 mg of highly purified protein (˃95%) from 1 L of bacterial culture. Isothermal titration calorimetry (ITC) analysis of any potential interaction of palmatine and berberine with the purified spike 1 protein was negative. Thus, it is possible the extract components work in a synergistic manner to prevent and treat COVID-19. Conclusions Berberis vulgaris root extract alkaloid components differ when alcohol or glycerine is used. Berberine and palmatine are the most abundant alkaloids but differ in the amount relative to each other in the extracts. The extract components could affect changes in a variety of human genes. Materials and Methods Ultra-High-Performance Liquid Chromatography—High-Definition quadrupole time-of-flight Mass Spectrometry was used to identify alkaloids. Databases were used to determine the genes that interact with the identified compounds. SARS-CoV-2 spike protein was expressed and purified. Purified protein was used in ITC with berberine and palmatine.

HIF-1α inhibition by MO-2097, a novel chiral-free benzofuran targeting hnRNPA2B1

IntroductionHypoxia-inducible factor 1 (HIF-1) is a transcriptional activator mediating adaptive responses to hypoxia. It is up-regulated in the tumor microenvironment and recognized as an effective anticancer drug target. Previously, we discovered that the natural compound moracin-O and its synthetic derivative MO-460 inhibited HIF-1α via hnRNPA2B1. ObjectivesThis study aimed to develop novel HIF-1 inhibitors for cancer chemotherapy by harnessing the potential of the natural products moracins-O and P. MethodsIn an ongoing search for novel HIF-1 inhibitors, a series of nature-inspired benzofurans with modifications on the chiral rings of moracins-O and P were synthesized. They showed improved chemical tractability and were evaluated for their inhibitory activity on HIF-1α accumulation under hypoxic conditions in HeLa CCL2 cells. The most potent derivative's chemical-based toxicities, binding affinities, and in vivo anti-tumorigenic effects were evaluated. Further, we examined whether our compound, MO-2097, exhibited anticancer effects in three-dimensional cultured organoids. ResultsHerein, we identified a novel synthetic chiral-free compound, MO-2097, with reduced structural complexity and increased efficiency. MO-2097 exhibited inhibitory effects on hypoxia-induced HIF-1α accumulation in HeLa CCL2 cells via inhibition of hnRNPA2B1 protein, whose binding affinities were confirmed by isothermal titration calorimetry analysis. In addition, MO-2097 demonstrated in vivo efficacy and biocompatibility in a BALB/c mice xenograft model. The immunohistochemistry staining of MO-2097-treated tissues showed decreased expression of HIF-1α and increased levels of apoptosis marker cleaved caspase 3, confirming in vivo efficacy. Furthermore, we confirmed that MO-2097 works effectively in cancer patient-based organoid models. ConclusionMO-2097 represents a promising new generation of chemotherapeutic agents targeting HIF-1α inhibition via hnRNPA2B1, requiring further investigation.

Enhanced Ca2+ binding to EF-hands through phosphorylation of conserved serine residues activates MpRBOHB and chitin-triggered ROS production.

NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca2+ binding and phosphorylation by various protein kinases, a mechanism linking Ca2+ binding and phosphorylation in the activity regulation remained elusive. Chitin-triggered ROS production required cytosolic Ca2+ elevation and Ca2+ binding to MpRBOHB in a liverwort Marchantia polymorpha. Heterologous expression analysis of truncated variants revealed that a segment of the N-terminal cytosolic region highly conserved among land plant RBOHs encompassing the two EF-hand motifs is essential for the activation of MpRBOHB. Within the conserved regulatory domain, we have identified two Ser residues whose phosphorylation is critical for the activation in planta. Isothermal titration calorimetry analyses revealed that phosphorylation of the two Ser residues increased the Ca2+ binding affinity of MpRBOHB, while Ca2+ binding is indispensable for the activation, even if the two Ser residues are phosphorylated. Our findings shed light on a mechanism through which phosphorylation potentiates the Ca2+ -dependent activation of MpRBOHB, emphasizing the pivotal role of Ca2+ binding in mediating the Ca2+ and phosphorylation-driven activation of MpRBOHB, which is likely to represent a fundamental mechanism conserved among land plant RBOHs.

Importance of aspartate 4 in the Mg2+ dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg2+ binding. Mg2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg2+ dependent repressed state of the catalytic PGK domain at neutral pH.

Carbazole diester strapped calix[4]pyrrole: Synthesis, anion binding and self-assembly based on ion pair recognition

A new carbazole strapped calix[4]pyrrole 1 has been synthesized and ion binding properties was investigated using 1H NMR, isothermal titration calorimetry and single crystal X-ray diffraction analyses. Compared to the parent octamethyl calix[4]pyrrole 2, receptor 1 showed enhanced binding towards halide anions. Solid state structural analysis revealed that receptor 1 is capable of binding various halide salts showing different binding modes depending on the bound ion pairs. Interestingly, complexes of tetrabutyammonium (TBA+), tetraethylammonium (TEA+) and tetramethylammonium (TMA+) salts of chloride anion displayed three distinctly different binding geometries in the solid state. As a result, the linear supramolecular polymers seen in the crystal lattices are also dramatically different for these three chloride complexes of 1. Combination of 1H NMR and solid state studies revealed that binding of anion in one condition do not translates to another completely. Receptor 1 also found to form 1/1 complexes with CsCl, CsBr and CsI salts and 1D coordination polymer chains are stabilized in the solid state. Overall, construction of four different 1D linear chains are described for chloride anion based on ion pair recognition. In addition, the solid state structure of ion pair complex with CsI salt is unprecedented in the calix[4]pyrrole chemistry. Moreover, under solid-to-liquid extraction condition, receptor 1 is able to solubilise CsF and CsCl ion pairs in chloroform in which these salts are otherwise insoluble.

Conformational entropy of hyaluronic acid contributes to taste enhancement

Natural taste/flavor enhancers are essential ingredients that could potentially address condiments overconsumption. For the first time, we report that hyaluronic acid (HA) could modulate taste perception, governed by the dynamic interactions among taste compounds, mucin, and HA. Various conformations of HA impact taste perception. The high molecular weight (Mw) of 1090 kDa HA inhibits the sense of taste due to its increased viscosity, which hinders the penetration of Na+ into the mucin layer. HA with low and medium Mw (100 kDa, 400 kDa) could enhance taste perception. Isothermal titration calorimetry analysis confirms the stronger binding between mucin and HA. The intensity of their interaction increases as the Mw of HA increases from 8 kDa to 400 kDa. Quartz crystal microbalance with dissipation characterization further indicates that the rigid conformation of 100 kDa HA facilitates the binding of Na+ with taste receptors, thereby enhancing taste perception. The flexible conformation of 400 kDa HA may conceal the taste receptor cells, reducing taste enhancement. Our work advances the understanding of conformational entropy of natural mucoadhesion and mucopenetration polymers, which lays the foundation for their potential use as taste enhancers.