Binding Studies Research Articles

Diorganotin(IV) derivatives of ONO tridentate Schiff bases: Synthesis, spectroscopic characterization, DFT studies, CT-DNA binding, and plasmid-DNA cleavage studies

Two Schiff bases 2-amino-N'-(5‑bromo-2-hydroxybenzylidene)-3-(4-hydroxyphenyl)propanehydrazide (H2L1) and 2-amino-N'-(5‑chloro-2-hydroxybenzylidene)-3-(4-hydroxyphenyl)propanehydrazide (H2L2) and their six diorganotin(IV) complexes (R = C4H9 (1, 4), CH3 (2, 5), and C8H17 (3, 6)) have been synthesized. Schiff bases (H2L1–2) and their R2SnL1–2 complexes (1–6) have been characterized by CHNS analysis, IR spectroscopy, 1H, 13C, 119Sn NMR, and ES-MS. Schiff bases (H2L1–2) act as a dianionic tridentate ligand coordinated to Sn through phenolic and enolic oxygen and azomethine nitrogen in R2SnL1–2 complexes (1–6). A density functional theory (DFT)-based quantum chemical calculation validated the structures of R2SnL1 (1–3) and R2SnL2 (4–6) at the B3LYP/6‐311G(df,pd)/Def2‐SVP(Sn) and B3LYP/6‐31G(d,p)/Def2‐SVP(Sn) levels of theory, respectively. At these levels of theories, comprehensive electronic structure calculations determined atomic charges at selected atoms. Additionally, a molecular electrostatic potential (MEP) map identified electrostatic potential-varying sites on the molecule's surface, and MEP maps used to identify regions where other molecules might interact or react with the molecules on them. Furthermore, a conceptual-DFT-based global reactivity descriptor determined the stability and reactivity behaviour of R2SnL1–2 complexes (1–6). H2L1–2 and R2SnL1–2 complexes (1–6) have binding constants in the range ∼105 M-1 with CT-DNA, as revealed by UV–visible and fluorescence spectral titrations. UV-visible, fluorescence spectroscopy, and CD studies suggested that H2L1–2 and their R2SnL1–2 complexes (1–6) interact with CT-DNA electrostatically or groove-bound. DNA cleavage studies reveal that H2L1–2 and their R2SnL1–2 complexes (1–6) have ability to cleave the plasmid-DNA from its supercoiled form (SC, I) to nicked form (NC, II). However, n-Bu2SnL2 (4) and n-Oct2SnL2 complex (6) have higher cleavage activity than Schiff bases and other R2SnL1–2 complexes (1–6).

Highly efficient and selective anticancer approach through acrolein-triggered cycloaddition chemistry in patient-derived xenografts: Mechanistic and preclinical investigation

The efficacy of anticancer treatments in the clinical setting can be improved by decreasing off-target effects. Chemistry-based methods are emerging as a promising strategy to overcome this limitation. We previously developed a prodrug strategy using mitomycin C (MMC) activated at the tumor site by the [3 + 2] cycloaddition of endogenous acrolein, which is overexpressed in various cancers. Herein, we report the results of mechanistic and preclinical studies using a prodrug of doxorubicin (DOX), which is used in the treatment of several cancers, but its dosage is restricted by off-target effects. First, we developed robust chemistry methods for the synthesis of large amounts of prodrug for animal experiments. The DOX prodrug showed high anticancer efficacy without off-target effects even at high doses. Pharmacokinetic/pharmacodynamic, biodistribution, and serum protein binding studies supported the in vivo anticancer efficacy of DOX derivatization with the hydrophobic 2,6-diisopropyl azidobenzylcarbamate protecting group. This enhanced albumin binding, thereby increasing circulatory residence (serum stability). The stabilized prodrug could only be activated at the tumor site by reacting with endogenous acrolein to gradually release the required amounts of drug. Inhibiting overproduction of the drug and its circulation back into the blood minimized off-target effects. Nonspecific organ accumulation was not observed, supporting the safety of the prodrug in vivo. DOX and MMC prodrugs showed selective anticancer efficacy against patient-derived samples from lung, colorectal, gastric, and breast cancers. The proposed in vivo chemical strategy should be tested in clinical trials in the future.

Arsenic Induced Oxidative Neural-Damages in Rat are Mitigated by Tea-Leave Extract via MMPs and AChE Inactivation, Shown by Molecular Docking and in Vitro Studies with Pure Theaflavin and AChE.

Chronic arsenic-exposure causes neuromuscular disorders and other health anomalies. Damage to DNA and cytoskeletal/extracellular matrix is brought on by reactive-oxygen-species (ROS)-induced intrinsic antioxidant depletion (thiols/urate). Therapeutic chelating-agents have multiple side-effects. The protection of (Camellia sinensis) tea-extract and role of uric-acid (UA) or allopurinol (urate-depletor) on arsenic-toxicity were verified in rat model. Camellia sinensis (CS dry-leaves), UA or allopurinol was supplemented to arsenic-intoxicated rats for 4-weeks. Purified theaflavins and their galloyl-ester were tested in-vitro on pure AChE (acetylcholinesterase) and their PDB/PubChem 3-D structures were utilized for in-silico binding studies. The primary chemical components were evaluated from CS-extracts. Biochemical analysis, PAGE-zymogram, DNA-stability comet analysis, HE-staining was performed in arsenic-exposed rat brain tissues. Animals exposed to arsenic showed symptoms of erratic locomotion, decreased intrinsic antioxidants (catalase/SOD1/uric acid), increased AChE, and malondialdehyde. Cerebellar and cerebrum tissue damages were shown with increased levels of matrix-metalloprotease (MMP2/9) and DNA damage (comets). Allopurinol- supplemented group demonstrated somewhat similar biochemical responses. In the CS-group brain tissues especially cerebellum is considerably protected which is evident from endogenous antioxidant and DNA and cytoskeleton protection with concomitant inactivation of MMPs and AChE. Present study indicates theaflavin-digallate (TFDG) demonstrated the highest inhibition of purified AChE (IC50 = 2.19 µg/ml with the lowest binding free-energy; -369.87 kcal/mol) followed by TFMG (IC50 = 3.86 µg/ml, -347.06 kcal/mol) suggesting their possible restoring effects of cholinergic response. Favorable responses in UA-group and adverse outcome in allo-group justify the neuro-protective effects of UA as an endogenous antioxidant. Role of flavon-gallate in neuro protection mechanism may be further studied.

DNA Interaction, Molecular Docking, Antimicrobial, Anticancer and Thermal Studies of Ternary Metal Complexes of N‐Methylbenzylamine and Ethylenediamine

ABSTRACTN‐Methylbenzylamine and ethylenediamine were utilized as primary and auxiliary ligands, respectively, alongside metal salts to synthesize ternary metal complexes: [FeCl3(Nmba)(en)], [CoCl2(Nmba)(en)(H2O)] and [CuCl2(Nmba)(en)(H2O)]. These complexes were thoroughly characterized using spectral and analytical techniques, revealing an octahedral geometry for all. Coats–Redfern calculations indicated their non‐spontaneous nature yet highlighted their thermal stability. DNA binding studies unveiled a groove binding mode for the complexes, with intrinsic binding constants (Kb) and Stern–Volmer quenching constant (Ksq) supporting their strong binding capabilities. Nuclease activity against pBR322 was assessed through gel electrophoresis. Additionally, docking studies using AutoDock 4.2 software provided insights into their binding affinities. In terms of biological activities, the Cu complex demonstrated superior cytotoxicity and antibacterial and antifungal properties compared to the other ternary metal complexes. This suggests its potential for further exploration in biomedical applications.

DNA binding studies and in-vitro anticancer studies of novel lanthanide complexes

Pancreatic cancer, is an aggressive type of cancer and the most common malignancy with a poor prognosis regarding metastatic disease (survival < 10 %). The development of Novel chemotherapeutic drugs holds significant prospects for practical applications. Here, this work focuses on the interaction between two lanthanide complexes, Yb-BZA and Er-BZA, with DNA, as well as their anticancer activity against pancreatic cancer. The relationship between complexes and DNA is revealed by fluorescence, absorption spectral titration, cyclic voltammetric (CV) experiments, indicating that the Yb-BZA and Er-BZA interact with FS-DNA by bind groove. Moreover, molecular docking technology was utilized to confirm the binding of Yb-BZA and Er-BZA with 1BNA and 4AV1. The cytotoxic effects of Yb-BZA and Er-BZA on cancer cells BxPC-3 were evaluated, Yb-BZA (IC50 = 6.459 μg/mL) is more effective than oxaliplatin (IC50 = 16.46 μg/mL) evaluated using cytotoxicity assay. Yb-BZA and Er-BZA has the potential to become a chemotherapy drug for pancreatic cancer cells.

The Development and Application of Tritium-Labeled Compounds in Biomedical Research.

With low background radiation, tritiate compounds exclusively emit intense beta particles without structural changes. This makes them a useful tool in the drug discovery arsenal. Thanks to the recent rapid progress in tritium chemistry, the preparation and analysis of tritium-labeled compounds are now much easier, simpler, and cheaper. Pharmacokinetics, autoradiography, and protein binding studies have been much more efficient with the employment of tritium-labeled compounds. This review provides a comprehensive overview of tritium-labeled compounds regarding their properties, synthesis strategies, and applications.

Metadynamics Study of Lipid-Mediated Antibacterial Toxin Binding to the EmrE Multiefflux Protein.

EmrE is a bacterial efflux protein in the small multidrug-resistant (SMR) family present in Escherichia coli. Due to its small size, 110 residues in each dimer subunit, it is an ideal model system to study ligand-protein-membrane interactions. Here in our work, we have calculated the free energy landscape of benzyltrimetylammonium (BTMA) and tetraphenyl phosphonium (TPP) binding to EmrE using the enhanced sampling method-multiple walker metadynamics. We estimate that the free energy of BTMA binding to EmrE is -21.2 ± 3.3 kJ/mol and for TPP is -43.6 ± 3.8 kJ/mol. BTMA passes through two metastable states to reach the binding pocket, while TPP has a more complex binding landscape with four metastable states and one main binding site. Our simulations show that the ligands interact with the membrane lipids at a distance 1 nm away from the binding site which forms a broad local minimum, consistent for both BTMA and TPP. This site can be an alternate entry point for ligands to partition from the membrane into the protein, especially for bulky and/or branched ligands. We also observed the membrane lipid and C-terminal 110HisA form salt-bridge interactions with the helix-1 residue 22LysB. Our free energy estimates and clusters are in close agreement with experimental data and give us an atomistic view of the ligand-protein-lipid interactions. Understanding the binding pathway of these ligands can guide us in future design of ligands that can alter or halt the function of EmrE.

Design and synthesis of two new thiosemicarbazide based Schiff base metal complexes of nickel (II): DNA binding study and cytotoxicity profile analysis

Two new nickel (II) substituted thiosemicarbazone Schiff base complexes [Ni(meph)2] (1) [where H2meph = (2E)-N-methyl-2-[1-(pyridin-2-yl)ethylidene]hydrazine-1-carbothioamide] and [Ni(hmm)2](NO3)2·2H2O (2) [where H2hmm = (2E)-2-[(2-hydroxyphenyl)methylidene]-N-methylhydrazine-1-carbothioamide] have been designed and synthesized by the condensation of 4-methyl-3-thiosemicarbazide with 2-acetylpyridine and salicylaldehyde respectively. Both the metal complexes 1 and 2 are characterized using different available spectroscopic techniques like FT-IR, UV–Vis spectroscopy, elemental analysis, and single crystal X-ray structure analysis. X-ray crystal structure analysis reveal that complex 1 and 2 are octahedral Ni(II) complexes. The calf-thymus CT-DNA-binding property of 1 and 2 has been evaluated by employing UV–Vis and fluorescence spectral titration. All the results show that CT-DNA binds with both nickel(II) complexes 1 and 2. In vitro cytotoxicity activity of complexes 1 and 2 toward A375 and MDA-MB-231 was evaluated using MTT assay and other methods which confirm that both complexes 1 and 2 behave as promising anti-cancer agents.

Inclusion complexes of novel formyl chromone Schiff bases with β-Cyclodextrin: Synthesis, characterization, DNA binding studies and in-vitro release study

The present study involved the synthesis of five novel Schiff bases (SB1-SB5) of formyl chromone and their inclusion complexes with β-cyclodextrin through kneading approach to enhance the solubility and stability of SBs. Characterization was conducted using FTIR, NMR, SEM, TEM, p-XRD, and Mass Spectrometry. UV fluorescence and pH stability studies confirmed the formation of the inclusion complex. Structural validation of complexes was conducted via molecular docking (PDB ID: 1BFN) and 50 ns MD simulation study. DFT studies were performed on SBs using B3LYP/6–31 + G(d,p) basis set. All SBs exhibited favorable ADME properties and high binding interactions were observed in molecular docking with ctDNA (PDB Id: 1BNA). Further, in-vitro UV absorption and fluorescence experiments demonstrated strong ctDNA interactions for all Schiff bases, with binding constants in the order of 105 M−1, indicating groove binding mode. Among the SBs, SB4 exhibited the highest affinity for DNA grooves, with a binding constant (Kb) of 1.7 × 106 M−1. However, the SB4/β-Cyd inclusion complex also interacted with DNA but with low binding constants compared to SB4. An in-vitro release study of SB4/β-Cyd, revealed 78.92 % dissolution of the inclusion complex, highlighting its potential for enhanced solubility and stability in biological systems.

Biosynthesis of silver nanoparticles using green tea aqueous leaf extract and their biological and chemotherapeutic activity

Green tea leaf extract-mediated silver nanoparticles (GT-AgNPs) were synthesized and characterized for their biological and chemotherapeutic activity. The synthesis process involved the reduction of silver ions by green tea leaf extract (GTLE), resulting in the formation of GT-AgNPs. Characterization techniques including UV–Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), and zeta potential analysis were employed to study the physicochemical properties of GT-AgNPs. The GT-AgNPs exhibited a characteristic color change and a noticeable shift in the surface plasma resonance (SPR) absorption peak at 458 nm, confirming their successful formation of nanoparticles. XRD analysis indicated a cubic structure with a crystallite size of 9 ± 1 nm. Both SEM and TEM images revealed that the spherical shape and size of the synthesized nanoparticles. The SEM analysis confirmed that the average grain size of GT-AgNPs is 20.27 ± 1 nm, while the TEM analysis determined the average particle size of 9 ± 0.025. GT-AgNPs have a negative zeta potential of -19.7 mV indicating a hydrodynamic diameter of 140.90 nm with a polydispersity index (PDI) of 25.8 %. XPS confirmed the oxidation state of Ag0/+1 in GT-AgNPs. The energy band gap (Eg) value of GT-AgNPs is 3.38 eV, indicating semiconducting properties, while fluorescence activity was observed at 506.17 nm. CT-DNA and BSA protein binding studies demonstrated the formation of CT-DNA-GT-AgNPs and BSA-GT-AgNPs complexes with binding constants of 1.366×1010 M−1 and 6.77×109 M−1, respectively. GT-AgNPs exhibited exceptional anti-oxidant activity by effectively scavenging stable 2, 2ʹ - diphenyl-1-picryl hydroxyl (DPPH) radicals. Cytotoxicity assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) demonstrate moderate activity against KB3 and AGS cancer cells while showing no detectable cytotoxicity in Caco-2 cells. This indicates a safe therapeutic window for human eukaryotic cells.

Rational design of 2-benzylsulfinyl-benzoxazoles as potent and selective indoleamine 2,3-dioxygenase 1 inhibitors to combat inflammation

Mimicking the transition state of tryptophan (Trp) and O2 in the enzymatic reaction is an effective approach to design indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors. In this study, we firstly assembled a small library of 2-substituted benzo-fused five membered heterocycles and found 2-sulfinyl-benzoxazoles with interesting IDO1 inhibitory activities. Next the inhibitory activity toward IDO1 was gradually improved. Several benzoxazoles showed potent IDO1 inhibitory activity with IC50 of 82–91 nM, and exhibited selectivity between IDO1 and tryptophan 2,3-dioxygenase (TDO2). Enzyme binding studies showed that benzoxazoles are reversible type II IDO1 inhibitors, and modeling studies suggested that the oxygen atom of the sulfoxide in benzoxazoles interacts with the iron atom of the heme group, which mimics the transition state of Fe-O-O-Trp complex. Especially, 10b can effectively inhibit the NO production in lipopolysaccharides (LPS) stimulated RAW264.7 cells, and it also shows good anti-inflammation effect on mice acute inflammation model of croton oil induced ear edema.

A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.

Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay. We demonstrate the capabilities of PPI-CONA by detecting low affinity ternary complex formation between the human CDC34A ubiquitin-conjugating (E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the CDC34A-ubiquitin interaction. We further exemplify PPI-CONA with an E2 enzyme binding study on CC0651 and a CDC34A binding specificity study of a series of CC0651 analogues. Our results indicate that CC0651 is highly selective toward CDC34A. We further demonstrate how PPI-CONA can be applied to screening very low affinity interactions. PPI-CONA holds potential for high-throughput screening for modulators of PPI targets and characterization of their affinity, specificity, and selectivity.

Investigating Active Site Binding of Ligands to High and Low Activity Carbonic Anhydrase Enzymes Using Native Mass Spectrometry.

Carbonic anhydrases (CAs) are a family of enzymes that play an important pH regulatory role in health and disease. While different CA isozymes have a high degree of structural similarity, they have variable enzymatic activity, with CA III being the least active and having less than 1% of the activity of CA II, the most active. Furthermore, ligand binding studies for CA III are limited, and a resulting lack of chemical probes impedes understanding of this CA isozyme in comparison to other CA family members where studies are abundant. Therefore, we employed native mass spectrometry (nMS), also known as intact mass spectrometry, to assess ligand binding to CA II and CA III and discovered two novel compounds that for the first time display strong binding to CA III. We present a new data visualization and quantification tool developed to display native mass spectra as an intuitive stacked heat map representation for rapidly interpreting the results of ligand-protein binding from nMS screening.

Galactose Glycopolymer-Grafted Silica Nanoparticles: Synthesis and Binding Studies with Lectin.

Weak binding of carbohydrates with protein receptors possesses serious drawbacks in the advancement of therapeutics; however, the development of strategies for multipoint interactions between carbohydrates and protein can overcome these challenges. One such method is developed in this work where glycopolymer-grafted silica nanoparticles with a large number of carbohydrate units are prepared for the interactions with multiple binding sites of the protein. First, a glycomonomer, β-d-galactose-hydroxyethyl methacrylate (β-GEMA), was synthesized in a two-step process by coupling β-d-galactose pentaacetate and hydroxyethyl methacrylate (HEMA), followed by deacetylation for the preparation of poly(β-GEMA) glycopolymers (GPs). Further, the poly(β-GEMA) chains were grafted onto the silica nanoparticle (SiNP) surface by utilizing the "grafting-from" strategy of surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare p(β-GEMA)-grafted SiNPs (GNPs). Five different chain lengths ranging from 10 to 40 kDa of the GPs and the GNPs were prepared, and various characterization techniques confirmed the formation of GPs and grafting of the GPs on the SiNP surface. The particle size of GNPs and the number of GPs grafted on the SiNP surface showed a strong dependence on the chain length of the GPs. Further, the GNPs were subjected to a binding study with β-galactose-specific protein peanut agglutinin (PNA). A much stronger binding in the case of GNPs was observed with an association constant ∼320 times and ∼53 times than that of the monomeric methyl-β-d-galactopyranoside and the GPs, respectively. Additionally, the binding of the PNA with GNPs and GPs was also studied with varying chain lengths to understand the effects of the chain length on the binding affinity. A clear increase in binding constants was observed in the case of GNPs with increasing chain length of grafted GPs, attributed to the enhanced enthalpic and entropic contributions. This work holds its uniqueness in these improved interactions between carbohydrates and proteins, which can be used for carbohydrate-based targeted therapeutics.

Pillar[n]arene-Based Fluorescence Turn-On Chemosensors for the Detection of Spermine, Spermidine, and Cadaverine in Saline Media and Biofluids.

Polyamines are essential analytes due to their critical role in various biological processes and human health in general. Due to their role as regulators for cell growth and proliferation (putrescine and spermine), as neuroprotectors, gero-, and cardiovascular protectors (spermidine), and as bacterial growth indicators (cadaverine), rapid, simple, and cost-effective methods for polyamine detection in biofluids are in demand. The present study focuses on the development and investigation of self-assembled and fluorescent host⋅dye chemo-sensors based on sulfonated pillar[5]arene for the specific detection of polyamines. Binding studies, as well as stability and functionality assessments of the turn-on chemosensors for selective polyamine detection in saline and biologically relevant media, are shown. Furthermore, the practical applicability of the developed chemo-sensors is demonstrated in biofluids such as human urine and saliva.

Single Chelator-Minibody Theranostic Agents for 89Zr PET Imaging and 177Lu Radiopharmaceutical Therapy of PSMA-Expressing Prostate Cancer.

Here we describe an anti-prostate-specific membrane antigen (PSMA) minibody (IAB2MA) conjugated to an octadentate, macrocyclic chelator based on four 1-hydroxypyridin-2-one coordinating units (Lumi804 [L804]) labeled with 89Zr (PET imaging) and 177Lu (radiopharmaceutical therapy), with the goal of developing safer and more efficacious treatment options for prostate cancer. Methods: L804 was compared with the current gold standard chelators, DOTA and deferoxamine (DFO), conjugated to IAB2MA for radiolabeling with 177Lu and 89Zr in cell binding, preclinical biodistribution, imaging, dosimetry, and efficacy studies in the PSMA-positive PC3-PIP tumor-bearing mouse model of prostate cancer. Results: Quantitative radiolabeling (>99% radiochemical yield) of L804-IAB2MA with 177Lu or 89Zr was achieved at ambient temperature in under 30 min, comparable to 89Zr labeling of DFO-IAB2MA. In contrast, DOTA-IAB2MA was radiolabeled with 177Lu for 30 min at 37°C in approximately 90% radiochemical yield, requiring further purification. Using europium(III) as a luminescent surrogate, high binding affinity of Eu-L804-IAB2MA to PSMA was demonstrated in PC3-PIP cells (dissociation constant, 4.6 ± 0.6 nM). All 4 radiolabeled constructs showed significantly higher levels of internalization after 30 min in the PC3-PIP cells than in PSMA-negative PC3-FLU cells. The accumulation of 177Lu- and 89Zr-L804-IAB2MA in PC3-PIP tumors and all organs examined (i.e., heart, liver, spleen, kidney, muscle, salivary glands, lacrimal glands, carcass, and bone) was significantly lower than that of 177Lu-DOTA-IAB2MA and 89Zr-DFO-IAB2MA at 96 and 72 h after injection, respectively. Generally, SPECT/CT and PET/CT imaging data showed no significant difference in the SUVmean of the tumors or muscle between the radiotracers. Dosimetry analysis via both organ-level and voxel-level dose calculation methods indicated significantly higher absorbed doses of 177Lu-DOTA-IAB2MA in tumors, kidney, liver, muscle, and spleen than of 177Lu-L804-IAB2MA. PC3-PIP tumor-bearing mice treated with single doses of 177Lu-L804-IAB2MA (18.4 or 22.2 MBq) exhibited significantly prolonged survival and reduced tumor volume compared with unlabeled minibody control. No significant difference in survival was observed between groups of mice treated with 177Lu-L804-IAB2MA or 177Lu-DOTA-IAB2MA (18.4 or 22.2 MBq). Treatment with 177Lu-L804-IAB2MA resulted in lower absorbed doses in tumors and less toxicity than that of 177Lu-DOTA-IAB2MA. Conclusion: 89Zr- and 177Lu-L804-IAB2MA may be a promising theranostic pair for imaging and therapy of prostate cancer.

Binding of yeast and human cytochrome c to cardiolipin nanodiscs at physiological ionic strength

Binding of cytochrome c (Cytc) to membranes containing cardiolipin (CL) is of considerable interest because of the importance of this interaction in the early stages of apoptosis. The molecular-level determinants of this interaction are still not well defined and there appear to be species-specific differences in Cytc affinity for CL-containing membranes. Many studies are carried out at low ionic strength far from the 100–150 mM ionic strength within mitochondria. Similarly, most binding studies are done at Cytc concentrations of 10 μM or less, much lower that the estimated range of 0.1 to 5 mM Cytc present in mitochondria. In this study, we evaluate binding of human and yeast Cytc to CL nanodiscs using size exclusion chromatography at 25 μM Cytc concentration and 100 mM ionic strength. We find that yeast Cytc affinity for CL nanodiscs is much stronger than that of human Cytc. Mutational analysis of the site A binding surface shows that lysines 86 and 87 are more important for yeast Cytc binding to CL nanodiscs than lysines 72 and 73, counter to results at lower ionic strength. Analysis of the electrostatic surface potential of human versus yeast Cytc shows that the positive potential due to lysines 86 and 87 and other nearby lysines (4, 5, 11, 89) is stronger than that due to lysines 72 and 73. In the case of human Cytc the positive potential around site A is less uniform and likely weakens electrostatic binding to CL membranes through site A.

Preclinical Assessment of a Cannabinoid CB2 Receptor Antagonist in a Murine Model of Cerebral Malaria

Malaria is a most important parasitic disease due to its highest impact worldwide. It results in around 200 million clinical cases and 0,5-1 million deaths per year, mainly due to cerebral malaria (CM), a life-threatening neurological syndrome that predominantly affects predominantly children under five years old. CM follows neurological alterations leading to the death if left untreated, and, even when it is treated, it is fatal in 15-20% of cases. Moreover, among the survivors, more than 10% of the children develop neurological sequelae. Consequently, there is an urgent need to find therapies to attenuate these neurological signs. Recent evidence has proposed the endocannabinoid system, which plays an important neuromodulatory function in the central nervous system (CNS), also including immunomodulation preferentially exerted by CB2 receptor. Previous studies have shown that the genetic ablation of this receptor improved mice survival against CM, suggesting a potential for the pharmacological treatment of CM with selective antagonists of this receptor. Considering this background, we investigated CM therapy by a classic CB2 antagonist SR144528 in a murine model of the disease. First, we carried out binding studies with SR144528 to confirm its pharmacodynamic profile (binding affinity [Ki] value = 2.34 ± 0.61 nM; and efficacy [IC50] = 96.17 ± 1.41 nM, at the CB2 receptor). Second, P. berghei ANKA infected C57BL/6 mice were treated daily with SR144528 and assessed for parasitemia growth and neurological alterations. 30% of the treated mice showed partial recovery of CM symptoms with 20% increased survival, but finally succumbing to hyperparasitemia and severe anemia. These preliminary preclinical results suggest that, although part of the CM course might be modulated by the pharmacological blockade of the CB2 receptor, other elements trigger the lethal outcome. Thus, while our hypothesis could not be completely validated in this CM model, we detail here all obtained results for further research.

Maximizing the Accuracy of Equilibrium Dissociation Constants for Affinity Complexes: From Theory to Practical Recommendations.

The equilibrium dissociation constant (Kd) is a major characteristic of affinity complexes and one of the most frequently determined physicochemical parameters. Despite its significance, the values of Kd obtained for the same complex under similar conditions often exhibit considerable discrepancies and sometimes vary by orders of magnitude. These inconsistencies highlight the susceptibility of Kd determination to large systematic errors, even when random errors are small. It is imperative to both minimize and quantitatively assess the systematic errors inherent in Kd determination. Traditionally, Kd values are determined through nonlinear regression of binding isotherms. This analysis utilizes three variables: concentrations of two reactants and a fraction R of unbound limiting reactant. The systematic errors in Kd arise directly from systematic errors in these variables. Therefore, to maximize the accuracy of Kd, this study thoroughly analyzes the sources of systematic errors within the three variables, including (i) non-additive signals to calculate R, (ii) mis-calibrated experimental instruments, (iii) inaccurate calibration parameters, (iv) insufficient incubation time, (v) unsaturated binding isotherm, (vi) impurities in the reactants, and (vii) solute adsorption onto surfaces. Through this analysis, we illustrate how each source contributes to inaccuracies in the determination of Kd and propose strategies to minimize these contributions. Additionally, we introduce a method for quantitatively assessing the confidence intervals of systematic errors in concentrations, a crucial step toward quantitatively evaluating the accuracy of Kd. While presenting original findings, this paper also reiterates the fundamentals of Kd determination, hence guiding researchers across all proficiency levels. By shedding light on the sources of systematic errors and offering strategies for their mitigation, our work will help researchers enhance the accuracy of Kd determination, thereby making binding studies more reliable and the conclusions drawn from such studies more robust.

Rigid and Flexible Bis-Porphyrinic Tweezers: Efficient Molecular Recognition of Bidentate Bases

Increasing efforts are devoted to the synthesis of cofacial bis-porphyrinic tweezers able to complex various bidentate guests through axial coordination of the Zn(II) porphyrins by the two nitrogen atoms. Elaboration of rigid receptors appeared up to now as the ideal way to pre-organize a bis-porphyrinic cavity and increase the stability of the formed host/guest complexes. However, the choice of using rigid spacer to attach the two porphyrins together may cost a lot as far as solubility and stability are concerned. The synthetic pathways necessary to achieve the synthesis of rigid systems are frequently long and tedious. We report here the synthesis and studies of new flexible bis-porphyrinic tweezers bearing uridine as linkers and offering pre-organized cavities able to welcome bidentate guests with extremely high association constants.As part of our studies concerning complexation of bidentate Lewis bases by bis-porphyrinic tweezers, we have synthesized two new bis-porphyrins bearing a flexible nucleosidic linker. Both uridine and 2’-deoxyuridine spacers were chosen on account of some observations we made a few years ago on the unexpected blocked conformations adopted by a pentaporphyrin consisting of four Zn(II) porphyrins attached to a central free-base porphyrin by four nucleosidic linkers. Since we observed no such blocked conformations in other flexible pentaporphyrins, we thought this might be due to the nucleosidic nature of the linkers. We thus synthesized two dimers, which differ by the attachment positions of the two porphyrins.The ability of these two tweezers to accomodate guests was investigated through ligand binding studies carried out in dichloromethane with DABCO as bidentate base. The complexation of DABCO by these dimers was monitored by UV-visible spectrophotometric titration in CH2Cl2. The binding constants between these two tweezers and DABCO were calculated from UV-visible spectroscopic data. These association constants are unexpectedly increased by more than one order of magnitude as compared to the association constants of the same bidentate ligand with a reference Zn(II) mono-porphyrin. This enhanced stability of the complexes may be ascribed to a pre-organization of the bis-porphyrinic tweezers forming a cavity, and provides convincing evidence that the bidentate base is inserted into the cavity of the dimers via host/guest interactions.We demonstrated that similar association constants can be obtained with flexible tweezers bearing uridine as linker and rigid tweezers. However, the stability of these flexible complexes isn’t as convincing as for our rigid tweezers bearing a tris-anthracenic spacer since the addition of an excess of DABCO destroys all the flexible complexes.Molecular modelling of one of the flexible tweezers and its 1/1 complex with DABCO is represented below, as well as our rigid tweezers bearing a tris-anthracenic spacer. Acknowledgements This work was supported by the CNRS and the French Ministry of Research.