Competitive Binding Studies Research Articles

Mechanism of dsDNA binding, enzyme inhibition, antioxidant activities, and molecular docking studies of taxifolin, daidzein, and S-equol.

This study investigated the binding mechanism of taxifolin (TA), daidzein (DA), and S-equol (SQ) flavonoids with fish sperm double helix DNA (dsDNA) under the simulated physiological pH condition using UV-Vis and photoluminescence spectroscopy, as well as viscometric methods. Binding constants (Kb) for the flavonoids to dsDNA were determined as 1.8 × 104 M-1 for SQ, 1.6 × 104 M-1 for DA and 1.7 × 104 M-1 for TA, indicating moderate affinity. The groove binding mode was confirmed by competitive binding studies with ethidium bromide or rhodamine B, UV-Vis spectrophotometry and viscosity evaluation. Additionally, the compounds showed high cholinesterase (ChE) inhibitory activity, with TA being the most potent, particularly against BChE (IC₅₀ = 2.93 μM) and AChE (IC₅₀ = 6.42 μM). Antioxidant activities were also evaluated using DPPH and ABTS assays, with TA showing the lowest IC₅₀ values. Additionally, molecular docking studies were performed to assess the interactions and binding affinities of all compounds with AChE and BChE enzymes. As a result, the studied compounds were found to prefer minor groove binding. This research analyzed the contribution of the structure-activities of natural flavones in terms of their biological properties, which is important for their future application against diseases.

Biophysical insight into the interaction mechanism of 4-bromo-N-(thiazol-2-yl)benzenesulfonamide and human serum albumin using multi-spectroscopic and computational studies

4-Bromo-N-(thiazol-2-yl)benzenesulfonamide (1) is enriched with bioactive components and is highlighted for its pharmacological properties. However, its pharmacokinetic characteristics are yet to be reported. The interaction of compound 1 with carrier proteins in the bloodstream is an important factor that affects its potential therapeutic efficacy. This study aimed to elucidate the pharmacokinetic mechanisms of compound 1 in relation to human serum albumin (HSA) using multi-spectroscopic and computational techniques. Its predicted drug-like properties revealed no mutagenicity, although potential hepatotoxicity and interactions with certain cytochrome P450 enzymes were observed. Spectroscopic analyses extensively provided the interaction between HSA and 1 through a static fluorescence quenching mechanism with spontaneous hydrophobic interactions and hydrogen bonding. The binding constant of the HSA‒1 complex was relatively moderate to strong at a level of 106 M−1. Various spectroscopic techniques including ultraviolet-visible, Fourier transform infrared, and circular dichroism spectroscopies indicated that its binding induced alteration in the α-helix content of HSA. Competitive binding and molecular docking studies designated the preferential binding of 1 to sub-structural domain IIA binding site I of HSA. Molecular dynamic simulations further illustrated the formation of a stable complex between 1 and HSA, accompanied by conformational changes in the protein. Importantly, esterase capacity of the HSA‒1 complex increased compared to the free HSA. Therefore, elucidation of the HSA‒1 binding mechanism provides valuable insights into the pharmacokinetics, suggesting potential benefits for the further development of 1 as a therapeutic agent.

A novel and selective fluorescent ligand for the study of adenosine A2B receptors.

Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

Characterization of an Estrogen Receptor α-Selective 18 F-Estradiol PET Tracer.

Objective Conventional imaging of cancer with modalities such as computed tomography or magnetic resonance imaging provides little information about the underlying biology of the cancer and consequently little guidance for systemic treatment choices. Accurate identification of aggressive cancers or those that are likely to respond to specific treatment regimens would allow more precisely tailored treatments to be used. The expression of the estrogen receptor α subunit is associated with a more aggressive phenotype, with a greater propensity to metastasize. We aimed to characterize the binding properties of an 18 F-estradiol positron emission tomography (PET) tracer in its ability to bind to the α and β forms of estrogen receptors in vitro and confirmed its binding to estrogen receptor α in vivo. Methods The 18 F-estradiol PET tracer was synthesized and its quality confirmed by high-performance liquid chromatography. Binding of the tracer was assessed in vitro by saturation and competitive binding studies to HEK293T cells transfected with estrogen receptor α ( ESR1 ) and/or estrogen receptor β ( ESR2 ). Binding of the tracer to estrogen receptor α in vivo was assessed by imaging of uptake of the tracer into MCF7 xenografts in BALB/c nu/nu mice. Results The 18 F-estradiol PET tracer bound with high affinity (94 nM) to estrogen receptor α, with negligible binding to estrogen receptor β. Uptake of the tracer was observed in MCF7 xenografts, which almost exclusively express estrogen receptor α. Conclusion 18 F-estradiol PET tracer binds in vitro with high specificity to the estrogen receptor α isoform, with minimal binding to estrogen receptor β. This may help distinguish human cancers with biological dependence on estrogen receptor subtypes.

Preparation and preliminary evaluation of a tritium-labeled allosteric P2X4 receptor antagonist

P2X4 receptors are ATP-gated cation channels that were proposed as novel drug targets due to their role in inflammation and neuropathic pain. Only few potent and selective P2X4 receptor antagonists have been described to date. Labeled tool compounds suitable for P2X4 receptor binding studies are lacking. Here, we present a novel allosteric P2X4 receptor antagonist possessing high potency in the low nanomolar range. We describe its tritium-labeling resulting in the P2X4-selective radiotracer [3H]PSB-OR-2020 with high specific activity (45 Ci/mmol; 1.67 TBq/mmol). A radioligand binding assay was developed using human embryonic kidney (HEK293) cell membranes recombinantly expressing the human P2X4 receptor. Competition binding studies with structurally diverse P2X4 receptor antagonists revealed different allosteric binding sites indicating that the new class of P2X4 receptor antagonists, to which PSB-OR-2020 belongs, interacts with an unprecedented allosteric site. [3H]PSB-OR-2020 may become a useful tool for research on P2X4 receptors and for promoting drug development.

Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope

Antigenic similarities between Zika virus (ZIKV) and other flaviviruses pose challenges to the development of virus-specific diagnostic tools and effective vaccines. Starting with a DNA-encoded one-bead-one-compound combinatorial library of 508,032 synthetic, non-natural oligomers, we selected and characterized small molecules that mimic ZIKV epitopes. High-throughput fluorescence-activated cell sorter-based bead screening was used to select molecules that bound IgG from ZIKV-immune but not from dengue-immune sera. Deep sequencing of the DNA from the "Zika-only" beads identified 40 candidate molecular structures. A lead candidate small molecule "CZV1-1" was selected that correctly identifies serum specimens from Zika-experienced patients with good sensitivity and specificity (85.3% and 98.4%, respectively). Binding competition studies of purified anti-CZV1-1 IgG against known ZIKV-specific monoclonal antibodies (mAbs) showed that CZV1-1 mimics a nonlinear, neutralizing conformational epitope in the domain III of the ZIKV envelope. Purified anti-CZV1-1 IgG neutralized infection of ZIKV in cell cultures with potencies comparable to highly specific ZIKV-neutralizing mAbs. This study demonstrates an innovative approach for identification of synthetic non-natural molecular mimics of conformational virus epitopes. Such molecular mimics may have value in the development of accurate diagnostic assays for Zika, as well as for other viruses.

Cage-To-Cage Transformations in Self-Assembled Coordination Cages Using "Acid/Base" or "Guest Binding-Induced Strain" as Stimuli.

Controlling supramolecular systems between different functional forms by utilizing acids/bases as stimuli is a formidable challenge, especially where labile coordination bonds are involved. A pair of acid/base responsive, interconvertible 1,5-enedione/pyrylium based Pd2L4-type cages are prepared that exhibit differential guest binding abilities towards disulfonates of varied sizes. A three-state switch has been achieved, where (i) a weakly coordinating base induced cage-to-cage transformation in the first step, (ii) a strongly coordinating base triggered cage disassembly as the second step, and (iii) the third step shows acid(strong) promoted generation of initial cage, thereby completing the cycle. To our surprise, binding of a specific disulfonate guest facilitated cage-to-cage transformations by inducing strain on the cage assembly thereby opening the labile pyrylium rings of the cage. Through a competitive guest binding study, we demonstrated the superior guest binding capability of the octacationic pyrylium-based cage over a similar-sized tetracationic cage. These results provide a reliable approach to reversibly modulate the guest binding properties of acid/base-responsive self-assembled coordination cages.

Cage‐To‐Cage Transformations in Self‐Assembled Coordination Cages Using “Acid/Base” or “Guest Binding‐Induced Strain” as Stimuli

AbstractControlling supramolecular systems between different functional forms by utilizing acids/bases as stimuli is a formidable challenge, especially where labile coordination bonds are involved. A pair of acid/base responsive, interconvertible 1,5‐enedione/pyrylium based Pd2L4‐type cages are prepared that exhibit differential guest binding abilities towards disulfonates of varied sizes. A three‐state switch has been achieved, where (i) a weakly coordinating base induced cage‐to‐cage transformation in the first step, (ii) a strongly coordinating base triggered cage disassembly as the second step, and (iii) the third step shows acid(strong) promoted generation of initial cage, thereby completing the cycle. To our surprise, binding of a specific disulfonate guest facilitated cage‐to‐cage transformations by inducing strain on the cage assembly thereby opening the labile pyrylium rings of the cage. Through a competitive guest binding study, we demonstrated the superior guest binding capability of the octacationic pyrylium‐based cage over a similar‐sized tetracationic cage. These results provide a reliable approach to reversibly modulate the guest binding properties of acid/base‐responsive self‐assembled coordination cages.

8-Bicycloalkyl-CPFPX derivatives as potent and selective tools for in vivo imaging of the A1 adenosine receptor

Imaging of the A1 adenosine receptor (A1R) by positron emission tomography (PET) with 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propyl-xanthine ([18F]CPFPX) has been widely used in preclinical and clinical studies. However, this radioligand suffers from rapid peripheral metabolism and subsequent accumulation of radiometabolites in the vascular compartment. In the present work, we prepared four derivatives of CPFPX by replacement of the cyclopentyl group with norbornane moieties. These derivatives were evaluated by competition binding studies, microsomal stability assays and LC-MS analysis of microsomal metabolites. In addition, the 18F-labeled isotopologue of 8-(1-norbornyl)-3-(3-fluoropropyl)-1-propylxanthine (1-NBX) as the most promising candidate was prepared by radiofluorination of the corresponding tosylate precursor and the resulting radioligand ([18F]1-NBX) was evaluated by permeability assays with Caco-2 cells and in vitro autoradiography in rat brain slices. Our results demonstrate that 1-NBX exhibits significantly improved A1R affinity and selectivity when compared to CPFPX and that it does not give rise to lipophilic metabolites expected to cross the blood-brain-barrier in microsomal assays. Furthermore, [18F]1-NBX showed a high passive permeability (Pc = 6.9 ± 2.9 × 10−5 cm/s) and in vitro autoradiography with this radioligand resulted in a distribution pattern matching A1R expression in the brain. Moreover, a low degree of non-specific binding (5%) was observed. Taken together, these findings identify [18F]1-NBX as a promising candidate for further preclinical evaluation as potential PET tracer for A1R imaging.

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

In vitro evaluation of [3H]PI-2620 and structural derivatives in non-Alzheimer's tauopathies

Alzheimer's disease (AD) and non-AD tauopathies such as chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) are characterized by the abnormal aggregation of three-repeat (3R) and/or four-repeat (4R) tau isoforms. Several tau-PET tracers have been applied for human imaging of AD and non-AD tauopathies including [18F]PI-2620. Our objective is to evaluate [3H]PI-2620 and two promising structural derivatives, [3H]PI-2014 and [3H]F-4, using in vitro saturation assays and competitive binding assays against new chemical entities based on this scaffold in human AD tissues for comparison with PSP, CBD and CTE tissues. Thin section autoradiography was employed to assess specific binding and distribution of [3H]PI-2620 and [3H]F-4 in fresh-frozen human post-mortem AD, PSP, CBD and CTE tissues. Immunohistochemistry was performed for phospho-tau (AT8) and 4R-tau (RD4). Homogenate filtration binding assays were performed for saturation analysis and competitive binding studies against [3H]PI-2620. All compounds bound with high affinity in AD tissue. In PSP tissue [3H]PI-2620 demonstrated the highest affinity (5.3 nM) and in CBD tissue [3H]F-4 bound with the highest affinity (9.4 nM). Over 40 fluorinated derivatives based on PI-2620 and F-4 were screened in AD and PSP tissue. Notably, compound 2 was the most potent derivative in PSP tissue (Ki = 7.3 nM). By autoradiography, [3H]PI-2620 and [3H]F-4 demonstrated positive signals similar in intensity in AD, PSP and CTE tissues that were displaced by homologous blockade. Binding of both radiotracers aligned with immunostaining for 4R-tau. This work demonstrates that [3H]PI-2620 and [3H]F-4 show promise for imaging 4R-tau aggregates in non-AD tauopathies. PI-2620 continues to serve as a structural scaffold for PET radiotracers with higher affinity for non-AD tau over AD tau.

Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence.

The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[125I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure-activity relationships, as the Ki values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[125I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT1 and MT2 receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[125I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT2 receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured Ki values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure-activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT2 receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT2-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor-ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure-activity relationships and with interactions taken at the receptor binding site.

New Pd(II)-pincer type complexes as potential antitumor drugs: synthesis, nucleophilic substitution reactions, DNA/HSA interaction, molecular docking study and cytotoxic activity.

Two new complexes of Pd(II), [Pd(L1)Cl]Cl (Pd1) and [Pd(L2)Cl]Cl (Pd2), (where L1 = N2,N6-bis(5-methylthiazol-2-yl)pyridine-2,6-dicarboxamide and L2 = N2,N6-di(benzo[d]thiazol-2-yl)pyridine-2.6-dicarboxamide) were synthesized. Characterization of the complexes was performed using elemental analysis, IR, 1H NMR spectroscopy and MALDI-TOF mass spectrometry. The nucleophilic substitution reactions of complexes with L-Methionine (L-Met), L-Cysteine (L-Cys) and guanosine-5'-monophosphate (5'-GMP) were studied by stopped-flow method at physiological conditions (pH = 7.2 and 37 °C). Complex Pd1 was more reactive than Pd2 in all studied reactions, while the order of reactivity of the selected ligands was: L-Met > L-Cys > 5'-GMP. The interaction of complexes with calf thymus-DNA (CT-DNA) was studied by Uv-Vis absorption and fluorescence emission spectroscopy. Competitive binding studies with intercalative agent ethidium bromide (EB) and minor groove binder Hoechst 33258 were performed as well. Both complexes interacted with DNA through intercalation and minor groove binding, where the latter was preferred. Additionally, the interaction of Pd1 and Pd2 complexes with human serum albumin (HSA) was studied employing fluorescence quenching spectroscopy. The results indicate a moderate binding affinity of complexes, with slightly stronger binding of the Pd1. Fluorescence competition experiments with site-markers (eosin Y and ibuprofen) for HSA were used to locate the binding site of Pd1 to the HSA. Additionally, the interaction with DNA and HSA was studied by molecular docking and the revealed results were in good agreement with the experimentally obtained ones. Pd1 complex exhibited cytotoxicity toward human (HCT116) and mouse cell lines (CT26) of colorectal cancer, mouse (4T1) and human (MDA-MB468) breast cancer lines and non-cancerous mouse mesenchymal stem cells (mMSC). In addition, Pd1 complex demonstrated significant selectivity towards cancer cells over non-cancerous mMSC, indicating a high potential to eliminate malignant cells without affecting normal cells. It induced apoptosis in CT26 cells, effectively arrested the cell cycle in the S phase, and selectively down-regulated cyclin D and cyclin E. Moreover, it can alter the expression of cell cycle regulators by increasing p21 and decreasing p-AKT. These findings confirm its ability to disrupt key tumor cell survival signals and suggest that the Pd1 complex is a potent candidate for effective cancer treatment.

Search for new biologically active compounds: in vitro studies of antitumor and antimicrobial activity of dirhodium(II,II) paddlewheel complexes.

Four neutral Rh1-Rh4 complexes of the general formula [Rh2(CH3COO)4L2], where L is an N-alkylimidazole ligand, were synthesized and characterized using various spectroscopic techniques, and in the case of Rh4 the crystal structure was confirmed. Investigation of the interactions of these complexes with HSA by fluorescence spectroscopy revealed that the binding constants Kb are moderately strong (∼104 M-1), and site-marker competition experiments showed that the complexes bind to Heme site III (subdomain IB). Competitive binding studies for CT DNA using EB and HOE showed that the complexes bind to the minor groove, which was also confirmed by viscosity experiments. Molecular docking confirmed the experimental data for HSA and CT DNA. Antimicrobial tests showed that the Rh2-Rh4 complexes exerted a strong inhibitory effect on G+ bacteria B. cereus and G- bacteria V. parahaemolyticus as well as on the yeast C. tropicalis, which showed a higher sensitivity compared to fluconazole. The cytotoxic activity of Rh1-Rh4 complexes tested on three cancer cell lines (HeLa, HCT116 and MDA-MB-231) and on healthy MRC-5 cells showed that all investigated complexes elicited more efficient cytotoxicity on all tested tumor cells than on control cells. Investigation of the mechanism of action revealed that the Rh1-Rh4 complexes inhibit cell proliferation via different mechanisms of action, namely apoptosis (increase in expression of the pro-apoptotic Bax protein and caspase-3 protein in HeLa and HCT116 cells; changes in mitochondrial potential and mitochondrial damage; release of cytochrome c from the mitochondria; cell cycle arrest in G2/M phase in both HeLa and HCT116 cells together with a decrease in the expression of cyclin A and cyclin B) and autophagy (reduction in the expression of the protein p62 in HeLa and HCT116 cells).

Infection of liver sinusoidal endothelial cells with Muromegalovirus muridbeta1 involves binding to neuropilin-1 and is dynamin-dependent.

Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles, including some viruses. Endocytosis in LSEC is mainly via the clathrin-coated pit mediated route, which is dynamin-dependent. LSEC can also be a site of infection and latency of betaherpesvirus, but mode of virus entry into these cells has not yet been described. In this study we have investigated the role of dynamin in the early stage of muromegalovirus muridbeta1 (MuHV-1, murid betaherpesvirus 1, murine cytomegalovirus) infection in mouse LSECs. LSEC cultures were freshly prepared from C57Bl/6JRj mouse liver. We first examined dose- and time-dependent effects of two dynamin-inhibitors, dynasore and MitMAB, on cell viability, morphology, and endocytosis of model ligands via different LSEC scavenger receptors to establish a protocol for dynamin-inhibition studies in these primary cells. LSECs were challenged with MuHV-1 (MOI 0.2) ± dynamin inhibitors for 1h, then without inhibitors and virus for 11h, and nuclear expression of MuHV-1 immediate early antigen (IE1) measured by immune fluorescence. MuHV-1 efficiently infected LSECs in vitro. Infection was significantly and independently inhibited by dynasore and MitMAB, which block dynamin function via different mechanisms, suggesting that initial steps of MuHV-1 infection is dynamin-dependent in LSECs. Infection was also reduced in the presence of monensin which inhibits acidification of endosomes. Furthermore, competitive binding studies with a neuropilin-1 antibody blocked LSEC infection. This suggests that MuHV-1 infection in mouse LSECs involves virus binding to neuropilin-1 and occurs via endocytosis.

Schiff bases containing 1,2,3-triazole group and phenanthroline: Synthesis, characterization, and investigation of DNA binding properties

In our study, the synthesis of Schiff base compounds C and D containing the phenanthroline structure and 1,2,3-triazole group was carried out in accordance with the literature. Structural characterization of the synthesized compounds was carried out using methods such as FT-IR, 1H, 13C NMR, UV–vis and Photoluminescence spectroscopy. Information regarding the interaction properties of the materials obtained within the scope of the study with the DNA structure was collected using UV–vis, viscosity tests and Photoluminescence spectroscopy methods. Data were collected about the DNA binding types, mode and energies of the Schiff base compounds synthesized within the scope of the study. As a result of the interaction of compounds C and D with the DNA structure, calibration equations, correlation coefficients (r2), DNA binding constants (Kb) and fluorescence spectrum extinction coefficient (Ksv) of EB-DNA were calculated. UV–vis, EB competitive binding and viscosity studies showed that compounds C and D interact with DNA via the groove binding mode. The Kb binding constants of substances C and D were calculated as 1.43×104 M−1 and 3.0×104 M−1, respectively, and the Ksv quenching constants were calculated as 5.34x104 M−1 and 0.6x104 M−1, respectively.

Fe(III) T1 MRI Probes Containing Phenolate or Hydroxypyridine-Appended Triamine Chelates and a Coordination Site for Bound Water.

Fe(III) complexes containing a triamine framework and phenolate or hydroxypyridine donors are characterized and studied as T1 MRI probes. In contrast to most Fe(III) MRI probes of linear chelates reported to date, the ligands reported here are pentadentate to give six-coordinate complexes with a coordination site for inner-sphere water. The crystal structure of the complex containing unsubstituted phenolate donors, Fe(L1)Cl, shows a six-coordinate iron center and contains a chloride ligand that is displaced in water. Two additional derivatives are sufficiently water-soluble for study as MRI probes, including a complex with a hydroxypyridine group, Fe(L2), and a hydroxybenzoic acid group, Fe(L3). The pH potentiometric titrations give protonation constants of 7.2 and 7.5 for Fe(L2) and Fe(L3), respectively, which are assigned to deprotonation of the bound water. Changes in the electronic absorbance spectra of the complexes as a function of pH are consistent with the deprotonation of phenol pendants at acidic pH values. However, the inner-sphere water ligand of Fe(L2) and Fe(L3) does not exchange rapidly on the NMR timescale at pH 6.0 or 7.4, as shown by variable-temperature 17O NMR spectroscopy. The pH-dependent proton relaxivity profiles show a maximum in relaxivity at a near-neutral pH, suggesting that exchange of the protons of the bound water is an important contribution. Competitive binding studies with ethylenediaminetetraacetic acid (EDTA) show effective stability constants for Fe(L2) and Fe(L3) at pH 7.4 with log K values of 21.1 and 20.5, respectively. These two complexes are kinetically inert in carbonate phosphate buffer at 37 °C for several hours but transfer iron to transferrin. Fe(L2) and Fe(L3) show enhanced contrast in T1-weighted imaging analyses in BALB/c mice. These studies show that Fe(L2) clears through mixed renal and hepatobiliary routes, while Fe(L3) has a similar pharmacokinetic clearance profile to a macrocyclic Gd(III) contrast agent.

Radiosynthesis, preclinical evaluation and pilot clinical PET imaging study of a 18F-labeled tracer targeting fibroblast activation protein

Fibroblast activation protein (FAP) is a promising molecular target for imaging in various types of cancers. Several 18F-labeled FAP inhibitor (FAPI) tracers have been evaluated in clinical study. However, these tracers display high physiological uptake in gallbladder and bile duct system. To overcome the limitation, we herein designed a novel radiotracer named 18F-FAPTG. 18F-FAPTG was produced with a non-decay-corrected radiochemical yield of 24.0 ± 6.0% and 22.0 ± 7.0% for manual and automatic synthesis, respectively. 18F-FAPTG exhibited high hydrophilicity and stability in vitro. The studies of cellular uptake, internalization, efflux properties and competitive binding to FAP of 18F-FAPTG indicated that the tracer showed high specificity, rapid internalization and low cellular efflux in FAP-positive cells. Biodistribution studies and microPET in mice bearing FAP-positive xenografts demonstrated extremely low uptake in the majority of other organs and main excretion of 18F-FAPTG through the urinary system. Furthermore, compared to 18F-FAPI-42, 18F-FAPTG showed significantly lower uptake in gallbladder, higher tumor uptake and longer tumor retention. In the pilot clinical study, 18F-FAPTG PET/CT demonstrated favorable tumor-to-background ratios in most organs and clearly displayed the malignant lesions. Our findings indicated that 18F-FAPTG had an advantage over 18F-FAPI-42 in PET imaging for cancers located in gallbladder the bile duct system. Thus, 18F-FAPTG could be an alternative to the currently available FAPI tracers.

99mTc]Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience

PurposeThe clinical success non-invasive imaging of CXCR4 expression using [68 Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of 99mTc-labeled cyclic pentapeptides based on the PentixaFor scaffold.MethodsSix mas3-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa3 linkers (L1–L6) as well as the corresponding HYNIC- and N4-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC50 and IC50inv) were carried out using Jurkat T cell lymphoma cells and [125I]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [99mTc]Tc-N4-L6-CPCR4 ([99mTc]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [99mTc]Tc-N4-L6-CPCR4 SPECT/planar imaging with individual dosimetry.ResultsOf the six mas3-conjugated peptides, mas3-L6-CPCR4 (mas3-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC50 = 5.0 ± 1.3 nM). Conjugation with N4 (N4-L6-CPCR4) further improved hCXCR4 affinity to 0.6 ± 0.1 nM. [99mTc]Tc-N4-L6-CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 ± 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [99mTc]Tc-N4-L6-CPCR4 (termed [99mTc]Tc-PentixaTec) was selected for first-in-human application. [99mTc]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1–3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging.ConclusionThe successive optimization of the amino acid composition of the linker structure and the N-terminal 99mTc-labeling strategies (mas3 vs HYNIC vs N4) has provided [99mTc]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use.Graphical abstract

PAR recognition by PARP1 regulates DNA-dependent activities and independently stimulates catalytic activity of PARP1.

Poly(ADP-ribosyl)ation is predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single-strand (SSB) and double-strand (DSB) DNA breaks are bona fide stimulators of PARP1 activity. However, PAR-mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT, and WGR, hitherto uncharacterized, as PAR reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared with PAR but bind with weak or no affinity to DNA breaks as standalone domains. Conversely, ZnF1 and ZnF2 of PARP1 recognize DNA breaks but bind weakly to PAR. In addition, PAR reader domains, together, exhibit a synergy to recognize PAR or PARylated protein. Further competition-binding studies suggest that PAR binding releases DNA from PARP1, and the WGR domain facilitates DNA release. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampered the DNA-dependent stimulation. Altogether, our work discovers dedicated high-affinity PAR reader domains of PARP1 and uncovers a novel mechanism of allosteric regulation of DNA-dependent and DNA-independent activities of PARP1 by its catalytic product PAR.