Displacement Binding Assays Research Articles

Novel Gd-DTPA-peptide for targeted breast tumor magnetic resonance imaging

The prevalence of breast cancer underscores the imperative for early diagnosis in guiding treatment decisions. This study introduces a novel contrast agent, Gd-DTPA-VGB3, derived from the peptide VGB3 targeting vascular endothelial growth factor receptor-1 (VEGFR1) and VEGFR2, to enhance the contrast of conventional drug Magnevist in breast tumor MRI. The MRI contrast agent was synthesized on rink amide resin via Fmoc strategy, incorporating amino acids, and coupling to diethylenetriaminepentaacetic acid (DTPA). Gadolinium (Gd)-DTPA-VGB3 displayed specific binding to VEGFR1/2 in a displacement binding assay. Gd-DTPA-VGB3 exhibited minimal cytotoxicity to normal MCF-10 cells while inhibiting 4T1 mammary carcinoma cell proliferation. Compared to Magnevist, Gd-DTPA-VGB3 demonstrated a 2.8-fold increase in contrast-to-noise ratio (CNR) (355 vs. 125). Gd-DTPA-VGB3 exhibited enhanced accumulation in 4T1 tumor-bearing mice, resulting in significant signal intensity improvement. The findings highlight Gd-DTPA-VGB3's specific binding to VEGFRs, substantiating its potential as a candidate for enhancing MRI contrast in breast cancer diagnostics.

Ibuprofen-Immobilized Thin Films: A Novel Approach to Improve the Clearance of Protein-Bound Uremic Toxins.

Chronic kidney disease (CKD), a pressing global health issue, affects millions and leads to end-stage renal disease (ESRD). Hemodialysis (HD) is a crucial treatment for ESRD, yet its limited efficiency in removing protein-bound uremic toxins (PBUTs) results in high morbidity and mortality rates. A high affinity of pharmaceutical drugs for human serum albumin (HSA) can be leveraged to compete effectively with PBUTs for the same HSA binding sites, thereby enabling them to be capable of displacing these toxins. One such drug is ibuprofen (IBF), known for its very high affinity for HSA and sharing the same binding site as indoxyl sulfate (IS). This study explores the development of IBF-immobilized cellulose acetate-based (CA-based) thin films. The films were created by reacting CA with IBF-modified silica precursors at varying concentrations. The presence of IBF in CA/TEOS/APTES-IBF-3 and CA/TEOS-IBF-25 films, containing 3 and 25 wt % IBF, respectively, was confirmed through 1H NMR spectra. Competitive displacement binding assays indicated that while the incorporation of 3 wt % IBF showed no significant enhancement in IS displacement, the 25 wt % IBF film increased the dialyzed IS by 1.3 when normalized to non-IBF films. Furthermore, there was a 1.2-fold decrease in the total percentage of IS, and the free percentage of IS increased 1.3 to 3.0 times. Although direct systemic infusion of IBF in HD patients achieves a 2.4 times higher removal of IS, it is impractical due to the risks it poses to ESRD patients. The IBF-immobilized films offer the advantage of localized binding, thus eliminating the need for systemic exposure. This innovative approach lays a foundation for developing more efficient HD membranes, aiming to address the challenging issue of PBUT elimination and potentially enhance the quality of life and treatment outcomes for ESRD patients.

Synthesis and preclinical evaluation of a heterodimeric radioligand targeting fibroblast activation protein and integrin-αvβ3.

Tumor progression is accompanied by intrinsic heterogeneity and different phenotypes, which implies a different expression of cell surface receptors. Fibroblast activation protein (FAP) and integrin αvβ3 are highly expressed in the cell surface of cancer-associated cells or cancer cells compared with normal cells. Therefore, a FAP/integrin αvβ3 bispecific heterodimer was developed for positron emission tomography (PET) diagnostic imaging and radiotherapy. The heterodimer DOTA-FAPI-RGD was labeled with the diagnostic radionuclide gallium-68 or the therapeutic radionuclide lutetium-177, with yields >80%, and high stability. The competitive displacement binding assay showed an IC50=6.8±0.6nM for DOTA-FAPI-RGD towards FAP and IC50=2.1±0.4nM towards integrin αvβ3. Radionuclide labeled DOTA-FAPI-RGD showed high specificity and rapid internalization into U87MG cells (FAP/αvβ3-positive) in vitro. Micro-PET and biodistribution studies of [68Ga]Ga-DOTA-FAPI-RGD in tumor-bearing mice demonstrated that a high and specific tumor uptake of the tracer and a fast body clearance, resulting in high contrast images. In addition to the imaging applications demonstrated in this study, the labeling of the heterodimeric ligand with the radionuclide lutetium-177 used in cancer treatment might allow the therapeutic application of this ligand.

Turning a Tumor Microenvironment Pitfall into Opportunity: Discovery of Benzamidoxime as PD-L1 Ligand with pH-Dependent Potency

PD-1/PD-L1 protein complex is attracting a great deal of interest as a drug target for the design of immune therapies able to block its assembly. Although some biologic drugs have entered clinical use, their poor response rate in patients are demanding further efforts to design small molecule inhibitors of PD-1/PD-L1 complex with higher efficacy and optimal physicochemical properties. Dysregulation of pH in the tumor microenvironment is indeed one of the key mechanisms promoting drug resistance and lack of response in cancer therapy. Integrating computational and biophysical approaches, herein we report a screening campaign that has led to identifying VIS310 as a novel ligand of PD-L1, with physicochemical properties enabling a pH-dependent binding potency. Additional optimization efforts by analogue-based screening have been instrumental to disclosing VIS1201, which exhibits improved binding potency against PD-L1 and is able to inhibit PD-1/PD-L1 complex formation in a ligand binding displacement assay. While providing preliminary structure-activity relationships (SARs) of a novel class of PD-L1 ligands, our results lay the foundation for the discovery of immunoregulatory small molecules resilient to tumor microenvironmental conditions for escaping drug-resistance mechanisms.

Displacement Binding Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

Displacement binding assays are nonfunctional assays mostly used with the aim of determining whether a certain compound (plant-derived or synthetic) can bind to a specific receptor with high affinity. Here, we describe the displacement binding assay that is carried out with a radioligand and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.

Nicotine and its metabolite cotinine target MD2 and inhibit TLR4 signaling

Nicotine is the principal alkaloid of tobacco often manufactured into cigarettes and belongs to a highly addictive class of drugs. Nicotine attenuates the neuroinflammation induced by microglial activation. However, the molecular target(s) underlying anti-inflammatory action of nicotine has not been fully understood. Considering the psychoactive substances morphine, cocaine, and methamphetamine act as xenobiotic-associated molecular patterns and can be specifically sensed by the innate immune receptor Toll-like receptor 4 (TLR4), here we sought to delineate whether nicotine and/or its metabolite cotinine may be recognized by the innate immune system via myeloid differentiation protein 2 (MD2), an accessory protein of TLR4 that is responsible for ligand recognition. MD2-intrinsic fluorescence titrations, surface plasmon resonance, and competitive displacement binding assays with curcumin (MD2 probe) demonstrated that both nicotine and cotinine targeted the lipopolysaccharide (LPS; TLR4 agonist) binding pocket of MD2 with similar affinities. The cellular thermal shift assay indicated that nicotine binding increased, while cotinine binding decreased, MD2 stability. These biophysical binding results were further supported by in silico simulations. In keeping with targeting MD2, both nicotine and cotinine inhibited LPS-induced production of nitric oxide and tumor necrosis factor alpha (TNF-α) and blocked microglial activation. Neither a pan nicotinic acetylcholine receptor (nAChR) inhibitor nor RNAi for nAChRs abolished the suppressive effect of nicotine- and cotinine-induced neuroinflammation. These data indicate that TLR4 inhibition by nicotine and cotinine at the concentrations tested in BV-2 cells is independent of classic neuronal nAChRs and validate that MD2 is a direct target of nicotine and cotinine in the inhibition of innate immunity.

Development of Heterobivalent Theranostic Probes Having High Affinity/Selectivity for the GRPR/PSMA.

In this study, we describe the development of heterobivalent [DUPA-6-Ahx-([111In]In-DO3A)-8-Aoc-BBN ANT] and [DUPA-6-Ahx-([177Lu]Lu-DO3A)-8-Aoc-BBN ANT] radiotracers that display very high selectivity/specificity for gastrin-releasing peptide receptor (GRPR)-/prostate-specific membrane antigen (PSMA)-expressing cells. These studies include metallation, purification, characterization, and in vitro and in vivo evaluation of the new small-molecule-/peptide-based radiopharmaceuticals having utility for imaging and potentially therapy. Competitive displacement binding assays using PC-3 cells and LNCaP cell membranes showed high binding affinity for the GRPR or the PSMA. Biodistribution studies showed favorable excretion pharmacokinetics with high tumor uptake in PC-3 or PC-3 prostatic inhibin peptide (PIP) tumor-bearing mice. For example, tumor accumulation at the 1 h time point ranged from (4.74 ± 0.90) to (7.51 ± 2.61)%ID/g. Micro-single-photon emission computed tomography (microSPECT) molecular imaging investigations showed very high uptake in tumors with minimal accumulation of tracers in the surrounding collateral tissues in xenografted mice at 4 h postintravenous injection. In conclusion, [DUPA-6-Ahx-([111In]In-DO3A)-8-Aoc-BBN ANT] and [DUPA-6-Ahx-([177Lu]Lu-DO3A)-8-Aoc-BBN ANT] tracers displayed favorable pharmacokinetic and excretion profiles with high uptake and retention in tumors.

Biological assays of BF2-naphthyridine compounds: Tyrosinase and acetylcholinesterase activity, CT-DNA and HSA binding property evaluations

The present work reports the biological assays between synthetic BF2-naphtyridine complexes and four proteins: human serum albumin (HSA), calf-thymus DNA (CT-DNA), tyrosinase and acetylcholinesterase enzymes via spectroscopic analysis at physiological conditions, combined with molecular docking simulations. The BF2-complexes presented spontaneous and moderate binding ability to HSA through the ground-state association (static fluorescence quenching mechanism). The main binding site is Sudlow's site I (subdomain IIA) and the binding does not perturb significantly both secondary and surface structure of HSA. Despite BF2-complexes showed good binding ability with HSA, these compounds presented weak intercalative ability with CT-DNA (the most conventional and simple model to preliminary studies), except in the case of 1 h, which suggested that the presence of electronic donor groups in both aromatic ring moieties of BF2-complex structure can increase the intercalative ability for DNA strands. Competitive binding displacement assays in the presence of methyl green and molecular docking calculations indicated that the studied compounds interact preferentially in the major groove of DNA. In addition, the assayed compounds presented the ability to activate or inhibit both tyrosinase (the decontrolled activity can induce melanoma carcinoma) or AChE (involved in reactions related to the function of neurotransmitters) enzymes.

Discovery of high affinity γ‐Hydroxybutyrate (GHB) Receptor Ligands for SSADH Deficiency, a Disorder of GABA Metabolism

ObjectiveSuccinic semialdehyde dehydrogenase deficiency (SSADHD) is the most prevalent inherited disorder of γ‐aminobutyric acid (GABA) metabolism. SSADHD is caused by mutations in the ALDH5A1 gene resulting in impaired enzyme activity. Functional deficiency of SSADH prevents conversion of succinic semialdehyde to succinic acid, resulting in excessive accumulation of neurotransmitters GABA and its analog γ‐hydroxybutyrate (GHB). The disease presents with non‐specific mild to moderate developmental delay, severe expressive language impairment, epilepsy, and neuropsychiatric problems. Blocking GHB receptors (GHBR) with the specific antagonist NCS‐382 has been shown to rescue SSADH‐deficient mice from premature lethality suggesting antagonism of GHB effects may be therapeutically beneficial to patients. Due to a lack of structural information for the GHBR, a virtual screening strategy using a ligand‐based pharmacophore model, followed by in vitro [3H]NCS‐382 binding displacement assay, was used to identify high‐affinity small molecules as potential GHBR antagonists. Here, we report the potential binding site map of GHBR and discovery of novel and structurally diverse high‐affinity GHBR ligands as potential lead compounds for further development as possible therapeutics in SSADHD.MethodsA feature‐ and geometry‐based pharmacophore model was developed in MOE software using a set of ~110 structurally diverse compounds with known experimental binding affinities for GHBR. The iteratively refined model exhibited high accuracy in distinguishing active compounds from inactive ones and was further validated with an external test set of 25 compounds with known GHBR affinities. The validated model was used to screen commercially available CNS‐focused libraries containing several hundreds of thousands of compounds. The top hit molecules with desirable physico‐chemical and pharmacokinetic properties were chosen for the [3H]NCS‐382 binding displacement assay. The in vitro assay results were used to establish structure‐activity relationships to guide further optimization of the pharmacophore model, whose refined version was used for subsequent screening.ResultsWe have obtained the potential binding site map of GHBR by developing and refining a ligand‐based pharmacophore model iteratively based on in vitro assay results. The refined pharmacophore model provides insights into structural features that are essential for binding to the GHB receptor. After three rounds of combined in silico and in vitro assessments, we discovered several structurally diverse lead compounds exhibiting low μM affinity to the GHB receptor.ConclusionsThis study represents a strategic application of ligand‐based pharmacophore model‐based virtual screening and in vitro [3H]NCS‐382 binding displacement assay to identify high affinity ligands for the putative GHB receptor. The obtained lead compounds are being subjected to further refinement of their GHBR binding activity prior to preclinical testing of their pharmacodynamic and pharmacokinetic profile.Support or Funding InformationNational Institutes of Health ‐ NINDS R41NS107099‐01Pharmacophore‐based virtual screening for GHBR ligandsFigure 1

Curious Results in the Prospective Binding Interactions of the Food Additive Tartrazine with β-Lactoglobulin.

The detailed characterizations of the binding interactions between food additive tartrazine (TZ) and β-lactoglobulin (β-LG) have been investigated through spectroscopic techniques combined with a molecular modeling study. A series of analyses, such as hyperchromic change in the UV-visible spectra, temperature-dependent quenching constant, time-resolved fluorescence, and Rayleigh scattering measurements, show that quenching of β-LG proceeds by a static quenching mechanism. TZ specifically binds with β-LG in a stoichiometry ratio of 1:1, and the observed binding constants (104, K) are 7.64, 9.13, 9.72, and 10.79 at 293, 298, 303, and 308 K, respectively. However, the curious results of binding constants (K) with temperature, encountered in the static quenching, have been well explained on the basis of Le Chatelier's principle. Thermodynamic data and pH-dependent studies along with the surface hydrophobicity binding displacement assay reveal that the durable mode of binding is chiefly entropy-driven, revealing noteworthy interactions of such ionic molecules with the hydrophobic part of β-LG. The modulation of protein conformation has been investigated through steady-state absorption spectroscopy, synchronous emission spectroscopy, circular dichroism, and dynamic light scattering studies. TZ acts as a potential inhibitor in fibrillogenesis. Furthermore, the molecular docking study offers accurate insights about the binding of TZ with β-LG, in consistence with the experimental results. This study would be helpful in pharmaceutical, food, and industrial engineering chemistry research.

Novel Dopamine D4 Receptor‐Selective Compounds Reveal Structure‐Activity Relationships that Engender Agonist Efficacy

The dopamine D4 receptor (D4R) plays important roles in cognition, attention, and decision making. Novel D4R‐selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer's disease and substance use disorders. To identify new D4R‐selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A‐412997 (2‐(4‐(pyridin‐2‐yl)piperidin‐1‐yl)‐N‐(m‐tolyl)acetamide). We report a series of eighteen novel D4R ligands with varied substitutions on the phenylpiperidinyl ring, linker region, and/or the arylamide moieties. Compounds were profiled using radioligand binding displacement assays, β‐arrestin recruitment assays, cAMP inhibition assays, and molecular dynamics computational modeling. We identified several novel high‐affinity (Ki ≤ 4.3 nM) and D4R‐selective (>100‐fold vs other D2‐like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. Group 1 modifications resulted in a gain of efficacy at D2Rs and D3Rs. Group 2 modifications resulted in lower partial agonist efficacy at D4R. Group 3 modifications resulted in high‐potency full antagonists at D4R. These compounds inform receptor‐ligand interactions that control efficacy at D2‐like receptors and may provide insights to targeted drug discovery leading to new compound development and new tools for a better understanding of the role of D4Rs in neuropsychiatric disorders.Support or Funding InformationSupport for this research was provided by High Point University, Fred Wilson School of Pharmacy. Additional support was provided by the National Institute of Neurological Disorders and Stroke‐Intramural Research Program, National Institute on Drug Abuse‐Intramural Research Program (Z1A DA000609), and Rowan University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dopamine D4 Receptor-Selective Compounds Reveal Structure-Activity Relationships that Engender Agonist Efficacy.

The dopamine D4 receptor (D4R) plays important roles in cognition, attention, and decision making. Novel D4R-selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer’s disease and substance use disorders. To identify new D4R-selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A-412997 (1, 2-(4-(pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide). Compounds were profiled using radioligand binding displacement assays, β-arrestin recruitment assays, cyclic AMP inhibition assays, and molecular dynamics computational modeling. We identified several novel D4R-selective (Ki ≤ 4.3 nM and >100-fold vs other D2-like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. These compounds highlight receptor–ligand interactions that control efficacy at D2-like receptors and may provide insights into targeted drug discovery, leading to a better understanding of the role of D4Rs in neuropsychiatric disorders.

Mutations in pheromone-binding protein3 contribute to pheromone response variations in Plutella xylostella (L.) (Lepidoptera: Plutellidae).

Diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most important crucifer pests. Commercial sex attractants have been developed to monitor and control P. xylostella. However, some studies have demonstrated a variety of pheromone responses of P. xylostella in different locations of the world. Soluble pheromone-binding proteins (PBPs), as a subfamily of odorant-binding proteins (OBPs), could selectively bind and transport pheromones across aqueous sensillar lymph to the surface of olfactory receptor neurons. It is worthy to study whether the mutation of PxylPBPs is one of the reasons for the different responses of sex attractors in different regions. In this study, P. xylostella males were collected from seven Chinese provinces, including Hainan, Guangdong, Yunnan, Fujian, Hunan, Zhejiang, and Hebei. PxylPBP1, PxylPBP2, and PxylPBP3 were cloned, and 3, 6, and 32 types of mutation pattern were identified, respectively. These mutation patterns were distributed in each province with different frequency. The results of fluorescence displacement binding assay and in silico simulation revealed that the three mutant PxylPBP3 were more sensitive to Z11-16:Ald than the reference protein (ACI28451). This result implied that mutation of PxylPBP3 may have contributed to regional differences in pheromone responses of P. xylostella. © 2019 Society of Chemical Industry.

Matched-pair, 86Y/90Y-labeled, bivalent RGD/bombesin antagonist, [RGD-Glu-[DO3A]-6-Ahx-RM2], as a potential theranostic agent for prostate cancer

IntroductionIn this study, we describe development of a true matched-pair theranostic agent that is able to target the αVβ3 integrin and the gastrin releasing peptide receptor (GRPR). We herein describe methods to metallate and characterize the new conjugate and to validate its biological efficacy by in vitro and in vivo methods. MethodsWe have previously described the development of [RGD-Glu-6Ahx-RM2] (where RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) that has been conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelating agent (BFCA) to afford [RGD-Glu-[DO3A]-6-Ahx-RM2] peptide. In this study, we have radiolabeled [RGD-Glu-[DO3A]-6-Ahx-RM2] peptide with 86Y or 90Y. Natural-metallated (natY) conjugates were assessed for binding affinity for the αVβ3 integrin or GRPR in human glioblastoma U87-MG and prostate PC-3 cell lines, respectively. The effective stability of the new tracers was also evaluated prior to in vivo evaluation in normal CF-1 mice and SCID mice bearing xenografted tumors. ResultsCompetitive displacement binding assays in PC-3 cells showed high binding affinity for the GRPR (IC50, 5.65 ± 0.00 nM). On the other hand, competitive displacement binding assays in U87-MG cells revealed only moderate binding to the αVβ3 integrin (IC50, 346 ± 5.30 nM). Biodistribution studies in PC-3 tumor-bearing mice [RGD-Glu-[[90Y]Y-DO3A]-6-Ahx-RM2] showed high tumor uptake (8.70 ± 0.35%ID/g at 1 h post-intravenous injection) and retention of tracer (5.28 ± 0.12%ID/g) at 24 h post-intravenous injection. Micro-positron emission tomography (microPET) in PC-3 tumor-bearing mice using [RGD-Glu-[[86Y]Y-DO3A]-6-Ahx-RM2] correlated well with biodistribution investigations over the various time points that were studied. ConclusionsThe [RGD-Glu-[[86Y]Y-DO3A]-6-Ahx-RM2] and [RGD-Glu-[[90Y]Y-DO3A]-6-Ahx-RM2] matched-pair conjugates described herein exhibit favorable microPET and pharmacokinetic profiles and merit further investigations for molecular imaging and/or therapeutic evaluation in larger animal models and potentially humans. Advances in knowledge and implications for patient careThe theranostic, heterobivalent, agents described herein perform comparably with other mono- and multivalent conjugates we have reported and offer the potential of improved sensitivity for detecting prostate cancer cells that might exhibit differing profiles of receptor expression on tumor cells in human patients.

D-Arg0-Bradykinin-Arg-Arg, a Latent Vasoactive Bradykinin B2 Receptor Agonist Metabolically Activated by Carboxypeptidases.

We previously reported hypotensive and vasodilator effects from C-terminally extended bradykinin (BK) sequences that behave as B2 receptor (B2R) agonists activated by vascular or plasma peptidases. D-Arg0-BK-Arg-Arg (r-BK-RR) is a novel prodrug peptide hypothetically activated by two catalytic cycles of Arg-carboxypeptidases (CPs) to release the direct agonist D-Arg0-BK. N-terminally extending the BK sequence with D-Arg0 in the latter peptide was meant to block the second kinin inactivation pathway in importance, aminopeptidase P. The affinity of r-BK and r-BK-RR for recombinant B2R was assessed using a [3H]BK binding displacement assay. Their pharmacology was evaluated in human isolated umbilical vein, a contractile bioassay for the B2R, in a morphological assay involving the endocytosis of B2R-green fusion protein (GFP) and in anesthetized rats instrumented to record hemodynamic responses to bolus intravenous injection of both peptides. r-BK exhibited an affinity equal to that of BK for the rat B2R, while r-BK-RR was 61-fold less potent. In the vein and the B2R-GFP internalization assay, r-BK was a direct agonist unaffected by the blockade of angiotensin converting enzyme (ACE) with enalaprilat, or Arg-CPs with Plummer’s inhibitor. However, the in vitro effects of r-BK-RR were reduced by these inhibitors, more so by enalaprilat. In anesthetized rats, r-BK and r-BK-RR were equipotent hypotensive agents and their effects were inhibited by icatibant (a B2R antagonist). The hypotensive effects of r-BK were potentiated by enalaprilat, but not influenced by the Arg-CPs inhibitor, which is consistent with a minor role of Arg-CPs in the metabolism of r-BK. However, in rats pretreated with both enalaprilat and Plummer’s inhibitor, the hypotensive responses and the duration of the hypotensive episode to r-BK were significantly potentiated. The hypotensive responses to r-BK-RR were not affected by enalaprilat, but were reduced by pre-treatment with the Arg-CPs inhibitor alone or combined with enalaprilat. Therefore, in vivo, Arg-CPs activity is dominant over ACE to regenerate the B2R agonist r-BK from r-BK-RR, a prodrug activator of the B2R. A B2R agonist activated only at the level of the microcirculation by resident peptidases could be developed as an intravenously infused drug for ischemic diseases.

Structure-Affinity Relationships and Structure-Kinetic Relationships of 1,2-Diarylimidazol-4-carboxamide Derivatives as Human Cannabinoid 1 Receptor Antagonists.

We report on the synthesis and biological evaluation of a series of 1,2-diarylimidazol-4-carboxamide derivatives developed as CB1 receptor antagonists. These were evaluated in a radioligand displacement binding assay, a [35S]GTPγS binding assay, and in a competition association assay that enables the relatively fast kinetic screening of multiple compounds. The compounds show high affinities and a diverse range of kinetic profiles at the CB1 receptor and their structure–kinetic relationships (SKRs) were established. Using the recently resolved hCB1 receptor crystal structures, we also performed a modeling study that sheds light on the crucial interactions for both the affinity and dissociation kinetics of this family of ligands. We provide evidence that, next to affinity, additional knowledge of binding kinetics is useful for selecting new hCB1 receptor antagonists in the early phases of drug discovery.

Conformationally rigid derivatives of WAY-267,464: Synthesis and pharmacology at the human oxytocin and vasopressin-1a receptors

WAY-267,464 (1) and twelve conformationally rigid analogues (3a-f–4a-f) were synthesised, characterised and evaluated in cellular assays with the aim of systematically exploring interactions with the oxytocin receptor (OTR). Each analogue was evaluated in radioligand binding displacement assays at both human OTR and arginine vasopressin 1a receptors (V1aR). Physiological characterisation was determined by whole cell IP1 accumulation assays on stably transfected human embryonic kidney (HEK) cells. Incorporation of the rigid, optionally substituted benzene ring abolished OTR activity and diminished V1aR pharmacology when compared to 1. A general trend was observed in V1aR affinity for the propyl analogues (3d-3f) which identified the ortho-substituted analogue as the best in series (Ki = 251 nM) followed by a decrease in affinity through the meta and para-derivatives (3e; Ki = 874 nM and 3f; Ki = 1756 nM respectively). This study confirms the importance of the central pharmacophoric motifs of WAY-267,464 and illuminates the differences in the binding pocket of the highly conserved OTR and V1aR.

Molecular imaging investigations of a 67Ga/64Cu labeled bivalent ligand, [RGD-Glu-(DO3A)-6-Ahx-RM2], targeting GRPR/α v β 3 biomarkers: a comparative study

Abstract In the present study, we report the metallation, characterization, in vitro and in vivo studies comparing 67Ga/64Cu-radiolabeled bivalent peptide ligands as targeting probes with the capability of targeting the α v β 3 integrin or the gastrin releasing peptide receptor (GRPR) for preclinical and clinical use in single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of prostate tumors. The ligand precursor, [RGD-Glu-6-Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2), was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA), purified by reversed-phase high-performance liquid chromatography (RP-HPLC), characterized via electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with 67Ga or 64Cu. The in vitro investigations of the binding affinities of the natural-metallated ligands for the GRPR or the α v β 3 integrin were conducted via competitive displacement binding assays in human prostate PC-3 and glioblastoma U87-MG cell lines. Following stability investigations via RP-HPLC, the in vivo evaluations of the Ga67/Cu64-radiolabeled ligands were performed in CF-1 mice and SCID mice bearing PC-3 tumors. The in vitro studies of the natural-metallated ligands showed high binding affinities for the GRPR (7.78 ± 2.42, 8.64 ± 2.16 nM; Ga, Cu respectively) and moderate binding affinity for the α v β 3 integrin receptor (307 ± 40.0, 308 ± 42.6 nM; Ga, Cu respectively). In vivo biodistribution studies displayed high tumor uptake (7.44 ± 1.09, 10.85 ±4.02% ID/g at 1 h post-intravenous injection; 67Ga, 64Cu respectively) and prolonged tumor retention (4.89 ± 1.11, 4.09 ±0.96% ID/g at 24 h post-intravenous injection; 67Ga, 64Cu respectively) in PC-3 tumor-bearing mice. Micro-single photon emission computed tomography (microSPECT) and micro-positron emission computed tomography (microPET) molecular imaging studies produced high-quality, high-contrast images in PC-3 tumor-bearing mice at 18 h post-intravenous injection. Both radiolabeled ligands show satisfactory tumor uptake and retention in PC-3 tumor-bearing mice. However, [RGD-Glu-(67Ga-DO3A)-6-Ahx-RM2] demonstrates superior pharmacokinetic profiles to [RGD-Glu-(64Cu-DO3A)-6-Ahx-RM2], presumably due to more favorable in vivo stability.

The Displacement Binding Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

Displacement binding assays are nonfunctional assays mostly used with the aim of determining whether a certain compound (plant-derived or synthetic) is able to bind to a specific receptor with high affinity. Here, we describe the displacement binding assay that is carried out with a radioligand and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.

Characterization and evaluation of DOTA-conjugated Bombesin/RGD-antagonists for prostate cancer tumor imaging and therapy

IntroductionHere we present the metallation, characterization, in vivo and in vitro evaluations of dual-targeting, peptide-based radiopharmaceuticals with utility for imaging and potentially treating prostate tumors by virtue of their ability to target the αVβ3 integrin or the gastrin releasing peptide receptor (GRPr). Methods[RGD-Glu-6Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA) purified via reversed-phase high-performance liquid chromatography (RP-HPLC), characterized by electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with 111In or 177Lu. Natural-metallated compounds were assessed for binding affinity for the αVβ3 integrin or GRPr in human glioblastoma U87-MG and prostate PC-3 cell lines and stability prior to in vivo evaluation in normal CF-1 mice and SCID mice xenografted with PC-3 cells. ResultsCompetitive displacement binding assays with PC-3 and U87-MG cells revealed high to moderate binding affinity for the GRPr or the αVβ3 integrin (IC50 range of 5.39±1.37 nM to 9.26±0.00 nM in PC-3 cells, and a range of 255±47 nM to 321±85 nM in U87-MG cells). Biodistribution studies indicated high tumor uptake in PC-3 tumor-bearing mice (average of 7.40±0.53% ID/g at 1h post-intravenous injection) and prolonged retention of tracer (mean of 4.41±0.91% ID/g at 24h post-intravenous injection). Blocking assays corroborated the specificity of radioconjugates for each target. Micro-single photon emission computed tomography (microSPECT) confirmed favorable radiouptake profiles in xenografted mice at 20h post-injection. Conclusions[RGD-Glu-[111In-DO3A]-6-Ahx-RM2] and [RGD-Glu-[177Lu- DO3A]-6-Ahx-RM2] show favorable pharmacokinetic and radiouptake profiles, meriting continued evaluation for molecular imaging in murine U87-MG/PC-3 xenograft models and radiotherapy studies with 177Lu and 90Y conjugates. Advances in Knowledge and Implications for Patient CareThese heterovalent, peptide-targeting ligands perform comparably with many mono- and multivalent conjugates with the potential benefit of increased sensitivity for detecting cancer cells exhibiting differential expression of target receptors.