Lipophilic Ligands Research Articles

ChemInform Abstract: Self‐Assembly and Functionalization of Lipophilic Metal—Triazole Complexes in Various Media

AbstractReview: 50 refs.

Nuclear Hormone Receptors and Female Reproduction

Nuclear Hormone Receptors (NHR) are, as the name implies, receptors located in the cell nucleus that have transcription modulating characteristics. Activated non-steroidal lipophilic ligands bind these receptors resulting in dimerisation of the ligands, DNA-binding and transcriptional regulation of target proteins that influence especially cell differentiation, metabolic homeostasis and embryogenesis. This review is based on publications derived from PubMed based pursuit of scientific literature in conjunction with the authors' experience. Here, a summary of NHR family members (RXR, PPAR, VDR, TR) first in respect to known general aspects such as ligands, binding domains, signalling mechanism and second focussing especially their influence on female reproduction is offered. Furthermore, crosstalk with other prominent signalling proteins important to trophoblast function [signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB), Akt/ phosphaidyl-3-kinase (PI3K), and Wnt, are described. Considering their attributes, it is not surprising that NHR family members play a central role in female reproduction by targeting cell differentiation, metabolic homeostasis and embryogenesis. However, it seems that crosstalk depends on stage of trophoblast differentiation.

Thermoregulated phase-transfer cobalt catalyst for production of linear higher alcohols from C11–12 internal olefins in aqueous/organic biphasic system

The thermoregulated phase-transfer cobalt catalyst, composed of a polyethylene glycol tailed phosphine ligand and cobalt carbonyl, has been applied for the conversion of normal C11–12 internal olefins into linear higher alcohols via hydroformylation and hydrogenation in an aqueous/organic biphasic system. Good catalyst activity (TOF=2.2h−1) in this biphasic system was obtained which was as high as that in the homogeneous system where a cobalt catalyst was modified by a lipophilic phosphine ligand. Easy catalyst recovery has been done by phase decanting, and the aqueous phase with the cobalt catalyst was used directly in recycling. In 4 times of recycling tests, the yield of alcohols decreased slightly, the leaching of cobalt into the organic phase was less than 2.7% each time and a total catalyst TON of around 150 was obtained.

Molecular design, synthesis and biological evaluation of 1,4-dihydro-4-oxoquinoline ribonucleosides as TcGAPDH inhibitors with trypanocidal activity

The 1,4-dihydro-4-oxoquinoline ribonucleoside, Neq135, is the first low micromolar trypanosomatidae inhibitor to show good ligand efficiency (0.28kcalmol−1atom−1) and good ligand lipophilicity efficiency (0.37kcalmol−1atom−1) when acting against Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase (TcGAPDH). This and other six ribonucleosides were synthesized using our in-house technology, and assayed against the GAPDH NAD+ site using isothermal titration calorimetry (ITC). Compound Neq135 had acceptable in vitro cytotoxicity, inhibited TcGAPDH with a Kiapp value of 16μM and killed the trypomastigote form of Trypanosoma cruzi Tulahuen strain with a concentration similar to that displayed by the control drug benznidazole. Neq135 is tenfold lower kinetic affinity against hGAPDH and does not kill Balb-c fibroblast nor spleen mouse cells. These results emphasize the possibility of integrating ligand- and target-based designs to uncover potent and selective TcGAPDH inhibitors that expands the opportunity for further medicinal chemistry endeavor towards NAD+ TcGAPDH site.

Studies on Antibacterial Mechanisms of Copper Complexes with 1,10-phenanthroline and Amino Acid on Escherichia coli

The antibacterial mechanisms of Cu(phen)2Cl2·6H2O, [Cu(phen)(Gly)(H2O)]Cl·3H2O, [Cu(phen)(L-Ser)(H2O)Cl] (1,10-phenanthroline (phen)) on Escherichia coli were investigated. In the inductively coupled plasma atomic emission spectroscopy experiments, it showed that lipophilic phen ligand can cause elevation of intracellular copper, but intracellular copper is not the decisive factor. The UV-vis and gel electrophoresis experiments reveal that the DNA binding and cleavage activity are decisive factors for the antibacterial action of these compounds. It is revealed by the cyclic voltammetry experiments that the redox potential was bound to the cleave activity.

Nuclear receptors in inflammation control: Repression by GR and beyond

Inflammation is a protective response of organisms to pathogens, irritation or injury. Primary inflammatory sensors activate an array of signaling pathways that ultimately converge upon a few transcription factors such as AP1, NFκB and STATs that in turn stimulate expression of inflammatory genes to ultimately eradicate infection and repair the damage. A disturbed balance between activation and inhibition of inflammatory pathways can set the stage for chronic inflammation which is increasingly recognized as a key pathogenic component of autoimmune, metabolic, cardiovascular and neurodegenerative disorders. Nuclear receptors (NRs) are a large family of transcription factors many of which are known for their potent anti-inflammatory actions. Activated by small lipophilic ligands, NRs interact with a wide range of transcription factors, cofactors and chromatin-modifying enzymes, assembling numerous cell- and tissue-specific DNA–protein transcriptional regulatory complexes with diverse activities. Here we discuss established and emerging roles and mechanisms by which NRs and, in particular, the glucocorticoid receptor (GR) repress genes encoding cytokines, chemokines and other pro-inflammatory mediators.

Abstract 3469: The expression and prognostic significance of neutrophil gelatinase-asssociated lipocalin (NGAL) in primary breast cancer.

Abstract Introduction: Neutrophil gelatinase-associated lipocalin (NGAL; also known as lipocalin 2), a member of the lipocalin family that transports small lipophilic ligands, has gained recent attention as both a potential biomarker and a modulator of human cancers including breast cancer. In this study, we examined the expression of NAGL in breast carcinoma and evaluated the prognostic value of its expression. Methods: Tissue microarray (TMA) blocks were made with formalin-fixed paraffin-embedded tissues from 207 breast cancer patients and the immunohistochemical staining was done using an anti-NGAL antibody. The correlation between NGAL expression and the clinicopathologic parameters such as age, tumor size, lymph node status, Ki-67 labeling index, histologic grade, hormone receptors and HER2/neu expression as well as clinical outcome was evaluated. Results: NGAL was expressed in 60 breast carcinomas (21.9%). A higher NGAL expression was noted in the younger (p=0.018) and premenopausal women (p=0.036). NGAL expression correlated with a high Ki-67 labeling index (p=0.001) and poor histologic grade (p=0.002). No significant association between NGAL expression and hormone receptor status and HER2/neu overexpression. With a median follow-up of 63 months, NGAL expression was associated with decreased disease-free survival, especially for patients with estrogen receptor negative group (p=0.013) and lymph node positive group (p=0.005), but not associated with overall survival. Conclusion: NGAL is expressed in approximately one-fifth of the breast carcinomas and its expression is correlated with several poor prognostic markers and decreased disease-free survival. This results show that NGAL expression may be a predictor of poor prognosis in primary human breast cancer, especially with more aggressive clinical features Citation Format: Lee-Su Kim, Hae Sung Kim. The expression and prognostic significance of neutrophil gelatinase-asssociated lipocalin (NGAL) in primary breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3469. doi:10.1158/1538-7445.AM2013-3469

On the Solubility and Lipophilicity of Metallacarborane Pharmacophores

Metallacarborane moieties have been identified as promising pharmacophores. The pharmaceutical use of such compounds is, however, complicated by their low solubility and tendency to self-assemble in aqueous solution. In this work, we estimated the solubilities of a vast series of metallacarboranes [cobalt bis(dicarbollide) derivatives] in pure water, saline, and saline with human serum albumin as a model of blood plasma. In addition, we determined the octanol-water partition coefficients (Pow) as a lipophilicity descriptor. Pow weakly correlates with the water solubility of metallacarboranes, whereas the ability of HSA to increase the solubility of metallacarboranes correlates well with their Pow values. Because metallacarboranes are known inhibitors of HIV protease, the possible correlation between Pow and the ability to inhibit HIV protease was investigated. Results from this study indicate that interaction of metallacarborane inhibitors with HIV protease is driven by specific binding rather than by promiscuous lipophilic interactions. The most promising candidates for further drug development were identified by ligand lipophilicity efficiency analysis.

A novel fluorescent ligand binding assay for free fatty acid receptor FFA1

Obtaining affinity estimates at FFA1 receptors using radioligand binding is made difficult by lipophilic ligands. We characterised the properties of a new fluorescent ligand 40Ag‐Cy5 (CellAura) using 293TR cells expressing FLAG‐FFA1. Whole cell competition binding experiments using 40AG‐Cy5 were performed on 96‐well plates, followed by imaging and granularity analysis on a confocal platereader. Curve fitting used GraphPadPrism.Both 40Ag‐Cy5 and its non‐fluroescent precursor 40Ag‐cg were agonists at FFA1 (Fluo4 measurements of intracellular Ca2+), with respective pEC50 values of 5.8±0.2 and 7.9±0.3. In binding experiments 40Ag‐Cy5 (100 nM) was displaced by synthetic ligands such as GW1100 (pIC50 6.0 ± 0.2) and GW9508 (pIC50 6.8 ± 0.2). However oleic acid (up to 100 μM) did not displace 40Ag‐Cy5 binding. By solving the Cheng‐Prusoff correction for GW1100 competition curves using 100nM or 1μM 40Ag‐Cy5 (GW1100 pIC50 5.6), we obtained pKi estimates for 40‐Ag Cy5 (6.2), and GW1100 (6.0; equivalent to its functional pKb). Fluorescence correlation spectroscopy experiments also confirmed 40Ag‐Cy5 binding to FFA1. Thus 40Ag‐Cy5 provides a novel fluorescent ligand which enables estimation of synthetic ligand affinities at FFA1.Supported by EPSRC and AstraZeneca.

Identification of RO4597014, a Glucokinase Activator Studied in the Clinic for the Treatment of Type 2 Diabetes.

To resolve the metabolite redox cycling associated with our earlier clinical compound 2, we carried out lead optimization of lead molecule 1. Compound 4 showed improved lipophilic ligand efficiency and demonstrated robust glucose lowering in diet-induced obese mice without a liability in predictive preclinical drug safety studies. Thus, it was selected as a clinical candidate and further studied in type 2 diabetic patients. Clinical data suggests no evidence of metabolite cycling, which is consistent with the preclinical profiling of metabolism.

A FUNCTIONAL APPROACH ON NEUTROPHIL GELATINASE ASSOCIATED LIPOCALIN (NGAL) - LIPOCALIN FAMILY

Lipocalins as biochemical markers of disease have been extensively used. The clinical indications relate to almost any field of medicine, such as inflammatory disease, cancer, lipid disorders, liver and kidney function. Lipocalins are mainly extracellular carriers of lipophilic molecules, though exceptions with properties like prostaglandin synthesis and protease inhibition are observed for specific lipocalins. Lipocalin 2, a member of the lipocalin family that carries small lipophilic ligands, has gained recent attention as both a potential biomarker and a modulator of human cancers. Neutrophil gelatinase-associated lipocalin, also known as lipocalin 2, has been originally identified as a 25 kDa protein covalently linked to neutrophil gelatinase B. It is an acute phase protein involved in diverse physiological processes with an important role in intracellular iron transport. Moreover, lipocalin 2 is involved in the physiopathology of neoplastic process. Lipocalin 2 is a protein that has garnered a great deal of interest in multidisciplinary fields over the last two decades since its discovery. This review focuses on lipocalin 2 and its function.

Quantifying Thiol Ligand Density of Self-Assembled Monolayers on Gold Nanoparticles by Inductively Coupled Plasma–Mass Spectrometry

Gold nanoparticles (GNPs) are often used as colloidal carriers in numerous applications owing to their low-cost and size-controlled preparation as well as their straightforward surface functionalization with thiol containing molecules forming self-assembling monolayers (SAM). The quantification of the ligand density of such modified GNPs is technically challenging, yet of utmost importance for quality control in many applications. In this contribution, a new method for the determination of the surface coverage of GNPs with thiol containing ligands is proposed. It makes use of the measurement of the gold-to-sulfur (Au/S) ratio by inductively coupled plasma mass spectrometry (ICP–MS) and its dependence on the nanoparticle diameter. The simultaneous ICP–MS measurement of gold and sulfur was carefully validated and found to be a robust method with a relative standard uncertainty of lower than 10%. A major advantage of this method is the independence from sample preparation; for example, sample loss during the washing steps is not affecting the results. To demonstrate the utility of the straightforward method, GNPs of different diameters were synthesized and derivatized on the surface with bifunctional (lipophilic) ω-mercapto-alkanoic acids and (hydrophilic) mercapto-poly(ethylene glycol) (PEG)n-carboxylic acids, respectively, by self-assembling monolayer (SAM) formation. Thereby, a size-independent but ligand-chain length-dependent ligand density was found. The surface coverage increases from 4.3 to 6.3 molecules nm–2 with a decrease of ligand chain length from 3.52 to 0.68 nm. Furthermore, no significant difference between the surface coverage of hydrophilic and lipophilic ligands with approximately the same ligand length was found, indicating that sterical hindrance is of more importance than, for example, intermolecular strand interactions of Van der Waals forces as claimed in other studies.

Molecular Dynamics Studies of Lipophilic Small Molecule TRPV1 Ligands in Lipid Bilayers

Many lipophilic small molecule ligands (drugs and natural compounds) are known to interact with ion channels, but often experimental approaches to address these interactions prove costly and time-consuming. Studies of these ligands using molecular dynamics (MD) simulations in an explicit lipid bilayer system can aid our understanding of their possible mechanisms of action with the target ion channels. Capsaicin, the active ingredient of chilli, and related vanilloid ligands specifically modify the activity of the TRPV1 ion channel and are thought to interact with the transmembrane (TM) region of the channel protein. However, many details of this interaction such as the orientation of capsaicin and the involvement of other domains of the channel remain unknown. In this study we employ MD simulations to define the interactions of capsaicin and its analogues with lipid bilayers. Our simulations have shown that capsaicin spontaneously partitions into phospholipid bilayers and preferentially localizes at the bilayer/water interface. They also suggest the possibility of capsaicin flip-flop from one side of the bilayer to the other. These and related results are discussed in the context of understanding the significance of the lipid bilayer environment to the orientation and kinetics of the interaction of vanilloid ligands with TRPV1.

Binding of Endogenous Cannabinoid Ligand 2-Arachidonoylglycerol to Cannabinoid Receptor CB2 via the Lipid Matrix

Endogenous cannabinoid ligand, 2-arachidonoylglycerol (2-AG), binds to and activates cannabinoid type-2 receptor CB2, controlling a variety of immune and hematopoietic cellular functions. Due to the high lipophilicity of cannabinoid ligands, there have been extensive discussions that after nonspecific incorporation into membrane, approach of the ligands to the binding pocket of CB2 may take place via lateral diffusion in lipid matrix. While previously reported microsecond-long molecular dynamics simulations showed molecular details in favor of this mechanism, no in-situ experimental evidence for the binding event has yet been made. Here we report on 2H NMR results that show exchange of deuterated 2-AG-d5 between the ligand binding site on CB2 and the surrounding lipid matrix at physiological conditions. Purified, recombinant CB2 was functionally reconstituted into liposomes containing four-fold molar excess of 2-AG-d5, and its deuterium resonances measured over the temperature range of 5-20°C. When the membranes contained CB2, the ligand resonances shifted upfield by 0.5 ppm and broadened significantly. Despite the excess amount of ligand, only one set of time-averaged resonances of the ligand was observed. The result suggests that 2-AG undergoes exchange between receptor-bound and free states on millisecond timescale. The resonance linewidth decreased with decreasing temperature as predicted from reduced exchange rates. The receptor binding was specific since only well-resolved signal of 2-AG in lipid matrix was observed when the binding pocket was blocked with a high affinity synthetic agonist CP-55,940. The experiments contrast to our previous experiments on CP-55,940 binding, where the free state of 2H-labeled CP-55,940-d6 in lipid matrix showed a highly-resolved signal under much slower exchange rate. The present work emphasizes the critical role of lipid matrix to provide a pathway and to regulate approach of lipophilic cannabinoid ligands to the receptor binding pocket.

Optimisation of biphenyl acetic acid inhibitors of diacylglycerol acetyl transferase 1 – the discovery of AZD2353

Focus on ligand efficiency, ligand lipophilicity efficiency, and conformational restriction led to the discovery of AZD2353.

In Vitro Measurement of Drug Efficiency Index to Aid Early Lead Optimization

The concepts of drug efficiency (D(eff) ) and Drug Efficiency Index (DEI) have been recently introduced as useful parameters to optimize the absorption, distribution, metabolism, elimination/excretion, and toxicity properties and in vivo efficacy potential of molecules during lead optimization and at pre-clinical stages. The available free drug concentration relative to dose depends on the compound's bioavailability, clearance, and the nonspecific binding to proteins and phospholipids. In this paper, we have demonstrated, using the data of over 115 known drug molecules, that the nonspecific binding can be determined in vitro very efficiently using biomimetic high-performance liquid chromatography measurements. DEI can therefore be estimated from in vitro measurements. The data show that high in vitro DEI values can be associated with lower efficacious dose. A strategy is described of how to use the DEI parameter during early lead optimization. An example is given to highlight the advantages of optimizing on DEI value rather than on potency alone. In order to facilitate the in silico compound design, correlation between in vitro DEI and in silico ligand efficiency parameters such as ligand lipophilicity efficiency has been revealed, suggesting the potential use of these efficiency-related parameters across lead optimization.

The discovery of a novel series of glucokinase activators based on a pyrazolopyrimidine scaffold

Glucokinase is a key enzyme in glucose homeostasis since it phosphorylates glucose to give glucose-6-phosphate, which is the first step in glycolysis. GK activators have been proven to lower blood-glucose, and therefore have potential as treatments for type 2 diabetes. Here the discovery of pyrazolopyrimidine GKAs is reported. An original singleton hit from a high-throughput screen with micromolar levels of potency was optimised to give compounds with nanomolar activities. Key steps in this success were the introduction of an extra side-chain, which increased potency, and changing the linking functionality from a thioether to an ether, which led to improved potency and lipophilic ligand efficiency. This also led to more stable compounds with improved profiles in biological assays.

Discovery of 3-Cyclopropylmethyl-7-(4-phenylpiperidin-1-yl)-8-trifluoromethyl[1,2,4]triazolo[4,3-a]pyridine (JNJ-42153605): A Positive Allosteric Modulator of the Metabotropic Glutamate 2 Receptor

Advanced leads from a series of 1,2,4-triazolo[4,3-a]pyridines with mGlu2 receptor PAM activity are reported. By modification of the analogous imidazo[1,2-a]pyridine series, the newly reported leads have improved potency, in vitro ADMET, and hERG as well as good in vivo PK profile. The optimization of the series focused on improving metabolic stability while controlling lipophilicity by introducing small modifications to the scaffold substituents. Analysis of this series combined with our previously reported mGlu2 receptor PAMs showed how lipophilic ligand efficiency was improved during the course of the program. Among the best compounds, example 20 (JNJ-42153605) showed a central in vivo efficacy by inhibition of REM sleep state at a dose of 3 mg/kg po in the rat sleep-wake EEG paradigm, a phenomenon shown earlier to be mGlu2 mediated. In mice, compound 20 reversed PCP-induced hyperlocomotion with an ED₅₀ of 5.4 mg/kg sc, indicative of antipsychotic activity.

Tuning the Activity of Platinum(IV) Anticancer Complexes through Asymmetric Acylation

Platinum(II) anticancer drug cisplatin is one of the most important chemotherapeutic agents in clinical use but is limited by its high toxicity and severe side effects. Platinum(IV) anticancer prodrugs can overcome these limitations by resisting premature aquation and binding to essential plasma proteins. Structure-activity relationship studies revealed a link between the efficacy of platinum(IV) complexes with the nature of their axial ligands, which can be modified to enhance the properties of the prodrug. The existing paradigm of employing platinum(IV) complexes with symmetrical axial carboxylate ligands does not fully exploit their vast potential. A new approach was conceived to control properties of platinum(IV) prodrugs using contrasting axial ligands via sequential acylation. We report a novel class of asymmetric platinum(IV) carboxylates based on the cisplatin template containing both hydrophilic and lipophilic ligands on the same scaffold designed to improve their aqueous properties and enhance their efficacy against cancer cells in vitro.

Systematic interaction analysis of human lipocalin-type prostaglandin D synthase with small lipophilic ligands

L-PGDS [lipocalin-type PG (prostaglandin) D synthase] is a multi-functional protein, acting as a PGD₂-producing enzyme and a lipid-transporter. In the present study, we focus on the function of L-PGDS as an extracellular transporter for small lipophilic molecules. We characterize the binding mechanism of human L-PGDS for the molecules, especially binding affinity stoichiometry and driving force, using tryptophan fluorescence quenching, ICD (induced circular dichroism) and ITC (isothermal titration calorimetry). The tryptophan fluorescence quenching measurements revealed that haem metabolites such as haemin, biliverdin and bilirubin bind to L-PGDS with significantly higher affinities than the other small lipophilic ligands examined, showing dissociation constant (K(d)) values from 17.0 to 20.9 nM. We focused particularly on the extra-specificities of haem metabolites and L-PGDS. The ITC and ICD data revealed that two molecules of the haem metabolites bind to L-PGDS with high and low affinities, showing K(d) values from 2.8 to 18.1 nM and from 0.209 to 1.63 μM respectively. The thermodynamic parameters for the interactions revealed that the contributions of enthalpy and entropy change were considerably different for each haem metabolite even when the Gibbs energy change was the same. Thus we believe that the binding energy of haem metabolites to L-PGDS is optimized by balancing enthalpy and entropy change.