Application of the guanidine–acylguanidine bioisosteric approach to NPY Y2 receptor antagonists: bivalent, radiolabeled and fluorescent pharmacological tools

Neuropeptide Y (NPY), a 36 amino acid peptide, is widely distributed in the central and peripheral nervous system, where it acts as a neurotransmitter and exhibits a large number of physiological functions, including the regulation of blood pressure, control of food intake, anxiety, pain and hormone secretion. In humans, these effects are mediated by four receptor subtypes (Y1, Y2, Y4, Y5), all belonging to class A of the superfamily of G-protein coupled receptors (GPCRs). The Y2R is expressed, e. g., in sympathetic nerve endings, in the renal tubules and in distinct brain regions such as the hippocampus and the hypothalamus. It is discussed to be involved in several human diseases, for instance, epilepsy, obesity and cancer, and therefore regarded as an attractive target in drug design. However, antagonistic pharmacological tools for the elucidation of the receptor´s function in health and disease are still missing. In 1999, the (S)-argininamide BIIE 0246, a C-terminal mimic of NPY, was reported the first highly potent and selective Y2R antagonist. NG-Acylation of BIIE 0246 was demonstrated to retain or even increase the Y2R affinity, thereby improving the pharmacokinetic properties. Thus, we prepared Nω-aminoacylated analogs as precursors towards radio- and fluorescence labeled as well as bivalent pharmacological tools according to the guanidine-acylguanidine bioisosteric approach. Such derivatives, bearing a free amino group, exhibited the highest Y2R affinities (Ki < 10 nM). Interestingly, masking the positive charge, e.g. by acylation, resulted in decreased affinities, presumably, due to the loss of an additional electrostatic interaction of the primary amine with the receptor. Bivalent ligands were synthesized starting from various amine precursors derived from BIIE 0246 by acylation with different aliphatic dicarboxylic acids. Only minor differences in affinities (Ki = 61-300 nM) were observed for the majority of the compounds, irrespective of the diversity in length and chemical nature of the spacer. However, bivalent ligand 4.6, constructed from an amine precursor bearing an additional amino group in the linker, showed a binding affinity (Ki = 21 nM) and antagonistic activity (KB = 15 nM) in the same range as the monovalent antagonists, corroborating the affinity-enhancing effect of a positive charge in the linker.   Fluorescence ligands were synthesized from the amine precursors by acylation with succinimidyl esters or by ring transformation of pyrylium dyes. In terms of retaining affinity, the pyrylium dyes were superior to the bulky cyanine, hemicyanine and Bodipy dyes. Whereas the majority of the cyanine and hemicyanine labeled fluorescent ligands proved to be suitable for the detection of Y2Rs at the cell membrane by confocal microscopy, rapid cellular uptake was observed for most of the Py-1 and Py-5 coupled antagonists. Based on the results from binding studies and confocal microscopy, four fluorescent Y2R antagonists (5.4, 5.15-5.17) were chosen for flow cytometric binding studies. These compounds turned out to be applicable as labeled standard ligands in saturation and competition binding experiments. Interestingly, kinetic studies revealed a pseudo-irreversible binding of 5.16 at the Y2R. The endogenous ligand NPY was not able to displace the novel red-fluorescent Y2R antagonists 5.4 and 5.16 even at very high concentrations. Acylation of the amine precursor (S)-3.48 with succinimidyl [2,3-3H]-propionate afforded the easily accessible, highly potent and selective tritiated Y2R antagonist [3H]-UR-PLN196 (A2: 43 nM, Ca2+ assay; KD: 43 nM, determined by kinetic studies), which was shown to be useful for the quantification of Y2R binding sites and for the radiochemical determination of Y2R binding affinities of small molecules. In addition, the novel radioligand was identified as an insurmountable antagonist against pNPY exhibiting pseudo-irreversible binding at the Y2R similar to the fluorescent labeled antagonist 5.16. In conclusion, this work presents a straight-forward synthetic route towards labeled argininamide-type Y2R antagonists related to BIIE 0246 with improved physicochemical properties. These compounds are useful pharmacological tools for the detection of the Y2R in vitro and for detailed investigations of the antagonistic binding mode, respectively, as well as for the identification and characterization of small molecule Y2R antagonists. Moreover, these tracers are suitable to replace labeled NPY standard ligands in binding assays due to their beneficial properties in terms of Y2 receptor subtype selectivity, antagonistic mode of action, stability and kinetics

[3H]UR-PLN196: A Selective Nonpeptide Radioligand and Insurmountable Antagonist for the Neuropeptide Y Y2 Receptor

Attachment of a [2,3-(3)H]propionyl group through an appropriate linker to the guanidine group of an (S)-argininamide-type neuropeptide Y (NPY) Y(2) receptor antagonist resulted in a subtype-selective radioligand

A Sensitive LC–MS/MS Method for the Quantification of 3-Hydroxybenzo[a]pyrene in Urine-Exposure Assessment in Smokers and Users of Potentially Reduced-Risk Products

Benzo[a]pyrene (BaP), a human carcinogen, is formed during the incomplete combustion of organic matter such as tobacco. A suitable biomarker of exposure is the monohydroxylated metabolite 3-hydroxybenzo[a]pyrene (3-OH-BaP). We developed a sensitive LC–MS/MS (liquid chromatography coupled with tandem mass spectrometry) method for the quantification of urinary 3-OH-BaP. The method was validated according to the US Food and Drug Administration (FDA) guideline for bioanalytical method validation and showed excellent results in terms of accuracy, precision, and sensitivity (lower limit of quantification (LLOQ): 50 pg/L). The method was applied to urine samples derived from a controlled clinical study to compare exposure from cigarette smoking to the use of potentially reduced-risk products. Urinary 3-OH-BaP concentrations were significantly higher in smokers of conventional cigarettes (149 pg/24 h) compared to users of potentially reduced-risk products as well as non-users (99% &lt; LLOQ in these groups). In conclusion, 3-OH-BaP is a suitable biomarker to assess the exposure to BaP in non-occupationally exposed populations and to distinguish not only cigarette smokers from non-smokers but also from users of potentially reduced-risk products

Identification of biomarkers specific to five different nicotine product user groups: Study protocol of a controlled clinical trial

Background: Assessing biomarker profiles in various body fluids is of large value to discern between the sole use of nicotine products. In particular, the assessment of the product compliance is required for long-term clinical studies. The objective of this study was the identification of biomarkers and biomarker patterns in body fluids, to distinguish between combustibles, heated tobacco products, electronic cigarettes, oral tobacco and oral/dermal nicotine products used for nicotine replacement therapy (NRT), as well as a control group of non-users. Methods: A controlled, single-center study was conducted with 60 healthy subjects, divided into 6 groups (5 nicotine product user groups and one non-user group) based on their sole use of the products of choice. The subjects were confined for 76 h, during which, free and uncontrolled use of the products was provided. Sample collections were performed according to the study time schedule provided in Table 2. The primary outcome will be validated through analysis of the collected biospecimens (urine, blood, saliva, exhaled breath and exhaled breath condensate) by means of untargeted omics approaches (i.e. exposomics, breathomics and adductomics). Secondary outcome will include established biomarker quantification methods to allow for the identification of typical biomarker patterns. Statistical analysis tools will be used to specifically discriminate different product use categories. Results/Conclusions: The clinical trial was successfully completed in May 2020, resulting in sample management and preparations for the quantitative and qualitative analyses. This work will serve as a solid basis to discern between biomarker profiles of different nicotine product user groups. The knowledge collected during this research will be required to develop prototype diagnostic tools that can reliably assess the differences and evaluate possible health risks of various nicotine products

Red-fluorescent argininamide-type NPY Y(1) receptor antagonists as pharmacological tools

Fluorescently labelled NPY Y1 receptor (Y1R) ligands were synthesized by connecting pyrylium and cyanine dyes with the argininamide-type Y1R antagonist core structure by linkers, covering a wide variety in length and chemical nature, attached to the guanidine group. The most promising fluorescent probes had Y1R affinities (radioligand binding) and antagonistic activities (calcium assay) in the one- to two-digit nanomolar range. These compounds turned out to be stable under assay conditions and to be appropriate for the detection of Y1Rs by confocal microscopy in live cells. To improve the signal-to-noise ratio by shifting the emission into the near infrared, a new benzothiazolium-type fluorescent cyanine dye (UR-DE99) was synthesized and attached to the parent antagonist via a carbamoyl linker yielding UR-MK131, a highly potent fluorescent Y1R probe, which was also successfully applied in flow cytometry

Application of the guanidine – acylguanidine bioisosteric approach to argininamide-type NPY Y2 receptor antagonists

Strongly basic groups such as guanidine moieties are crucial structural elements but compromising drug-likeness of numerous biologically active compounds including ligands of G-protein coupled receptors (GPCRs). As part of a project focusing on the search for guanidine bioisosteres, argininamide-type neuropeptide Y (NPY) Y2 receptor (Y2R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, NG-acylated argininamides were preferably obtained by guanidinylation using tailor-made mono Boc-protected N-acyl-S-methylisothioureas. The compounds were investigated for Y2R antagonism (calcium assays), Y2R affinity and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the NG-substituted (S)-argininamides showed Y2R antagonistic activities and binding affinities comparable to the parent compound, whereas NG-acylated or -carbamoylated analogs containing a terminal amine were superior (Y2R: Ki and KB values in the low nanomolar range). This demonstrates that the basicity of the compounds, although being by 4-5 orders lower than that of guanidines, suffices to form key interactions with acidic amino acids of the Y2R. The acylguanidines bind with high affinity and selectivity to Y2R compared to Y1, Y4, and Y5 receptors. As derivatization of the amino group is tolerated, these compounds are considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y2R. The results support the concept of bioisosteric guanidine-acylguanidine exchange as a broadly applicable approach to retain pharmacological activity regardless of reduced basicity

Collaborative Method Performance Study of the Measurement of Nicotine, Its Metabolites, and Total Nicotine Equivalents in Human Urine.

Background: Biomarkers of tobacco exposure have a central role in studies of tobacco use and nicotine intake. The most significant exposure markers are nicotine itself and its metabolites in urine. Therefore, it is important to evaluate the performance of laboratories conducting these biomarker measurements.Methods: This report presents the results from a method performance study involving 11 laboratories from 6 countries that are currently active in this area. Each laboratory assayed blind replicates of seven human urine pools at various concentrations on three separate days. The samples included five pools blended from smoker and nonsmoker urine sources, and two additional blank urine samples fortified with pure nicotine, cotinine, and hydroxycotinine standards. All laboratories used their own methods, and all were based on some form of liquid chromatography/tandem mass spectrometry.Results: Overall, good agreement was found among the laboratories in this study. Intralaboratory precision was good, and in the fortified pools, the mean bias observed was &lt; + 3.5% for nicotine, approximately 1.2% for hydroxycotinine, and less than 1% for cotinine (1 outlier excluded in each case). Both indirect and direct methods for analyzing the glucuronides gave comparable results.Conclusions: This evaluation indicates that the experienced laboratories participating in this study can produce reliable and comparable human urinary nicotine metabolic profiles in samples from people with significant recent exposure to nicotine.Impact: This work supports the reliability and agreement of an international group of established laboratories measuring nicotine and its metabolites in urine in support of nicotine exposure studies. Cancer Epidemiol Biomarkers Prev; 27(9); 1083-90. ©2018 AACR