Small-Molecule Ligands as Challenge for Positron Emission Tomography of Peptide Receptors in Neurons and Microglia of the Brain

Neuropeptide and chemokine receptors of the G protein-coupled receptor (GPCR) family belong to different classes and subgroups providing different docking sites and special binding behavior at extracellular and also transmembrane domains for small molecules potentially suitable for positron emission tomography (PET). The contribution gives an overview updating developments of small-molecule, nonpeptide ligands at a selection of peptide and chemokine receptors, expressed in neurons and microglia of the brain, regarding the last five years. Orexin 1 and orexin 2 receptors (OX1R; OX2R) and neuropeptide Y1 and Y2 receptors (NPY1R, NPY2R) were chosen as representatives of Class A neuropeptide receptors, chemokine receptor CX3C (CX3CR1) as Class A, protein-activated receptor, highly expressed in activated microglia, and corticotropin releasing factor receptor 1 (CRFR1) as representative Class B1 receptor. Structural differences between binding domains and their endogenous ligands as well as parallel expression in different types of cells and generally low density of these receptors in brain tissue are factors making the search for selective and sensitive ligands more difficult than for classical GPCR receptors. Main progress in ligand development is observed for NPY receptor antagonists and orexin receptor antagonists. For orexin receptors, search for suitable ligands can be supported with modelling approaches, as recently the complete molecular structure of these receptors is available. Small molecules, binding at CRFR1, as for other Class B1 receptor ligands, in PET and investigations of pharmacodynamics the GPCRs, CX3CR1 is focused as target of PET during inflammation of brain and spinal cord. Differences in Microglial CX3CR Signaling Obesity Suscepti-M.

croglia in neurogenic niches, the possibilities of transformation of brain cells and opportunities of support of tissue repair by transplantation of microglia in selected brain regions or spinal cord [33] [34] [35]. Moreover, it is not finally clear if the dogma is true that microglia population of the brain recruits new cells only from itself. For instance, Özen et al. postulated 2014 related to experiments in mice that also pericytes can be progenitor cells of cerebral microglia [36]. Furthermore, exit of microglia from and return to spinal cord had been reported in an experimental mouse model resulting in changed, "peripherally experienced" microglia cells in the spinal cord tissue [34].
Efforts in the development of novel small-molecule ligands are directed also to some neuropeptide receptors of both classes of GPCRs identified either expressed in neurons and in microglia, simultaneously, or predominantly in activated microglia, during the last decade. The chemokine receptor accepted as one of the biomarkers of activated microglia is CX3CR1 [17] [51] [52] [53]. It can be enhanced by the tenfold in diverse inflammatory processes in comparison to macrophages, neurons or astrocytes [16]. The extent of expression of CX3CR1 has been determined, predominantly, with mRNA measurements [20]. Further representatives of Class A receptors, however, with neuropeptides as endogenous ligands and small-molecule candidates for positron emission tomography (PET) imaging are the neuropeptide Y1 (NPY1) and neuropeptide Y2 (NPY2) receptors for which quantitative data on receptor density (receptor protein) in the brain are available [54] [55]. Expression in microglia has been reported [21] even if the percentage of contributions to PET images or in specific brain regions and the quantitative data on the degree of expression of the receptor proteins in comparison to neurons are yet not available. Orexin receptors, also Class A GPCRs, belong to the more than 40 receptors, in which the 3D crystal structure has been ascertained [48], meanwhile. Whereas the orexin 1 receptor (OX1R) seems to be expressed relatively specific in the brain and spinal cord [18], the main attention in research on orexin receptors is directed to orexin 2 receptors with view to its role in wake-sleep cycle and the therapeutic application of receptor antagonists in insomnia which get increasing attention, especially, because of strong adverse effects of the so called Z-drugs (like zolpidem) [56] [57].
Applications, especially for potential OX1R PET tracers, could be identified in traumatically injured brain tissue due to increased migration of microglia [19].   Table 2), respectively, which already show sub-nanomolar affinity and appropriate log P and molecular weight.
Series of substitutions at the guanidino moieties supplied several further high affinity compounds.
However, already, the parent compounds had molecular weights of 500 kDa.  Table 2), suitable for labelling with C-11 or F-18 were in a range which no longer meets the requirements to molecular size proposed by Lipinski et al. [68] for a drug diffusible at the BBB.
A potential application for investigations of the spinal cord where the brain spinal cord barrier (BSCB) provides higher permeability in comparison to BBB [46] remains to be elucidated, especially, for conditions connected with inflammatory processes.
Recently, Kawamura et al. [69] presented ureido phenyl dihydroxypyridine dicarboxylates, which are available as [ 11    The authors investigated in first in vivo experiments the distribution of the compounds in mice with Elacridar-an inhibitor of permeability glycoprotein (Pgp) and of breast cancer resistance protein (BCRP). The methylated BMS 193858 showed better uptake into the brain. Elacridar increased the uptake of the test substance in lung, heart, muscle and brain although the total accumulation in the brain stood below 1% of the injected dose during the observation period. Uptake of the desmethylated compound was increased only in the liver and the heart [69].
Regarding binding-site structure and density-related PET, NPY2 receptors are found in the brain with high incidence. However, functional approach has to World Journal of Neuroscience take in account that NPY2 binding sites are also presynaptic receptors providing by this way a more complex response on potential therapeutics. Simultaneously, this requires more differentiated approaches to interpretation of changes in density.
A first real breakthrough with relevance for availability of a NPY2 receptor PET tool was the study by Winterdahl   could not be transferred in brain PET clinical imaging also due to a too high molecular weight resulting in pseudo-peptidic properties and, finally, in abolishment of the diffusion through the BBB. The IC 50 of 3.3 nM in competitive binding assays with [ 125 I] NPY corresponded at least to a moderate affinity beyond the sub-nanomolar scope [72]. Finally, the description by Dautzenberg et al. [74] (Johnson &Johnson Research and Development, Hofmann la Roche.) as an irreversibly binding, unsurmountable ligand prevented BIE246 as a potential brainPET tracer.   For NPY1 receptors the problem of selectivity, especially versus NPY4 receptors, belongs to the questions to be asked also in molecular modelling models.
Resolving this will define and complete not only preliminary pharmacophore models but also important differences in the action of endogenous ligands and differences between diverse GPCR classes [76].

Orexin Receptors
Orexin receptors have been suggested to play special roles in neuroprotection (OX1R), wake sleep-cycle, pro-arousal effects and feeding behavior (OX2R) [ [84]. Orexin A is reported to inhibit activation of the TCC, while Orexin B, 10 fold more active at the OX2R than at OX1R, is supposed to activate the TCC as well as premonitory autonomic symptoms of the migraine attack. Dysregulations of the orexin system resulting in higher orexin concentration in the cerebrovascular liquor of patients are suggested to be involved in occurrence of depressive periods in humans [85].
Signal transmission has been described for OX1R, predominantly, mediated by G q protein and OX2R via G s and G q or G i . In vitro OX1R and OX2R are able to formation of dimeres and able of induction of G q , G s or G i protein signaling [85]. Potentially, this can induce also diverse functional interferences between these and other receptors in vivo.
First approved orexin receptor antagonist with dual affinity to OX1R and OX2R (DORA: dual orexin receptor antagonists) was disclosed 2014 with suvorexant. Its analogue almorexant had been confirmed to remain without improvement of wakefulness in OX2R knock-out mice [86] [87], although some effect on REM (rapid eye movement) sleep was observed [87]. Compared to the hypnotics commonly used clinically, the approach via ligands of orexin receptors is relatively new in the field of hypnotics and the idea of involvement in therapy of migraine even newer. While potential therapeutics, hitherto, predominantly are dual orexin receptor ligands like suvorexant, also more and more scaffolds of selective OXR antagonists (SORA) are published. The OX1R is the receptor type described to be expressed by a larger extent in pathological conditions especially, following traumatic injury also due to its presence in microglia cells and migration of microglia into foci of injured tissue [19].  Table   2). However, the selectivity was at least rather limited because the compound showed interactions also with some receptors of the amine cluster as well as with purine receptors [88].   The ligands, in the focus of the study by Yin et al. [94], were the selective  showing higher affinity.  At least, however, an affinity of the tracers in sub-nanomolar range would be preferable even if physicochemical properties can be optimal. Indeed, Wang et al. [99] showed binding to different brain areas with the [ 11 C] labelled compound CW4 ( Figure 5, {30}) (Table 2) in the brain of baboons [99]. Contributions of microglia and neurons had not been discriminated during these experiments.
Recently, Skudlarek et al. [106] described several series of potential drug candidates with more or less suitable pharmacokinetic properties and receptor affinities ( Figure 5, {31} {32} {33}). Although the general content and motivation remains the treatment of insomnia with antagonists of OX2R, the group provides many suggestions to pharmacophore development of ligands not only for OX2R but also for OX1R. A comparison of ligand binding mechanisms was performed at an engineered receptor construct using the third intracellular loop of the hOX1R [94].
A series of cyclo propane carboxamide structures published 2013 by Oi et al. [107] showed with inverse frequency rather a role as Pgp substrate and increased lipophilic features. One compound (9a) (Figure 5, {29}) with log P of 3.29 and log D 2.97 was shown with appropriate binding in the brain of a Pgp knock-out rat.
A Pgp flux ratio of 3.11, in the border range of appropriate values, avoids a use without support of Pgp inhibitors. Related structures were proposed by Gao et al. [108] as OX2R antagonists. Roecker et al. [79] developed the isoquinoline compound 53 as a candidate for labelling with a positron emitter ( Figure 5, {34}). With a suitable log P and high affinity binding it could acquire also sufficient properties as a PET tracer.

CX3CR1 (Fractalkine Receptor)
During the last five years more and more attention has been paid to a special role of microglia and its CX3C (chemokine CX3C) receptor 1 subtype (fraktalkine receptor) in induction of obesity and the immunological response mediated by this receptor under conditions of high fat diet [109] as well as sex differences in the CX3CL-CX3CR signaling of CX3CR1 knock-in mice [110]. The receptor is suggested to be responsible for the enhanced sensitivity of male animals associated with high fat diet. Although quantitative data on the expression of receptor protein in microglia membranes are not available, approaches have been done earlier, in 1998 by Nishiyori et al. [17], with mRNA in situ hybridization studies in rat brain ( Table 1). The results showed high levels of receptor mRNA in microglia cells compared to neurons and astrocytes while fractalkine mRNA was very high in neurons in comparison to astrocytes and microglia [17]. The receptor has been reported, meanwhile, with tenfold increased density in microglia compared to macrophages, neurons or astrocytes [16] [17]. In the periphery, CX3CR1-expressing monocytes have been demonstrated as immune surveillant of blood vessels. Such cells were able to invade into the tissue if irritants were present [111]. Determination of small RNAs for specific signature proteins allows the differentiation between glia subtypes of different stages of development World Journal of Neuroscience or diseases, at subcellular level [6]. CX3CR1 plays not a critical role for short-term survival, but was demonstrated to show enhanced expression on microglia associated with injuries of CNS and spinal cord [53] [54]. Microglia and monocyte-derived macrophages promote in spinal cord lesions inflammatory responses. Freria et al. [53] demonstrated in CX3CR1-/-mice that the deficiency of CX3CR1 interrupts pro-inflammatory signal chains of these cells and support repair and sprouting processes in neuronal synapses. Earlier, the soluble and the membrane-anchored forms of CX3CL-the endogenous ligand of the receptor, had been described. The knock-out of CX3CR1 resulted in decrease of atherogenesis but also enhancement of inflammation in the brain and development of pain as well as age related macular degeneration [112] [113].
The membrane-anchored CX3CL1 (fractalkine) contributes to binding of leukocytes expressing CX3CR1 without involvement of adhesion proteins or integrines in the periphery. This suggests therapeutic potential of CX3CR1 inhibition after spinal injury. Whether in vivo imaging would can supply information on healing processes using PET remains to be elucidated.
First trials for the development of CX3CR1 antagonists were focused on chemokine analogues usually characterized by molecular weights between 8 and 10 kDa [112] [113].
To date, the number of potentially effective small molecules available for labeling of CX3CR1 is very limited [42] [43] [51].
Karlström et al. [42] and Cederblad et al. [43] Table 2). Two series of compounds were developed including neutral amino thiazolopyrimidines (series A) and acidic thiazolopyrimidinones (series B) which were further tested also for their selectivity for CX3CR1. Generally, membrane permeability was higher for series B compounds. However, for compound 18a (AZD8797; series A) was found appropriate selectivity for CX3CR1 in comparison to some other chemokine receptors, but also a more intensive interaction with adenosine A1 receptors (K i of 132 nM) and a 33 fold selectivity. Selectivity versus receptors of the amine cluster was 200 fold for CX3CR1 [42].  Also the crystal structure of adenosine A1 receptors is in progress [114], but not yet complete, what hampers the discrimination of structural features of the receptors responsible for the deficit in selectivity small-molecule ligands of CX3CR1.
Some investigations with AZD8797 as drug in animal disease models are promising. Ridderstad-Wollberg et al. [115] observed in a first therapeutic experiment in Agouti mice suffering from EAE (experimental autoimmune encephalitis) a common model of multiple sclerosis, effective action of the drug if applied before start of the disease [115].

CRF-Receptor-1
CRFR and its endogenous ligands play a central role in the hypothalamic-pituitary-axis but are located and acting also widespread in the body. The CRF1 subtype of the corticotropin releasing factor receptor is most frequently target of drugs for therapeutic purposes and expressed by larger extent than CRF2 subtype. Diverse concepts had been developed for the involvement of the CRF1 receptor in the treatment of anxiety, depression, stress and posttraumatic disturbances. Some of the drugs proposed for such purposes had been labelled also with positron emitting isotopes, i.  [116].
The general expectations to the role of corticotropin releasing factor receptor ligands in future therapies of mental diseases returned to reality of structural constraints for these receptors, during the last years [117]. Simultaneously, a crucial role of CRF receptors in stress response and contributions to protection against stress remained beyond doubt. Spierling and Zorilla resumed 2017 [118] on the general behavioral approach to treat affective and anxiety diseases rather a lack of success and account that outcome, especially, to problems in physico- There are only few studies using molecular dynamics for the proof of mechanism in this field. Bai et al. [60] and Hollenstein et al. [59] described 2013 and 2014 a first simulation of CRFR1 binding at the TMDs of the receptor with the allosteric antagonist CP376395 (Figure 7 {42}). Doré et al. [61] and Seidel et al. [116] published first studies with attention to the complete receptor structure World Journal of Neuroscience  Doré et al. [61] identified the binding mode of CP376395 in a mutagenesis approach as an allosteric interaction at the transmembrane domain.
Although the most drug developments focus on the role of the receptor in neuronal cells, some investigations asked also for potential roles and hyper-expression of the CRFR1 in microglia [23] [33] [34] [64]. Such approaches are concerned, currently, rather with spinal cord diseases or pathophysiological reactions than with brain diseases [23] [33] [64].  [58] could not confirm, finally, the suitability of the tracer also in brain PET imaging of primates and monkey.
During the last years several groups proposed further lead structures and modified physicochemical properties for better uptake across the BBB. Density of CRF1 receptors is the highest in pituitary gland (302 fmol/mg protein) [120].
However, localizations with B max interesting for PET in vivo imaging have been found also in cerebral cortex and cerebellum [120]. Lodge et al. [120] Table 2) were tested in vitro and in vivo in rat and monkey. But the promising binding potentials of in vitro investigations could not be confirmed in vivo [120]. The authors presume three possible reasons for this mismatch, which are: potential concurrence of endogenous peptides at the binding site of the receptor in vivo with slow binding related to relatively fast elimination of the exogenous ligand; allosteric modulation of the receptor in vivo and potentially stressful conditions during the in vivo measurements influencing the binding behavior [120].
Stehouwer et al. [121] created a series of dihydropryridin-pyrazinones ( Figure   8; Table 2) and designed two of these as optimized PET tracers with high affinity to CRF1R and log P 2.2. First investigation in cynomouglus brain, finally, resulted in a low brain uptake. Inhibition of Pgp was not further tested. In 2017, the group proposed the fluoalkyl pyrazinon BMS 6650553 [122] [123] (Figure 8 {52}) ( Table 2) as a further development derived from the original series.  Also questions on variations in the regional role of microglia [124], especially, between cerebellum and cortex get first detailed answers with some scientific dynamics [125] [126] [127]. First studies in mice demonstrated that microglias of the cerebellum are less ramified than in cerebral cortex but are distributed more sparsely compared to the cortex. Stowell et al. [45] postulated that changed morphology and occurrence together with some functional properties of microglia could alter the efficacy of surveillance of the neuronal population in the cerebellum. Ayata et al. [125] postulated that microglia are responsible for a higher degree of cell death in cerebellum compared to cortical regions of the brain.

Conclusions and Challenges
They suggested that a decrease of cell death can trigger a change of the pheno-  [127] and functional properties of microglia similar to such in neurodegenerative diseases. The high phagocytotic activity typical for cerebellar microglia is suppressed in striatum and hippocampus. Finally, the "new" distribution of the cellular population in the brain reflects not only new local priorities of neurons but also of non-neuronal cells, especially of microglia.
A functional aspect which meets one key role of this part of cell population is the increasing inflammatory property of microglia during aging [128]. This can result in a hypersensitive response to stress in aged patients and alters requirements to therapy under such conditions [126] as well as for the tolerance to implants [129] and gives the item personalized therapy an even higher priority.
The density of microglial neuropeptide receptors or even of the chemokine receptor CX3CR is described, as well as hitherto, half-quantitatively with antibodies or mRNA measurements (Table 1) which hampers access to the quantitative comparison of neuronal and microglia receptors. Trials to test correlations between expression of receptor protein and formation of mRNA have been undertaken rather for classical receptors like dopamine receptors by Araki et al. [130]. The authors found a good correlations between dopamine 1 receptor mRNA and the receptor protein.
Although microglia cultures are not easy to obtain and to maintain, meanwhile, they are available in several working groups opening the possibilities to obtain quantitative data also on receptor protein expression. Moreover, trascriptoms of special subtypes of microglia are determined in regional tissue sections, allowing detailed evaluation of functional specificities in the respective brain regions.
For orexin receptors and CRF receptor subtype 1, densities in the range of 100 fmol/mg protein have been measured. Sullivan et al. [58] suggested this range as potentially sufficient for PET visualization with reference to a similar extent for serotonin receptors, where PET has been performed with success.
The developed small-molecule candidates of PET tracers for CRFR1, orexin receptors and NPY1 and 2 receptors could be helpful tools for such evaluations even before they are introduced as PET diagnostics. However, the complex binding behavior of endogenous agonists of CRFR1 might be a challenge for modelling of small-molecule ligands as candidates for drugs or diagnostic tools.
The more complicate question of a specific CX3CR1 antagonist with appropriate physicochemical properties necessary for brain PET might need rather the interdisciplinary approach of molecular modelling. This would be also a tribute to the complex structure of the endogenous ligands of chemokine receptors.
Moreover, modelling in present crystal structures of CRFR1 and orexin receptors could facilitate understanding and pharmacophore development of Class A World Journal of Neuroscience and B1 receptor ligands as allosteric or orthosteric effectors. A special challenge to in vivo visualization methods stays mapping of cerebellar structures [131] and exact identification of its regional binding places with view to both, classical GPCR s and receptors with more complicate extracellular binding domains.

Conflicts of Interest
The author declares no conflicts of interest regarding the publication of this paper.