Minor Changes in Erythrocyte Osmotic Fragility in Trace Amine-Associated Receptor 5 (TAAR5) Knockout Mice

Trace amine-associated receptors (TAARs) are a group of G protein-coupled receptors that are expressed in the olfactory epithelium, central nervous system, and periphery. TAAR family generally consists of nine types of receptors (TAAR1-9), which can detect biogenic amines. During the last 5 years, the TAAR5 receptor became one of the most intriguing receptors in this subfamily. Recent studies revealed that TAAR5 is involved not only in sensing socially relevant odors but also in the regulation of dopamine and serotonin transmission, emotional regulation, and adult neurogenesis by providing significant input from the olfactory system to the limbic brain areas. Such results indicate that future antagonistic TAAR5-based therapies may have high pharmacological potential in the field of neuropsychiatric disorders. TAAR5 is known to be expressed in leucocytes as well. To evaluate potential hematological side effects of such future treatments we analyzed several hematological parameters in mice lacking TAAR5. In these mutants, we observed minor but significant changes in the osmotic fragility test of erythrocytes and hematocrit levels. At the same time, analysis of other parameters including complete blood count and reticulocyte levels showed no significant alterations in TAAR5 knockout mice. Thus, TAAR5 gene knockout leads to minor negative changes in the erythropoiesis or eryptosis processes, and further research in that field is needed. The impact of TAAR5 deficiency on other hematological parameters seems minimal. Such negative, albeit minor, effects of TAAR5 deficiency should be taken into account during future TAAR5-based therapy development.     

TAAR1 Expression in Human Macrophages and Brain Tissue: A Potential Novel Facet of MS Neuroinflammation

TAAR1 is a neuroregulator with emerging evidence suggesting a role in immunomodulation. Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. Here, we investigate TAAR1 expression in human primary monocytes, peripherally-derived macrophages, and MS brain tissue. RT-qPCR was used to assess TAAR1 levels in MS monocytes. Using a previously validated anti-human TAAR1 antibody and fluorescence microscopy, TAAR1 protein was visualized in lipopolysaccharide-stimulated or basal human macrophages, as well as macrophage/microglia populations surrounding, bordering, and within a mixed active/inactive MS lesion. In vivo, TAAR1 mRNA expression was significantly lower in MS monocytes compared to age- and sex-matched healthy controls. In vitro, TAAR1 protein showed a predominant nuclear localization in quiescent/control macrophages with a shift to a diffuse intracellular distribution following lipopolysaccharide-induced activation. In brain tissue, TAAR1 protein was predominantly expressed in macrophages/microglia within the border region of mixed active/inactive MS lesions. Considering that TAAR1-mediated anti-inflammatory effects have been previously reported, decreased mRNA in MS patients suggests possible pathophysiologic relevance. A shift in TAAR1 localization following pro-inflammatory activation suggests its function is altered in pro-inflammatory states, while TAAR1-expressing macrophages/microglia bordering an MS lesion supports TAAR1 as a novel pharmacological target in cells directly implicated in MS neuroinflammation.     

Genetic Deletion of Trace-Amine Associated Receptor 9 (TAAR9) in Rats Leads to Decreased Blood Cholesterol Levels

In the last two decades, interest has grown significantly in the investigation of the role of trace amines and their receptors in mammalian physiology and pathology. Trace amine-associated receptor 9 (TAAR9) is one of the least studied members of this receptor family with unidentified endogenous ligands and an unknown role in the central nervous system and periphery. In this study, we generated two new TAAR9 knockout (TAAR9-KO) rat strains by CRISPR-Cas9 technology as in vivo models to evaluate the role of TAAR9 in mammalian physiology. In these mutant rats, we performed a comparative analysis of a number of hematological and biochemical parameters in the blood. Particularly, we carried out a complete blood count, erythrocyte osmotic fragility test, and screening of a panel of basic biochemical parameters. No significant alterations in any of the hematological and most biochemical parameters were found between mutant and WT rats. However, biochemical studies revealed a significant decrease in total and low-density lipoprotein cholesterol levels in the blood of both strains of TAAR9-KO rats. Such role of TAAR9 in cholesterol regulation not only brings a new understanding of mechanisms and biological pathways of lipid exchange but also provides a new potential drug target for disorders involving cholesterol-related pathology, such as atherosclerosis.     

Trace Amine-Associated Receptor 1 (TAAR1) Is a Positive Prognosticator for Epithelial Ovarian Cancer

Trace amine-associated receptor 1 (TAAR1) is a G_αs- protein coupled receptor that plays an important role in the regulation of the immune system and neurotransmission in the CNS. In ovarian cancer cell lines, stimulation of TAAR1 via 3-iodothyronamine (T₁AM) reduces cell viability and induces cell death and DNA damage. Aim of this study was to evaluate the prognostic value of TAAR1 on overall survival of ovarian carcinoma patients and the correlation of TAAR1 expression with clinical parameters. Ovarian cancer tissue of n = 156 patients who were diagnosed with epithelial ovarian cancer (serous, n = 110 (high-grade, n = 80; low-grade, n = 24; unknown, n = 6); clear cell, n = 12; endometrioid, n = 21; mucinous, n = 13), and who underwent surgery at the Department of Obstetrics and Gynecology, University Hospital of the Ludwig-Maximilians University Munich, Germany between 1990 and 2002, were analyzed. The tissue was stained immunohistochemically with anti-TAAR1 and evaluated with the semiquantitative immunoreactive score (IRS). TAAR1 expression was correlated with grading, FIGO and TNM-classification, and analyzed via the Spearman’s rank correlation coefficient. Further statistical analysis was obtained using nonparametric Kruskal-Wallis rank-sum test and Mann-Whitney-U-test. This study shows that high TAAR1 expression is a positive prognosticator for overall survival in ovarian cancer patients and is significantly enhanced in low-grade serous carcinomas compared to high-grade serous carcinomas. The influence of TAAR1 as a positive prognosticator on overall survival indicates a potential prognostic relevance of signal transduction of thyroid hormone derivatives in epithelial ovarian cancer. Further studies are required to evaluate TAAR1 and its role in the development of ovarian cancer.     

Enhanced Aggression,Reduced Self-Grooming Behavior and Altered 5-HT Regulation in the Frontal Cortex in Mice Lacking Trace Amine-Associated Receptor 1 (TAAR1)

The Trace Amine-Associated Receptor 1 (TAAR1) is one of the six functional receptors belonging to the family of monoamine-related G protein-coupled receptors (TAAR1-TAAR9) found in humans. However, the exact biological mechanisms of TAAR1 central and peripheral action remain to be fully understood. TAAR1 is widely expressed in the prefrontal cortex and several limbic regions, interplaying with the dopamine system to modulate the reward circuitry. Recent clinical trials suggest the efficacy of TAAR1 agonists as potential novel antipsychotic agents. Here, we characterize behavioral and neurochemical phenotypes of TAAR1 knockout mice, focusing on aggression and self-grooming behavior that both strongly depend on the monoaminergic signaling and cortico-striatal and cortico-limbic circuits. Overall, we report increased aggression in these knockout mice in the resident-intruder test, accompanied by reduced self-grooming behavior in the novelty-induced grooming test, and by higher cortical serotonin (5-HT) tissue levels. Further studies are necessary to explore whether TAAR1-based therapies can become potential novel treatments for a wide range of neuropsychiatric disorders associated with aggression.     

Studies of New Fused Benzazepine as Selective Dopamine D3 Receptor Antagonists Using 3D-QSAR, Molecular Docking and Molecular Dynamics

In recent years, great interest has been paid to the development of compounds with high selectivity for central dopamine (DA) D3 receptors, an interesting therapeutic target in the treatment of different neurological disorders. In the present work, based on a dataset of 110 collected benzazepine (BAZ) DA D3 antagonists with diverse kinds of structures, a variety of in silico modeling approaches, including comparative molecular field analysis (CoMFA), comparative similarity indices analysis (CoMSIA), homology modeling, molecular docking and molecular dynamics (MD) were carried out to reveal the requisite 3D structural features for activity. Our results show that both the receptor-based (Q(2) = 0.603, R(2) (ncv) = 0.829, R(2) (pre) = 0.690, SEE = 0.316, SEP = 0.406) and ligand-based 3D-QSAR models (Q(2) = 0.506, R(2) (ncv) =0.838, R(2) (pre) = 0.794, SEE = 0.316, SEP = 0.296) are reliable with proper predictive capacity. In addition, a combined analysis between the CoMFA, CoMSIA contour maps and MD results with a homology DA receptor model shows that: (1) ring-A, position-2 and R(3) substituent in ring-D are crucial in the design of antagonists with higher activity; (2) more bulky R(1) substituents (at position-2 of ring-A) of antagonists may well fit in the binding pocket; (3) hydrophobicity represented by MlogP is important for building satisfactory QSAR models; (4) key amino acids of the binding pocket are CYS101, ILE105, LEU106, VAL151, PHE175, PHE184, PRO254 and ALA251. To our best knowledge, this work is the first report on 3D-QSAR modeling of the new fused BAZs as DA D3 antagonists. These results might provide information for a better understanding of the mechanism of antagonism and thus be helpful in designing new potent DA D3 antagonists.     

Discovery of Highly Potent Dual Orexin Receptor Antagonists via a Scaffold‐Hopping Approach

Starting from suvorexant (trade name Belsomra), we successfully identified interesting templates leading to potent dual orexin receptor antagonists (DORAs) via a scaffold‐hopping approach. Structure–activity relationship optimization allowed us not only to improve the antagonistic potency on both orexin 1 and orexin 2 receptors (Ox1 and Ox2, respectively), but also to increase metabolic stability in human liver microsomes (HLM), decrease time‐dependent inhibition of cytochrome P450 (CYP) 3A4, and decrease P‐glycoprotein (Pgp)‐mediated efflux. Compound 80 c [{(1S,6R)‐3‐(6,7‐difluoroquinoxalin‐2‐yl)‐3,8‐diazabicyclo[4.2.0]octan‐8‐yl}(4‐methyl‐[1,1′‐biphenyl]‐2‐yl)methanone] is a potent and selective DORA that inhibits the stimulating effects of orexin peptides OXA and OXB at both Ox1 and Ox2. In calcium‐release assays, 80 c was found to exhibit an insurmountable antagonistic profile at both Ox1 and Ox2, while displaying a sleep‐promoting effect in rat and dog models, similar to that of the benchmark compound suvorexant.     

Therapeutic Perspectives of HIV-Associated Chemokine Receptor (CCR5 and CXCR4) Antagonists in Carcinomas

The interaction between malignant cells and the tumor microenvironment is critical for tumor progression, and the chemokine ligand/receptor axes play a crucial role in this process. The CXCR4/CXCL12 and CCR5/CCL5 axes, both related to HIV, have been associated with the early (epithelial–mesenchymal transition and invasion) and late events (migration and metastasis) of cancer progression. In addition, these axes can also modulate the immune response against tumors. Thus, antagonists against the receptors of these axes have been proposed in cancer therapy. Although preclinical studies have shown promising results, clinical trials are needed to include these drugs in the oncological treatment protocols. New alternatives for these antagonists, such as dual CXCR4/CCR5 antagonists or combined therapy in association with immunotherapy, need to be studied in cancer therapy.     

The Predicted Ensemble of Low‐Energy Conformations of Human Somatostatin Receptor Subtype 5 and the Binding of Antagonists

Human somatostatin receptor subtype 5 (hSSTR5) regulates cell proliferation and hormone secretion. However, the identification of effective therapeutic small‐molecule ligands is impeded because experimental structures are not available for any SSTR subtypes. Here, we predict the ensemble of low‐energy 3D structures of hSSTR5 using a modified GPCR Ensemble of Structures in Membrane BiLayer Environment (GEnSeMBLE) complete sampling computational method. We find that this conformational ensemble displays most interhelical interactions conserved in class A G protein‐coupled receptors (GPCRs) plus seven additional interactions (e.g., Y2.43–D3.49, T3.38–S4.53, K5.64–Y3.51) likely conserved among SSTRs. We then predicted the binding sites for a series of five known antagonists, leading to predicted binding energies consistent with experimental results reported in the literature. Molecular dynamics (MD) simulation of 50 ns in explicit water and lipid retained the predicted ligand‐bound structure and formed new interaction patterns (e.g. R3.50–T6.34) consistent with the inactive μ‐opioid receptor X‐ray structure. We suggest more than six mutations for experimental validation of our prediction. The final predicted receptor conformations and antagonist binding sites provide valuable insights for designing new small‐molecule drugs targeting SSTRs.     

The Phenoxyalkyltriazine Antagonists for 5-HT6 Receptor with Promising Procognitive and Pharmacokinetic Properties In Vivo in Search for a Novel Therapeutic Approach to Dementia Diseases

Among the serotonin receptors, one of the most recently discovered 5-HT₆ subtype is an important protein target and its ligands may play a key role in the innovative treatment of cognitive disorders. However, none of its selective ligands have reached the pharmaceutical market yet. Recently, a new chemical class of potent 5-HT₆ receptor agents, the 1,3,5-triazine-piperazine derivatives, has been synthesized. Three members, the ortho and meta dichloro- (1,2) and the unsubstituted phenyl (3) derivatives, proved to be of special interest due to their high affinities (1,2) and selectivity (3) toward 5-HT₆ receptor. Thus, a broader pharmacological profile for 1–3, including comprehensive screening of the receptor selectivity and drug-like parameters in vitro as well as both, pharmacokinetic and pharmacodynamic properties in vivo, have been investigated within this study. A comprehensive analysis of the obtained results indicated significant procognitive-like activity together with beneficial drug-likeness in vitro and pharmacokinetics in vivo profiles for both, (RS)-4-[1-(2,3-dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (2) and (RS)-4-(4-methylpiperazin-1-yl)-6-(1-phenoxypropyl)-1,3,5-triazin-2-amine (3), but insensibly predominant for compound 2. Nevertheless, both compounds (2 and 3) seem to be good Central Nervous System drug candidates in search for novel therapeutic approach to dementia diseases, based on the 5-HT₆ receptor target.     

Blockade of Serotonin 5-HT6 Receptor Constitutive Activity Alleviates Cognitive Deficits in a Preclinical Model of Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is a common inherited disorder caused by mutations of the NF1 gene that encodes the Ras-GTPase activating protein neurofibromin, leading to overactivation of Ras-dependent signaling pathways such as the mTOR pathway. It is often characterized by a broad range of cognitive symptoms that are currently untreated. The serotonin 5-HT₆ receptor is a potentially relevant target in view of its ability to associate with neurofibromin and to engage the mTOR pathway to compromise cognition in several cognitive impairment paradigms. Here, we show that constitutively active 5-HT₆ receptors contribute to increased mTOR activity in the brain of Nf1^+/− mice, a preclinical model recapitulating some behavioral alterations of NF1. Correspondingly, peripheral administration of SB258585, a 5-HT₆ receptor inverse agonist, or rapamycin, abolished deficits in long-term social and associative memories in Nf1^+/− mice, whereas administration of CPPQ, a neutral antagonist, did not produce cognitive improvement. These results show a key influence of mTOR activation by constitutively active 5-HT₆ receptors in NF1 cognitive symptoms. They provide a proof of concept that 5-HT₆ receptor inverse agonists already in clinical development as symptomatic treatments to reduce cognitive decline in dementia and psychoses, might be repurposed as therapies alleviating cognitive deficits in NF1 patients.     

Description of a Novel Mechanism Possibly Explaining the Antiproliferative Properties of Glucocorticoids in Duchenne Muscular Dystrophy Fibroblasts Based on Glucocorticoid Receptor GR and NFAT5

Glucocorticoids are drugs of choice in Duchenne muscular dystrophy (DMD), prolonging patients’ ambulation. Their mode of action at the protein level is not completely understood. In DMD, muscle tissue is replaced by fibrotic tissue produced by fibroblasts, reducing mobility. Nuclear factor of activated T-cells 5 (NFAT5) is involved in fibroblast proliferation. By treating one DMD fibroblast cell culture and one of unaffected skeletal muscle fibroblasts with methylprednisolone (MP) or hydrocortisone (HC) for 24 h or 12 d, the antiproliferative properties of glucocorticoids could be unraveled. NFAT5 localization and expression was explored by immunocytochemistry (ICC), Western blotting (WB) and RT-qPCR. NFAT5 and glucocorticoid receptor (GR) colocalization was measured by ImageJ. GR siRNA was used, evaluating GR’s influence on NFAT5 expression during MP and HC treatment. Cell proliferation was monitored by IncuCyte ZOOM. In DMD fibroblasts, treatment with MP for 24 h induced dots (ICC) positive for NFAT5 and colocalizing with GR. After 12 d of MP or HC in DMD fibroblasts, NFAT5 expression was decreased (RT-qPCR and WB) and growth arrest was observed (Incucyte ZOOM), whereas NFAT5 expression and cell growth remained unchanged in unaffected skeletal muscle fibroblasts. This study may help understand the antiproliferative properties of glucocorticoids in DMD fibroblasts.     

The Three Dimensional Quantitative Structure Activity Relationships (3D-QSAR) and Docking Studies of Curcumin Derivatives as Androgen Receptor Antagonists

Androgen receptor antagonists have been proved to be effective anti-prostate cancer agents. 3D-QSAR and Molecular docking methods were performed on curcumin derivatives as androgen receptor antagonists. The bioactive conformation was explored by docking the potent compound 29 into the binding site of AR. The constructed Comparative Molecular Field Analysis (CoMFA) and Comparative Similarity Indices Analysis (CoMSIA) models produced statistically significant results with the cross-validated correlation coefficients q(2) of 0.658 and 0.567, non-cross-validated correlation coefficients r(2) of 0.988 and 0.978, and predicted correction coefficients r(2) (pred) of 0.715 and 0.793, respectively. These results ensure the CoMFA and CoMSIA models as a tool to guide the design of novel potent AR antagonists. A set of 30 new analogs were proposed by utilizing the results revealed in the present study, and were predicted with potential activities in the developed models.     

Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH–DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH–DR5-B can be considered as a promising candidate for therapeutic applications.     

Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling

Recently, Toll-like receptors (TLRs), a family of pattern recognition receptors, are reported as potential modulators for neuropathic pain; however, the desired mechanism is still unexplained. Here, we operated on the sciatic nerve to establish a pre-clinical rodent model of chronic constriction injury (CCI) in Sprague-Dawley rats, which were assigned into CCI and Decompression groups randomly. In Decompression group, the rats were performed with nerve decompression at post-operative week 4. Mechanical hyperalgesia and mechanical allodynia were obviously attenuated after a month. Toll-like receptor 5 (TLR5)-immunoreactive (ir) expression increased in dorsal horn, particularly in the inner part of lamina II. Additionally, substance P (SP) and isolectin B4 (IB4)-ir expressions, rather than calcitonin-gene-related peptide (CGRP)-ir expression, increased in their distinct laminae. Double immunofluorescence proved that increased TLR5-ir expression was co-expressed mainly with IB4-ir expression. Through an intrathecal administration with FLA-ST Ultrapure (a TLR5 agonist, purified flagellin from Salmonella Typhimurium, only the CCI-induced mechanical hyperalgesia was attenuated dose-dependently. Moreover, we confirmed that mu-opioid receptor (MOR) and phospho-protein kinase Cα (pPKCα)-ir expressions but not phospho-protein kinase A RII (pPKA RII)-ir expression, increased in lamina II, where they mostly co-expressed with IB4-ir expression. Go 6976, a potent protein kinase C inhibitor, effectively reversed the FLA-ST Ultrapure- or DAMGO-mediated attenuated trend towards mechanical hyperalgesia by an intrathecal administration in CCI rats. In summary, our current findings suggest that nerve decompression improves CCI-induced mechanical hyperalgesia that might be through the cross-talk of TLR5 and MOR in a PKCα-dependent manner, which opens a novel opportunity for the development of analgesic therapeutics in neuropathic pain.     

BioS2Net: Holistic Structural and Sequential Analysis of Biomolecules Using a Deep Neural Network

Background: For decades, the rate of solving new biomolecular structures has been exceeding that at which their manual classification and feature characterisation can be carried out efficiently. Therefore, a new comprehensive and holistic tool for their examination is needed. Methods: Here we propose the Biological Sequence and Structure Network (BioS2Net), which is a novel deep neural network architecture that extracts both sequential and structural information of biomolecules. Our architecture consists of four main parts: (i) a sequence convolutional extractor, (ii) a 3D structure extractor, (iii) a 3D structure-aware sequence temporal network, as well as (iv) a fusion and classification network. Results: We have evaluated our approach using two protein fold classification datasets. BioS2Net achieved a 95.4% mean class accuracy on the eDD dataset and a 76% mean class accuracy on the F184 dataset. The accuracy of BioS2Net obtained on the eDD dataset was comparable to results achieved by previously published methods, confirming that the algorithm described in this article is a top-class solution for protein fold recognition. Conclusions: BioS2Net is a novel tool for the holistic examination of biomolecules of known structure and sequence. It is a reliable tool for protein analysis and their unified representation as feature vectors.