Bacteriophage-Derived Double-Stranded RNA Exerts Anti-SARS-CoV-2 Activity In Vitro and in Golden Syrian Hamsters In Vivo.

Bacteriophage-derived dsRNA, known as Larifan, is a nationally well-known broad-spectrum antiviral medication. This study aimed to ascertain the antiviral activity of Larifan against the novel SARS-CoV-2 virus. Larifan's effect against SARS-CoV-2 in vitro was measured in human lung adenocarcinoma (Calu3) and primary human small airway epithelial cells (HSAEC), and in vivo in the SARS-CoV-2 infection model in golden Syrian hamsters. Larifan inhibited SARS-CoV-2 replication both in vitro and in vivo. Viral RNA copy numbers and titer of infectious virus in the supernatant of Calu3 cells dropped significantly: p = 0.0296 and p = 0.0286, respectively. A reduction in viral RNA copy number was also observed in HSAEC, especially when Larifan was added before infection (p = 0.0218). Larifan markedly reduced virus numbers in infected hamsters' lungs post-infection, with a more pronounced effect after intranasal administration (p = 0.0032). The administration of Larifan also reduced the amount of infections virus titer in the lungs (p = 0.0039). Improvements in the infection-induced pathological lesion severity in the lungs of animals treated with Larifan were also demonstrated. The inhibition of SARS-CoV-2 replication in vitro and the reduction in the viral load in the lungs of infected hamsters treated with Larifan alongside the improved lung histopathology suggests a potential use of Larifan in also controlling the COVID-19 disease in humans.

The Specificity and Broad Multitarget Properties of Ligands for the Free Fatty Acid Receptors FFA3/GPR41 and FFA2/GPR43 and the Related Hydroxycarboxylic Acid Receptor HCA2/GPR109A.

The paradigm of ligand-receptor interactions postulated as "one compound-one target" has been evolving; a multi-target, pleiotropic approach is now considered to be realistic. Novel series of 1,4,5,6,7,8-hexahydro-5-oxoquinolines, pyranopyrimidines and S-alkyl derivatives of pyranopyrimidines have been synthesized in order to characterise their pleiotropic, multitarget activity on the FFA3/GPR41, FFA2/GPR43, and HCA2/GPR109A receptors. Hexahydroquinoline derivatives have been known to exhibit characteristic activity as FFA3/GPR41 ligands, but during this study we observed their impact on FFA2/GPR43 and HCA2/GPR109A receptors as well as their electron-donating activity. Oxopyranopyrimidine and thioxopyranopyrimidine type compounds have been studied as ligands of the HCA2/GPR109A receptor; nevertheless, they exhibited equal or higher activity towards FFA3/GPR41 and FFA2/GPR43 receptors. S-Alkyl derivatives of pyranopyrimidines that have not yet been studied as ligands of GPCRs were more active towards HCA2/GPR109A and FFA3/GPR41 receptors than towards FFA2/GPR43. Representative compounds from each synthesized series were able to decrease the lipopolysaccharide-induced gene expression and secretion of proinflammatory cytokines (IL-6, TNF-α) and of a chemokine (MCP-1) in THP-1 macrophages, resembling the effect of HCA2/GPR109A ligand niacin and the endogenous ligand propionate. This study revealed groups of compounds possessing multitarget activity towards several receptors. The obtained data could be useful for further development of multitarget ligands.

Antibacterial and Anti-Inflammatory Potential of Polyherbal Formulation Used in Chronic Wound Healing.

OBJECTIVE: Polyherbal formulations Jathyadi Thailam and Jatyadi Ghritam (JT) are used in Indian traditional medicine for diabetic chronic wounds, fistula, fissure, eczema, and burn management. We aimed to investigate the antibacterial and anti-inflammatory properties of crude hexane and ethanol extracts of JT formulations. METHODS: Antibacterial activity of JT extracts was tested to estimate minimum inhibitory concentrations (MICs) against nine reference bacterial strains, including one methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant (MDR) Pseudomonas aeruginosa, and clinical strains of methicillin-susceptible S.aureus (MSSA), all involved in diabetic foot infection. The anti-inflammatory activity of plant extracts was evaluated in LPS-treated macrophage cells by measuring the mRNA levels and secretion of inflammatory mediators. RESULTS: The antibacterial activity of JT extracts was higher against Gram (+) bacteria, with the MICs varying from 1.95 to 62.5 mg/mL. Gram (-) bacteria were only susceptible to ethanol extracts of JT. Plant extracts were found to be the most active against the reference and clinical strains of MSSA, MRSA, and biofilm-forming S. epidermidis. JT extracts efficiently inhibited in a dose-dependent manner the mRNA expression and protein secretion of proinflammatory cytokines IL-6 and IL-1ß, and chemokines MCP-1 and CXCL10 in LPS-challenged macrophages. CONCLUSION: In the present study, we have shown that extracts of JT formulations possess potent antibacterial and anti-inflammatory properties that could be involved in chronic wound healing activity and has the potential to be used as external add-on therapy in the management of multidrug-resistant bacterial infections at the wound.

Metformin Strongly Affects Gut Microbiome Composition in High-Fat Diet-Induced Type 2 Diabetes Mouse Model of Both Sexes.

Effects of metformin, the first-line drug for type 2 diabetes therapy, on gut microbiome composition in type 2 diabetes have been described in various studies both in human subjects and animals. However, the details of the molecular mechanisms of metformin action have not been fully understood. Moreover, there is a significant lack of information on how metformin affects gut microbiome composition in female mouse models, depending on sex and metabolic status in well controlled experimental setting. Our study aimed to examine metformin-induced alterations in gut microbiome diversity, composition, and functional implications of high-fat diet-induced type 2 diabetes mouse model, using, for the first time in mice study, the shotgun metagenomic sequencing that allows estimation of microorganisms at species level. We also employed a randomized block, factorial study design, and including 24 experimental units allocated to 8 treatment groups to systematically evaluate the effect of sex and metabolic status on metformin interaction with microbiome. We used DNA obtained from fecal samples representing gut microbiome before and after ten weeks-long metformin treatment. We identified 100 metformin-related differentially abundant species in high-fat diet-fed mice before and after the treatment, with most of the species relative abundances increased. In contrast, no significant changes were observed in control diet-fed mice. Functional analysis targeted to carbohydrate, lipid, and amino acid metabolism pathways revealed 14 significantly altered hierarchies. We also observed sex-specific differences in response to metformin treatment. Males experienced more pronounced changes in metabolic markers, while in females the extent of changes in gut microbiome representatives was more marked, indicated by 53 differentially abundant species with more remarkable Log fold changes compared to the combined-sex analysis. The same pattern manifested regarding the functional analysis, where we discovered 5 significantly affected hierarchies in female groups but not in males. Our results suggest that both sexes of animals should be included in future studies focusing on metformin effects on the gut microbiome.

Pituispheres Contain Genetic Variants Characteristic to Pituitary Adenoma Tumor Tissue.

The most common type of pituitary neoplasms is benign pituitary adenoma (PA). Clinically significant PAs affect around 0.1% of the population. Currently, there is no established human PA cell culture available and when PA tumor cells are cultured they form two distinct types depending on culturing conditions either free-floating aggregates also known as pituispheres or cells adhering to the surface of cell plates and displaying mesenchymal stem-like properties. The aim of this study was to trace the origin of sphere-forming and adherent pituitary cell cultures and characterize the potential use of these surgery derived cell lines as PA model. We carried out a paired-end exome sequencing of patients' tumor and germline DNA using Illumina NextSeq followed by characterization of corresponding PA cell cultures. Variation analysis revealed a low amount of somatic mutations (mean = 5.2, range 3-7) in exomes of PAs. Somatic mutations of the primary surgery material can be detected in the exomes of respective pituispheres, but not in exomes of respective mesenchymal stem-like cells. For the first time, we show that the genome of pituispheres represents genome of PA while mesenchymal stem cells derived from the PA tissue do not contain mutations characteristic to PA in their genome, therefore, most likely representing normal cells of pituitary or surrounding tissues. This finding indicates that pituispheres can be used as a human model of PA cells, but combination of cell culturing techniques and NGS needs to be employed to adjust for disability to propagate spheres in culturing conditions.

Medication for Acromegaly Reduces Expression of MUC16, MACC1 and GRHL2 in Pituitary Neuroendocrine Tumour Tissue.

Acromegaly is a disease mainly caused by pituitary neuroendocrine tumor (PitNET) overproducing growth hormone. First-line medication for this condition is the use of somatostatin analogs (SSAs), that decrease tumor mass and induce antiproliferative effects on PitNET cells. Dopamine agonists (DAs) can also be used if SSA treatment is not effective. This study aimed to determine differences in transcriptome signatures induced by SSA/DA therapy in PitNET tissue. We selected tumor tissue from twelve patients with somatotropinomas, with half of the patients receiving SSA/DA treatment before surgery and the other half treatment naive. Transcriptome sequencing was then carried out to identify differentially expressed genes (DEGs) and their protein-protein interactions, using pathway analyses. We found 34 upregulated and six downregulated DEGs in patients with SSA/DA treatment. Three tumor development promoting factors MUC16, MACC1, and GRHL2, were significantly downregulated in therapy administered PitNET tissue; this finding was supported by functional studies in GH3 cells. Protein-protein interactions and pathway analyses revealed extracellular matrix involvement in the antiproliferative effects of this type of the drug treatment, with pronounced alterations in collagen regulation. Here, we have demonstrated that somatotropinomas can be distinguished based on their transcriptional profiles following SSA/DA therapy, and SSA/DA treatment does indeed cause changes in gene expression. Treatment with SSA/DA significantly downregulated several factors involved in tumorigenesis, including MUC16, MACC1, and GRHL2. Genes that were upregulated, however, did not have a direct influence on antiproliferative function in the PitNET cells. These findings suggested that SSA/DA treatment acted in a tumor suppressive manner and furthermore, collagen related interactions and pathways were enriched, implicating extracellular matrix involvement in this anti-tumor effect of drug treatment.

Comparison of the effects of bacteriophage-derived dsRNA and poly(I:C) on ex vivo cultivated peripheral blood mononuclear cells.

Double-stranded RNA (dsRNA), regardless of the origin and nucleotide sequence, exhibits multiple biological activities, including the establishment of an antiviral state and modulation of the immune response. Both involve the stimulation of innate immunity primarily via the release of pro-inflammatory cytokines, which in turn shapes the adaptive immune response. In this study, we compared the immune response triggered by two different dsRNAs: 1) a well-known synthetic dsRNA-poly (I:C); and 2) bacteriophage-derived dsRNA (bf-dsRNA) that is a replicative form of ssRNA bacteriophage f2. Human peripheral blood mononuclear cells (PBMCs) from 61 heathy volunteers were stimulated ex vivo with both dsRNAs. Subsequently, activation markers on the main lymphocyte subpopulations were analysed by flow cytometry and the production of 29 different cytokines and chemokines was measured by Luminex xMAP technology. The effect of bf-dsRNA on ex vivo cultivated PBMCs is similar to that induced by poly(I:C), albeit with subtle dissimilarities. Both treatments increased expression of the lymphocyte CD38 marker and intracellular IFN-γ in CD8+ T and natural killer (NK) cells, as well as the CD95 marker on the main lymphocyte subpopulations. Poly(I:C) was a stronger inducer of IL-6, IL-1ß, and CCL4, whereas bf-dsRNA induced higher levels of IFN-α2, CXCL10, and CCL17. These differences might contribute to a distinct clinical manifestation when used as vaccine adjuvants, and bf-dsRNA may have more profound activity against several types of bacteria.

Crystal structure and metabolic activity of 4-(thien-2-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carb-oxy-lic acid eth-oxy-carbonyl-phenyl-methyl-ester.

In the title compound, C25H25NO5S, which exhibits metabolism-regulating activity, the 1,4-di-hydro-pyridine ring adopts a flattened boat conformation while the cyclo-hexenone ring is in an envelope conformation. Mol-ecules in the crystal are assembled into C(6) chains along the a-axis direction via N-H⋯O hydrogen bonds. The thienyl fragment is disordered over two sets of sites in a 0.7220 (19):0.2780 (19) ratio.

Hydroxycarboxylic Acid Receptor Ligands Modulate Proinflammatory Cytokine Expression in Human Macrophages and Adipocytes without Affecting Adipose Differentiation.

Members of the hydroxycarboxylic acid receptor (HCA1-3) family are mainly expressed in adipocytes and immune cells. HCA2 ligand, niacin, has been used for decades as lipid-modifying drug. Recent studies suggest that HCA ligands can be involved in the modulation of inflammatory processes. In this study, we evaluated the effects of HCA1-3 ligands on adipose differentiation and cytokine expression in human adipocytes and macrophages. Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes were induced to differentiate into adipocytes for 8 d in the presence or absence of HCA ligands and evaluated for lipid accumulation and adipogenic gene expression. The inhibitory effects of the ligands on the expression and production of cytokines were measured in lipopolysaccharide (LPS)-stimulated adipocytes and THP-1 macrophage cells. Preadipocytes treated with HCA ligands showed no changes in the capacity to differentiate into adipocytes and no significant alteration in peroxisome proliferator activated receptor γ (PPARγ) or its target gene expression. HCA2-3 ligands significantly downregulated LPS-induced expression of interleukin (IL)-6 (53-64%), tumor necrosis factor-α (TNF-α) (55-69%) and IL-8 (51-59%) in adipocytes and macrophages. IL-1ß inhibition (58-68%) by HCA2-3 ligands was observed only in adipocytes. Furthermore, LPS increased the expression of HCA2-3 in adipocytes and macrophages and this expression was decreased by treatment with their ligands. These results suggest that HCA ligands modulated LPS-mediated pro-inflammatory gene expression in both macrophages and adipocytes without affecting adipogenesis. Therefore, targeting HCA2 and HCA3 would be beneficial in treating inflammation conditions associated with atherosclerosis and obesity.

Synthesis and evaluation of (E)-2-(5-phenylpent-2-en-4-ynamido)cyclohex-1-ene-1-carboxylate derivatives as HCA2 receptor agonists.

Novel series of compounds consisting of 2-amidocyclohex-1-ene carboxylate and phenyl parts which are connected by enyne (compounds 2a-f), but-1-yne (compounds 4a-j), and phenylethylene (compounds 5a-f) linkers as HCA2 full agonists were designed and their functional activity using cAMP assay and binding affinity using radioligand (3H-niacin) binding assay were evaluated. In general, compounds of all three series exhibit similar HCA2 binding and activation profile. However, the activity is strongly dependent on the substituent at the aromatic part of the structure. Among the structures evaluated, the highest affinity and potency in all series were exhibited by compounds containing 4-hydroxy and/or 2-chloro or 2-fluoro substituents. The most active compounds in the enyne and but-1-yne series in the cAMP assay are 2-fluoro,4-hydroxy and 2-chloro,4-hydroxy phenyl derivatives 2f, 4f, and 4g showing potency similar to the previously described 4-hydroxy-biphenyl analogue 5c.

Functional Characteristics of Multipotent Mesenchymal Stromal Cells from Pituitary Adenomas.

Pituitary adenomas are one of the most common endocrine and intracranial neoplasms. Although they are theoretically monoclonal in origin, several studies have shown that they contain different multipotent cell types that are thought to play an important role in tumor initiation, maintenance, and recurrence after therapy. In the present study, we isolated and characterized cell populations from seven pituitary somatotroph, nonhormonal, and lactotroph adenomas. The obtained cells showed characteristics of multipotent mesenchymal stromal cells as observed by cell morphology, cell surface marker CD90, CD105, CD44, and vimentin expression, as well as differentiation to osteogenic and adipogenic lineages. They are capable of growth and passaging under standard laboratory cell culture conditions and do not manifest any hormonal cell characteristics. Multipotent mesenchymal stromal cells are present in pituitary adenomas regardless of their clinical manifestation and show no considerable expression of somatostatin 1-5 and dopamine 2 receptors. Most likely obtained cells are a part of tissue-supportive cells in pituitary adenoma microenvironment.

Noninvasive optical diagnostics of enhanced green fluorescent protein expression in skeletal muscle for comparison of electroporation and sonoporation efficiencies.

We highlight the options available for noninvasive optical diagnostics of reporter gene expression in mouse tibialis cranialis muscle. An in vivo multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) expression, providing information on location and duration of EGFP expression and allowing quantification of EGFP expression levels. For EGFP coding plasmid (pEGFP-Nuc Vector, 10 µg/50 ml 10 µg/50 ml ) transfection, we used electroporation or ultrasound enhanced microbubble cavitation [sonoporation (SP)]. The transcutaneous EGFP fluorescence in live mice was monitored over a period of one year using the described parameters: area of EGFP positive fibers, integral intensity, and mean intensity of EGFP fluorescence. The most efficient transfection of EGFP coding plasmid was achieved, when one high voltage and four low voltage electric pulses were applied. This protocol resulted in the highest short-term and long-term EGFP expression. Other electric pulse protocols as well as SP resulted in lower fluorescence intensities of EGFP in the transfected area. We conclude that noninvasive multispectral imaging technique combined with fluorescence spectroscopy point measurements is a suitable method to estimate the dynamics and efficiency of reporter gene transfection in vivo.

Replacement of short segments within transmembrane domains of MC2R disrupts retention signal.

The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1-24) were estimated during their stand-alone expression and coexpression with MRAP. Our results indicate that both the motif that determines the ligand-recognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segment of the second extracellular loop is involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-).

Synthesis and evaluation of (E)-2-(acrylamido)cyclohex-1-enecarboxylic acid derivatives as HCA1, HCA2, and HCA3 receptor agonists.

2-(3-(Naphthalen-2-yl)propanamido)cyclohex-1-enecarboxylic acid and its 6-hydroxynaphthalen-2-yl analogue are well-known hydroxyl-carboxylic acid (HCA) receptor HCA2 agonists. A series of novel aryl derivatives of 2-amidocyclohex-1-ene carboxylic acid that contained rigidity elements, such as an E-double bond, triple bond, and trans or cis-substituted cyclopropane rings, instead of the saturated ethane linker in the amide part of the molecules were designed and synthesized, and the derivatives' potency for the activation of HCA1, HCA2, and HCA3 receptors by 3'-5'-cyclic adenosine monophosphate (cAMP) assay were evaluated. The SAR studies revealed that the rigidifying of appropriate molecules enabled modulation of the potency and selectivity of the HCA2 receptor activation.

The role of common and rare MC4R variants and FTO polymorphisms in extreme form of obesity.

Melanocortin 4 receptor (MC4R) is an important regulator of food intake and number of studies report genetic variations influencing the risk of obesity. Here we explored the role of common genetic variation from MC4R locus comparing with SNPs from gene FTO locus, as well as the frequency and functionality of rare MC4R mutations in cohort of 380 severely obese individuals (BMI > 39 kg/m(2)) and 380 lean subjects from the Genome Database of Latvian Population (LGDB). We found correlation for two SNPs--rs11642015 and rs62048402 in the fat mass and obesity-associated protein (FTO) with obesity but no association was detected for rs17782313 located in the MC4R locus in these severely obese individuals. We sequenced the whole gene MC4R coding region in all study subjects and found five previously known heterozygous non-synonymous substitutions V103I, I121T, S127L, V166I and I251L. Expression in mammalian cells showed that the S127L, V166I and double V103I/S127L mutant receptors had significantly decreased quantity at the cell surface compared to the wild type MC4R. We carried out detailed functional analysis of V166I that demonstrated that, despite low abundance in plasma membrane, the V166I variant has lower EC50 value upon αMSH activation than the wild type receptor, while the level of AGRP inhibition was decreased, implying that V166I cause hyperactive satiety signalling. Overall, this study suggest that S127L may be the most frequent functional MC4R mutation leading to the severe obesity in general population and provides new insight into the functionality of population based variants of the MC4R.

Ex vivo cytokine production in peripheral blood mononuclear cells after their stimulation with dsRNA of natural origin.

Double-stranded RNA (dsRNA) is a pathogen-associated molecular pattern, known for its ability to induce antiviral response and enhance communication between cells mediating innate and adaptive immune responses. The aim of this study was to characterize the effect of the dsRNA-containing product Larifan on the production of a wide spectrum of cytokines and chemokines in ex vivo cultivated peripheral blood mononuclear cells. Concentrations of 29 different cytokines were detected by a Luminex® 200™ System using three Milliplex MAP Multiplex Assay Kits. Larifan caused strong induction of chemokine macrophage inflammatory protein 1ß, I-309, and TARC, proinflammatory cytokines IL-6, tumor necrosis factor -α, granulocyte macrophage colony-stimulating factor, anti-inflammatory IL-10, and cellular immunity mediating factors IL-23 and interferon-γ. Considerable suppression of IL-16 and chemokine stromal cell-derived factor 1 a+b and interferon gamma-induced protein 10 was also observed. The network of molecules responding to the presence of Larifan revealed the pleiotropic effect this product exerts on immune response.

Design and evaluation of substrate-based octapeptide and non substrate-based tetrapeptide inhibitors of dengue virus NS2B-NS3 proteases.

A series of 45 peptide inhibitors was designed, synthesized, and evaluated against the NS2B-NS3 proteases of the four subtypes of dengue virus, DEN-1-4. The design was based on proteochemometric models for Michaelis (Km) and cleavage rate constants (kcat) of protease substrates. This led first to octapeptides showing submicromolar or low micromolar inhibitory activities on the four proteases. Stepwise removal of cationic substrate non-prime side residues and variations in the prime side sequence resulted finally in an uncharged tetrapeptide, WYCW-NH2, with inhibitory Ki values of 4.2, 4.8, 24.4, and 11.2 µM for the DEN-1-4 proteases, respectively. Analysis of the inhibition data by proteochemometric modeling suggested the possibility for different binding poses of the shortened peptides compared to the octapeptides, which was supported by results of docking of WYCW-NH2 into the X-ray structure of DEN-3 protease.

Melanoma risk associated with MC1R gene variants in Latvia and the functional analysis of rare variants.

To evaluate the association of melanocortin 1 receptor gene (MC1R) variants with melanoma risk in a Latvian population, the MC1R gene was sequenced in 200 melanoma patients and 200 control persons. A functional study of previously uncharacterized, rare MC1R variants was also performed. In total, 26 different MC1R variants, including two novel variants Val165Ile and Val188Ile, were detected. The highest risk of melanoma was associated with the Arg151Cys variant (odds ratio (OR) 4.47, 95% confidence interval (CI) 2.19-9.14, P<0.001). A gene dosage effect was observed, with melanoma risk for carriers of two variants being twice (OR 3.98, 95% CI 2.15-7.38, P<0.001) that of carriers of one variant (OR 1.98, 95% CI 1.26-3.11, P=0.003). After stratification according to the pigmentation phenotype, the risk of melanoma remained in groups with otherwise protective phenotypes. Functional analyses of eight previously uncharacterized MC1R variants revealed that a subset of them is functionally relevant. Our results support the contribution of MC1R variants to a genetic predisposition to melanoma in Latvia.

Evidence for constitutive dimerization of niacin receptor subtypes.

The recently deorphanized niacin receptor subtypes NIACR1 (GPR109A) and NIACR2 (GPR109B) play an essential role in the regulation of metabolic processes and immune reactions. Both receptors belong to the G-protein-coupled receptor (GPCR) family, whose members have traditionally been treated as monomeric entities, but now appear to exist and function as both homodimers and heterodimers. In this study, a close physical interaction is shown between the highly homologous niacin receptor subtypes, NIACR1 and NIACR2, using bioluminescence resonance energy transfer (BRET(2)) in living cells. The extent of homo- and hetero-dimerization of the niacin receptors did not vary after activation of the receptors with selective agonists, indicating that the dimerization state of NIACR1 and NIACR2 is not regulated by ligand binding. Moreover, detection of niacin receptor dimers in both plasma membrane- and endoplasmic reticulum-enriched fractions suggests that they are formed early in the biosynthetic pathway. Taken together, these results demonstrate that niacin receptor dimerization is a constitutive process occurring early during biosynthesis.