Synthesis of novel andostane-N-cyclohexyl-17-carboxamides, and their effect on the 5α-reductase isoform 2, the androgen receptor, and androgen-dependent glands

Background: Benign Prostatic Hyperplasia (BPH), and Prostate Cancer (PCa) are androgen-dependent diseases. PCa is associated with excessive signalling of the androgen receptor (AR) due to the binding of 5α-DHT and T. BPH is related to high levels of 5α-dihydrotestosterone (5α-DHT), biosynthesized from testosterone (T) by 5α-reductase (5RD5A). The inhibition of 5RD5A and the blockage of AR are targets for their treatment. In this study, the synthesis and determination of biological activity of the new N-cyclohexyl-3β-hydroxyandrosta-5,16-diene-17-carboxamide (6), N-cyclohexyl-3-oxoandrosta-4,6,16-triene-17-carboxamide (7), and N-cyclohexyl-3-oxoandrosta-4,16-diene-17-carboxamide (8) were carried out to find new drugs to improve these afflictions. Methods: The synthesis of 6 to 8 was confirmed by spectroscopic and spectrometric analyses. Competitive binding assays determined the affinity of 6 to 8 to the AR. The inhibitory activity of 5RD5A isoform 2 (5RD5A2) (IC50) was established by the conversion of [3H]-T to [3H]-5α-DHT and it was compared with finasteride (FIN). The pharmacological effect of 6 to 8 was determined on the weight of the prostate and seminal vesicles glands of castrated hamsters treated with T, and on the diameter size of their flank organs. Results: Compounds 7 and 8 bound lightly (ca. 15 %) to AR. Comparing to FIN (IC50 = 8.5 nM), 6 to 8 (IC50 = 0.169, 0.105 and 0.155 nM, respectively) showed higher potency as inhibitors of 5RD5A2. Compound 6 decreased the prostate and seminal vesicles weight, as well as the hamsters' diameter flank organs. However, 7 only decreased the diameter of flank organs. Surprisingly, 8 increased these pharmacological parameters. Conclusion: Androstane-17-caboxamide 6 is a 5RD5A2 inhibitor that reduces the weight of androgen-dependent glands such as the prostate, suggesting it could be a lead for new drugs to treat BPH and PCa.

Function and Characterization Analysis of BodoOBP8 from Bradysia odoriphaga (Diptera: Sciaridae) in the Recognition of Plant Volatiles and Sex Pheromones

The belowground pest Bradysia odoriphaga (Diptera: Sciaridae) has a sophisticated and sensitive olfactory system to detect semiochemical signals from the surrounding environment. In particular, odorant-binding proteins (OBPs) are crucial in capturing and transporting these semiochemical signals across the sensilla lymph to the corresponding odorant receptors. In this study, we cloned a full-length cDNA sequence of BodoOBP8 from B. odoriphaga. Real-time PCR (qRT-PCR) analysis revealed that BodoOBP8 has the highest expression levels in males, with more pronounced expression in the male antennae than in other tissues. In this study, the recombinant protein BodoOBP8 was successfully expressed by a bacterial system to explore its function. Competitive binding assays with 33 host plant volatiles and one putative sex pheromone (n-heptadecane) revealed that purified BodoOBP8 strongly bound to two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and to n-heptadecane; the corresponding dissolution constants (Ki) were 4.04, 6.73, and 4.04 μM, respectively. Molecular docking indicated that Ile96, Ile103, Ala107, and Leu111, located in the hydrophobic cavity of BodoOBP8, are the key residues mediating the interaction of BodoOBP8 with two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and n-heptadecane. These results show that BodoOBP8 plays a role in the recognition of plant volatiles and sex pheromones, suggesting its application as a molecular target for the screening of B. odoriphaga attractants and repellents and facilitating a new mechanism of B. odoriphaga control.

Radiolabeled 6-(2, 3-Dichlorophenyl)-N4-methylpyrimidine-2, 4-diamine (TH287): A Potential Radiotracer for Measuring and Imaging MTH1

MTH1 (MutT homolog 1) or NUDT1 (Nudix Hydrolase 1), also known as oxidized purine nucleoside triphosphatase, has potential as a biomarker for monitoring cancer progression and quantifying target engagement for relevant therapies. In this study, we validate one MTH1 inhibitor TH287 as a PET MTH1 radiotracer. TH287 was radiolabeled with tritium and the binding of [3H]TH287 to MTH1 was evaluated in live glioblastoma cells (U251MG) through saturation and competitive binding assays, together with in vitro enzymatic assays. Furthermore, TH287 was radiolabeled with carbon-11 for in vivo microPET studies. Saturation binding assays show that [3H]TH287 has a dissociation constant (Kd) of 1.97 ± 0.18 nM, Bmax of 2676 ± 122 fmol/mg protein for U251MG cells, and nH of 0.98 ± 0.02. Competitive binding assays show that TH287 (Ki: 3.04 ± 0.14 nM) has a higher affinity for MTH1 in U251MG cells compared to another well studied MTH1 inhibitor: (S)-crizotinib (Ki: 153.90 ± 20.48 nM). In vitro enzymatic assays show that TH287 has an IC50 of 2.2 nM in inhibiting MTH1 hydrolase activity and a Ki of 1.3 nM from kinetics assays, these results are consistent with our radioligand binding assays. Furthermore, MicroPET imaging shows that [11C]TH287 gets into the brain with rapid clearance from the brain, kidney, and heart. The results presented here indicate that radiolabeled TH287 has favorable properties to be a useful tool for measuring MTH1 in vitro and for further evaluation for in vivo PET imaging MTH1 of brain tumors and other central nervous system disorders.

Citronellal perception and transmission by Anopheles gambiae s.s. (Diptera: Culicidae) females

Anopheles gambiae s.s. is a key vector of Plasmodium parasites. Repellents, which may be a promising alternative to pesticides used to control malaria mosquitoes. Although citronellal is a known mosquito repellent, its repellency characteristics are largely unknown. Determining the specific odorant-binding proteins (OBPs) and odorant receptors (ORs) that detect and transfer the citronellal molecule in A. gambiae s.s. will help to define the mode of action of this compound. In this research, we assessed the repellent activity of citronellal in A. gambiae s.s. using a Y-tube olfactory meter, screened candidate citronellal-binding OBPs and ORs using reverse molecular docking, clarified the binding properties of predicted proteins for citronellal using fluorescence competition binding assay. Results showed that citronellal had a dosage effect on repelling A. gambiae s.s.. The 50% repellent rate was determined to be 4.02 nmol. Results of simulated molecular docking showed that the only proteins that bound tightly with citronellal were AgamOBP4 and AgamORC7. Fluorescence competitive binding assays confirmed the simulations. This research determined that citronellal was captured by AgamOBP4 and transmitted to AgamORC7 in A. gambiae s.s.. Our study will be beneficial in the further understanding the repellent mechanism of citronellal against A. gambiae s.s..

Characterization of MK6240, a tau PET tracer, in autopsy brain tissue from Alzheimer’s disease cases

Purpose MK6240 is a second-generation tau PET tracer designed to detect the neurofibrillary tangles in the brains of patients with Alzheimer’s disease (AD). The aim of the study was to characterize 3H-MK6240 in AD and control brain tissue and to compare its binding properties with those of first-generation tau PET tracers. Methods Saturation binding assays with 3H-MK6240 were carried out in the temporal and parietal cortices of AD brains to determine the maximum number of binding sites (Bmax) and the dissociation constants (Kd) at these sites. Competitive binding assays were carried out between 3H-MK6240 and unlabelled MK6240, AV-1451 (aka T807, flortaucipir) and THK5117, and between 3H-THK5351 and unlabelled MK6240. Regional binding studies with 3H-MK6240 were carried out in homogenates from six AD and seven control brains and, using autoradiography, on large frozen sections from two AD brains and one control brain. Results The saturation binding assays gave Bmax and Kd values of 59.2 fmol/mg and 0.32 nM in the temporal cortex and 154.7 fmol/mg and 0.15 nM in the parietal cortex. The competitive binding assays revealed two binding sites with affinities in the picomolar and nanomolar range shared by 3H-MK6240 and all the tested unlabelled compounds. There were no binding sites in common between 3H-THK5351 and unlabelled MK6240. Regional binding of 3H-MK6240 was significantly higher in AD brain tissue than in controls. Binding in brain tissue from AD patients with early-onset AD was significantly higher than in brain tissue from patients with late-onset AD. Binding of 3H-MK6240 was not observed in off-target regions. Autoradiography showed high regional cortical binding in the two AD brains and very low binding in the control brain. Conclusions 3H-MK6240 has a high binding affinity for tau deposits in AD brain tissue but also has different binding characteristics from those of the first-generation tau tracers. This confirms the complexity of tau tracer binding on tau deposits with different binding affinities for different binding sites.

Characterization of the Expression and Functions of Two Odorant-Binding Proteins of Sitophilus zeamais Motschulsky (Coleoptera: Curculionoidea)

Odorant-binding proteins (OBPs) are important in insect chemical communication. The objective of this research was to identify the functions of two OBPs in Sitophilus zeamais. qRT-PCR and western blot (WB) were performed to investigate the expression profiles at the transcript and protein levels, respectively. Fluorescence competitive binding assays were used to measure the ability of the OBPs to bind to host volatiles, and a Y-tube olfactometer was used to verify the results (attraction/no response) via behavioral experiments. The RNAi was used to verify the function by knocking down the ability of proteins to bind odorants. qRT-PCR showed the highest expression SzeaOBP1 and SzeaOBP28 at the low-instar larva (LL) and eclosion adult (EA) stages, respectively. WB showed that both SzeaOBP1 and SzeaOBP28 were highly expressed in the EA stage. Fluorescence competitive binding assays indicated that SzeaOBP1 exhibited extremely high binding affinity with cetanol. SzeaOBP28 exhibited a pronounced binding affinity for 4-hydroxy-3-methoxybenzaldehyde. The behavioral experiment showed that the adult S. zeamais responded strongly to 4-hydroxy-3-methoxybenzaldehyde and valeraldehyde from Sorghum bicolor. The RNAi knockdown individuals displayed behavioral differences between normal insects and dsRNA (SzeaOBP1)-treated insects. We infer that they both have functions in perception and recognition of host volatiles, whereas SzeaOBP28 may also have other functions.

Structural and Biological Characterizations of Novel High-Affinity Fluorescent Probes with Overlapped and Distinctive Binding Regions on CXCR4

CXC-type chemokine receptor 4 (CXCR4) is well known as a co-receptor for cellular entry and infection of human immunodeficiency virus type 1 (HIV-1). As an important member of the G protein-coupled receptor (GPCR) family, CXCR4 also mediates a variety of cellular processes and functions, such as cell chemotaxis, proliferation, and calcium signal transductions. Identification and characterization of molecular ligands or probes of CXCR4 have been an intensive area of investigations as such ligands or probes are of significant clinical values for the studies and treatments of HIV-1 infection and other human diseases mediated by the receptor. The crystal structures of CXCR4 in complex with different ligands have revealed two distinctive binding regions or subpockets. Thus, understanding the interactions of diverse ligands with these distinctive CXCR4 binding regions has become vital for elucidating the relationship between binding modes and biological mechanisms of ligand actions. Peptidic CVX15 is the only ligand that has been validated to bind one of these distinctive binding regions (or so called the major subpocket) of CXCR4. Therefore, in this study, we developed an efficient probe system including two high-affinity peptidic fluorescent probes, designated as FITC-CVX15 and FITC-DV1, with the aim of targeting distinctive CXCR4 subpockets. We conducted rational design and chemical characterization of the two CXCR4-specific probes and examined their application in biological experiments including competitive binding assays, flow cytometry analysis, and confocal imaging. Especially these two probes were applied in parallel CXCR4 competitive binding assays to detect and analyze potential binding modes of diverse CXCR4 ligands, together with molecular docking and simulations. Our results have indicated that these peptidic fluorescent probe systems provide novel ligand detecting tools, as well as present a new approach for analyzing distinctive binding modes of diverse CXCR4 ligands.

Inhibitory Anti-Peroxidasin Antibodies in Pulmonary-Renal Syndromes

BackgroundGoodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP.MethodsWe used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score.ResultsWe detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease.ConclusionsAnti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.