Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.

Estimation of the lipophilicity of purine-2,6-dione-based TRPA1 antagonists and PDE4/7 inhibitors with analgesic activity.

Lipophilicity is one of the principal QSAR parameters which influences among others the pharmacodynamics and pharmacokinetic properties of a drug candidates. In this paper, the lipophilicity of 14 amide derivatives of 1,3-dimethyl-2,6-dioxopurin-7-yl-alkylcarboxylic acids as multifunctional TRPA1 channel antagonists and phosphodiesterase 4/7 inhibitors with analgesic activity were investigated, using reversed-phase thin-layer chromatography method. It was observed that the retention behavior of the analyzed compounds was dependent on their structural features i.e. an aliphatic linker length, a kind of substituent at 8 position of purine-2,6-dione scaffold as well as on a substitution in a phenyl group. The experimental parameters (RM0) were compared with computationally calculated partition coefficient values by Principal Component Analysis (PCA). To verify the influence of lipophilic parameter of the investigated compounds on their biological activity the Kruskal-Wallis test was performed. The lowest lipophilicity was observed for the compounds with weak PDE4/7 inhibitory potency. The differences between the lipophilicity of potent inhibitors and inactive compounds were statistically significant. It was found that the presence of more lipophilic propoxy- or butoxy- substituents as well as the elongation of the aliphatic chain to propylene one between the purine-2,6-dione core and amide group were preferable for desired multifunctional activity.

Interactive Molecular Dynamics in Virtual Reality Is an Effective Tool for Flexible Substrate and Inhibitor Docking to the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the SARS-CoV-2 virus is one focus of drug development efforts for COVID-19. Here, we show that interactive molecular dynamics in virtual reality (iMD-VR) is a useful and effective tool for creating Mpro complexes. We make these tools and models freely available. iMD-VR provides an immersive environment in which users can interact with MD simulations and so build protein complexes in a physically rigorous and flexible way. Recently, we have demonstrated that iMD-VR is an effective method for interactive, flexible docking of small molecule drugs into their protein targets (Deeks et al. PLoS One 2020, 15, e0228461). Here, we apply this approach to both an Mpro inhibitor and an oligopeptide substrate, using experimentally determined crystal structures. For the oligopeptide, we test against a crystallographic structure of the original SARS Mpro. Docking with iMD-VR gives models in agreement with experimentally observed (crystal) structures. The docked structures are also tested in MD simulations and found to be stable. Different protocols for iMD-VR docking are explored, e.g., with and without restraints on protein backbone, and we provide recommendations for its use. We find that it is important for the user to focus on forming binding interactions, such as hydrogen bonds, and not to rely on using simple metrics (such as RMSD), in order to create realistic, stable complexes. We also test the use of apo (uncomplexed) crystal structures for docking and find that they can give good results. This is because of the flexibility and dynamic response allowed by the physically rigorous, atomically detailed simulation approach of iMD-VR. We make our models (and interactive simulations) freely available. The software framework that we use, Narupa, is open source, and uses commodity VR hardware, so these tools are readily accessible to the wider research community working on Mpro (and other COVID-19 targets). These should be widely useful in drug development, in education applications, e.g., on viral enzyme structure and function, and in scientific communication more generally.

Screening of an Epigenetic Drug Library Identifies 4-((hydroxyamino)carbonyl)-N-(2-hydroxyethyl)-N-Phenyl-Benzeneacetamide that Reduces Melanin Synthesis by Inhibiting Tyrosinase Activity Independently of Epigenetic Mechanisms.

The aim of this study was to identify novel antimelanogenic drugs from an epigenetic screening library containing various modulators targeting DNA methyltransferases, histone deacetylases, and other related enzymes/proteins. Of 141 drugs tested, K8 (4-((hydroxyamino)carbonyl)-N-(2-hydroxyethyl)-N-phenyl-benzeneacetamide; HPOB) was found to effectively inhibit the α-melanocyte-stimulating hormone (α-MSH)-induced melanin synthesis in B16-F10 murine melanoma cells without accompanying cytotoxicity. Additional experiments showed that K8 did not significantly reduce the mRNA and protein level of tyrosinase (TYR) or microphthalmia-associated transcription factor (MITF) in cells, but it potently inhibited the catalytic activity TYR in vitro (IC50, 1.1-1.5 µM) as compared to ß-arbutin (IC50, 500-700 µM) or kojic acid (IC50, 63 µM). K8 showed copper chelating activity similar to kojic acid. Therefore, these data suggest that K8 inhibits cellular melanin synthesis not by downregulation of TYR protein expression through an epigenetic mechanism, but by direct inhibition of TYR catalytic activity through copper chelation. Metal chelating activity of K8 is not surprising because it is known to inhibit histone deacetylase (HDAC) 6 through zinc chelation. This study identified K8 as a potent inhibitor of cellular melanin synthesis, which may be useful for the treatment of hyperpigmentation disorders.

Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism.

Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.

Conformational changes in the activation loop of mitochondrial glutaminase C: A direct fluorescence readout that distinguishes the binding of allosteric inhibitors from activators.

The first step in glutamine catabolism is catalysis by the mitochondrial enzyme glutaminase, with a specific isoform, glutaminase C (GAC), being highly expressed in cancer cells. GAC activation requires the formation of homotetramers, promoted by anionic allosteric activators such as inorganic phosphate. This leads to the proper orientation of a flexible loop proximal to the dimer-dimer interface that is essential for catalysis (i.e. the "activation loop"). A major class of allosteric inhibitors of GAC, with the prototype being bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and the related molecule CB-839, binds to the activation loop and induces the formation of an inactive tetramer (two inhibitors bound per active tetramer). Here we describe a direct readout for monitoring the dynamics of the activation loop of GAC in response to these allosteric inhibitors, as well as allosteric activators, through the substitution of phenylalanine at position 327 with tryptophan (F327W). The tryptophan fluorescence of the GAC(F327W) mutant undergoes a marked quenching upon the binding of BPTES or CB-839, yielding titration profiles that make it possible to measure the binding affinities of these inhibitors for the enzyme. Allosteric activators like phosphate induce the opposite effect (i.e. fluorescence enhancement). These results describe direct readouts for the binding of the BPTES class of allosteric inhibitors as well as for inorganic phosphate and related activators of GAC, which should facilitate screening for additional modulators of this important metabolic enzyme.

Crystal structure of EGFR T790M/C797S/V948R in complex with EAI045.

Lung cancer is the leading cause of cancer deaths. Epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small cell lung cancers (NSCLCs), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the anti-EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib initially, but soon develop resistance to them in about half of the cases due to the emergence of the gatekeeper mutation T790M. The third-generation TKIs such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to the EGFR kinase through Cys 797, but ultimately lose their efficacy upon emergence of the C797S mutation that abolishes the covalent bonding. Therefore to develop new TKIs to overcome EGFR drug-resistant mutants harboring T790M/C797S is urgently demanded. EAI001 and EAI045 are a new type of EGFR TKIs that bind to EGFR reversibly and not relying on Cys 797. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR L858R/T790M and L858R/T790M/C797S. Here we report the crystal structure of EGFR T790M/C797S/V948R in complex with EAI045, and compare it to EGFR T790M/V948R in complex with EAI001. The complex structure reveals why EAI045 binds tighter to EGFR than does EAI001, and why EAI001 and EAI045 prefer binding to EGFR T790M. The knowledge may facilitate future drug development studies targeting this very important cancer target.

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors.

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79 ±â€¯6% yield (n = 9), and 18F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6 ±â€¯1.6% (n = 5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with 18F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.

Creation of a histone deacetylase 6 inhibitor and its biological effects [corrected].

We report the development of a potent, selective histone deacetylase 6 (HDAC6) inhibitor. This HDAC6 inhibitor blocks growth of normal and transformed cells but does not induce death of normal cells. The HDAC6 inhibitor alone is as effective as paclitaxel in anticancer activity in tumor-bearing mice.

Improved Ocular Delivery of Nepafenac by Cyclodextrin Complexation.

Nepafenac is a nonsteroidal anti-inflammatory drug (NSAID), currently only available as 0.1% ophthalmic suspension (Nevanac®). This study utilized hydroxypropyl-ß-cyclodextrin (HPBCD) to increase the water solubility and trans-corneal permeation of nepafenac. The nepafenac-HPBCD complexation in the liquid and solid states were confirmed by phase solubility, differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR) analyses. Nepafenac 0.1% ophthalmic solution was formulated using HPBCD (same pH and osmolality as that of Nevanac®) and pig eye trans-corneal permeation was studied versus Nevanac®. Furthermore, nepafenac content in cornea, sclera, iris, lens, aqueous humor, choroid, ciliary body, retina, and vitreous humor was studied in a continuous isolated pig eye perfusion model in comparison to the suspension and Nevanac®. Permeation studies using porcine corneas revealed that the solution formulation had a permeation rate 18 times higher than Nevanac®. Furthermore, the solution had 11 times higher corneal retention than Nevanac®. Drug distribution studies using porcine eyes revealed that the solution formulation enables detectable levels in various ocular tissues while the drug was undetectable by Nevanac®. The ocular solution formulation had a significantly higher drug concentration in the cornea compared to the suspension or Nevanac®.

A Computational Investigation of the Equilibrium Constants for the Fluorescent and Chemiluminescent States of Coelenteramide.

In spite of recent advances in understanding the mechanism of coelenterate bioluminescence, there is no consensus about which coelenteramide specie and/or state are the light emitter. In this study, a systematic investigation of the geometries and spectra of all possible light emitters has been performed at the TD ωB97XD/6-31+G(d) level of theory, including various fluorescent and chemiluminescent states in vacuum, in a hydrophobic environment and in aqueous solution. To deduce the most probable form of the fluorescent and chemiluminescent coelenteramide emitter, the equilibrium constants for the fluorescent and chemiluminescent states connecting the various species have been calculated. ωB97XD gives a qualitatively good description of fluorescent and chemiluminescent structures. Coelenteramide is formed in a "dark" chemiluminescent state and must evolve to a bright fluorescent state. Moreover, the photoacidity of the phenol group is significantly higher in the fluorescent state than in the chemiluminescent state, which allows the formation of phenolate coelenteramide and clarifies its role as the bioluminescent emitter.

Side Fenestrations Provide an "Anchor" for a Stable Binding of A1899 to the Pore of TASK-1 Potassium Channels.

A1899 is a potent and selective inhibitor of the two-pore domain potassium (K2P) channel TASK-1. It was previously reported that A1899 acts as an open-channel blocker and binds to residues of the P1 and P2 regions, the M2 and M4 segments, and the halothane response element. The recently described crystal structures of K2P channels together with the newly identified side fenestrations indicate that residues relevant for TASK-1 inhibition are not purely facing the central cavity as initially proposed. Accordingly, the TASK-1 binding site and the mechanism of inhibition might need a re-evaluation. We have used TASK-1 homology models based on recently crystallized K2P channels and molecular dynamics simulation to demonstrate that the highly potent TASK-1 blocker A1899 requires binding to residues located in the side fenestrations. Unexpectedly, most of the previously described residues that interfere with TASK-1 blockade by A1899 project their side chains toward the fenestration lumina, underlining the relevance of these structures for drug binding in K2P channels. Despite its hydrophobicity, A1899 does not seem to use the fenestrations to gain access to the central cavity from the lipid bilayer. In contrast, binding of A1899 to residues of the side fenestrations might provide a physical "anchor", reflecting an energetically favorable binding mode that after pore occlusion stabilizes the closed state of the channels.

Structure-activity and in vivo evaluation of a novel lipoprotein lipase (LPL) activator.

Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitutions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10mg/kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the HFD induced fat accumulation in the liver. Our study has provided insights into the structural and functional characteristics of this novel LPL activator.

Fluorescent coelenteramide-containing protein as a color bioindicator for low-dose radiation effects.

The study addresses the application of fluorescent coelenteramide-containing proteins as color bioindicators for radiotoxicity evaluation. Biological effects of chronic low-dose radiation are under investigation. Tritiated water (200 MBq/L) was used as a model source of low-intensive ionizing radiation of beta type. 'Discharged obelin,' product of bioluminescent reaction of marine coelenterate Obelia longissimi, was used as a representative of the coelenteramide-containing proteins. Coelenteramide, fluorophore of discharged obelin, is a photochemically active molecule; it produces fluorescence forms of different color. Contributions of 'violet' and 'blue-green' forms to the visible fluorescence serve as tested parameters. The contributions depend on the coelenteramide's microenvironment in the protein, and, hence, evaluate distractive ability and toxicity of radiation. The protein samples were exposed to beta radiation for 18 days, and maximal dose accumulated by the samples was 0.28 Gy, being close to a tentative limit of a low-dose interval. Increase of relative contribution of 'violet' fluorescence under exposure to the beta irradiation was revealed. High sensitivity of the protein-based test system to low-dose ionizing radiation (to 0.03 Gy) was demonstrated. The study develops physicochemical understanding of radiotoxic effects. Graphical abstract Coelenteramide-containing protein (discharged obelin) changes fluorescence color under exposure to low-dose ionizing radiation of tritium.

Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca(2+)-regulated photoprotein berovin.

Bright bioluminescence of ctenophores is caused by Ca(2+)-regulated photoproteins. Although these photoproteins are functionally identical to and share many properties of cnidarian photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e. lose the ability to bioluminesce on exposure to light over the entire absorption spectrum. In addition, the degree of identity of their amino acid sequences with those of cnidarian photoproteins is only 29.4%. This suggests that the residues involved in bioluminescence of ctenophore and cnidarian photoproteins significantly differ. Here we describe the bioluminescent properties of berovin mutants with substitution of the residues located in the photoprotein internal cavity. Since the spatial structure of berovin bound with a substrate is not determined yet, to identify these residues we have modeled it with an accommodated substrate using the structures of some cnidarian Ca(2+)-regulated photoproteins with bound coelenterazine or coelenteramide as templates in order to obtain an adequate sampling and to take into account all possible conformers and variants for ligand-protein docking. Based on the impact of substitutions on the bioluminescent properties and model structures we speculate that within the internal cavity of ctenophore photoproteins, coelenterazine is bound as a 2-peroxy anion adduct which is stabilized owing to Coulomb interaction with a positively charged guanidinium group of Arg41 paired with Tyr204. In this case, the bioluminescence reaction is triggered by only calcium-induced conformational changes leading to the disturbance of charge-charge interaction.

Design and evaluation of novel glutaminase inhibitors.

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.

Metabolomic comparison between cells over-expressing isocitrate dehydrogenase 1 and 2 mutants and the effects of an inhibitor on the metabolism.

The R132H and R172K mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have neomorphic activity of generating 2-hydroxyglutarate (2-HG) which has been implicated in the oncogenesis. Although similarities in structure and enzyme activity for the two isotypic mutations have been suggested, the difference in their cellular localization and biochemical properties suggests differential effects on the metabolic oncogenesis. Using U87 cells transfected with either wild-type (WT) and mutant (MT) IDH genes, the MT-IDH1 and MT-IDH2 cells were compared with NMR-based metabolomics. When normalized with the respective WT-IDH cells, the general metabolic shifts of MT-IDH1 and IDH2 were almost opposite. Subsequent analysis with LC-MS and metabolic pathway mapping showed that key metabolites in pentose phosphate pathway and tricarboxylic acid cycle are disproportionately altered in the two mutants, suggesting different activities in the key metabolic pathways. Notably, lactate level was lower in MT-IDH2 cells which produced more 2-HG than MT-IDH1 cells, indicating that the Warburg effects can be overridden by the production of 2-HG. We also found that the effect of a mutant enzyme inhibitor is mainly reduction of the 2-HG level rather than general metabolic normalization. Overall, the metabolic alterations in the MT-IDH1 and 2 can be different and seem to be commensurate with the degree of 2-HG production. The R132H and R172K mutations of isocitrate dehydrogenase 1 and 2, respectively, (IDH1 and IDH2) have neomorphic activity of generating 2-hydroxyglutarate (2-HG) which has been implicated in oncogenesis. The mutant cell's metabolic shifts from the respective wild type cells were almost opposite, with lactate level being lower in the IDH2 mutant only, implicating an overridden Warburg effect. The metabolic effect of an IDH1 mutant inhibitor was limited to 2-HG lowering.

Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type.

Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations.

FLZ attenuates learning and memory deficits via suppressing neuroinflammation induced by LPS in mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in which neuroinflammation plays an important role. FLZ is a novel synthetic derivative of natural squamosamide. Previous studies demonstrated that FLZ had neuroprotective effects on AD models and showed strong anti-inflammatory property in Parkinson's disease models. However, whether the neuroprotective effects of FLZ on AD are associated with its anti-inflammatory property is still not fully elucidated. In this study, we aimed to investigate the ability of FLZ in modulating inflammation. The results showed that FLZ significantly improved memory deficits and alleviated neuronal damage as well as neuronal loss in the hippocampus of mice intracerebroventricular injected with lipopolysaccharide (LPS). Mechanistic studies revealed that the neuroprotective effects of FLZ were due to the suppression of neuroinflammation induced by LPS, as indicated by inactivation of astrocytes and microglia, reduced production of tumor necrosis factor-α, interleukin-1ß, and nitric oxide, as well as decreased expression of cyclooxygenase-2 and inducible nitric oxide synthase. The beneficial effects of FLZ on AD were further supported by the finding that FLZ attenuated ß-amyloid production through inhibiting ß-amyloid precursor protein cleaving enzyme 1 expression. These results suggested that anti-inflammatory agent could be useful for the treatment of AD.

Identification of the antiarrhythmic drugs amiodarone and lorcainide as potent H3 histamine receptor inverse agonists.

Use of molecular pharmacology to reprofile older drugs discovered before the advent of recombinant technologies is a fruitful method to elucidate mechanisms of drug action, expand understanding of structure-activity relationships between drugs and receptors, and in some cases, repurpose approved drugs. The H3 histamine receptor is a G-protein-coupled receptor (GPCR) primarily expressed in the central nervous system where among many things it modulates cognitive processes, nociception, feeding and drinking behavior, and sleep/wakefulness. In binding assays and functional screens of the H3 histamine receptor, the antiarrhythmic drugs lorcainide and amiodarone were identified as potent, selective antagonists/inverse agonists of human and rat H3 histamine receptors, with relatively little or no activity at over 20 other monoamine GPCRs, including H1, H2, and H4 receptors. Potent antagonism of H3 receptors was unique to amiodarone and lorcainide of 20 antiarrhythmic drugs tested, representing six pharmacological classes. These results expand the pharmacophore of H3 histamine receptor antagonist/inverse agonists and may explain, in part, the effects of lorcainide on sleep in humans.