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1.
Eur J Med Chem ; 185: 111785, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31669851

RESUMO

Multi-target-directed ligands seem to be an interesting approach to the treatment of complex disorders such as Alzheimer's disease. The aim of the present study was to find novel multifunctional compounds in a non-imidazole histamine H3 receptor ligand library. Docking-based virtual screening was applied for selection of twenty-six hits which were subsequently evaluated in Ellman's assay for the inhibitory potency toward acetyl- (AChE) and butyrylcholinesterase (BuChE). The virtual screening with high success ratio enabled to choose multi-target-directed ligands. Based on docking results, all selected ligands were able to bind both catalytic and peripheral sites of AChE and BuChE. The most promising derivatives combined the flavone moiety via a six carbon atom linker with a heterocyclic moiety, such as azepane, piperidine or 3-methylpiperidine. They showed the highest inhibitory activities toward cholinesterases as well as well-balanced potencies against H3R and both enzymes. Two derivatives were chosen - 5 (IC50 = 0.46 µM (AChE); 0.44 µM (BuChE); Ki = 159.8 nM (H3R)) and 17 (IC50 = 0.50 µM (AChE); 0.76 µM (BuChE); Ki = 228.2 nM (H3R)), and their inhibition mechanism was evaluated in kinetic studies. Both compounds displayed non-competitive mode of AChE and BuChE inhibition. Compounds 5 and 17 might serve as good lead structures for further optimization and development of novel multi-target anti-Alzheimer's agents.

2.
Phys Rev E ; 100(4-1): 042612, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31771000

RESUMO

We theoretically study the active motion of self-diffusiophoretic Janus particles (JPs) using the Onsager-Casimir reciprocal relations. The linear and angular velocity of a single JP are shown to respectively result from a coupling of electrochemical forces to the fluid flow fields induced by a force and torque on the JP. A model calculation is provided for half-capped JPs catalyzing a chemical reaction of solutes at their surface by reducing the continuity equations of the reacting solutes to Poisson equations for the corresponding electrochemical fields. We find that an anisotropic surface reactivity alone is enough to give rise to active linear motion of a JP, whereas active rotation only occurs if the JP is not axisymmetric. In the absence of specific interactions with the solutes, the active linear velocity of the JP is shown to be related to the stoichiometrically weighted sum of the friction coefficients (or hydrodynamic radii) of the reacting solutes. Our reciprocal treatment further suggests that a specific interaction with the solutes is required to observe far-field diffusiophoretic interactions between JPs, which rely on an interfacial solute excess at the JP surface. Most notably, our approach applies beyond the boundary-layer approximation and accounts for both the diffusio- and electrophoretic nature of active motion.

3.
Chem Biol Drug Des ; 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31661597

RESUMO

Recently, multi-target directed ligands have been of research interest for multifactorial disorders such as Alzheimer's disease (AD). Since H3 receptors (H3 Rs) and cholinesterases are involved in pathophysiology of AD, identification of dual-acting compounds capable of improving cholinergic neurotransmission is of importance in AD pharmacotherapy. In the present study, H3 R antagonistic activity combined with anticholinesterase properties of two previously computationally identified lead compounds, that is, compound 3 (6-chloro-N-methyl-N-[3-(4-methylpiperazin-1-yl)propyl]-1H-indole-2-carboxamide) and compound 4 (7-chloro-N-[(1-methylpiperidin-3-yl)methyl]-1,2,3,4-tetrahydroisoquinoline-2-carboxamide), was tested. Moreover, molecular docking and binding free energy calculations were conducted for binding mode and affinity prediction of studied ligands toward cholinesterases. Biological evaluations revealed inhibitory activity of ligands in nanomolar (compound 3: H3 R EC50  = 0.73 nM; compound 4: H3 R EC50  = 31 nM) and micromolar values (compound 3: AChE IC50  = 9.09 µM, BuChE IC50  = 21.10 µM; compound 4: AChE IC50  = 8.40 µM, BuChE IC50  = 4.93 µM) for H3 R antagonism and cholinesterase inhibition, respectively. Binding free energies yielded good consistency with cholinesterase inhibitory profiles. The results of this study can be used for lead optimization where dual inhibitory activity on H3 R and cholinesterases is needed. Such ligands can exert their biological activity in a synergistic manner resulting in higher potency and efficacy.

4.
Bioorg Med Chem ; 27(20): 115079, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31500943

RESUMO

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules.

5.
Chem Biol Interact ; 312: 108775, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31369746

RESUMO

Postnatal exposure to valproic acid (VPA) in rodents induces autism-like neurobehavioral defects which are comparable to the motor and cognitive deficits observed in humans with autism spectrum disorder (ASD). Histamine H3 receptor (H3R) and acetylcholine esterase (AChE) are involved in several cognitive disorders such as Alzheimer's disease, schizophrenia, anxiety, and narcolepsy, all of which are comorbid with ASD. Therefore, the present study aimed at evaluating effect of the novel dual-active ligand E100 with high H3R antagonist affinity and balanced AChE inhibition on autistic-like repetitive behavior, anxiety parameters, locomotor activity, and neuroinflammation in a mouse model of VPA-induced ASD in C57BL/6 mice. E100 (5, 10, and 15 mg/kg) dose-dependently and significantly ameliorated repetitive and compulsive behaviors by reducing the increased percentages of nestlets shredded (all P < 0.05). Moreover, pretreatment with E100 (10 and 15 mg/kg) attenuated disturbed anxiety levels (P < 0.05) but failed to restore the hyperactivity observed in the open field test. Furthermore, pretreatment with E100 (10 mg/kg) the increased microglial activation, proinflammatory cytokines and expression of NF-κB, iNOS, and COX-2 in the cerebellum as well as the hippocampus (all P < 0.05). These results demonstrate the ameliorative effects of E100 on repetitive compulsive behaviors in a mouse model of ASD. To our knowledge, this is the first in vivo demonstration of the effectiveness of a potent dual-active H3R antagonist and AChE inhibitor against autistic-like repetitive compulsive behaviors and neuroinflammation, and provides evidence for the role of such compounds in treating ASD.


Assuntos
Transtorno do Espectro Autista/patologia , Comportamento Animal/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Animais , Transtorno do Espectro Autista/induzido quimicamente , Transtorno do Espectro Autista/tratamento farmacológico , Inibidores da Colinesterase/uso terapêutico , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipocampo/metabolismo , Antagonistas dos Receptores Histamínicos H3/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Transcrição RelA/metabolismo , Ácido Valproico/toxicidade
6.
Proc Natl Acad Sci U S A ; 116(35): 17280-17289, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31350353

RESUMO

Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub's C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.

7.
Bioorg Chem ; 91: 103071, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31362197

RESUMO

A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents proved to be potent histamine H3 receptor (hH3R) ligands in the nanomolar concentration range. While paying attention to their alkyl linker length, derivatives with a six methylene linker tend to be more potent than their five methylene homologues. Moreover, in the case of both phenoxyacetyl- and phenoxypropionyl- derivatives, an eight methylene linkers possess lower activity than their seven methylene homologues. However, in global analysis of collected data on the influence of alkyl linker length, a three methylene homologues appeared to be of highest hH3R affinity among all described 4-pyridylpiperazine derivatives from our group up to date. In the case of biphenyl and benzophenone derivatives, compounds with para- substituted second aromatic ring were of higher affinity than their meta analogues. Interestingly, benzophenone derivative 18 showed the highest affinity among all tested compounds (hH3R Ki = 3.12 nM). The likely protein-ligand interactions, responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at H3R, as well as drug-like properties of selected ligands were evaluated using in vitro methods.

8.
J Chem Phys ; 150(21): 214901, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31176313

RESUMO

Mixtures of active and passive colloids show an intriguing dynamics of self-assembling, which is driven by the active component. Self-phoretic active colloids generate sinks in a chemical concentration field that cause passive colloids to drift toward active colloids by diffusiophoresis. The strength of this effective attraction is governed by the diffusiophoretic parameter, which determines the drift velocity. Simulating the Langevin dynamics of the colloids, we determine the state diagram for increasing diffusiophoretic strength and fixed active velocity. Three main states are distinguished. For weak attraction, passive particles are first scattered in the simulation box and then form a colloidal cloud around its center. Increasing the diffusiophoretic parameter further, passive particles oscillate between the cloud and a compact cluster, which embeds active colloids. Ultimately, in the third state, all particles collapse into a single stable cluster. In the collapse regime, the clustering dynamics of the largest cluster follows a logistic function and the mean cluster velocity vs cluster size decays with a power law. Throughout this article, we discuss our simulation results with regard to the experiments of Singh et al., Adv. Mater. 29(32), 1701328 (2017).

9.
World J Gastroenterol ; 25(23): 2846-2862, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31249444

RESUMO

Cytochromes P450s (CYPs) are terminal enzymes in CYP dependent monooxygenases, which constitute a superfamily of enzymes catalysing the metabolism of both endogenous and exogenous substances. One of their main tasks is to facilitate the excretion of these substances and eliminate their toxicities in most phase 1 reactions. Endogenous substrates of CYPs include steroids, bile acids, eicosanoids, cholesterol, vitamin D and neurotransmitters. About 80% of currently used drugs and environmental chemicals comprise exogenous substrates for CYPs. Genetic polymorphisms of CYPs may affect the enzyme functions and have been reported to be associated with various diseases and adverse drug reactions among different populations. In this review, we discuss the role of some critical CYP isoforms (CYP1A1, CYP2D6, CYP2J2, CYP2R1, CYP3A5, CYP3A7, CYP4F3, CYP24A1, CYP26B1 and CYP27B1) in the pathogenesis or aetiology of ulcerative colitis concerning gene polymorphisms. In addition, their significance in metabolism concerning ulcerative colitis in patients is also discussed showing a clear underestimation in genetic studies performed so far.

10.
Bioorg Med Chem ; 27(14): 3194-3200, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31176569

RESUMO

Inspired by marine compounds the derivatization of the natural pyrrolo[2,3-d]pyrimidine lead scaffold led to a series of novel compounds targeting the histamine H3 receptor. The focus was set on improved binding towards the receptor and to establish an initial structure-activity relationship for this compound class based on the lead structure (compound V, Ki value of 126 nM). As highest binding affinities were found with 1,4-bipiperidines as basic part of the ligands, further optimization was focused on 4-([1,4'-bipiperidin]-1'-yl)-pyrrolo[2,3-d]pyrimidines. Related pyrrolo[2,3-d]pyrimidines that were isolated from marine sponges like 4-amino-5-bromopyrrolo[2,3-d]pyrimidine (compound III), showed variations in halogenation pattern, though in a next step the impact of halogenation at 2-position was evaluated. The chloro variations did not improve the affinity compared to the dehalogenated compounds. However, the simultaneous introduction of lipophilic cores with electron-withdrawing substitution patterns in 7-position and dehalogenation at 2-position (11b, 12b) resulted in compounds with significantly higher binding affinities (Ki values of 7 nM and 6 nM, respectively) than the initial lead structure compound V. The presented structures allow for a reasonable structure-activity relationship of pyrrolo[2,3-d]pyrimidines as histamine H3 receptor ligands and yielded novel lead structures within the natural compound library against this target.

11.
Bioorg Med Chem ; 27(15): 3463-3468, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31248707

RESUMO

3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising derivatives were identified as apoptosis inducers and, emphasizing the multikinase inhibition potential, as sphingosine kinase 2 inhibitors. Our measurements provide additional insights into the structure-activity relationship of meriolin derivatives, suggesting derivatives bearing a pyridine moiety with amino groups in 2-position as most active anticancer compounds and thus as highly promising candidates for future in vivo studies.

12.
Soft Matter ; 15(28): 5685-5694, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31246219

RESUMO

We present a hydrodynamic study of a monolayer of squirmer model microswimmers confined to a boundary by strong gravity using the simulation method of multi-particle collision dynamics. The squirmers interact with each other via their self-generated hydrodynamic flow fields and thereby form a variety of fascinating dynamic states when density and squirmer type are varied. Weak pushers, neutral squirmers, and pullers have an upright orientation. With their flow fields they push neighbors away and thereby form a hydrodynamic Wigner fluid at lower densities. Furthermore, states of fluctuating chains and trimers, of kissing, and at large densities a global cluster exist. Finally, pushers at all densities can tilt against the wall normal and their in-plane velocities align to show swarming. It turns into chaotic swarming for strong pushers at high densities. We characterize all these states quantitatively.

13.
PLoS One ; 14(6): e0218820, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237914

RESUMO

In this study, we report a ligand-guided homology modeling approach allowing the analysis of relevant binding site residue conformations and the identification of two novel histamine H3 receptor ligands with binding affinity in the nanomolar range. The newly developed method is based on exploiting an essential charge interaction characteristic for aminergic G-protein coupled receptors for ranking 3D receptor models appropriate for the discovery of novel compounds through virtual screening.

14.
Structure ; 27(7): 1124-1136.e4, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31130485

RESUMO

The pseudo-atomic structural model of human pyruvate dehydrogenase complex (PDHc) core composed of full-length E2 and E3BP components, calculated from our cryoelectron microscopy-derived density maps at 6-Å resolution, is similar to those of prokaryotic E2 structures. The spatial organization of human PDHc components as evidenced by negative-staining electron microscopy and native mass spectrometry is not homogeneous, and entails the unanticipated formation of local clusters of E1:E2 and E3BP:E3 complexes. Such uneven, clustered organization translates into specific duties for E1-E2 clusters (oxidative decarboxylation and acetyl transfer) and E3BP-E3 clusters (regeneration of reduced lipoamide) corresponding to half-reactions of the PDHc catalytic cycle. The addition of substrate coenzyme A modulates the conformational landscape of PDHc, in particular of the lipoyl domains, extending the postulated multiple random coupling mechanism. The conformational and associated chemical landscapes of PDHc are thus not determined entirely stochastically, but are restrained and channeled through an asymmetric architecture and further modulated by substrate binding.

15.
Int J Mol Sci ; 20(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974892

RESUMO

Human ß-defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2-histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-α (TNF-α), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP.


Assuntos
Queratinócitos/metabolismo , Líquen Plano Bucal/metabolismo , Transdução de Sinais , Regulação para Cima , beta-Defensinas/biossíntese , Linhagem Celular Tumoral , Feminino , Histamina/metabolismo , Humanos , Queratinócitos/patologia , Líquen Plano Bucal/patologia , Masculino , Fator de Necrose Tumoral alfa/metabolismo
16.
Pharmacol Ther ; 200: 69-84, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31028835

RESUMO

Since the discovery of the histamine H3 receptor in 1983, tremendous advances in the pharmacological aspects of H3 receptor antagonists/inverse agonists have been accomplished in preclinical studies. At present, there are several drug candidates that reached clinical trial studies for various indications. However, entrance of these candidates to the pharmaceutical market is not free from challenges, and a variety of difficulties is engaged with their developmental process. In this review, the potential role of H3 receptors in the pathophysiology of various central nervous system, metabolic and allergic diseases is discussed. Thereafter, the current status for H3 receptor antagonists/inverse agonists in ongoing clinical trial studies is reviewed and obstacles in developing these agents are emphasized.

17.
Int J Mol Sci ; 20(4)2019 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-30813468

RESUMO

Intraocular pressure (IOP) has a tendency to fluctuate throughout the day, reaching its peak in the early morning in healthy subjects or glaucoma patients. Likewise, histamine tone also fluctuates over time, being lower at nighttime. Numerous studies have demonstrated a correlation between short-term IOP fluctuation and glaucoma progression; however, it has not yet been determined whether histamine plays a role in IOP fluctuations. The aim of this research was to establish the distribution of the histamine receptor proteins and respective mRNAs in the eye by western blot, immunohistochemistry and RT-PCR in New Zealand rabbits. Furthermore, we used a transient ocular hypertension (OHT) model induced by injection of 50 µL of 5% hypertonic saline into the vitreous and a stable OHT model (100 µL 0.1% carbomer in the anterior chamber) to address the potential IOP-lowering ability of H3 receptor (H3R) antagonists (ciproxifan, DL76 and GSK189254). IOPs were performed with a Tono-Pen at baseline and 60, 120 and 240 min post treatment after transient OHT induction and, every day for 12 days in the stable OHT model. All histamine receptor subtypes were localized in the rabbit retina and ciliary body/trabecular meshwork. All the treatments lowered IOP in a dose-dependent fashion between 0.3% and 1%. More specifically, the effects were maximal with ciproxifan at 60 min post-dose (IOP60 change = -18.84 ± 4.85 mmHg, at 1%), remained stable until 120 min (IOP120 change = -16.38 ± 3.8 mmHg, at 1%) and decayed thereafter to reach baseline values at 240 min. These effects were highly specific and dependent on histamine release as pre-treatment with imetit (H3R agonist, 1%) or pyrilamine (H1R antagonist, 1%) largely blocked ciproxifan-mediated effects. Color Doppler ultrasound examination was performed to evaluate changes in ophtalmic artery resistivity index (RI) before and after repeated dosing with DL 76, GSK189254, ciproxifan and timolol. Chronic treatments with H3R antagonists and timolol improved the vascular performance of ophthalmic arteries and reduced retinal ganglion cell death. Oxidative stress was also reduced and measured 8-Hydroxy-2'-deoxyguanosine (8OHdG) expression, and by dihidroethydium (DHE) staining. These results demonstrated that the histamine system participates in IOP regulation and that H3R antagonists could represent a future promising therapy for glaucoma. Further studies should be focused on the long-term IOP circadian fluctuations.


Assuntos
Glaucoma/tratamento farmacológico , Glaucoma/fisiopatologia , Antagonistas dos Receptores Histamínicos H3/uso terapêutico , Pressão Intraocular , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/fisiopatologia , Animais , Corioide/efeitos dos fármacos , Corioide/metabolismo , Corioide/patologia , Modelos Animais de Doenças , Glaucoma/genética , Antagonistas dos Receptores Histamínicos H3/farmacologia , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Pressão Intraocular/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Modelos Biológicos , Hipertensão Ocular/genética , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Fatores de Tempo
18.
Eur J Pharmacol ; 848: 112-120, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30703360

RESUMO

Growing evidence recommends incorporating the concept of drug-target residence times within drug development and screening programs. For many targets, systematic research for binding kinetics is emerging and reported, as in case of the histamine H3 receptor. Alternatively, fluorescent methods based on Foerster resonance energy transfer have been reported recently but application of fluorescence polarization to kinetics of unlabeled ligands is not known to us. Thus, we established a radiolabel-free, real-time resolving method that is compatible to high-throughput-screening programs with the objective to explore the underlying binding kinetics. This method takes benefit of bodilisant as H3 receptor ligand. Thereby, we detected short residence times around 5 min for the H3 receptor ligands ciproxifan, clobenpropit, thioperamide as well as pitolisant. Monitoring association rates, remarkably slower association rate constants were examined for ciproxifan and thioperamide when compared to those of pitolisant or clobenpropit. The affinities for the ligands derived by the kinetic approach differ from affinity estimates in literature using radiolabeled agonists in displacement assays. Further investigation raised exceptional pharmacological properties, consistent with occurrence of secondary binding sites at the H3 receptor. Validation of resulting affinity constants was successfully performed by displacement assays based on fluorescence polarization with bodilisant.


Assuntos
Polarização de Fluorescência/métodos , Agonistas dos Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos H3/metabolismo , Receptores Histamínicos H3/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Humanos , Ligantes , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia
19.
Soft Matter ; 15(9): 1988-1998, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30714602

RESUMO

A flowing pair of particles in inertial microfluidics gives important insights into understanding and controlling the collective dynamics of particles like cells or droplets in microfluidic devices. They are applied in medical cell analysis and engineering. We study the dynamics of a pair of solid particles flowing through a rectangular microchannel using lattice Boltzmann simulations. We determine the inertial lift force profiles as a function of the two particle positions, their axial distance, and the Reynolds number. Generally, the profiles strongly differ between particles leading and lagging in flow and the lift forces are enhanced due to the presence of a second particle. At small axial distances, they are determined by viscous forces, while inertial forces dominate at large separations. We identify cross-streamline pairs as stable fixed points in the lift force profiles and argue that same-streamline configurations are only one-sided stable. Depending on the initial conditions, the two-particle lift forces in combination with the Poiseuille flow give rise to three types of unbound particle trajectories, called moving-apart, passing, and swapping, and one type of bound trajectory, where the particles perform damped oscillations towards the cross-stream line configuration. The damping rate scales with Reynolds number squared, since inertial forces are responsible for driving the particles to their steady-state positions.

20.
Bioorg Med Chem ; 27(7): 1254-1262, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30792106

RESUMO

This study focuses on the design, synthesis, molecular modeling and biological evaluation of a novel group of alkyl-1,3,5-triazinyl-methylpiperazines. New compounds were synthesized and their affinities for human histamine H4 receptor (hH4R) were evaluated. Among them, 4-(cyclohexylmethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (14) exhibited hH4R affinity with a Ki of 160 nM and behaved as antagonist in functional assays: the cellular aequorin-based assay (IC50 = 32 nM) and [35S]GTPγS binding assay (pKb = 6.67). In addition, antinociceptive activity of 14in vivo was observed in Formalin test (in mice) and in Carrageenan-induced acute inflammation test (in rats).

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