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Microorganisms use chemotaxis, regulated by internal complex chemical pathways, to swim along chemical gradients to find better living conditions. Artificial microswimmers can mimic such a strategy by a pure physical process called diffusiophoresis, where they drift and orient along the gradient in a chemical density field. Similarly, for other forms of taxis in nature such as photo- or thermotaxis the phoretic counterpart exists. In this Account, we concentrate on the chemotaxis of self-phoretic active colloids. They are driven by self-electro- and diffusiophoresis at the particle surface and thereby acquire a swimming speed. During this process, they also produce nonuniform chemical fields in their surroundings through which they interact with other colloids by translational and rotational diffusiophoresis. In combination with active motion, this gives rise to effective phoretic attraction and repulsion and thereby to diverse emergent collective behavior. A particular appealing example is dynamic clustering in dilute suspensions first reported by a group from Lyon. A subtle balance of attraction and repulsion causes very dynamic clusters, which form and resolve again. This is in stark contrast to the relatively static clusters of motility-induced phase separation at larger densities. To treat chemotaxis in active colloids confined to a plane, we formulate two Langevin equations for position and orientation, which include translational and rotational diffusiophoretic drift velocities. The colloids are chemical sinks and develop their long-range chemical profiles instantaneously. For dense packings, we include screening of the chemical fields. We present a state diagram in the two diffusiophoretic parameters governing translational, as well as rotational, drift and, thereby, explore the full range of phoretic attraction and repulsion. The identified states range from a gaslike phase over dynamic clustering states 1 and 2, which we distinguish through their cluster size distributions, to different types of collapsed states. The latter include a full chemotactic collapse for translational phoretic attraction. Turning it into an effective repulsion, with increasing strength first the collapsed cluster starts to fluctuate at the rim, then oscillates, and ultimately becomes a static collapsed cloud. We also present a state diagram without screening. Finally, we summarize how the famous Keller-Segel model derives from our Langevin equations through a multipole expansion of the full one-particle distribution function in position and orientation. The Keller-Segel model gives a continuum equation for treating chemotaxis of microorganisms on the level of their spatial density. Our theory is extensible to mixtures of active and passive particles and allows to include a dipolar correction to the chemical field resulting from the dipolar symmetry of Janus colloids.

This work describes the microwave assisted synthesis of twelve novel histamine H3 receptor ligands. They display pyrrolo[2,3-d]pyrimidine derivatives with rigidized aliphatic amines as warheads. The compounds were screened for H3R and H4R binding affinities in radioligand displacement assays and the most potent compounds were evaluated for H3R binding properties in vitro and in docking studies. The combination of a rigidized H3R warhead and the pyrrolo[2,3-d]pyrimidine scaffold resulted in selective activity at the H3 receptor with a pKi value of 6.90 for the most potent compound. A bipiperidine warhead displayed higher affinity than a piperazine or morpholine motif, while a naphthyl moiety in the arbitrary region increased affinity compared to a phenyl derivative. The compounds can be starting points for novel, simply synthesized histamine H3 receptor ligands.

Histamine H3 receptor (H3R) is largely expressed in the CNS and modulation of the H3R function can affect histamine synthesis and liberation, and modulate the release of many other neurotransmitters. Targeting H3R with antagonists/inverse agonists may have therapeutic applications in neurodegenerative disorders, gastrointestinal and inflammatory diseases. This prompted us to design and synthesize azole-based H3R ligands, i.e. having oxadiazole- or thiazole-based core structures. While ligands of oxadiazole scaffold were almost inactive, thiazole-based ligands were very potent and several exhibited binding affinities in a nanomolar concentration range. Ligands combining 4-cyanophenyl moiety as arbitrary region, para-xylene or piperidine carbamoyl linkers, and/or pyrrolidine or piperidine basic heads were found to be the most active within this series of thiazole-based H3R ligands. The most active ligands were in silico screened for ADMET properties and drug-likeness. They fulfilled Lipinski's and Veber's rules and exhibited potential activities for oral administration, blood-brain barrier penetration, low hepatotoxicity, combined with an overall good toxicity profile.

Cryo-electron microscopy (cryo-EM) of non-crystalline single particles is a biophysical technique that can be used to determine the structure of biological macromolecules and assemblies. Historically, its potential for application in drug discovery has been heavily limited by two issues: the minimum size of the structures it can be used to study and the resolution of the images. However, recent technological advances - including the development of direct electron detectors and more effective computational image analysis techniques - are revolutionizing the utility of cryo-EM, leading to a burst of high-resolution structures of large macromolecular assemblies. These advances have raised hopes that single-particle cryo-EM might soon become an important tool for drug discovery, particularly if they could enable structural determination for 'intractable' targets that are still not accessible to X-ray crystallographic analysis. This article describes the recent advances in the field and critically assesses their relevance for drug discovery as well as discussing at what stages of the drug discovery pipeline cryo-EM can be useful today and what to expect in the near future.

Cryo-electron microscopy (cryo-EM) can solve structures of highly dynamic macromolecular complexes. To characterize less well defined regions in cryo-EM images, cross-linking coupled with mass spectrometry (CX-MS) provides valuable information on the arrangement of domains and amino acids. CX-MS involves covalent linkage of protein residues close to each other and identifying these connections by mass spectrometry. Here, we summarise the advances of CX-MS and its integration with cryo-EM for structural reconstruction.

Histamine is an important mediator of biological functions and present in high amounts in inflammatory skin lesions which are characterised by a marked infiltration of myeloid derived cell populations. The aim of the study was to investigate the expression and function of histamine receptors, with a focus on the histamine H4 receptor (H4R) in detail during the differentiation process from monocytes to macrophages and on fully differentiated M1 macrophages. Quantitative PCR, ELISA technique, and flow cytometry were applied to analyze expression levels of histamine receptors, of CXCL10, CCL4, CCL3, or IL-23 and of the macrophage differentiation marker CD68, respectively. We demonstrated that monocytes and fully differentiated M1 macrophages express H1R-, H2R-, and H4R mRNA which were differentially regulated during the differentiation process and in IFN-Ƴ and LPS classically activated M1 macrophages. The H3R mRNA was not expressed. During in vitro differentiation from monocytes to macrophages, the H4R agonist ST-1006 modified the M1 phenotype by up-regulating the macrophage differentiation marker CD68, by down-regulating the production of CXCL10, and by changing the morphology. In fully differentiated M1 macrophages, histamine or ST-1006 decreased the IFN-Ƴ- and LPS-induced CCL4 mRNA expression and protein production, whereas CCL3 or IL-23 production was not regulated via H4R. We describe novel immunomodulatory functions of the H4R during the differentiation process of human monocyte-derived macrophages and in fully differentiated M1 macrophages. The down-regulation of Th1-related chemokines during the differentiation process or in classically activated macrophages via H4R may contribute to decreased migration of immune cells to the site of inflammation. This may have implications for the treatment of allergic diseases with H4R ligands regulating the dysbalance of Th2/Th1 polarizations in these disorders.

Summary The identification of the high-risk human papilloma viruses (HPVs) as a cause of cervical cancer offered the possibility for the development of a HPV vaccine. Twenty years after this identification of the HPV types, the first HPV vaccine came to the market. There are three HPV vaccines today on the market, all containing the virus-like particles (VLPs) of the HPV types 16 and 18, which are considered to cause 77 % of all cancers caused by HPVs. In addition, two of the vaccines contain two low-risk HPV types (6 and 11)-quadrivalent or the same as low-risk types and additional high-risk HPV types (31, 32, 45, 52, 58)-nonavalent vaccines. The cervical cancer protection efficacy of the vaccines is very high, around 100%. The VLPs of the 6- and 11-type offer efficient protection against genital warts. Unfortunately, the implementation of the vaccination is actually not so high despite all scientific and medical facts, but their rates in Europe are steadily increasing reaching about 90% in one country after another. In Serbia, all of the three vaccines are on the market but are highly underused. Actually, there is no national program and the Serbian vaccination rates are very low. High vaccination rates in Serbia need to be achieved as a goal of prevention of cervical cancer.

During the past decades, histamine H3 receptors have received widespread attention in pharmaceutical research due to their involvement in pathophysiology of several diseases such as neurodegenerative disorders. In this context, blocking of these receptors is of paramount importance in progression of such diseases. In the current investigation, novel histamine H3 receptor ligands were designed by exploiting scaffold-hopping drug-design strategy. We inspected the designed molecules in terms of ADME properties, drug-likeness, as well as toxicity profiles. Additionally molecular docking and dynamics simulation studies were performed to predict binding mode and binding free energy calculations, respectively. Among the designed structures, we selected compound d2 and its demethylated derivative as examples for synthesis and affinity measurement. In vitro binding assays of the synthesized molecules demonstrated that d2 has lower binding affinity (Ki = 2.61 μM) in radioligand displacement assay to hH3R than that of demethylated form (Ki = 12.53 μM). The newly designed compounds avoid of any toxicity predictors resulted from extended in silico and experimental studies, can offer another scaffold for histamine H3R antagonists for further structure-activity relationship studies.

Photoresponsive surfactants provide a unique microfluidic driving mechanism. Since they switch between two molecular shapes under illumination and thereby affect surface tension of fluid interfaces, Marangoni flow along the interface occurs. To describe the dynamics of the surfactant mixture at a planar interface, we formulate diffusion-advection-reaction equations for both surfactant densities. They also include adsorption from and desorption into the neighboring fluids and photoisomerization by light. We then study how the interface responds when illuminated by spots of light. Switching on a single light spot, the density of the switched surfactant spreads in time and assumes an exponentially decaying profile in steady state. Simultaneously, the induced radial Marangoni flow reverses its flow direction from inward to outward. We use this feature to set up specific feedback rules, which couple the advection velocities sensed at the light spots to their intensities. As a result two neighboring spots switch on and off alternately. Extending the feedback rule to light spots arranged on the vertices of regular polygons, we observe periodic switching patterns for even-sided polygons, where two sets of next-nearest neighbors alternate with each other. A triangle and pentagon also show regular oscillations, while heptagon and nonagon exhibit irregular oscillations due to frustration. While our findings are specific to the chosen set of parameters, they show how complex patterns at photoresponsive fluid interfaces emerge from simple feedback coupling.

The anaphylatoxin C3a triggers inflammation by binding to its specific G-protein-coupled C3a receptor (C3aR). Since the number of C3aR, which is expressed on the cell surface, affects the response to C3a, we investigated the expression levels of C3aR on human M2 macrophages in allergic situations where high levels of the Th2 cytokine IL-4 and histamine are present in a local microenvironment. The histamine H1 receptor (H1R), H2R and the H4R mRNA expressions were induced or up-regulated during the differentiation process of M2 macrophages. The presence of histamine or agonists targeting the H1R, H2R and, in particular, the H4R during in vitro differentiation from monocytes to macrophages modified the M2 phenotype by regulating the macrophage differentiation marker CD68 and CD163 expressions. In ­addition, the C3aR expression was also down-regulated by ­ST-1006 during this process. Histamine and ST-1006 down-regulated the expression of C3aR with different time kinetics on fully differentiated M2 macrophages. By analysing C3a-induced IL-6 mRNA expression, we observed a diminished response to C3a in ST-1006-treated M2 macrophages when compared to un-treated cells. Expression of C3 was not affected by histamine, whereas IL-4 strongly down-regulated C3aR and C3 expressions. Our data suggests that down-regulation of C3aR expression by mediators present in allergic situations such as IL-4 or histamine has an anti-inflammatory impact by reducing the sensitivity to C3a-induced down-stream signaling, thereby contributing to the regulation of local inflammatory responses in the skin.

Alzheimeŕs disease (AD) is the most prominent neurodegenerative disorder with high medical need. Protein-protein-interactions (PPI) interactions have a critical role in AD where β-amyloid structures (Aβ) build toxic oligomers. Design of disease modifying multi target directed ligand (MTDL) has been performed, which disable PPI on the one hand and on the other hand, act as procognitive antagonists at the histamine H3 receptor (H3R). The synthetized compounds are structurally based on peptidomimetic amino acid-like structures mainly as keto, diketo-, or acyl variations of a piperazine moiety connected to an H3R pharmacophore. Most of them showed low nanomolar affinities at H3R and some with promising affinity to Aβ-monomers. The structure–activity relationships (SAR) described offer new possibilities for MTDL with an optimized profile combining symptomatic and potential causal therapeutic approaches in AD.

Simulations of flow fields around microscopic objects typically require methods that both solve the Navier-Stokes equations and also include thermal fluctuations. One such method popular in the field of soft-matter physics is the particle-based simulation method of multi-particle collision dynamics (MPCD). However, in contrast to the typically incompressible real fluid, the fluid of the traditional MPCD methods obeys the ideal-gas equation of state. This can be problematic because most fluid properties strongly depend on the fluid density. In a recent article, we proposed an extended MPCD algorithm and derived its non-ideal equation of state and an expression for the viscosity. In the present work, we demonstrate its accuracy and efficiency for the simulations of the flow fields of single squirmers and of the collective dynamics of squirmer rods. We use two exemplary squirmer-rod systems for which we compare the outcome of the extended MPCD method to the well-established MPCD version with an Andersen thermostat. First, we explicitly demonstrate the reduced compressibility of the MPCD fluid in a cluster of squirmer rods. Second, for shorter rods, we show the interesting result that in simulations with the extended MPCD method, dynamic swarms are more pronounced and have a higher polar order. Finally, we present a thorough study of the state diagram of squirmer rods moving in the center plane of a Hele-Shaw geometry. From a small to large aspect ratio and density, we observe a disordered state, dynamic swarms, a single swarm, and a jammed cluster, which we characterize accordingly.

Alzheimer’s disease (AD) is a complex and incurable illness that requires the urgent approval of new effective drugs. However, since 2003, no new molecules have shown successful results in clinical trials, thereby making the common “one compound – one target” paradigm questionable. Recently, the multitarget-directed ligand (MTDL) approach has gained popularity, as compounds targeting at least two biological targets may be potentially more effective in treating AD. On the basis of these findings, we designed, synthesized, and evaluated through biological assays a series of derivatives of alicyclic amines linked by an alkoxy bridge to an aromatic lipophilic moiety of [1,1ʹ-biphenyl]-4-carbonitrile. The research results revealed promising biological activity of the obtained compounds toward the chosen targets involved in AD pathophysiology; the compounds showed high affinity (mostly low nanomolar range of Ki values) for human histamine H3 receptors (hH3R) and good nonselective inhibitory potency (micromolar range of IC50 values) against acetylcholinesterase from electric eel (eeAChE) and equine serum butyrylcholinesterase (eqBuChE). Moreover, micromolar/submicromolar potency against human monoamine oxidase B (hMAO B) was detected for some compounds. The study identified compound 5 as a multiple hH3R/eeAChE/eqBuChE/hMAO B ligand (5: hH3R Ki = 9.2 nM; eeAChE IC50 = 2.63 µM; eqBuChE IC50 = 1.30 µM; hMAO B IC50 = 0.60 µM). Further in vitro studies revealed that compound 5 exhibits a mixed type of eeAChE and eqBuChE inhibition, good metabolic stability, and moderate hepatotoxicity effect on HepG2 cells. Finally, compound 5 showed a beneficial effect on scopolamine-induced memory impairments, as assessed by the passive avoidance test, thus revealing the potential of this compound as a promising agent for further optimization for AD treatment.

The aim was to evaluate the activity of seven medicinal, anti-inflammatory plants at the hH4R with focus on defined chemical compounds from Curcuma longa. Activities were analyzed with membrane preparations from Sf9 cells, transiently expressing the hH4R, Gαi2 and Gβ1γ2 subunits. From the methanolic extract of C. longa curcumin (1), demethoxycurcumin (2) and bis(4-hydroxy-cinnamoyl)methane (3) were isolated, purified with HPLC (elution-time 10.20, 9.66, 9.20 min, respectively) and together with six additional extracts, were characterized via radioligand binding studies at the hH4R. Compounds from C. longa were the most potent ligands at the hH4R. They exhibited estimated K i values of 4.26–6.26 µM (1.57–2.31 µg/mL) (1); 6.66––8.97 µM (2.26–3.04 µg/mL) (2) and 10.24–14.57 µM (3.16–4.49 µg/mL) (3) (95% CI). The estimated K i value of the crude extract of curcuma was 0.50–0.81 µg/mL. Fractionated curcumin and the crude extract surpassed the effect of pure curcumin with a K i value of 5.54 µM or 2.04 µg/mL [95% CI (4.47–6.86 µM), (1.65–2.53 µg/mL)]. Within this study, defined compounds of C. longa were recognized as potential ligands and reasonable lead structures at the hH4R. The mode of anti-inflammatory action of curcumin was further elucidated and the role of extracts in traditional phytomedicine was strengthened.

Multifactorial neurodegenerative disorders such as Alzheimer's disease (AD) are considered a growing public health problem due the rising incidence and low effectiveness of current treatments [6]. Since pharmacotherapy based on a single target has been insufficient for drug development in complex diseases, the emerging multi-target approach is a promising strategy for the search of new anti-AD drug candidates. Herein described natural isoquinoline alkaloids were investigated for multi-target activity on key mechanisms associated with the AD's pathogenesis, i.e. cholinergic depletion, beta amyloid (Aβ) aggregation and oxidative stress. Alkaloid isolation from root extract of Zanthoxylum rigidum was carried out using multi-step chromatography and TLC-bioautography against acetylcholinesterase (AChE) giving eight purified isoquinoline alkaloids. Isolated compounds were tested for inhibitory activity against cholinesterase (AChE and BChE), monoamine oxidase (MAO-A and B) and Aβ aggregation. Our study revealed two benzophenanthridine alkaloids, nitidine (5) and avicine (7), as the most potent multi-target candidates. Both showed dual cholinesterase inhibition, being more active against AChE over BChE, with IC

Taking into account that multidrug resistance (MDR) is the main cause for chemotherapeutic failure in cancer treatment, the ability of novel histamine H3 receptor ligands to reverse the cancer MDR was evaluated, using the ABCB1 efflux pump inhibition assay in mouse MDR T-lymphoma cells. The most active compounds displayed significant cytotoxic and antiproliferative effects as well as a very potent MDR efflux pump inhibitory action, 3-5-fold stronger than that of reference inhibitor verapamil. Although these compounds possess weak antagonistic properties against histamine H3 receptors, they are valuable pharmacological tools in the search for novel anticancer molecules. Furthermore, for the most active compounds, an insight into mechanisms of action using either, the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp, was performed.

Cell 180(6), 1130 - 1143.e20 (2020). doi:10.1016/j.cell.2020.02.034, Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a �� subunit of the 2.6 megadalton ��6-��6 S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The �� subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The �� subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS., Published by Elsevier, New York, NY

Abstract. We present the results of hydrodynamic simulations using the method of multi-particle collision dynamics for a system of squirmer microswimmers moving under the influence of gravity at low Reynolds numbers. In addition, the squirmers are bottom-heavy so that they experience a torque which aligns them along the vertical. The squirmers interact hydrodynamically by the flow fields of a stokeslet and rotlet, which are initiated by the acting gravitational force and torque, respectively, and by their own flow fields. By varying the ratio of swimming to bulk sedimentation velocity and the torque, we determine state diagrams for the emergent collective dynamics of neutral squirmers as well as strong pushers and pullers. For low swimming velocity and torque we observe conventional sedimentation, while the sedimentation profile becomes inverted when their values are increased. For neutral squirmers we discover convective rolls of circulating squirmers between both sedimentation states, which sit at the bottom of the system and are fed by plumes made of collectively sinking squirmers. At larger torques porous clusters occur that spawn single squirmers. The two latter states can also occur transiently starting from a uniform squirmer distribution and then disappear in the long-time limit. For strong pushers and pullers only weak plume formation is observed. Graphical abstract

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya&reg, ), which acts as a functional S1P1 antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P1 activation profile and a sustained S1P1 internalization in cultures of S1P1-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional antagonism. In vivo, both compounds induced a profound lymphopenia in mice. Finally, these substances showed efficacy in the EAE model, where they reduced clinical symptoms of the disease, and, on the molecular level, they reduced the T-cell infiltration and several inflammatory mediators in the brain and spinal cord. In summary, these data suggest that S1P1-selective compounds may have an advantage over fingolimod and siponimod, not only in MS but also in other autoimmune diseases.

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.

Histamine and dopamine neuronal pathways display interesting overlapping in the CNS, especially in the limbic areas, making them very attractive to designing drugs with synergistic and/or additive effects. The roles of these systems to treat schizophrenia, drug addiction, Parkinson’s and Alzheimer’s diseases, among others are widely known. The LINS01 compounds were previously reported as histamine H3 receptor (H3R) antagonists and some of them are under evaluation in rodent memory models. Considering their pharmacological potential and similarities to literature dopamine D2 receptor (D2R) and dopamine D3 receptor (D3R) ligands, this work aimed to evaluate these compounds as ligands these receptors by using [3H]spiperone displacement assays. A set of 11 compounds containing the dihydrobenzofuranyl-piperazine core with substituents at 5-position of dihydrobenzofuran ring and at the piperazine nitrogen was examined. The compounds showed low to moderate affinities at both, D2R and D3R. N-Phenyl compounds LINS01005 (1d), LINS01011 (1h), LINS01012 (1i) and LINS01016 (1k) showed the highest affinities in the set to D3R (Ki 0.3–1.5 µM), indicating that N-phenylpiperazine moiety increases the affinity to this receptor subtype with some selectivity, since they showed lower affinities to D2R (Ki 1.3–5.5 µM). With the LINS01 compounds showing moderate binding affinity, new lead structures for optimization with regards to combined H3R and D2R/D3R-ligands are provided. Histamine and dopamine neuronal pathways display interesting overlapping in the CNS, and thus LINS01 compounds previously reported as histamine H3 receptor antagonists were evaluated as dopamine D2R and D3R ligands. The compounds showed micromolar affinities to both receptors

Since the discovery and early characterization of the histamine H

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52–7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem–loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 A and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2–branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site. The cryo-EM structure of human U2 small nuclear ribonucleoprotein (snRNP) offers insights into what rearrangements are required for this snRNP to be stably incorporated into the spliceosome, and the role that the DEAD-box ATPase PRP5 may have in these rearrangements.

Epilepsy is a chronic neurological disorder characterized by irregular, excessive neuronal excitability, and recurrent seizures that affect millions of patients worldwide. Currently, accessible antiepileptic drugs (AEDs) do not adequately support all epilepsy patients, with around 30% patients not responding to the existing therapies. As lifelong epilepsy treatment is essential, the search for new and more effective AEDs with an enhanced safety profile is a significant therapeutic goal. Seizures are a combination of electrical and behavioral events that can induce biochemical, molecular, and anatomic changes. Therefore, appropriate animal models are required to evaluate novel potential AEDs. Among the large number of available animal models of seizures, the acute pentylenetetrazole (PTZ)-induced myoclonic seizure model is the most widely used model assessing the anticonvulsant effect of prospective AEDs, whereas chronic PTZ-kindled seizure models represent chronic models in which the repeated administration of PTZ at subconvulsive doses leads to the intensification of seizure activity or enhanced seizure susceptibility similar to that in human epilepsy. In this review, we summarized the memory deficits accompanying acute or chronic PTZ seizure models and how these deficits were evaluated applying several behavioral animal models. Furthermore, major advantages and limitations of the PTZ seizure models in the discovery of new AEDs were highlighted. With a focus on PTZ seizures, the major biochemicals, as well as morphological alterations and the modulated brain neurotransmitter levels associated with memory deficits have been illustrated. Moreover, numerous medicinal compounds with concurrent anticonvulsant, procognitive, antioxidant effects, modulating effects on several brain neurotransmitters in rodents, and several newly developed classes of compounds applying computer-aided drug design (CADD) have been under development as potential AEDs. The article details the in-silico approach following CADD, which can be utilized for generating libraries of novel compounds for AED discovery. Additionally, in vivo studies could be useful in demonstrating efficacy, safety, and novel mode of action of AEDs for further clinical development.

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.

Histamine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Histamine Receptors [75, 163]) are activated by the endogenous ligand histamine. Marked species differences exist between histamine receptor orthologues [75]. The human and rat H3 receptor genes are subject to significant splice variance [12]. The potency order of histamine at histamine receptor subtypes is H3 = H4 > H2 > H1 [163]. Some agonists at the human H3 receptor display significant ligand bias [171]. Antagonists of all 4 histamine receptors have clinical uses: H1 antagonists for allergies (e.g. cetirizine), H2 antagonists for acid-reflux diseases (e.g. ranitidine), H3 antagonists for narcolepsy (e.g. pitolisant/WAKIX; Registered) and H4 antagonists for atopic dermatitis (e.g. ZPL-3893787; Phase IIa) [163] and vestibular neuritis (AUV) (SENS-111 (Seliforant, previously UR-63325), entered and completed vestibular neuritis (AUV) Phase IIa efficacy and safety trials, respectively) [205, 8].

We formulate the theory for steering an active particle with optimal travel time between two locations and apply it to the Mexican hat potential without brim. For small heights the particle can cross the potential barrier, while for large heights it has to move around it. Thermal fluctuations in the orientation strongly affect the path over the barrier. Then we consider a smart active particle and apply reinforcement learning. We show how the active particle learns in repeating episodes to move optimally. The optimal steering is stored in the optimized action-value function, which is able to rectify thermal fluctuations., 7 pages, 6 figures

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.

We study capillary condensation in a bath consisting of active Brownian particles (ABPs) and the forces acting on the capillary close to the motility-induced phase separation (MIPS). The capillary is modelled as two parallel rods of high aspect ratio which are fixed in space. We consider a bath of ABPs having a self-propulsion speed much larger than the critical speed necessary for MIPS to occur. For a given particle speed, we gradually increase the packing fraction of ABPs, starting from a homogeneous dilute phase of ABPs and going towards the binodal of MIPS. Depending on the packing fraction of ABPs and capillary width, we find that the effective force between the capillary rods can be either attractive or repulsive. In fact, with increasing width it shows damped oscillations as long as capillary condensation occurs. We analyze them in detail by studying the distribution of particle distances from the inner and outer wall of the capillary, respectively. In addition, we examine the capillary in the active bath prepared under conditions close to the critical point. We do not observe signs of the presence of long-range Casimir interactions., 7 pages, 8 figures

Recently, multi-target directed ligands have been of research interest for multifactorial disorders such as Alzheimer's disease (AD). Since H

Ubiquitin-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 ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitin’s C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin 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 ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and 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 ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new 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 ubiquitin binding sites within large multiprotein complexes.SIGNIFICANCE STATEMENTUbiquitin-mediated interactions influence numerous biological processes. These are often transient or a part of multivalent interactions. Therefore, unmasking these interactions remains a significant challenge for large, complicated enzymes such as the Anaphase-Promoting Complex/Cyclosome (APC/C), a multisubunit RING E3 ubiquitin (Ub) ligase. APC/C activity regulates numerous facets of biology by targeting key regulatory proteins for Ub-mediated degradation. Using a series of Ub variants (UbVs), we identified a new Ub-binding site on the APC/C that preferentially binds to K48-linked Ub chains. More broadly, we demonstrate a workflow that can be exploited to uncover Ub-binding sites within ubiquitylation machinery and other associated regulatory proteins to interrogate the complexity of the Ub code in biology.

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).

A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents proved to be potent histamine H

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.

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., 11 pages, 14 figures, supplemental material not included

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 H

Human &beta, 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&ndash, 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-&alpha, (TNF-&alpha, ), 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.

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.

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 H

A series of 4-pyridylpiperazine derivatives with varying regulatory region substituents proved to be potent histamine H3 receptor (H3R) ligands in the nanomolar concentration range. The most influential modification that affected the affinity toward the H3R appeared by introducing electron-withdrawing moieties into the distal aromatic ring. In order to finally discuss the influence of the characteristic 4-pyridylpiperazine moiety on H3R affinity, two Ciproxifan analogues 2 and 3 with a slight modification in their basic part were obtained. The replacement of piperazine in 3 with piperidine in compound 2, led to slightly reduced affinity towards the H3R (Ki = 3.17 and 7.70 nM, respectively). In fact, 3 showed the highest antagonistic properties among all compounds in this series, hence affirming our previous assumptions, that the 4-pyridylpiperazine moiety is the key element for suitable interaction with the human histamine H3 receptor. While its structural replacement to piperidine is also tolerated for H3R binding, the heteroaromatic 4-pyridyl moiety seems to be essential for proper ligand-receptor interaction. The putative protein-ligand interactions responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at the H3R, as well as drug-like properties of ligands were evaluated using in vitro methods. Moreover, pharmacological in vivo test results of compound 9 (structural analogue of Abbott’s A-331440) clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound.

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2–U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 A, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 A from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP. The cryo-EM structure of the splicing intermediate known as the C* complex from human. Recent years have seen substantial progress in understanding the structure of various intermediates of the splicing process. Two groups, led by Reinhard Luhrmann and Kiyoshi Nagai, now describe the cryo-electron microscopy structures (from human and yeast cells, respectively) of the splicing intermediate known as the C* complex. The notable feature observed in this complex, relative to the preceding catalytic intermediate (the C complex), is a remodelling that positions the branch-site adenosine and the branched intron out of the catalytic core, opening up space for the 3′ exon to dock in preparation for exon ligation.

5-Lipoxygenase (5-LO, EC1.13.11.34) has been implicated in the pathogenesis of inflammatory and immune diseases. Recently, aminothiazole comprising inhibitors have been discovered for this valuable target. Yet, the molecular mode of action of this class of substances is only poorly understood. Here, we present the detailed molecular mechanism of action of the compound class and the in vitro pharmacological profile of two lead compounds ST-1853 and ST-1906. Mechanistic studies with recombinant proteins as well as intact cell assays enabled us to define this class as a novel type of 5-LO inhibitors with unique characteristics. The parent compounds herein presented a certain reactivity concerning oxidation and thiol binding: Unsubstituted aminophenols bound covalently to C159 and C418 of human 5-LO. Yet, dimethyl substitution of the aminophenol prevented this reactivity and slowed down phase II metabolism. Both ST-1853 and ST-1906 confirmed their lead likeness by retaining their high potency in physiologically relevant 5-LO activity assays, high metabolic stability, high specificity and non-cytotoxicity.

Nuclear magnetic resonance (NMR) spectroscopy is a widely used analytical technique for molecular structure determination, and is highly valued in the fields of chemistry, biochemistry, and medicinal chemistry. The importance of NMR methods in the European (PhEur) and United States Pharmacopeia (USP) is steadily growing. However, undergraduates often have problems becoming familiar with handling the complex data. We have developed a simple experiment in which undergraduates, who are learning 1H NMR spectroscopy for the first time, investigate natural amino acids, and determine their structure and identity using low-field 1H NMR measurements and simple COSY experiments. These students see and learn the connection between the chemical shift of the αC-proton and the isoelectric point of the amino acid. They engage with the spectroscopic topic by acquiring their own spectra, and processing and interpreting the data. Understanding important natural amino acids and their physicochemical character is highly rele...

Marketed pain relief drugs with one to three biologically active components, as well as mixtures of these ingredients, were qualitatively and quantitatively analyzed in an undergraduate student lab using a compact, low-field 1H NMR spectrometer. The students successfully analyzed more than 50 self-made sample mixtures with two or three components as well as the two marketed tablet formulations containing acetylsalicylic acid/l-ascorbic acid, or acetylsalicylic acid/paracetamol (acetaminophen)/caffeine. The NMR-based quantification is an attractive application of the technique, as well as a helpful introduction to NMR spectroscopic applications in life sciences. Problem-based learning on NMR techniques on commonly known drugs provided students the opportunity to develop and improve their skills in solving 1H NMR problems.

Phenytoin (PHT), valproic acid, and modern antiepileptic drugs (AEDs), eg, remacemide, loreclezole, and safinamide, are only effective within a maximum of 70%-80% of epileptic patients, and in many cases the clinical use of AEDs is restricted by their side effects. Therefore, a continuous need remains to discover innovative chemical entities for the development of active and safer AEDs. Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. To determine the potential of H3Rs ligands as new AEDs, we recently reported that no anticonvulsant effects were observed for the (S)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propanamide (1). In continuation of our research, we asked whether anticonvulsant differences in activities will be observed for its R-enantiomer, namely, (R)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propaneamide (2) and analogs thereof, in maximum electroshock (MES)-, pentylenetetrazole (PTZ)-, and strychnine (STR)-induced convulsion models in rats having PHT and valproic acid (VPA) as reference AEDs. Unlike the S-enantiomer (1), the results show that animals pretreated intraperitoneally (ip) with the R-enantiomer 2 (10 mg/kg) were moderately protected in MES and STR induced models, whereas proconvulsant effect was observed for the same ligand in PTZ-induced convulsion models. However, animals pretreated with intraperitoneal doses of 5, 10, or 15 mg/kg of structurally bulkier (R)-enantiomer (3), in which 3-piperidinopropan-1-ol in ligand 2 was replaced by (4-(3-(piperidin-1-yl)propoxy)phenyl)methanol, and its (S)-enantiomer (4) significantly and in a dose-dependent manner reduced convulsions or exhibited full protection in MES and PTZ convulsions model, respectively. Interestingly, the protective effects observed for the (R)-enantiomer (3) in MES model were significantly greater than those of the standard H3R inverse agonist/antagonist pitolisant, comparable with those observed for PHT, and reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine. Comparisons of the observed antagonistic in vitro affinities among the ligands 1-6 revealed profound stereoselectivity at human H3Rs with varying preferences for this receptor subtype. Moreover, the in vivo anticonvulsant effects observed in this study for ligands 1-6 showed stereoselectivity in different convulsion models in male adult rats.