Publications by authors named "Sheraz Gul"

100 Publications

Gap junction-mediated cell-cell interaction between transplanted mesenchymal stem cells and vascular endothelium in stroke.

Stem Cells 2021 Mar 11. Epub 2021 Mar 11.

Department of Regenerative Medicine Research, Foundation for Biomedical Research and Innovation at Kobe, Hyogo, Japan.

We have shown previously that transplanted bone marrow mononuclear cells (BM-MNC), which are a cell fraction rich in hematopoietic stem cells, can activate cerebral endothelial cells via gap junction-mediated cell-cell interaction. In the present study, we investigated such cell-cell interaction between mesenchymal stem cells (MSC) and cerebral endothelial cells. In contrast to BM-MNC, for MSC we observed suppression of vascular endothelial growth factor uptake into endothelial cells and transfer of glucose from endothelial cells to MSC in vitro. The transfer of such a small molecule from MSC to vascular endothelium was subsequently confirmed in vivo and was followed by suppressed activation of macrophage/microglia in stroke mice. The suppressive effect was absent by blockade of gap junction at MSC. Furthermore, gap junction-mediated cell-cell interaction was observed between circulating white blood cells and MSC. Our findings indicate that gap junction-mediated cell-cell interaction is one of the major pathways for MSC-mediated suppression of inflammation in the brain following stroke and provides a novel strategy to maintain the blood-brain barrier in injured brain. Furthermore, our current results have the potential to provide a novel insight for other ongoing clinical trials that make use of MSC transplantation aiming to suppress excess inflammation, as well as other diseases such as COVID-19 (coronavirus disease 2019).
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http://dx.doi.org/10.1002/stem.3360DOI Listing
March 2021

The discovery of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines.

Eur J Med Chem 2021 Jan 5;209:112871. Epub 2020 Oct 5.

Leicester School of Allied Health Sciences, De Montfort University, The Gateway, Leicester, LE1 9BH, UK. Electronic address:

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense which seriously affects human health in Africa. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work herein describes the design and syntheses of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines, with compound 13, the 4-(2-methoxyphenyl)-6-(pyridine-3-yl)pyrimidin-2-amine demonstrating an IC value of 0.38 μM and a promising off-target ADME-Tox profile in vitro. In silico molecular target investigations showed rhodesain to be a putative candidate, supported by STD and WaterLOGSY NMR experiments, however, in vitro evaluation of compound 13 against rhodesain exhibited low experimental inhibition. Therefore, our reported library of drug-like pyrimidines present promising scaffolds for further antikinetoplastid drug development for both phenotypic and target-based drug discovery.
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http://dx.doi.org/10.1016/j.ejmech.2020.112871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762786PMC
January 2021

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

J Am Chem Soc 2020 08 5;142(33):14249-14266. Epub 2020 Aug 5.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm 106 91, Sweden.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.
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http://dx.doi.org/10.1021/jacs.0c05613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457426PMC
August 2020

Intravenous Bone Marrow Mononuclear Cells Transplantation in Aged Mice Increases Transcription of Glucose Transporter 1 and Na/K-ATPase at Hippocampus Followed by Restored Neurological Functions.

Front Aging Neurosci 2020 11;12:170. Epub 2020 Jun 11.

Department of Regenerative Medicine Research, Foundation for Biomedical Research and Innovation at Kobe, Hyogo, Japan.

We recently reported that intravenous bone marrow mononuclear cell (BM-MNC) transplantation in stroke improves neurological function through improvement of cerebral metabolism. Cerebral metabolism is known to diminish with aging, and the reduction of metabolism is one of the presumed causes of neurological decline in the elderly. We report herein that transcription of glucose transporters, monocarboxylate transporters, and Na/K-ATPase is downregulated in the hippocampus of aged mice with impaired neurological functions. Intravenous BM-MNC transplantation in aged mice stimulated the transcription of glucose transporter 1 and Na/K-ATPase α1 followed by restoration of neurological function. As glucose transporters and Na/K-ATPases are closely related to cerebral metabolism and neurological function, our data indicate that BM-MNC transplantation in aged mice has the potential to restore neurological function by activating transcription of glucose transporter and Na/K-ATPase. Furthermore, our data indicate that changes in transcription of glucose transporter and Na/K-ATPase could be surrogate biomarkers for age-related neurological impairment as well as quantifying the efficacy of therapies.
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http://dx.doi.org/10.3389/fnagi.2020.00170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301702PMC
June 2020

Untangling the sequence of events during the S → S transition in photosystem II and implications for the water oxidation mechanism.

Proc Natl Acad Sci U S A 2020 06 20;117(23):12624-12635. Epub 2020 May 20.

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S, S, S, and S, showing that a water molecule is inserted during the S → S transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S → S transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, Q and Q, are observed. At the donor site, tyrosine Y and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of O(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S → S transition mirrors the appearance of O electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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http://dx.doi.org/10.1073/pnas.2000529117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293653PMC
June 2020

In-vitro safety and off-target profile of the anti-parasitic arylmethylaminosteroid 1o.

Sci Rep 2020 05 5;10(1):7534. Epub 2020 May 5.

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and Pharmacology (TMP), Theodor-Stern-Kai 7, 60596, Frankfurt/Main, Germany.

Parasite-mediated diseases like malaria and schistosomiasis are growing health problems worldwide and novel drug candidates are urgently needed. In this study, the in-vitro safety profile of steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni with an IC value of 5 nM, was characterized. We assessed viability/proliferation, apoptosis and cell cycle tests to determine the cytotoxic profile of sc1o in cancer cells. The mutagenic potential was determined with the AMES test. To identify off-target effects we investigated whether sc1o interacts with safety-relevant molecules such as cytochrome P450 (CYP) enzymes, phosphodiesterases (PDE), histone deacteylases (HDAC) and human ether-a-go-go related gene (hERG). Furthermore, to predict the potential bioavailability of sc1o, its effect on Caco-2 cell barrier integrity, by measurement of the transepithelial electrical resistance (TEER), was determined. Sc1o at 25 µM reduced cell viability, probably through cell-cycle arrest, but did not induce apoptosis in cancer cells. No adverse off-target effects nor mutagenic potential of sc1o were observed. Furthermore, sc1o did not disturb the integrity of the cell barrier, but exhibited low membrane permeability, apparently due to cell adherence. In conclusion, sc1o up to 10 µM showed a good in-vitro safety profile.
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http://dx.doi.org/10.1038/s41598-020-64382-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200784PMC
May 2020

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases.

Inorg Chem 2020 May 20;59(9):6000-6009. Epub 2020 Apr 20.

Department of Chemistry, 1102 Natural Science II, University of California, Irvine, California 92697, United States.

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the SE mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic Fe complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from SE. Two aqua ligands complete the primary coordination sphere of the Fe center with both involved in hydrogen bond networks within Sav. The corresponding Fe protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The Fe protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.
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http://dx.doi.org/10.1021/acs.inorgchem.9b03791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7219546PMC
May 2020

Operando X-ray absorption spectroscopy of hyperfine β-FeOOH nanorods modified with amorphous Ni(OH) under electrocatalytic water oxidation conditions.

Chem Commun (Camb) 2020 May 7;56(38):5158-5161. Epub 2020 Apr 7.

Toyota Central R&D Labs., Inc., 41-1 Yokomichi, Nagakute, Aichi 480-1192, Japan.

Operando X-ray absorption spectroscopy was employed to study an active electrocatalyst, hyperfine β-FeOOH nanorods (∅ 3 × 15 nm) surface-modified with amorphous Ni hydroxide. The nearest neighbor structure and valence of Fe ions did not change under water oxidation conditions, while changes in the nearest neighbor ordering of Ni ions and a reversible transition to Ni were observed in accordance with the electrical bias for the reaction.
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http://dx.doi.org/10.1039/d0cc00692kDOI Listing
May 2020

Lead Optimization of Phthalazinone Phosphodiesterase Inhibitors as Novel Antitrypanosomal Compounds.

J Med Chem 2020 04 26;63(7):3485-3507. Epub 2020 Mar 26.

Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium.

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound resulted in being a potent TbrPDEB1 inhibitor with interesting activity against in a phenotypic screen. Derivative was studied in an acute mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and . Compound presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00985DOI Listing
April 2020

Bone Marrow Mononuclear Cells Activate Angiogenesis via Gap Junction-Mediated Cell-Cell Interaction.

Stroke 2020 04 19;51(4):1279-1289. Epub 2020 Feb 19.

From the Department of Regenerative Medicine Research, Institute of Biomedical Research and Innovation, Kobe, Japan (A.K.-T., Y. Okinaka, Y.T., Y. Ogawa, M.M., J.B., A.T.).

Background and Purpose- Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells and have been widely used in experimental therapies for patients with ischemic diseases. Activation of angiogenesis is believed to be one of major BM-MNC mode of actions, but the essential mechanism by which BM-MNCs activate angiogenesis have hitherto been elusive. The objective of this study is to reveal the mechanism how BM-MNCs activate angiogenesis. Methods- We have evaluated the effect of direct cell-cell interaction between BM-MNC and endothelial cell on uptake of VEGF (vascular endothelial growth factor) into endothelial cells in vitro. Cerebral ischemia model was used to evaluate the effects of direct cell-cell interaction with transplanted BM-MNC on endothelial cell at ischemic tissue. Results- The uptake of VEGF into endothelial cells was increased by BM-MNC, while being inhibited by blockading the gap junction. Low-molecular-weight substance was transferred from BM-MNC into endothelial cells via gap junctions in vivo, followed by increased expression of hypoxia-inducible factor-1α and suppression of autophagy in endothelial cells. The concentration of glucose in BM-MNC cytoplasm was significantly higher than in endothelial cells, and transfer of glucose homologue from BM-MNC to endothelial cells was observed. Conclusions- Our findings demonstrated cell-cell interaction via gap junction is the prominent pathway for activation of angiogenesis at endothelial cells after ischemia and provided novel paradigm that energy source supply by stem cell to injured cell is one of the therapeutic mechanisms of cell-based therapy. Visual Overview- An online visual overview is available for this article.
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http://dx.doi.org/10.1161/STROKEAHA.119.028072DOI Listing
April 2020

Collecting data through high throughput early toxicity and off-target liability assays to rapidly identify limitations of novel thyromimetics.

Data Brief 2020 Apr 31;29:105206. Epub 2020 Jan 31.

Department of Pharmacy, University of Pisa, Pisa, 56126, Italy.

In order to rapidly identify the phenotypic profile and possible off-target liability effects of novel synthesized thyromimetics for selection of lead compounds for further optimization studies, we performed screening on a new small library of synthetic thyromimetics. A comprehensive panel of early toxicity assays comprising cytotoxicity on 4 different cell lines (osteosarcoma, U2OS; lung fibroblast, hTERT; human breast adenocarcinoma, MCF7; human embryonic kidney, HEK293), ERG liability, cytochrome P450 inhibition (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 isoforms), and off-target liability against selected proteins (Aurora B kinase and phosphodiesterase PDE4C1) and epigenetic enzymes (HDAC4, HDAC6, HDAC8, HDAC9 & SIRT7). All the compounds were screened at 10 μM in at least triplicate using well-established assays with readouts in luminescence or fluorescence polarization mode. The raw data were processed using Microsoft Excel and the Z' for each assay was calculated (acceptable Z' >0.40). The processed and normalized data were organized in tables and visualized using spider plots. The results which are reported in the present manuscript can be used in prediction studies of early toxicity and off-target liabilities of other thyromimetics using methods. The data reported herein support our research article entitled "Design, synthesis and biological evaluation of novel TRβ selective agonists sustained by ADME-Toxicity analysis" by Runfola M., Sestito S., et al. [1].
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http://dx.doi.org/10.1016/j.dib.2020.105206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015999PMC
April 2020

Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA compound library screening campaign.

Eur J Med Chem 2020 Mar 10;189:112047. Epub 2020 Jan 10.

Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy. Electronic address:

The LIBRA compound library is a collection of 522 non-commercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives.
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http://dx.doi.org/10.1016/j.ejmech.2020.112047DOI Listing
March 2020

Design, synthesis and biological evaluation of novel TRβ selective agonists sustained by ADME-toxicity analysis.

Eur J Med Chem 2020 Feb 23;188:112006. Epub 2019 Dec 23.

Department of Pharmacy, University of Pisa, Pisa, 56126, Italy; Interdepartmental Research Centre for Biology and Pathology of Aging, University of Pisa, Pisa, Italy. Electronic address:

Although triiodothyronine (T3) induces several beneficial effects on lipid metabolism, its use is hampered by toxic side-effects, such as tachycardia, arrhythmia, heart failure, bone and muscle catabolism and mood disturbances. Since the α isoform of thyroid hormone receptors (TRs) is the main cause of T3-related harmful effects, several efforts have been made to develop selective agonists of the β isoform that could induce some beneficial effects (i.e. lowering triglyceride and cholesterol levels reducing obesity and improving metabolic syndrome), while overcoming most of the adverse T3-dependent side effects. Herein, we describe the drug discovery process sustained by ADME-Toxicity analysis that led us to identify novel agonists with selectivity for the isoform TRβ and an acceptable off-target and absorption, distribution metabolism, excretion and toxicity (ADME-Tox) profile. Within the small series of compounds synthesized, derivatives 1 and 3, emerge from this analysis as "potentially safe" to be engaged in preclinical studies. In in vitro investigation proved that both compounds were able to reduce lipid accumulation in HepG2 and promote lipolysis with comparable effects to those elicited by T3, used as reference drug. Moreover, a preliminary in vivo study confirmed the apparent lack of toxicity, thus suggesting compounds 1 and 3 as new potential TRβ-selective thyromimetics.
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http://dx.doi.org/10.1016/j.ejmech.2019.112006DOI Listing
February 2020

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

Proc Natl Acad Sci U S A 2020 01 18;117(1):300-307. Epub 2019 Dec 18.

Department of Biology, Washington University in St. Louis, St. Louis, MO 63130;

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on F - F difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.
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http://dx.doi.org/10.1073/pnas.1912041116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955287PMC
January 2020

Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics.

Eur J Med Chem 2019 Dec 5;183:111676. Epub 2019 Sep 5.

University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy. Electronic address:

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC 1.9 μM for Leishmania infantum, 3.4 μM for L. donovani, 6.7 μM for L. major), Trypanosoma cruzi (EC 7.5 μM) and T. brucei (EC 0.8 μM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.
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http://dx.doi.org/10.1016/j.ejmech.2019.111676DOI Listing
December 2019

Clot-Derived Contaminants in Transplanted Bone Marrow Mononuclear Cells Impair the Therapeutic Effect in Stroke.

Stroke 2019 10 12;50(10):2883-2891. Epub 2019 Sep 12.

From the Department of Regenerative Medicine Research, Institute of Biomedical Research and Innovation, Kobe, Japan (Y.O., A.K.-T., Y.T., Y.O., J.B., A.T.).

Background and Purpose- The beneficial effects of bone marrow mononuclear cell (BM-MNC) transplantation in preclinical experimental stroke have been reliably demonstrated. However, only overall modest effects in clinical trials were observed. We have investigated and reported a cause of the discrepancy between the preclinical and clinical studies. Methods- To investigate the possible cause of low efficacy of BM-MNC transplantation in experimental stroke, we have focused on blood clot formation, which is not uncommon in human bone marrow aspirates. To evaluate the effects of clot-derived contaminants in transplanted BM-MNC on stroke outcome, a murine stroke model was used. Results- We show that BM-MNC separated by an automatic cell isolator (Sepax2), which does not have the ability to remove clots, did not attenuate brain atrophy after stroke. In contrast, manually isolated, clot-free BM-MNC exerted therapeutic effects. Clot-derived contaminants were also transplanted intravenously to poststroke mice. We found that the transplanted contaminants were trapped at the peristroke area, which were associated with microglial/macrophage activation. Conclusions- Clot-derived contaminants in transplanted BM-MNC nullify therapeutic effects in experimental stroke. This may explain neutral results in clinical trials, especially in those using automated stem cell separators that lack the ability to remove clot-derived contaminants. Visual Overview- An online visual overview is available for this article.
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http://dx.doi.org/10.1161/STROKEAHA.119.026669DOI Listing
October 2019

XANES and EXAFS of dilute solutions of transition metals at XFELs.

J Synchrotron Radiat 2019 Sep 7;26(Pt 5):1716-1724. Epub 2019 Aug 7.

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94704, USA.

This work has demonstrated that X-ray absorption spectroscopy (XAS), both Mn XANES and EXAFS, of solutions with millimolar concentrations of metal is possible using the femtosecond X-ray pulses from XFELs. Mn XAS data were collected using two different sample delivery methods, a Rayleigh jet and a drop-on-demand setup, with varying concentrations of Mn. Here, a new method for normalization of XAS spectra based on solvent scattering that is compatible with data collection from a highly variable pulsed source is described. The measured XANES and EXAFS spectra of such dilute solution samples are in good agreement with data collected at synchrotron sources using traditional scanning protocols. The procedures described here will enable XFEL-based XAS on dilute biological samples, especially metalloproteins, with low sample consumption. Details of the experimental setup and data analysis methods used in this XANES and EXAFS study are presented. This method will also benefit XAS performed at high-repetition-rate XFELs such as the European XFEL, LCLS-II and LCLS-II-HE.
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http://dx.doi.org/10.1107/S1600577519007550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730626PMC
September 2019

Iron detection and remediation with a functionalized porous polymer applied to environmental water samples.

Chem Sci 2019 Jul 5;10(27):6651-6660. Epub 2019 Jun 5.

Department of Chemistry , University of California , Berkeley , CA 94720 , USA . Email:

Iron is one of the most abundant elements in the environment and in the human body. As an essential nutrient, iron homeostasis is tightly regulated, and iron dysregulation is implicated in numerous pathologies, including neuro-degenerative diseases, atherosclerosis, and diabetes. Endogenous iron pool concentrations are directly linked to iron ion uptake from environmental sources such as drinking water, providing motivation for developing new technologies for assessing iron(ii) and iron(iii) levels in water. However, conventional methods for measuring aqueous iron pools remain laborious and costly and often require sophisticated equipment and/or additional processing steps to remove the iron ions from the original environmental source. We now report a simplified and accurate chemical platform for capturing and quantifying the iron present in aqueous samples through use of a post-synthetically modified porous aromatic framework (PAF). The ether/thioether-functionalized network polymer, PAF-1-ET, exhibits high selectivity for the uptake of iron(ii) and iron(iii) over other physiologically and environmentally relevant metal ions. Mössbauer spectroscopy, XANES, and EXAFS measurements provide evidence to support iron(iii) coordination to oxygen-based ligands within the material. The polymer is further successfully employed to adsorb and remove iron ions from groundwater, including field sources in West Bengal, India. Combined with an 8-hydroxyquinoline colorimetric indicator, PAF-1-ET enables the simple and direct determination of the iron(ii) and iron(iii) ion concentrations in these samples, providing a starting point for the design and use of molecularly-functionalized porous materials for potential dual detection and remediation applications.
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http://dx.doi.org/10.1039/c9sc01441aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624977PMC
July 2019

Activation of Caspase-6 Is Promoted by a Mutant Huntingtin Fragment and Blocked by an Allosteric Inhibitor Compound.

Cell Chem Biol 2019 09 25;26(9):1295-1305.e6. Epub 2019 Jul 25.

Centre for Molecular Medicine and Therapeutics (CMMT), Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada.

Aberrant activation of caspase-6 (C6) in the absence of other hallmarks of apoptosis has been demonstrated in cells and tissues from patients with Huntington disease (HD) and animal models. C6 activity correlates with disease progression in patients with HD and the cleavage of mutant huntingtin (mHTT) protein is thought to strongly contribute to disease pathogenesis. Here we show that the mHTT fragment generated by C6 cleavage interacts with the zymogen form of the enzyme, stabilizing a conformation that contains an active site and is prone to full activation. This shift toward enhanced activity can be prevented by a small-molecule inhibitor that blocks the interaction between C6 and mHTT. Molecular docking studies suggest that the inhibitor binds an allosteric site in the C6 zymogen. The interaction of mHTT with C6 may therefore promote a self-reinforcing, feedforward cycle of C6 zymogen activation and mHTT cleavage driving HD pathogenesis.
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http://dx.doi.org/10.1016/j.chembiol.2019.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754302PMC
September 2019

Effect of oxygen deficiency on the excited state kinetics of WO and implications for photocatalysis.

Chem Sci 2019 Jun 9;10(22):5667-5677. Epub 2019 May 9.

Department of Chemistry , Imperial College London , London , SW7 2AZ , UK . Email: ; Email:

Oxygen vacancies are widely used to tune the light absorption of semiconducting metal oxides, but a photophysical framework describing the impact of such point defects on the dynamics of photogenerated charges, and ultimately on catalysis, is still missing. We herein use WO as a model material and investigate the impact of significantly different degrees of oxygen deficiency on its excited state kinetics. For highly oxygen-deficient films, photoelectron spectroscopy shows an over 2 eV broad distribution of oxygen vacancy states within the bandgap which gives rise to extended visible light absorption. We examine the nature of this distribution using first-principles defect calculations and find that defects aggregate to form clusters rather than isolated vacancy sites. Using transient absorption spectroscopy, we observe trapping of photogenerated holes within 200 fs after excitation at high degrees of oxygen deficiency, which increases their lifetime at the expense of oxidative driving force. This loss in driving force limits the use of metal oxides with significant degrees of sub-stoichiometry to photocatalytic reactions that require low oxidation power such as pollutant degradation, and highlights the need to fine-tune vacancy state distributions for specific target reactions.
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http://dx.doi.org/10.1039/c9sc00693aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563783PMC
June 2019

Structural Insights into the Development of Cycloguanil Derivatives as Pteridine-Reductase-1 Inhibitors.

ACS Infect Dis 2019 07 1;5(7):1105-1114. Epub 2019 May 1.

Department of Biotechnology, Chemistry and Pharmacy-Department of Excellence 2018-2020 , University of Siena , via Aldo Moro 2 , 53100 Siena , Italy.

Cycloguanil is a known dihydrofolate-reductase (DHFR) inhibitor, but there is no evidence of its activity on pteridine reductase (PTR), the main metabolic bypass to DHFR inhibition in trypanosomatid parasites. Here, we provide experimental evidence of cycloguanil as an inhibitor of PTR1 (PTR1). A small library of cycloguanil derivatives was developed, resulting in and having IC values of 692 and 186 nM, respectively, toward PTR1. Structural analysis revealed that the increased potency of and is due to the combined contributions of hydrophobic interactions, H-bonds, and halogen bonds. Moreover, cell-growth-inhibition tests indicated that is also effective on . The simultaneous inhibition of DHFR and PTR1 activity in is a promising new strategy for the treatment of human African trypanosomiasis. For this purpose, 1,6-dihydrotriazines represent new molecular tools to develop potent dual PTR and DHFR inhibitors.
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http://dx.doi.org/10.1021/acsinfecdis.8b00358DOI Listing
July 2019

SAR Studies and Biological Characterization of a Chromen-4-one Derivative as an Anti- Agent.

ACS Med Chem Lett 2019 Apr 29;10(4):528-533. Epub 2019 Jan 29.

University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy.

Chemical modulation of the flavonol 2-(benzo[d][1,3]dioxol-5-yl)-chromen-4-one (), a promising anti-Trypanosomatid agent previously identified, was evaluated through a phenotypic screening approach. Herein, we have performed structure-activity relationship studies around hit compound . The pivaloyl derivative () showed significant anti- activity (EC = 1.1 μM) together with a selectivity index higher than 92. The early ADME-tox properties (cytotoxicity, mitochondrial toxicity, cytochrome P450 and ERG inhibition) were determined for compound and its derivatives, and these led to the identification of some liabilities. The 1,3-benzodioxole moiety in the presented compounds confers better in vivo pharmacokinetic properties than those of classical flavonols. Further studies using different delivery systems could lead to an increase of compound blood levels.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466517PMC
April 2019

Enhancement of Benzothiazoles as Pteridine Reductase-1 Inhibitors for the Treatment of Trypanosomatidic Infections.

J Med Chem 2019 04 9;62(8):3989-4012. Epub 2019 Apr 9.

Dipartimento di Scienze della Vita , University of Modena and Reggio Emilia , Via Campi 103 , 41125 Modena , Italy.

2-Amino-benzo[ d]thiazole was identified as a new scaffold for the development of improved pteridine reductase-1 (PTR1) inhibitors and anti-trypanosomatidic agents. Molecular docking and crystallography guided the design and synthesis of 42 new benzothiazoles. The compounds were assessed for Trypanosoma brucei and Leishmania major PTR1 inhibition and in vitro activity against T. brucei and amastigote Leishmania infantum. We identified several 2-amino-benzo[ d]thiazoles with improved enzymatic activity ( TbPTR1 IC = 0.35 μM; LmPTR1 IC = 1.9 μM) and low μM antiparasitic activity against T. brucei. The ten most active compounds against TbPTR1 were able to potentiate the antiparasitic activity of methotrexate when evaluated in combination against T. brucei, with a potentiating index between 1.2 and 2.7. The compound library was profiled for early ADME toxicity, and 2-amino- N-benzylbenzo[ d]thiazole-6-carboxamide (4c) was finally identified as a novel potent, safe, and selective anti-trypanocydal agent (EC = 7.0 μM). Formulation of 4c with hydroxypropyl-β-cyclodextrin yielded good oral bioavailability, encouraging progression to in vivo studies.
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http://dx.doi.org/10.1021/acs.jmedchem.8b02021DOI Listing
April 2019

Structural isomers of the S state in photosystem II: do they exist at room temperature and are they important for function?

Physiol Plant 2019 May 15;166(1):60-72. Epub 2019 Mar 15.

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

In nature, an oxo-bridged Mn CaO cluster embedded in photosystem II (PSII), a membrane-bound multi-subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light-induced charge separations in the reaction center of PSII. The Mn CaO cluster accumulates four oxidizing equivalents to enable the four-electron four-proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S-states, S  - S in the Kok cycle. One important question related to the catalytic mechanism of the oxygen-evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S-states and if such equilibria are essential for the mechanism of the O-O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S state of PSII with structural data collected of the S , S and S states by serial crystallography at neutral pH (∼6.5) using an X-ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S state, the room temperature crystallography data can be well-described by just one S structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.
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http://dx.doi.org/10.1111/ppl.12947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478542PMC
May 2019

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform.

SLAS Discov 2019 03;24(3):346-361

3 Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Hamburg, Germany.

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.
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http://dx.doi.org/10.1177/2472555218823171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484532PMC
March 2019

Tetranuclear [MnMnO] Complexes as Spectroscopic Models of the S State of the Oxygen Evolving Complex in Photosystem II.

J Am Chem Soc 2018 12 30;140(49):17175-17187. Epub 2018 Nov 30.

Department of Chemistry and Chemical Engineering , California Institute of Technology , 1200 E California Blvd MC 127-72 , Pasadena , California 91125 , United States.

Despite extensive biochemical, spectroscopic, and computational studies, the mechanism of biological water oxidation by the oxygen evolving complex (OEC) of Photosystem II remains a subject of significant debate. Mechanistic proposals are guided by the characterization of reaction intermediates such as the S state, which features two characteristic EPR signals at g = 2 and g = 4.1. Two nearly isoenergetic structural isomers have been proposed as the source of these distinct signals, but relevant structure-electronic structure studies remain rare. Herein, we report the synthesis, crystal structure, electrochemistry, XAS, magnetic susceptibility, variable temperature CW-EPR, and pulse EPR data for a series of [MnMnO] cuboidal complexes as spectroscopic models of the S state of the OEC. Resembling the oxidation state and EPR spectra of the S state of the OEC, these model complexes show two EPR signals, a broad low field signal and a multiline signal, that are remarkably similar to the biological system. The effect of systematic changes in the nature of the bridging ligands on spectroscopy were studied. Results show that the electronic structure of tetranuclear Mn complexes is highly sensitive to even small geometric changes and the nature of the bridging ligands. Our model studies suggest that the spectroscopic properties of the OEC may also react very sensitively to small changes in structure; the effect of protonation state and other reorganization processes need to be carefully assessed.
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http://dx.doi.org/10.1021/jacs.8b09961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6574184PMC
December 2018

Structures of the intermediates of Kok's photosynthetic water oxidation clock.

Nature 2018 11 7;563(7731):421-425. Epub 2018 Nov 7.

Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle. The model comprises four (meta)stable intermediates (S, S, S and S) and one transient S state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (MnCaO) cluster in the oxygen-evolving complex. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S→S state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O release. Thus, our results exclude peroxo-bond formation in the S state, and the nucleophilic attack of W3 onto W2 is unlikely.
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http://dx.doi.org/10.1038/s41586-018-0681-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485242PMC
November 2018

Probing the oxidation state of transition metal complexes: a case study on how charge and spin densities determine Mn L-edge X-ray absorption energies.

Chem Sci 2018 Sep 17;9(33):6813-6829. Epub 2018 Jul 17.

Institute for Methods and Instrumentation for Synchrotron Radiation Research , Helmholtz-Zentrum Berlin für Materialien und Energie GmbH , Albert-Einstein-Strasse 15 , 12489 Berlin , Germany . Email:

Transition metals in inorganic systems and metalloproteins can occur in different oxidation states, which makes them ideal redox-active catalysts. To gain a mechanistic understanding of the catalytic reactions, knowledge of the oxidation state of the active metals, ideally , is therefore critical. L-edge X-ray absorption spectroscopy (XAS) is a powerful technique that is frequently used to infer the oxidation state a distinct blue shift of L-edge absorption energies with increasing oxidation state. A unified description accounting for quantum-chemical notions whereupon oxidation does not occur locally on the metal but on the whole molecule and the basic understanding that L-edge XAS probes the electronic structure locally at the metal has been missing to date. Here we quantify how charge and spin densities change at the metal and throughout the molecule for both redox and core-excitation processes. We explain the origin of the L-edge XAS shift between the high-spin complexes Mn(acac) and Mn(acac) as representative model systems and use theory to uncouple effects of oxidation-state changes from geometric effects. The shift reflects an increased electron affinity of Mn in the core-excited states compared to the ground state due to a contraction of the Mn 3d shell upon core-excitation with accompanied changes in the classical Coulomb interactions. This new picture quantifies how the metal-centered core hole probes changes in formal oxidation state and encloses and substantiates earlier explanations. The approach is broadly applicable to mechanistic studies of redox-catalytic reactions in molecular systems where charge and spin localization/delocalization determine reaction pathways.
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http://dx.doi.org/10.1039/c8sc00550hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115617PMC
September 2018

In vitro and in silico analysis of the effects of D receptor antagonist target binding kinetics on the cellular response to fluctuating dopamine concentrations.

Br J Pharmacol 2018 11 21;175(21):4121-4136. Epub 2018 Sep 21.

Department of Pharmacology, Leiden Academic Centre for Drug Research, Leiden, Netherlands.

Background And Purpose: Target binding kinetics influence the time course of the drug effect (pharmacodynamics) both (i) directly, by affecting the time course of target occupancy, driven by the pharmacokinetics of the drug, competition with endogenous ligands and target turnover, and (ii) indirectly, by affecting signal transduction and homeostatic feedback. For dopamine D receptor antagonists, it has been hypothesized that fast receptor binding kinetics cause fewer side effects, because part of the dynamics of the dopaminergic system is preserved by displacement of these antagonists.

Experimental Approach: Target binding kinetics of D receptor antagonists and signal transduction after dopamine and D receptor antagonist exposure were measured in vitro. These data were integrated by mechanistic modelling, taking into account competitive binding of endogenous dopamine and the antagonist, the turnover of the second messenger cAMP and negative feedback by PDE turnover.

Key Results: The proposed signal transduction model successfully described the cellular cAMP response for 17 D receptor antagonists with widely different binding kinetics. Simulation of the response to fluctuating dopamine concentrations revealed that a significant effect of the target binding kinetics on the dynamics of the signalling only occurs at endogenous dopamine concentration fluctuations with frequencies below 1 min .

Conclusions And Implications: Signal transduction and feedback are important determinants of the time course of drug effects. The effect of the D receptor antagonist dissociation rate constant (k ) is limited to the maximal rate of fluctuations in dopamine signalling as determined by the dopamine k and the cAMP turnover.
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http://dx.doi.org/10.1111/bph.14456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177617PMC
November 2018

Development and implementation of a cell-based assay to discover agonists of the nuclear receptor REV-ERBα.

J Biol Methods 2018 25;5(3):e94. Epub 2018 Jun 25.

Fraunhofer Institute for Molecular Biology and Applied Ecology, ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany.

The nuclear receptors are transcription factors involved in the regulation of a variety of physiological processes whose activity can be modulated by binding to relevant small molecule ligands. Their dysfunction has been shown to play a role in disease states such as diabetes, cancer, inflammatory diseases, and hormonal resistance ailments, which makes them interesting targets for drug discovery. The nuclear receptor REV-ERBα is involved in regulating the circadian rhythm and metabolism. Its natural ligand is heme and there is significant interest in identifying novel synthetic modulators to serve as tools to characterize its function and to serve as drugs in treating metabolic disorders. To do so, we established a mammalian cell-based two-hybrid assay system capable of measuring the interaction between REV-ERBα and its co-repressor, nuclear co-repressor 1. This assay was validated to industry standard criteria and was used to screen a subset of the LOPAC library and 29568 compounds from a diverse compound library. Profiling of the primary hits in a panel of counter and selectivity assays confirmed that REV-ERBα activity can be modulated pharmacologically and chemical scaffolds have been identified for optimization.
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http://dx.doi.org/10.14440/jbm.2018.244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706147PMC
June 2018