Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1

2011 ◽  
Vol 392 (5) ◽  
Author(s):  
Ingrid L. Cockburn ◽  
Eva-Rachele Pesce ◽  
Jude M. Pryzborski ◽  
Michael T. Davies-Coleman ◽  
Peter G.K. Clark ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Small Molecule ◽  
Drug Target ◽  
Molecular Probes ◽  
Chaperone Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Chaperone Function ◽  
Hsp70 Chaperone ◽  
Small Molecule Modulators

Abstract Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC50 values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

scholarly journals Functionally Selective Inhibition of the Oxytocin Receptor by Retosiban in Human Myometrial Smooth Muscle

Endocrinology ◽  
2020 ◽  
Vol 161 (2) ◽  
Author(s):  
Paul J Brighton ◽  
Michael J Fossler ◽  
Siobhan Quenby ◽  
Andrew M Blanks
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Small Molecule ◽  
Selective Inhibition ◽  
Oxytocin Receptor ◽  
Human Myometrium ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Prostaglandin Production ◽  
Downstream Signaling

Abstract Novel small molecule inhibitors of the oxytocin receptor (OTR) may have distinct pharmacology and mode of action when compared with first-generation oxytocin antagonists when used for the prevention of preterm birth. The aim was to determine the mechanism of action of small molecule OTR antagonists retosiban and epelsiban compared with the currently used peptide-based compound atosiban. Human myometrial samples were obtained at cesarean section and subjected to pharmacological manipulations to establish the effect of antagonist binding to OTR on downstream signaling. Retosiban antagonism of oxytocin action in human myometrium was potent, rapid, and reversible. Inhibition of inositol 1,4,5-trisphosphate (IP3) production followed single-site competitive binding kinetics for epelsiban, retosiban, and atosiban. Retosiban inhibited basal production of IP3 in the absence of oxytocin. Oxytocin and atosiban but not retosiban inhibited forskolin, and calcitonin stimulated 3′,5′-cyclic adenosine 5′-mono-phosphate (cAMP) production. Inhibition of cAMP was reversed by pertussis toxin. Oxytocin and atosiban, but not retosiban and epelsiban, stimulated extracellular regulated kinase (ERK)1/2 activity in a time- and concentration-dependent manner. Oxytocin and atosiban stimulated cyclo-oxygenase 2 activity and subsequent production of prostaglandin E2 and F2α. Prostaglandin production was inhibited by rofecoxib, pertussin toxin, and ERK inhibitor U0126. Oxytocin but not retosiban or atosiban stimulated coupling of the OTR to Gα q G-proteins. Oxytocin and atosiban but not retosiban stimulated coupling of the OTR to Gα i G-proteins. Retosiban and epelsiban demonstrate distinct pharmacology when compared with atosiban in human myometrial smooth muscle. Atosiban displays agonist activity at micromolar concentrations leading to stimulation of prostaglandin production.

scholarly journals A5, a New Small-Molecule Inhibitor of CD4 D1 Obtained from a Computer-Aided Screening Method, Contributes to the Inhibition of CD4+ T-cell Function

2007 ◽  
Vol 12 (6) ◽  
pp. 800-808 ◽  
Author(s):  
He Xiao ◽  
Jian-Nan Feng ◽  
Zu-Yin Yu ◽  
Lei Zhang ◽  
Ming Yu ◽  
...  
Keyword(s):  
T Cell ◽  
Small Molecule ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Screening Method ◽  
Interleukin 2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Molecule Inhibitor

In this study, the authors apply a computer-based strategy to screen thousands of small-molecule, nonpeptidic organic compounds in the Available Chemicals Directory database and to select a series of potential candidates as ligands of the proposed CD4 D1 surface pocket. Then, several cell-based models are used to determine the actual biological functions of these compounds. A small molecule designated A5 ( N-((pyridine-4-yl)methylene)thiophene-2-carbohydrazide) was obtained by a virtual screening followed by 3 cell-based functional assays. The results show that A5 could specifically block the CD4—major histocompatibility complex II binding in a rosetting assay, inhibit the mixed lymphocyte reaction—induced T-cell proliferation in a concentration-dependent manner, and reduce the PMA plus ionomycin—stimulated interleukin-2 secretion from peripheral blood mononuclear cells. ( Journal of Biomolecular Screening 2007:800-808)

scholarly journals GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

2020 ◽  
Author(s):  
Ulrike Leurs ◽  
Anders B. Klein ◽  
Ethan D. McSpadden ◽  
Nane Griem-Krey ◽  
Sara M. Ø. Solbak ◽  
...  
Keyword(s):  
Small Molecule ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Neuroprotective Effects ◽  
Concentration Dependent Manner ◽  
Chemical Proteomics ◽  
Differential Scanning Fluorimetry ◽  
Selective Ligands ◽  
Dependent Protein ◽  
Thermal Stability Of

ABSTRACTCa2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation1,2. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers3-6. Recent findings have suggested that the hub is also an allosteric determinant of kinase activity7, and is thus an emerging target for therapies to correct CaMKIIα dysregulation8,9. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKIIα hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite γ-hydroxybutyrate (GHB)10 we reveal that CaMKIIα is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 Å crystal structure of CaMKIIα with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKIIα hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKIIα-selective ligands have therapeutic potential.

scholarly journals Proteostasis collapse, a hallmark of aging, hinders the chaperone-Start network and arrests cells in G1

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
David F Moreno ◽  
Kirsten Jenkins ◽  
Sandrine Morlot ◽  
Gilles Charvin ◽  
Attila Csikasz-Nagy ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Cellular Senescence ◽  
Cell Senescence ◽  
Chaperone Activity ◽  
Yeast Cells ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Severe Reduction ◽  
G1 Cyclin ◽  
Nuclear Levels

Loss of proteostasis and cellular senescence are key hallmarks of aging, but direct cause-effect relationships are not well understood. We show that most yeast cells arrest in G1 before death with low nuclear levels of Cln3, a key G1 cyclin extremely sensitive to chaperone status. Chaperone availability is seriously compromised in aged cells, and the G1 arrest coincides with massive aggregation of a metastable chaperone-activity reporter. Moreover, G1-cyclin overexpression increases lifespan in a chaperone-dependent manner. As a key prediction of a model integrating autocatalytic protein aggregation and a minimal Start network, enforced protein aggregation causes a severe reduction in lifespan, an effect that is greatly alleviated by increased expression of specific chaperones or cyclin Cln3. Overall, our data show that proteostasis breakdown, by compromising chaperone activity and G1-cyclin function, causes an irreversible arrest in G1, configuring a molecular pathway postulating proteostasis decay as a key contributing effector of cell senescence.

scholarly journals Evidence for a Bacterial Lipopolysaccharide-Recognizing G-Protein-Coupled Receptor in the Bacterial Engulfment by Entamoeba histolytica

2013 ◽  
Vol 12 (11) ◽  
pp. 1433-1438 ◽  
Author(s):  
Matthew T. Brewer ◽  
Prince N. Agbedanu ◽  
Mostafa Zamanian ◽  
Tim A. Day ◽  
Steve A. Carlson
Keyword(s):  
G Protein ◽  
Entamoeba Histolytica ◽  
Drug Target ◽  
Bacterial Flora ◽  
Bacterial Lipopolysaccharide ◽  
Dependent Manner ◽  
G Protein Coupled Receptor ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
G Protein Coupled

ABSTRACT Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae . We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

Small-Molecule In Vitro Inhibitors of the Coronavirus Spike – ACE2 Protein-Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

2020 ◽  
Author(s):  
Damir Bojadzic ◽  
Oscar Alcazar ◽  
Jinshui Chen ◽  
Peter Buchwald
Keyword(s):  
Small Molecule ◽  
Protein Interaction ◽  
Organic Dyes ◽  
Chemical Space ◽  
Dependent Manner ◽  
Sunset Yellow ◽  
Concentration Dependent Manner ◽  
Viral Attachment ◽  
Protein Protein Interaction

ABSTRACTInhibitors of the protein-protein interaction (PPI) between the SARS-CoV-2 spike protein and ACE2, which acts as a ligand-receptor pair that initiates the viral attachment and cellular entry of this coronavirus causing the ongoing COVID-19 pandemic, are of considerable interest as potential antiviral agents. While blockade of such PPIs with small molecules is more challenging than with antibodies, small-molecule inhibitors (SMIs) might offer alternatives that are less strain- and mutation-sensitive, suitable for oral or inhaled administration, and more controllable / less immunogenic. Here, we report the identification of SMIs of this PPI by screening our compound-library that is focused on the chemical space of organic dyes. Among promising candidates identified, several dyes (Congo red, direct violet 1, Evans blue) and novel drug-like compounds (DRI-C23041, DRI-C91005) inhibited the interaction of hACE2 with the spike proteins of SARS-CoV-2 as well as SARS-CoV with low micromolar activity in our cell-free ELISA-type assays (IC50s of 0.2-3.0 μM); whereas, control compounds, such as sunset yellow FCF, chloroquine, and suramin, showed no activity. Protein thermal shift assays indicated that the SMIs identified here bind SARS-CoV-2-S and not ACE2. Selected promising compounds inhibited the entry of a SARS-CoV-2-S expressing pseudovirus into ACE2-expressing cells in concentration-dependent manner with low micromolar IC50s (6-30 μM). This provides proof-of-principle evidence for the feasibility of small-molecule inhibition of PPIs critical for coronavirus attachment/entry and serves as a first guide in the search for SMI-based alternative antiviral therapies for the prevention and treatment of diseases caused by coronaviruses in general and COVID-19 in particular.

scholarly journals Screening for Small Molecule Modulators of Trypanosoma brucei Hsp70 Chaperone Activity Based upon Alcyonarian Coral-Derived Natural Products

Marine Drugs ◽  
2020 ◽  
Vol 18 (2) ◽  
pp. 81
Author(s):  
Sarah K. Andreassend ◽  
Stephen J. Bentley ◽  
Gregory L. Blatch ◽  
Aileen Boshoff ◽  
Robert A. Keyzers
Keyword(s):  
Small Molecules ◽  
Trypanosoma Brucei ◽  
Essential Role ◽  
Protozoan Parasite ◽  
Chaperone Activity ◽  
Mammalian Cell Line ◽  
Structure Activity ◽  
Hsp70 Chaperone ◽  
Small Molecule Modulators ◽  
J Protein

The Trypanosoma brucei Hsp70/J-protein machinery plays an essential role in survival, differentiation, and pathogenesis of the protozoan parasite, and is an emerging target against African Trypanosomiasis. This study evaluated a set of small molecules, inspired by the malonganenones and nuttingins, as modulators of the chaperone activity of the cytosolic heat inducible T. brucei Hsp70 and constitutive TbHsp70.4 proteins. The compounds were assessed for cytotoxicity on both the bloodstream form of T. b. brucei parasites and a mammalian cell line. The compounds were then investigated for their modulatory effect on the aggregation suppression and ATPase activities of the TbHsp70 proteins. A structure–activity relationship for the malonganenone-class of alkaloids is proposed based upon these results.

scholarly journals A selective small molecule glucagon-like peptide-1 secretagogue acting via depolarization-coupled Ca2+ influx

2009 ◽  
Vol 201 (3) ◽  
pp. 361-367 ◽  
Author(s):  
Jun-ichi Eiki ◽  
Kaori Saeki ◽  
Norihiro Nagano ◽  
Tomoharu Iino ◽  
Mari Yonemoto ◽  
...  
Keyword(s):  
Small Molecule ◽  
Underlying Mechanism ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Compound A ◽  
Fetal Rat ◽  
Small Molecule Compound ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca2+ release from intracellular stores, but to extracellular Ca2+ influx. The response was inhibited by the presence of either L-type Ca2+ channel blockers or K+ ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Gαs- nor Gαq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca2+ influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.

scholarly journals Trehalose Restrains the Fibril Load towards α-Lactalbumin Aggregation and Halts Fibrillation in a Concentration-Dependent Manner

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 414
Author(s):  
Sania Bashir ◽  
Ishfaq Ahmad Ahanger ◽  
Anas Shamsi ◽  
Mohamed F. Alajmi ◽  
Afzal Hussain ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Rayleigh Scattering ◽  
Binding Pocket ◽  
Dependent Manner ◽  
Thermal Aggregation ◽  
Concentration Dependent Manner ◽  
In Silico Studies ◽  
Vis Spectroscopy ◽  
Amyloid Fibrillation

Protein aggregation and misfolding are some of the most challenging obstacles, customarily studied for their association with amyloid pathologies. The mechanism of amyloid fibrillation development is a dynamic phenomenon involving various factors such as the intrinsic properties of protein and the physical and chemical environmental conditions. The purpose of this study was to see the thermal aggregation profile of alpha-lactalbumin (α-LA) and to delineate the effect of trehalose on its aggregation profile. α-LA was subjected to thermal aggregation at high concentrations. UV-Vis spectroscopy, a turbidity assay, intrinsic fluorescence, Rayleigh scattering and a thioflavin T (ThT) assay explained the steady outcomes that 1 M trehalose repressed α-LA aggregation in the most effective way followed by 0.75 M and 0.5 M and to a significantly lesser degree by 0.25 M. Multi spectroscopic obser Sania Bashir ations were further entrenched by microscopy. Transmission electron microscopy confirmed that in the presence of its higher concentration, trehalose hinders fibril development in α-LA. In vitro studies were further validated by in silico studies. Molecular docking analysis indicated that trehalose occupied the binding pocket cavity of α-LA and offered several significant interactions, including H-bonds with important residues. This study provides a platform for trehalose in the therapeutic management of protein aggregation-related diseases.

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