Papers by Ngoc Tram Anh Chau
Journal of medicinal chemistry, Jan 20, 2016
The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). Th... more The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin localization to the plasma membrane via the PH domain and implicate this mechanism in the inhibition of CME. We have used a computational approach of binding site identification, docking, and interaction energy calculations to design and synthesize a new library of aminopyrimidine analogues targeting site-2 of the pleckstrin homology (PH) domain. The optimized analogues showed low micromolar inhibition against both dynamin I (IC50 = 10.6 ± 1.3 to 1.6 ± 0.3 μM) and CME (IC50(CME) = 65.9 ± 7.7 to 3.7 ± 1.1 mM), which makes this series among the more potent inhibitors of dynamin and CME yet reported. In CME and growth inhibition cell-based assays, the data obtained was consistent with dynamin inhibition. CEREP ExpresS profiling identified off-target effects at the cholecystokinin, dopamine D2, histamine H1 and H2, melanocortin, me...
Org. Biomol. Chem., 2016
The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-... more The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop® 2 family of clathrin inhibitors is described herein.
1 Pendidikan Perencanaan Demographic Aspects of Educational Planning Ta Ngoc Chau, 1969
Organic & Biomolecular Chemistry, 2015
Fragment-basedin silicoscreening against dynamin I (dynI) GTPase activity identified the 1,8-naph... more Fragment-basedin silicoscreening against dynamin I (dynI) GTPase activity identified the 1,8-naphthalimide framework as a potential scaffold for the design of new inhibitors targeting the GTP binding pocket of dynI.
European journal of medicinal chemistry, Jan 6, 2014
Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI)... more Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4-benzoquinone 1 as a 273 ± 106 μM inhibitor. In silico lead optimization and focused library-led synthesis resulted in the development of four discrete benzoquinone/naphthoquinone based compound libraries comprising 54 compounds in total. Sixteen analogues were more potent than lead 1, with 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone (45) and 2,5-bis(4-carboxyanilino)-1,4-benzoquinone (49) the most active with IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 μM respectively. Molecular modelling suggested a number of hydrogen bonding and hydrophobic interactions were involved in stabilization of 49 within the dynI GTP binding site. Six of the most active inhibitors were evaluated for potential inhibition of clathrin-mediated endocytosis (CME). Quinone 45 was the most effective CME inhibitor with an IC50(CME) of 36 ± 16 μM.
Traffic (Copenhagen, Denmark), Jan 18, 2015
Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediate... more Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 μM range, the most potent being trifluoperazine (IC50 2.6 ± 0.7 μM). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8-10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed phenothiazine-derived APDs were lipid-competitive, while haloperidol was uncompetitive with lipid. Accordingly the drugs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin CME in cells at related potencies. Structure-activity relations...
Traffic, 2013
Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lip... more Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC 50 ∼ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC 50 = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposomestimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC 50 of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.
Journal of Neuroscience, 2009
Synaptic vesicles (SVs) are retrieved by more than one mode in central nerve terminals. During mi... more Synaptic vesicles (SVs) are retrieved by more than one mode in central nerve terminals. During mild stimulation, the dominant SV retrieval pathway is classical clathrin-mediated endocytosis (CME). During elevated neuronal activity, activity-dependent bulk endocytosis (ADBE) predominates, which requires activation of the calcium-dependent protein phosphatase calcineurin. We now report that calcineurin dephosphorylates dynamin I in nerve terminals only above the same activity threshold that triggers ADBE. ADBE was arrested when the two major phospho-sites on dynamin I were perturbed, suggesting that dynamin I dephosphorylation is a key step in its activation. Dynamin I dephosphorylation stimulates a specific dynamin I-syndapin I interaction. Inhibition of this interaction by competitive peptides or by site-directed mutagenesis exclusively inhibited ADBE but did not affect CME. The results reveal that the phospho-dependent dynamin-syndapin interaction recruits ADBE to massively increase SV endocytosis under conditions of elevated neuronal activity.
Molecular Pharmacology, 2007
Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phos... more Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiT-MAB inhibited dynamin GTPase activity, with a K i of 940 Ϯ 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophos-phorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells. Endocytosis in eukaryotic cells services the uptake of extracellular material and the recycling of membrane components. Multiple forms of endocytosis exist that regulate a variety of different cellular processes, such as regulation of cell surface receptor expression and signaling, cell fate determination, cell migration, antigen presentation, and synaptic transmission (Liu and Robinson, 1995; Kaksonen et al., 2006). Two of the best characterized pathways are receptormediated endocytosis (RME) and synaptic vesicle endocytosis (SVE). Both use many proteins and lipid-cofactors (Cousin and Robinson, 2001; Le Roy and Wrana, 2005). RME is activated by binding of hormones or growth factor ligands to specific cell surface receptors, for example, transferrin (Tf) and epidermal growth factor (EGF). RME usually occurs via clathrin-coated pits, and it requires the ubiquitously ex-This work was supported by grants from the National Health and Medical Research Council (Australia) and The Wellcome Trust (Ref. 062841). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
Journal of Natural Products, 2009
Journal of Medicinal Chemistry, 2010
Herein we report the synthesis of discrete iminochromene (&am... more Herein we report the synthesis of discrete iminochromene ("iminodyn") libraries (14-38) as potential inhibitors of dynamin GTPase. Thirteen iminodyns were active (IC(50) values of 260 nM to 100 microM), with N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (17), N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (22), and N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (23) (IC(50) values of 330 +/- 70, 450 +/- 50, and 260 +/- 80 nM, respectively) being the most potent. Five of the most potent iminodyns all inhibited dynamins I and II approximately equally. Iminodyn-22 displayed uncompetitive inhibition with respect to GTP. Selected iminodyns were evaluated for their ability to block receptor mediated endocytosis (RME, mediated by dynamin II) and synaptic vesicle endocytosis (SVE, mediated by dynamin I), with 17 showing no activity while 22 returned RME and SVE IC(50) values of 10.7 +/- 4.5 and 99.5 +/- 1.7 microM, respectively. The iminodyns reported herein represent a new chemical class of the first nanomolar potent dynamin inhibitors that are also effective endocytosis inhibitors.
Journal of Medicinal Chemistry, 2009
Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of ... more Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 +/- 0.3 microM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC(50) approximately 15 microM; RME IC(50) approximately 80 microM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.
Journal of Medicinal Chemistry, 2010
We report the development of a homology model for the GTP binding domain of human dynamin I based... more We report the development of a homology model for the GTP binding domain of human dynamin I based on the corresponding crystal structure of Dictyostelium discoidum dynamin A. Virtual screening identified 2-[(2-biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-4-chlorobenzoic acid (1) as a ∼170 μM potent inhibitor. Homology modeling-and focused library-led synthesis resulted in development of a series of active compounds (the "pthaladyns") with 4-chloro-2-(2-(4-(hydroxymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (29), a 4.58 (0.06 μM dynamin I GTPase inhibitor. Pthaladyn-29 displays borderline selectivity for dynamin I relative to dynamin II (∼5-10 fold). Only pthaladyn-23 (dynamin I IC 50 17.4 (5.8 μM) was an effective inhibitor of dynamin I mediated synaptic vesicle endocytosis in brain synaptosomes with an IC 50 of 12.9 (5.9 μM. This compound was also competitive with respect to Mg 2þ 3 GTP. Thus the pthaladyns are the first GTP competitive inhibitors of dynamin I and II GTPase and may be effective new tools for the study of neuronal endocytosis.
Journal of Medicinal Chemistry, 2013
Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-... more Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4(5H)-one isostere (19, IC 50(dyn I) = 7.7 μM), reduced under flow chemistry conditions (20, IC 50(dyn I) = 5.2 μM) or replaced by a simple amine. The latter provided a basis for a high yield library of compounds via a reductive amination by flow hydrogenation. Two compounds, 24 (IC 50 (dyn I) = 0.56 μM) and 25 (IC 50(dyn I) = 0.76 μM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I and II in vitro and in cells (IC 50(CME) = 1.9 μM). It also showed 4.4-fold selectivity for dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date.
Journal of Medicinal Chemistry, 2014
We reported the first small molecule inhibitors of the interaction between the clathrin N-termina... more We reported the first small molecule inhibitors of the interaction between the clathrin N-terminal domain (TD) and endocyctic accessory proteins (i.e., clathrin inhibition1). Initial screening of a ∼17 000 small molecule ChemBioNet library identified 1. Screening of an existing in-house propriety library identified four substituted 1,8-napthalimides as ∼80-120 μM clathrin inhibitors. Focused library development gave 3-sulfo-N-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (18, IC50 ≈ 18 μM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (26, 27, 28, 34 with IC50 values of 22, 16, 15, and 15 μM respectively) with a further four (24, 25, 32, 33) more active than 18 with IC50 values of 10, 6.9, 12, and 10 μM, respectively. Docking studies rationalized the structure-activity relationship (SAR) with the biological data. 3-Sulfo-N-benzyl-1,8-naphthalimide, potassium salt (25) with an IC50 ≈ 6.9 μM, is the most potent clathrin terminal domain-amphiphysin inhibitor reported to date.
Journal of Biological Chemistry, 2011
The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic d... more The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a TM , was sufficient to abolish BoNT/ A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/ A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms. Among neurotoxins acting presynaptically (1-3), botulinum neurotoxins (BoNTs) 4 comprise a group of highly lethal toxins consisting of seven serotypes (BoNT/A-G) produced by the anerobic bacteria, Clostridium botulinum. Due to the extreme * This work was supported by the Australian National Health and Medical Research Council. Dyngo-4a TM and Dyngo TM are trademarks of the authors' institutions and affiliates, the Children's Medical Research Institute and Newcastle Innovation, Ltd. (the commercial arm of the University of Newcastle), which have entered into a commercial license agreement with Ascent Scientific, Ltd. (Bristol, United Kingdom) to market and distribute Dyngo-4a as a research tool globally.
Contemporary Sociology, 1975
ChemMedChem, 2009
Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospho... more Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospholipid-stimulated GTPase activity of dynamin I. Analogues of Bis-T have significant potential as a biological probes for the dissection of endocytic pathways. Bis-T-derived compounds were synthesised and evaluated for their ability to inhibit the GTPase activity of dynamin I. Two analogues (23 and 24) represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibition.Two azidobenzyl amide (4 and 23) and one 3-trifluoromethyl-3H-diazirin-3-ylphenyl (24) analogues of bis-tyrphostin (1, Bis-T) were synthesised as potential photoaffinity labels for the elucidation of the binding site of compound 1 in dynamin I. Of the two azidobenzyl amide analogues (4 and 23), the terminally substituted 23 retained dynamin I GTPase inhibition (IC(50)=6.4+/-2.8 microM) whilst 4, which was substituted on the central carbon of the amide linker, displayed no activity. Analogue 24 also retained inhibitory activity (IC(50)=36+/-9 microM). Photoaffinity labelling experiments did not unequivocally elucidate the binding pocket of compound 1. However, compounds 23 and 24 represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibitory activity, providing a new direction for analogue synthesis.
Cell, 2011
Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the inter... more Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptormediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.
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Papers by Ngoc Tram Anh Chau