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Identification of Nanomolar Lectin Ligands by a Glycodendrimer Microarray.

ABSTRACT: Carbohydrate-protein interactions play key roles in a wide variety of biological processes. These interactions are usually weak, with dissociation constants in the low millimolar to high micromolar range. Nature uses multivalency to reach high avidities via the glycoside cluster effect. Capitalizing on this effect, numerous synthetic multivalent glycoconjugates have been described and used as ligands for carbohydrate-binding proteins. However, valency is only one of the several parameters governing the binding mechanisms that are different for every biological receptor, making it almost impossible to predict. In this context, ligand optimization requires the screening of a large number of structures with different valencies, rigidities/flexibilities, and architectures. In this article, we describe a screening platform based on a glycodendrimer array and its use to determine the key parameters for high-affinity ligands of lectin. Several glycoclusters and glycodendrimers displaying varying numbers of ?-N-acetylgalactosamine residues were covalently attached on glass slides, and their bindings were studied with the fluorophore-functionalized Helix pomatia agglutinin (HPA) used as a lectin model. This technique requires minimal quantities of glycoconjugate compared to those for other techniques and affords useful information on the binding strength. Building of the glycodendrimer array and quantification of the interactions with HPA are described.

SUBMITTER: Laigre E

PROVIDER: S-EPMC6210076 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Identification of Nanomolar Lectin Ligands by a Glycodendrimer Microarray.

Laigre Eugénie E Tiertant Claire C Goyard David D Renaudet Olivier O

ACS omega 20181025 10

Carbohydrate-protein interactions play key roles in a wide variety of biological processes. These interactions are usually weak, with dissociation constants in the low millimolar to high micromolar range. Nature uses multivalency to reach high avidities via the glycoside cluster effect. Capitalizing on this effect, numerous synthetic multivalent glycoconjugates have been described and used as ligands for carbohydrate-binding proteins. However, valency is only one of the several parameters govern ...[more]

PMID: 30411056

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Project description:Novel allosteric modulators of the dopamine transporter (DAT) have been identified. We have shown previously that SRI-9804 [N-(diphenylmethyl)-2-phenyl-4-quinazolinamine], SRI-20040 [N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine], and SRI-20041 [N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine] partially inhibit [(125)I]RTI-55 ([(125)I]3β-(4'-iodophenyl)tropan-2β-carboxylic acid methyl ester) binding and [(3)H]dopamine ([(3)H]DA) uptake, slow the dissociation rate of [(125)I]RTI-55 from the DAT, and allosterically modulate d-amphetamine-induced, DAT-mediated DA release. We synthesized and evaluated the activity of >500 analogs of these ligands and report here on 36 selected compounds. Using synaptosomes prepared from rat caudate, we conducted [(3)H]DA uptake inhibition assays, DAT binding assays with [(3)H]WIN35428 ([(3)H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane), and DAT-mediated release assays with either [(3)H]MPP(+) ([(3)H]1-methyl-4-phenylpyridinium) or [(3)H]DA. We observed three groups of [(3)H]DA uptake inhibitors: 1) full-efficacy agents with a one-site fit, 2) full-efficacy agents with a two-site fit, and 3) partial-efficacy agents with a one-site fit-the focus of further studies. These agents partially inhibited DA, serotonin, and norepinephrine uptake, yet were much less potent at inhibiting [(3)H]WIN35428 binding to the DAT. For example, SRI-29574 [N-(2,2-diphenylethyl)-2-(imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine] partially inhibited DAT uptake, with an IC50 = 2.3 ± 0.4 nM, without affecting binding to the DAT. These agents did not alter DAT-mediated release of [(3)H]MPP(+) in the absence or presence of 100 nM d-amphetamine. SRI-29574 had no significant effect on the d-amphetamine EC50 or Emax value for DAT-mediated release of [(3)H]MPP(+). These studies demonstrate the existence of potent DAT ligands that partially block [(3)H]DA uptake, without affecting DAT binding or d-amphetamine-induced [(3)H]MPP(+) release. These compounds may prove to be useful probes of biogenic amine transporter function as well as novel therapeutics.

Dataset Information

Identification of Nanomolar Lectin Ligands by a Glycodendrimer Microarray.

Publications

Identification of Nanomolar Lectin Ligands by a Glycodendrimer Microarray.

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