Dataset Information

Target deconvolution in Plasmodium falciparum using pulldowns

ABSTRACT: Parasite lysates were centrifuged (20,000 g, 4°C, 15 min). Beads (blank and with linker attached) were washed 3× with water then twice with lysis buffer. The lysate (~2 mg total protein) was first incubated with 2 mg blank beads for 30 min at 4°C with rotating agitation. The lysate was divided into two then incubated with either 1% DMSO or 100 µM DDD01510706 (competitor) for 30 min at 4°C with agitation. Finally, lysates were incubated with 2 mg of compound-bound beads for 1 h at 4°C with agitation. The beads were then washed 3× with wash buffer (0.8% (w/v) octyl β-D-glucopyranoside, 50 mM Tris pH 8.0, 5 mM EDTA, 1 mg/mL BSA) and 2× Tris-buffered saline (TBS; 50 mM Tris-Cl pH 7.5, 150 mM NaCl). Samples were run 1.5 cm into a Bis-Tris 10% (w/v) acrylamide gel and stained with Coomassie quick reagent for 30 min. The entire gel bands were removed and subjected to in-gel reduction with 10 mM dithiothreitol, alkylation with 50 mM iodoacetamide and digestion with 12.5 μg/mL trypsin (Pierce) for >16 h at 37°C. Recovered tryptic peptides were then vacuum dried prior to analysis

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Plasmodium Falciparum (isolate 3d7)

SUBMITTER: Victoriano Corpas-Lopez

LAB HEAD: Susan Wyllie

PROVIDER: PXD033740 | Pride | 2022-10-15

REPOSITORIES: Pride

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Publications

Toolkit of Approaches To Support Target-Focused Drug Discovery for <i>Plasmodium falciparum</i> Lysyl tRNA Synthetase.

Milne Rachel R Wiedemar Natalie N Corpas-Lopez Victoriano V Moynihan Eoin E Wall Richard J RJ Dawson Alice A Robinson David A DA Shepherd Sharon M SM Smith Robert J RJ Hallyburton Irene I Post John M JM Dowers Karen K Torrie Leah S LS Gilbert Ian H IH Baragaña Beatriz B Patterson Stephen S Wyllie Susan S

ACS infectious diseases 20220829 9

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (<i>Pf</i>KRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target en ...[more]

PMID: 36037410

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