Post-translational import of protein into the endoplasmic reticulum of a trypanosome: an in vitro system for discovery of anti-trypanosomal chemical entities

Abstract

HAT (human African trypanosomiasis), caused by the protozoan parasite Trypanosoma brucei, is an emerging disease for which new drugs are needed. Expression of plasma membrane proteins [e.g. VSG (variant surface glycoprotein)] is crucial for the establishment and maintenance of an infection by T. brucei. Transport of a majority of proteins to the plasma membrane involves their translocation into the ER (endoplasmic reticulum). Thus inhibition of protein import into the ER of T. brucei would be a logical target for discovery of lead compounds against trypanosomes. We have developed a TbRM (T. brucei microsome) system that imports VSG_117 post-translationally. Using this system, MAL3-101, equisetin and CJ-21,058 were discovered to be small molecule inhibitors of VSG_117 translocation into the ER. These agents also killed bloodstream T. brucei in vitro; the concentrations at which 50% of parasites were killed (IC50) were 1.5 microM (MAL3-101), 3.3 microM (equisetin) and 7 microM (CJ-21,058). Thus VSG_117 import into TbRMs is a rapid and novel assay to identify 'new chemical entities' (e.g. MAL3-101, equisetin and CJ-21,058) for anti-trypanosome drug development.

Figure 1. Cell-free protein import into T. brucei ER membranes

(A) Protocol for post-translational import of VSG. Depicted are the various steps, duration and temperatures at which the reactions took place. CHX, cycloheximide; PK, proteinase K; RRL, rabbit reticulocyte lysate. (B) Import of VSG_117 into TbRMs. VSG_117 mRNA was translated in rabbit reticulocyte lysate for 15 min and then cycloheximide (50 µg/ml final concentration) was added. Cytosol from T. brucei (Tb) was also added to the reactions analysed in lanes 4, 5 and 6. Reaction mixtures were incubated with TbRMs (one equivalent) for 45 min at 37°C, followed by proteinase K digestion (300 µg/ml final concentration) on ice for 60 min. Proteins were resolved by SDS/PAGE and detected by phosphorimaging. Lane 1, VSG_117 translated in reticulocyte lysate; lane 2, VSG_117 digested with proteinase K; lane 3, VSG_117 digested with proteinase K in the presence of NP-40 (NP40) (2 %); lane 4, VSG_117 incubated with TbRMs; lane 5, VSG_117 incubated with TbRMs and digested with proteinase K (PK); lane 6, VSG_117 was translocated into TbRMs and then permeabilized with 2% NP-40 during proteinase K digestion. (C) Translocation of VSG_MVAT7 into TbRMs. Lane 1, VSG_MVAT7 translated in reticulocyte lysate; lane 2, VSG_MVAT7 produced in reticulocyte lysate and digested with proteinase K; lane 3, VSG_MVAT7 incubated with TbRMs; lane 4, VSG_MVAT7 incubated with TbRMs and then challenged with proteinase K; lane 5, VSG_MVAT7 incubated with TbRMs, permeabilized with NP-40 (2%) and digested with proteinase K. (D) A signal peptide is required for VSG import into TbRMs: VSG_117₅₀₀ ΔSP translocation. VSG_117₅₀₀ ΔSP mRNA was translated in a rabbit reticulocyte lysate with T. brucei cytosol (1.5 equivalents) for 60 min and treated with cycloheximide (50 µg/ml final concentration). VSG_117₅₀₀ ΔSP was incubated with TbRMs (one equivalent) for 60 min at 37°C and digested with proteinase K (20 µg/ml final concentration) for 60 min on ice. Proteins were separated by SDS/PAGE and radiolabelled polypeptides were detected by phosphorimaging. Lane 1, untreated VSG_117₅₀₀ ΔSP; lane 2, VSG_117₅₀₀ ΔSP treated with proteinase K; lane 3, VSG_117₅₀₀ ΔSP incubated with TbRM; lane 4, VSG_117₅₀₀ ΔSP incubated with TbRM and treated with proteinase K. The rectangular brackets underneath the sets of bars denote those data points that were directly compared for quantification, and the asterisks denote instances where no signal was detected. Results are representative of four experiments that produced similar results.

Figure 2. Sucrose flotation of [³⁵S]methionine-labelled VSG_117 after incubation with TbRMs

(A) Floatation of VSG_117 in 2.3 M sucrose in the absence (−TbRM, top panel) or presence (+TbRM, bottom panel) of TbRMs. [³⁵S]Methionine-labelled VSG_117 was translated in a rabbit reticulocyte lysate and incubated with (+) or without (−) TbRMs. Reaction mixtures were loaded on to a 2.3 M sucrose cushion with layers of 1.5 M and 0.25 M sucrose above (see the Materials and methods section for details). After centrifugation, each sucrose layer was retrieved, proteins were precipitated, separated by SDS/PAGE, detected with a phosphorimager and quantified. Lane 1, proteins from the 0.25 M sucrose layer; lane 2, proteins obtained from 1.5 M sucrose layer; lane 3, proteins obtained from the 2.3 M sucrose layer; and lane 4, proteins from the 2.3 M sucrose cushion and those that pelleted through the cushion. (B) Quantification of the results in (A). Asterisks (*) represent 0% VSG_117 detected. The images of the bands from lanes 1 and 2 and 3 and 4 were adjusted separately to detect relatively weak bands. Raw data (i.e. not visually adjusted bands) were used for quantification using QuantityOne software. Results are representative of four experiments that produced similar results.

Figure 3. MAL3-101, equisetin and CJ-21,058 inhibit VSG translocation into TbRM

(A) Schematic diagram of the protocol used for import of full-length VSG_117. CHX, cycloheximide; PK, proteinase K; RRL, rabbit reticulocyte lysate. (B) VSG_117 mRNA was translated in rabbit reticulocyte lysate with 1.5 equivalents of T. brucei cytosol pre-treated with MAL3-101 (3 µM), MAL3-51 (10 µM), CJ-21,058 (20 µM) or equisetin (50 µM) for 60 min. The reaction mixtures were treated with cycloheximide (50 µg/ml final concentration) and were incubated with TbRMs (one equivalent). After incubation at 37°C for 60 min, reaction mixtures were transferred to an ice-water bath and treated with proteinase K (30 µg/ml final concentration) for 60 min. Proteins were resolved by SDS/PAGE and detected by phosphorimaging. Lane 1, VSG_117 incubated with TbRMs; lane 2, VSG_117 digested with proteinase K after incubation with TbRMs; lane 3, VSG_117 incubated with MAL3-101 in the presence of TbRMs; lane 4, VSG_117 and TbRMs incubated with MAL3-101 and then digested with proteinase K; lane 5, VSG_117 incubated with MAL3-51 and TbRMs; lane 6, VSG_117 incubated with TbRMs in the presence of MAL3-51 and digested with proteinase K; lane 7, VSG_117 incubated with TbRMs; lane 8, VSG_117 incubated with equisetin and digested with proteinase K; lane 9, VSG_117 incubated with CJ-21,058 and TbRMs; lane 10, VSG_117 incubated with TbRMs and CJ-21,058 and digested with proteinase K. For quantification, only the upper band corresponding to the full-length VSG_117 (indicated by an arrow) was taken into consideration. Furthermore, each proteinase-digested sample was compared to a control experiment containing the drug but without proteinase addition. Thus lane 2 is compared to lane 1, while lane 4 is compared to lane 3 etc. Results are representative of four experiments that produced similar results.

Figure 4. Structures of MAL3-101, MAL3-51, CJ-21,058 and equisetin

The structures of these compounds have been published previously [–14].

Figure 5. Trypanocidal effect of translocation inhibitors

Cultured bloodstream T. brucei CA427 was seeded in medium at a density of 10⁴ cells/ml in 96-well plates. The indicated concentrations of MAL3-101, MAL3-51, CJ-21,058 and equisetin were added to the cells (duplicate cultures) and were incubated for 24 h. In control studies (i.e. no drug), equal volumes of DMSO were added to the cultures. The cell density was determined after 24 h, and the results were plotted. Results are means ± S.D. (n = 4).

Figure 6. Effect of MAL3-101, equisetin and CJ-21,058 on a human cell line

HeLa cells (10⁴ cells/well in a 96-well plate) were treated with one of the following agents: MAL3-101 (10 µM), MAL3-51 (10 µM), CJ-21,058 (12 µM), equisetin (20 µM), etoposide (20 µM), DMSO (5 µM) or mock-treated for 24 or 48 h. Cell viability was determined with a propidium iodide exclusion assay (see the Materials and methods section for details). Results are means ± S.D. (n = 4)