Myristate labelling of GPI anchor biosynthesis using YnC12

The previous analysed rhodanine-N-acetic ester derivatives 14v and 14a showed strong in-hibitory effects on the incorporation of myristic acid derivativeYnC12 in T. brucei, most likely by inhibition of DPMS and/or the first mannosyltransferase as shown by a cell-free GPI-synthesis assay (Chapter 3.5.1 and 3.5.3). In this experiments, the analogous N-allyl rhodanine series (Table 38) was assessed for their ability to inhibit incorporation of the myristic acidYnC12 in GPI-VSG proteins. Therefore, T. brucei was incubated with the N-allyl derivatives40x and 40c for 3 h, before parasite density was adjusted to equal amounts for both inhibitors andYnC12 was added. T. brucei was incubated for a further 4 h to ensure incorporation of the myristic acid in the GPI-anchor. Next, the crude proteins were precipitated and the fluorescent tag38

Table 38: Anti-parasitic activity of chosen N-allyl rhodanine derivatives.

N S O

S

R

GI₅₀ [µM]

# R T. brucei T. cruzi L. infantum HL60 40x OBn 14.9 ± 3.2 >100 >100 >100 40c CH₃ 12.9 ± 0.8 >100 >100 81.0 ± 2.3

was introduced via Click-chemistry, followed by visualisation of fluorescent protein bands on a SDS-gel (Figure 54).

The positive control in the presence of DMSO and YnC12 showed a strong fluorescent band at 50 kDa and a wide weak band at 57 kDa. As discussed previously it is assumed that the intense protein band at 50 kDa may represent a GPI-VSG precursor protein, which may not be fully glycosylated (Chapter 3.5.3). The allyl-derivative 40x with a 3-benzyloxy substituent reduced the intensity of the 50 kDa band by about 50 %. However, in comparison to the analogous rhodanine-N-acetic ester derivative14v the effect of 40x to inhibit myristate incorporation was reduced after incubation of 3 h. The methyl substituted N-allyl derivative40c showed comparable results to the analogous rhodanine-N-acetic ester14a, as both showed only weak inhibition ofYnC12 incorporation. Although, the fluorescent signal bands appeared reduced in Figure 54 for 40c, Coomassie staining showed that a smaller quantity of protein was loaded to the SDS gel, explaining the reduced fluorescent intensity.

YnC12 labelling after 24 h incubation with 40x and 40c

The allyl-derivative40x had a moderate effect on incorporation of the myristic acid analogue YnC12⁹ in T. brucei after an incubation period of 3 h, as can be seen in Figure 54. The meta methyl substituted allyl derivative40c did not show any effect on myristate incorporation in GPI-VSG-proteins. In order to study the inhibition effect of 40x and 40c over a longer time period, the incubation time with T. brucei was increased to 24 h. After 24 h incubation with compound40c an increase in toxicity against T. brucei was observed, although earlier activity assays against T. brucei did not show any toxicity at this inhibitory concentration.

Therefore, 40c could not be evaluated for its inhibitory effect on YnC12 labelling in the GPI bound VSG proteins. However, the remaining 3-benzyloxy derivative 40x did not show any

9YnC12 was kindly provided by Megan Wright and Dr. Ed Tate (Imperial College London, London, UK).

4 N-allyl rhodanine derivatives

in-gel fluorescence in SDS gel observed under TAMRA settings

Coomassie-stained SDS gel

Figure 54: SDS gels after 3 h incubation with 40x and 40c; lane 1: 40x, lane 2: 40c, full gels see Chapter 11.

anti-trypanosomal effects after 24 h and labelling withYnC12 was performed. After lysis of the parasites and the introduction of the tamra-fluorophore38, the proteins were separated on a SDS gel and analysed by in-gel fluoresence and Coomassie staining.

The inhibitor 40x resulted in a significant reduction of incorporation of YnC12 in the GPI bound VSG proteins (Figure 55). However the rhodanine-N-acetic ester analogue14v showed a complete inhibition ofYnC12 incorporation, suggesting that the N-allyl functionality caused the reduction in activity and therefore suggesting that a carboxylic moiety is preferable for inhibitingYnC12 incorporation in T. brucei.

YnC15 labelling in T. brucei and influence of N-allyl derivatives

The inhibitory effect of derivative40c on YnC12 incorporation in T. brucei could not be studied after 24 h due to increased trypanocidal effects. Unfortunately, these experiments could not be repeated at lower concentrations of 40c and YnC12, as all of the myristic acid analogue YnC12 had been used. However, in order to overcome this problem, the pentadecylic fatty acid analogueYnC15¹⁰was used in combination with lower concentration of40c.

Indeed, after 24 h of incubation with T. brucei, a decrease in anti-trypanosomal activity was observed and labelling withYnC15 could be performed with both 40x and 40c treated trypanosomes. However, the corresponding gel (Figure 55) showed only unspecific binding of

10YnC15 was kindly provided by Megan Wright (Prof. Dr. Ed Tate, Imperial College London, London, UK).

in-gel fluorescence with TAMRA setting Coomassie-staining;

Figure 55: SDS gels after 24 h incubation with 40x; lane 1: 40x, full gels see Chapter 11.

the tamra-fluorophore with trypanosomal proteins in the negative control (absence ofYnC15) and all treated trypanosome cultures. Therefore confirming the specificity of myristate incor-poration in GPI-anchor bound VSG proteins.^[203,205]

YnC15 labelling in HL60 cells and influence of N-allyl derivatives 40x and 40c

After showing that40x inhibited incorporation of the mysristic acid analogue YnC12 in the GPI bound VSG proteins in T. brucei, studies on the effect ofYnC12 in HL60 cells were performed.

Therefore, T. brucei was incubated with the derivatives40x and 40c for 24 h, after which cells were lysed, proteins tagged with the tamra-fluorophore38 via Click-chemistry and visualised on a SDS gel using in-gel fluorescence and Coomassie staining. The negative control, which was performed in the absence ofYnC12 showed unspecific binding of the tamra-fluorophore with HL60 proteins (Figure 57). The same unspecific binding was observed in proteins treated withYnC12 and inhibitors (DMSO, 40x and 40c).

This experiment could not provide an answer about the molecular target in HL60 cells of the N-allyl-derivatives40x and in particular 40c, as this derivative showed toxicity at GI₅₀ 81.0 µM against HL60 cells.

4.4.2 Flow cytometry analysis of T. brucei transferrin receptors for indication of GPI