Análisis de la pobreza en México (2014)

Introduction to cell-free GPI-synthesis assay

Initially the DPMS inhibitors in Table 2 were screened to evaluate their trypanocidal activity against T. brucei (Table 25).^[68]With the exception of the unsubstituted 5-benzylidene deriva-tive2a (GI₅₀ 56.0 µM) none of the other inhibitors showed any effect on T. brucei growth at 100 µM, although they inhibited DPMS, a validated anti-trypanosomal protein, with good to moderate residual activities.^[68] In addition to these studies a cell free-GPI synthesis assay on trypanosome enzymes was performed.⁷ Briefly, radio-labelled GDP-[³H]-Man was sub-jected to cell-free membranes of T. brucei in the presence of 1 mM of rhodanine-inhibitor.^[68]

This membrane layer contains all enzymes involved in GPI anchor biosynthesis and is able to synthesise the GPI-anchor until the substrate ethanolamine-P-Manα1-2Manα1-6Manα1-4GlcNH₂-PI.^[68] Intermediates of the GPI-anchor synthesis can be extracted and separated by high performance thin layer chromatography (HPTLC).^[68]The radio-labelled intermediates of the GPI-anchor synthesis can be visualised by fluorography and this TLC-plate can be analysed by comparison of substrate band intensities (Table 25).^[68] In order to explain GPI-anchor synthesis in the cell-free system, individual steps will be explained in further detail for the control (DMSO, Table 25). The first band on the HPTLC is representing Dol-P-Man, whose synthesis is catalysed by DPMS from GDP-[³H]-Man and dolichylphosphate. A weak

7This assays were performed by Dr. Terry Smith from the University of St. Andrews, St Andrews, Scotland, UK

intensity of this band in combination with weaker consecutive bands is indicative for DPMS inhibition. However, frequently this band will appear with a decreased fluorescent intensity, but is accompanied by intensive consecutive bands, indicating that the substrate Dol-P-Man is faster consumed than re-synthesised.⁸ In the consecutive step the mannosyltransferase 1 (MT-1) catalyses the transfer of Dol-P-Man onto GlcNH2-PI, resulting in the formation of the second band (M1) on the HPTLC.^[65] The next mannose residue is installed by the α1-6 mannosyltransferase (MT-2) resulting in the formation of Manα1-6Manα1-4GlcNH2-PI (M2) on the HPTLC plate.^[65] The third mannose is transferred by an α1-2-mannosyl transferase from Dol-P-Man to generate Manα1-2Manα1-6Manα1-4GlcNH₂-PI (M3).^[65] Next the inositol acyl-transferase acylates M3 to generate the substrate Manα1-2Manα1-6Manα1-4GlcNH₂-(acyl)PI (aM3).^[65] The final step performed by the cell-free system is the ethanol amine phosphate transfer to form the substrate ethanolamine-P-Manα1-2Manα1-6Manα1-4GlcNH₂-PI (A’) (Fig-ure 42). Inhibition of individual enzymes by rhodanine derivatives will lead to accumulation of

O

Figure 42: Substrates of cell-free GPI-synthesis assay, M1: Manα1-4GlcNH₂-PI; M2:

Manα1-6Manα1-4GlcNH₂-PI; M3: Manα1-2Manα1-6Manα1-4GlcNH₂-PI (M3);

aM3: Manα1-2Manα1-6Manα1-4GlcNH₂-(acyl)PI; A’: ethanolamine-P-Manα1-2Manα1-6Manα1-4GlcNH2-PI.

the individual substrates of that particular enzyme and can therefore be identified by this cell-free assay.

8Oral communication with Dr. Terry Smith, St. Andrews University

3 Rhodanine-N-acetic acid derivatives

Comparison of DPMS activity and activity in cell-free GPI-synthesis assay and in vitro activity

A chosen subset of rhodanine-N-acetic acid derivatives was screened in this cell-free assay of GPI-anchor synthesis in order to identify potential targets within the GPI-anchor biosynthesis pathway. Particularly interesting were the derivatives shown in Table 25, as residual DPMS activity data was already available.^[68] The DPMS activity correlated well with the cell-free assay, as low residual DPMS activity resulted in weak substrate bands upstream from Dol-P-Man. The high intensity of the Dol-P-Man band might be explained by accumulation of substrate over time, as none of the inhibitors abolished DPMS synthesis completely. This hypothesis is supported by derivative2f, a weak DPMS inhibitor, resulting in a diminished Dol-P-Man band followed by a weak M1 band. While2f inhibits DPMS weakly, less Dol-P-Man substrate is accumulated, resulting in a weak band. However, it may also be possible that the derivatives inhibit additionally the first mannosyltransferases MT-1 and therefore cause decrease in subsequent substrate bands. To this point it is unclear if one or more enzymes were inhibited by these rhodanine-N-acetic acid derivatives. However, particularly exciting was the observation that2c, the free acid analogue of the ester derivative 14v, which has been shown by myristate labelling and flow cytometry analysis to inhibit GPI-anchor biosynthesis, indeed is a good DPMS and possible MT-1 inhibitor.

It is likely, that the ester derivative has the same molecular target, as the ester moiety potentially serves as a pro-drug and increases membrane diffusion, while being hydrolysed within the cell. This hypothesis was supported by low µM activity of the ester derivatives against the bloodstream form of T. brucei in vitro (Table 25). Indeed, the most promising inhibitor was 14v as it showed a good selectivity index of SI > 23, while most of the other ester-derivatives showed increased toxicity towards HL60 cells.

Methyl and trifluoromethyl substituted rhodanine-N-acetic acid and their activity in the cell-free GPI-synthesis assay

Previously, the rhodanine-N-acetic ester derivative14a has been shown by myristate labelling and flow cytometry analysis to inhibit GPI-anchor biosynthesis. In the following research study, the activity against isolated enzymes of the GPI-anchor synthesis is narrowed down and should allow the identification of the molecular target(s) within the GPI-anchor biosyn-thesis. The free carboxylic acid derivative 2m was screened in the cell-free GPI-synthesis assay as it is assumed that the ester moiety is cleaved upon entering into the parasitic cell.

However, it may also be likely that the ester derivative is the active agent. The free acid analogue2m showed the strongest inhibition in this series, resulting in complete inhibition of the first mannosyltransferase (MT-1) and possible also DPMS (Table 26). This radio-labelled assay possibly identified the enzymes inhibited in the GPI-anchor biosynthesis, resulting in

Table 25: cell-free GPI assay for rhodanine-N-acetic acid derivatives; lane 1: 2a, lane 2: 2ah, lane 3: 2c, lane 4: 2e, lane 5: 2g, lane 6: 2f; in vitro activity against T. brucei; Blue highlighted bands: DPMS inhibition; Green highlighted bands: MT-1 inhibition.

N S S

O R¹O

O

R² R³ R⁴

R¹=H R¹=Et

GI₅₀ [µM] GI₅₀ [µM]

# R² R³ R⁴ res. DPMS^[68][%] T. brucei # T. brucei HL60

2e OH H H 23 ± 8 >100 14p 1.7 ± 0.1 4.1 ± 0.7

2c H OBn H 23 ± 3 >100 14v 4.4 ± 1.1 >100

2f H OH H 90 ± 5 >100 14q 4.8 ± 0.9 9.9 ± 0.5

2a H H H 42 ± 5 56.0 ± 3.3 14i 8.3 ± 0.7 38.0 ± 1.8

2ah H H OBn 10 ± 2 >100 14w 17.0 ± 0.9 >100

2g H H OH 70 ± 4 >100 14r 17.6 ± 0.1 n.a.

decreased incorporation of myristate in metabolic labelling experiments and a reduction in up-take of FITC-labelled transferrin. All three assays showed similar results, but the radio-label assay narrowed the inhibition of GPI-anchor biosynthesis down to MT-1 and DPMS. Shift-ing the methyl group on the 5-benzylidene moiety in para-position (2n) abolished its activity against MT-1. There was a weak band for DPMS, but this was not indicative for DPMS inhi-bition, as Dol-P-Man has been synthesised but was used in subsequent steps, explaining the intense bands of consecutive substrates. Transferring the methyl substituent in ortho-position in derivative2o decreased MT-1 inhibition. Interestingly, in the trifluromethyl series the meta-and para-substituted analogues showed good MT-1 inhibition, whereas the ortho substituent did not show any inhibition of GPI-anchor synthesis. The anti-trypanosomal activity against T.

brucei in vitro correlated well with GPI-anchor synthesis inhibition. However, trifluoromethyl substituted analogues showed increasing toxicity against HL60 cells. The most promising candidate in this series was the meta-methyl substituted ester analogue14a as it showed low µM activity against T. brucei, while having a good SI of 21.

3 Rhodanine-N-acetic acid derivatives

Table 26: cell-free GPI assay for rhodanine-N-acetic acid derivative-2; lane 1: 2o, lane 2: 2m, lane 3: 2n, lane 4: 2r, lane 5: 2p, lane 6: 2q; in vitro activity against T. brucei; Blue highlighted bands: DPMS inhibition; Green highlighted bands: MT-1 inhibition.

N S S

O R¹O

O

R² R³ R⁴

R¹=H R¹=Et

GI50 [µM] GI50 [µM]

# R² R³ R⁴ T. brucei # T. brucei HL60 SI

2o CH₃ H H >100 14g 1.3 ± 0.1 12.7 ± 0.9 10 2m H CH₃ H >100 14a 1.5 ± 0.3 31.0 ± 0.8 21 2n H H CH3 >100 14n 15.6 ± 1.4 29.3 ± 8.3 2 2r CF₃ H H >100 14k 1.4 ± 0.1 9.3 ± 3.7 7 2p H CF₃ H >100 14l 1.7 ± 0.1 11.3 ± 3.7 7 2q H H CF3 >100 14d 1.4 ± 0.1 11.8 ± 0.5 8

Anti-trypanosomal rhodanine-N-acetic acid derivative with no activity in cell-free GPI-synthesis

Interestingly, one of the most potent inhibitors against T. brucei growth in vitro did not show any effect on enzymes of the GPI-anchor biosynthesis as is to be seen in the results of the cell-free GPI-synthesis assay with2u (structural analogue of 14t, Table 27). Also, the pyridine derivative 26d was inactive in the GPI-synthesis assay, whereas its structural ethyl ester analogue 26h displayed anti-trypanosomal activity in the low µM range. The catechol derivative 14t may inhibit oxide-reductases in parasites, as several other rhodanine-catechol derivatives have been reported as molecular probes for bacterial NAD(P)H-dependant oxidoreductases.^[107,108]

However, the exact mode of action of these derivatives is unknown at the present time, but using the fluorescence properties of2u in combination with native protein gels (western blots), the molecular target could potentially be identified.^[108]

Table 27: Cell-free GPI assay for rhodanine-N-acetic acid derivatives-3; R¹=H; lane 1: 2u, lane 2: 2s, lane 3: 2h, lane 4: 26c, lane 5: 26d; in vitro activity against T. brucei;

Blue highlighted bands: DPMS inhibition; Green highlighted bands: MT-1 inhibition.

N S S

O R¹O

O

R² R³ R⁴

R¹=H R¹=Et

GI₅₀ [µM] GI₅₀ [µM]

# R² R³ R⁴ T. brucei # T. brucei HL60 SI

2u H OH OH >100 14t 1.6 ± 0.1 >100 63

2s H H SO₂Me >100 n.a.

2h H H Cl >100 n.a.

26c 3-pyridinyl >100 26g 1.5 ± 0.2 51.5 ± 2.5 34 26d 4-pyridinyl >100 26h >100 >100 >1