Regioselective Covalent Immobilization of Catalytically Active Glutathione S‑Transferase on Glass Slides

The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His₆-tagged glutathione S-transferase (His₆-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His₆-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω-alkyne and azide moieties and covalently attached to the surface by a Cu­(I)-catalyzed Huisgen [3 + 2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in <i>A</i>₃₄₀ upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His₆-GSTase-CVIA-PE) or azide (His₆-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His₆-GSTase-CVIA-F)

Screening work flow.

<p>The different steps, the most relevant assay conditions and the go/no-go criteria of the screening campaign are indicated in boxes. The figures on the right refer to the number of compounds screened and that subsequently advanced during the campaign. From 144 compounds, 22 compounds lowered assay signal ≥ 45% for at least one TryS. From these 22, 7 BDA were false positive and the remaining 15 compounds were confirmed as enzyme inhibitors. Two of them are <b>AI</b> with potency in the submicromolar range against <i>Li</i>TryS. AI (P), 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives (P, paullone); APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives; BZ, benzofuroxan derivatives; BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives.</p

Biological activity of compounds against infective <i>Trypanosoma brucei brucei</i> with downregulated expression of trypanothione synthetase (TryS).

<p><b>A)</b> Western blot analysis of cell extracts from 2x10⁷ <i>T</i>. <i>b</i>. <i>brucei</i> from the wildtype (WT), 48 h tetracycline-induced (+) and non-induced (-) TryS-RNAi cell line. Two hundred ng of recombinant <i>Tb</i>TryS was loaded as control. Bands from the molecular weight marker are indicated on left. The picture at the bottom shows the abundance of TryS for each condition as estimated by densitometry and expressed relative to the level of the WT cell line. <b>B)</b> Ponceau staining of the Western blot membrane that served as normalization control of protein load for each condition. <b>C)</b> Cytotoxicity (%) ± S.D. (n = 2) for tetracycline-induced (+) and non-induced (-) TryS-RNAi <i>T</i>. <i>b</i>. <i>brucei</i> treated with 5 μM nifurtimox or 100 nM EAP1-47.</p

Inhibitors of tritryp trypanothione synthetase (TryS) identified in this work and their biological activity.

<p>Inhibitors of tritryp trypanothione synthetase (TryS) identified in this work and their biological activity.</p

Structure of compounds affecting tritryp trypanothione synthetase activity.

<p><b>AI (P)</b>, 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives, paullones derivatives, (FS-554 and MOL2008), five APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives (ZEA10, ZEA35, ZEA40, ZEA41 and ZVR159), eight BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives (EAP1-47, EAP1-63, APC1-67, APC1-87, APC1-89, APC1-99, APC1-101 and APC1-111), seven BBHPP, 1-(benzo[<i>d</i>]thiazol-2-yl)-4-benzoyl-3-hydroxy-5-phenyl-1<i>H</i>-pyrrol-2(5<i>H</i>)-one derivatives (AD81, AD84, ADMRC158, ADKPN160, ADKPN161, ADKPN164 and ADKPN165), three BZ, benzofuroxan derivatives (J18, J20 and J31) and one PD, 1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione derivatives [(<i>Z</i>)-8-(2-(2,4-dihydroxybenzylidene)hydrazinyl)-7-(2-hydroxy-3-phenoxy propyl)-1,3-dimethyl-1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione, TC227]. iPr, tBu, OBn, Mo and Ph, correspond to an isopropyl, tert-butyl, O-benzyl, 4 -morpholinyl and phenyl substitution, respectively.</p

Trypanothione dependent redox metabolism.

<p>The chemical structure of trypanothione (<i>N</i>¹,<i>N</i>⁸-bis(glutathionyl)spermidine; T(SH)₂) is depicted at the center. Synthesis: trypanothione synthetase catalyzes the ligation of two molecules of gluthatione to one of spermidine using the energy provided by two ATP molecules. Regeneration: trypanothione reductase maintains trypanothione in the reduced state at expenses of NADPH, which can be supplied by the oxidative phase of the pentose phosphate pathway <i>via</i> glucose 6-phosphate dehydrogenase. Utilization: reduced trypanothione is involved in multiple functions such as the detoxification of xenobiotics, cell proliferation, defense against oxidants and protein thiol-redox homeostasis. The multipurpose oxidoreductase tryparedoxin plays an important role catalyzing electron transfer from T(SH)₂ to different molecular targets (e.g. peroxidases, ribonucleotide reductase and protein disulfides). G6P: glucose-6-phosphate, 6PGL: 6-phosphogluconolactone, T(SH)₂: reduced trypanothione, TS_2[ox]: oxidized trypanothione, NDPs: nucleosides diphosphate, dNDP: deoxinucleosides diphosphate, E^-: electrophilic species, TS-E: trypanothione-electrophile adduct, ROOH: hydroperoxide, ONOOH: peroxynitrite, NO₂^-: nitrite.</p