Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors

Abstract

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.

Fig. 1. Synthetic substrate analogues of GlcNAc-PI. Their abbreviated names and the m/z values of their [M-H]^–1 pseudomolecular ions are indicated.

Fig. 2. Molecular models of GlcNAcα1-6d-myo-inositol-1-HPO₄-lipid and GlcNAcβ1-6d-myo-inositol-1-HPO₄-lipid. The energy-minimized structures (using an unrestrained MOPAC molecular dynamics simulation at 300 K) show how similar the two isomers are with respect to the relative positions of the GlcNAc residue and the phosphodiester group. For simplicity, the lipid group in these models is represented as a simple alkyl chain.

Fig. 3. GlcNR-PI analogues as substrates for the trypanosomal GlcNAc-PI de-N-acetylase. (A) The trypanosomal cell-free system was incubated with GDP-[³H]Man and NEM alone (lane 1), or together with either GlcN-PI, GlcNAc-PI or GlcNBz-PI at the concentrations indicated (lanes 2–5). (B) The trypanosomal cell-free system was incubated with GDP-[³H]Man and NEM alone (lane 1), or together with the compounds indicated at a final concentration of 10 µM (lanes 2–8). In both cases, the radiolabelled glycolipid products were analysed by HPTLC and fluorography. The products are: DPM, dolichol-phosphate-mannose; M1–3, Man_1-3GlcN-PI; aM2–3, Man_2-3GlcN-(acyl)PI; A′, EtNP-Man₃GlcN-PI; C′, EtNP-Man₃GlcN-(acyl)PI.

Fig. 4. Inhibition of GPI biosynthesis in the trypanosomal cell-free system by GlcNR-PI substrate analogues. (A) The trypanosomal cell-free system was incubated with GDP-[³H]Man and NEM either alone (lane 1) or together with GlcNAc-PI (lane 2), or with GlcNAc-PI after pre-incubation with the compounds indicated (lanes 3–7). (B) The trypanosomal cell-free system was incubated with GlcN[³H]Ac-PI alone (squares) or in the presence of equimolar GlcNAc-PI (diamonds), GlcNBz-PI (circles), GlcNMe₂-PI (triangles) or GlcNCONH₂-PI (crosses), and the release of [³H]acetate was measured against time. (C) The trypanosomal cell-free system was incubated with GDP-[³H]Man and NEM either alone (lane 1) or with GlcNAc-PI (lane 2) or GlcNCONH₂-PI (lane 3), or with GlcNAc-PI after pre-incubation with various concentrations of GlcNCONH₂-PI (lanes 4–8).

Fig. 5. GlcNR-PI inhibitors of trypanosome GPI biosynthesis inhibit MT-1 as well as GlcNAc-PI de-N-acetylase. The trypanosomal cell-free system was incubated with GDP-[³H]Man and NEM either alone (lane 2) or with GlcN-PI (lane 1), or with GlcN-PI after pre-incubation with the compounds indicated (lanes 3–8).

Fig. 6. The reversible and irreversible inhibition of trypanosome GPI biosynthesis by GlcNR-PI analogues. (A) The trypanosomal cell-free system was incubated with GDP-[³H]Man and NEM alone (lane 1) or with GlcN-PI (lane 2), or with GlcN-PI after pre-incubation with the compounds indicated (lanes 3–6). Alternatively, membranes were pre-incubated with GDP-[³H]Man and NEM alone (lanes 7 and 8), or with GlcN-[L]-PI (lane 9), GlcNCONH₂-PI (lane 10), GlcNBn-PI (lane 11) or GlcNMe₂-PI (lane 12) prior to membrane washing (+) and incubation with GlcN-PI (lanes 8–12). (B) The trypanosomal cell-free system was incubated with GDP-[³H]Man and NEM alone (lane 1) or with GlcN-PI (lane 2), or with GlcN-PI after pre-incubation for 5 min with GlcNCONH₂-PI (lane 3). The same membranes were also pre-incubated with (lanes 4–7) or without (lane 8) GlcNCONH₂-PI, and subsequently washed with HEPES incorporation buffer alone (lanes 4 and 8) or with buffer containing DTT, hydroxylamine (NH₂OH) or hydrazine (NH₂NH₂) (lanes 5–7), prior to incubation with GlcN-PI.

Fig. 7. Inhibition of HeLa cell GPI biosynthesis by GlcNR-PI analogues. (A) The HeLa cell-free system was incubated with GDP-[³H]Man alone (lane 1) or with GlcNAc-PI (lanes 2 and 6), or with GlcNAc-PI after pre-incubation with the compounds indicated (lanes 3–5). The products are: DPM, dolichol-phosphate-mannose; H2, Man₁GlcN-(acyl)PI; ENDO and EXOG H5, endogenous and exogenous EtNP-Man₁GlcN-(acyl)PI derived from endogenous GlcN-PI or exogenous synthetic GlcNAc-PI, respectively. (B) The HeLa cell-free system was incubated with GlcN[³H]Ac-PI alone (squares) or in the presence of equimolar GlcNAc-PI (diamonds), GlcNBn-PI (circles), GlcNMe₂-PI (triangles) or GlcNCONH₂-PI (crosses), and the release of [³H]acetate was measured against time.

Fig. 8. Selective inhibition of the trypansome de-N-acetylase by GlcNCONH₂-β-PI and GlcNCONH₂-(2-O-octyl)-PI. The trypanosome (A, C and E) and HeLa cell (B, D and F) cell-free systems were incubated with GlcN[³H]Ac-PI after pre-incubation with various concentrations of GlcNCONH₂-PI (A and B), GlcNCONH₂-β-PI (C and D) or GlcNCONH₂-(2-O-octyl)-PI (E and F), and the release of [³H]acetate was measured against time.

Fig. 9. A proposed mechanism for the action of GlcNAc-PI de-N-acetylase. (A) A proposed mechanism for cleavage of the acetyl function from GlcNAc-PI via the formation of an O-acetyl enzyme intermediate. (B) A proposed mechanism for inhibition of the de-N-acetylase by GlcNCONH₂-PI. Note: the data do not exclude the possibility that the active site residue is a hydroxy-amino acid instead of Cys.