H-D-Ala-Gly-Gly-OH

The crystal-state conformations of three protected tripeptides, four tetrapeptides, and one pentapeptide, heavily based on the chiral C(alpha)-methylated alpha-amino acids Iva, (alpha Me)Nva, and (Me)Val, were assessed by X-ray diffraction analyses. The eight peptide sequences are as follows: Z-(D-Iva)2-D-Val-OMe, Z-D-Iva-L-Iva-Gly-OtBu, Z-L-Pro-D-Iva-L-Iva-Gly-OtBu, Z-L-Pro-L-Iva-D-Iva-Gly-OtBu, Z-Aib-[L-(alpha Me)Nva]2-OtBu, Ac-[L-(alpha Me)Val]3-D-(alpha Me)Val-OtBu, Z-[L-(alpha Me)Val]4-OH, and Z-L-Ala-[L-(alpha Me)Nva]4-OtBu. Two independent molecules were observed in the asymmetric units of Z-D-Iva-L-Iva-Gly-OtBu and Z-Aib-[L-(alpha Me)Nva]2-OtBu, while three independent molecules were seen in Z-L-Ala-[L-(alpha Me)Nva]4-OtBu. All peptides are folded in a single or multiple beta-turn conformations. Interestingly: (i) a water bridge within the N-terminal beta-turn is seen in Z-L-Pro-L-Iva-D-Iva-Gly-OtBu (dihydrate), and (ii) the hydroxyl group of the C-terminal carboxyl functionality of Z-[L-(alpha Me)Val]4-OH generates an oxy-analogue of a beta-turn. The N-terminal beta-turn is missing in molecules A and B, but it does occur, although poorly stabilized, in molecule C, of Z-L-Ala-[L-(alpha Me)Nva]4-OtBu.

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.