Figure 1
From: Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor
Design and pharmacological evaluation of TRP601. (a) Substrate binding region of caspase-2 (Casp2) in complex with TRP601. The quinolin-2-carbonyl-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH2- moiety is shown in sticks representation with the atoms represented as follows: gray=carbon; white=hydrogen; blue=nitrogen; and red=oxygen. The S–C covalent bond between the Cys-155 residue and the C terminus CH2 of TRP601 is represented in yellow. The electric interactions (hydrogen bonds and salts bridges) are represented by the dashed, green lines. The enzyme residues that interact with the inhibitor are shown by the wireframe representation. Right panel: the table shows the minimal energy (kcal/mol, Emin) of the Casp2–TRP601 complex resulting from electric or Coulombic component (Ec) together with repulsion–attraction component (Evw). The interaction energy of the Casp2–TRP601 complex is −147.28 kcal/mol. Owing to the C–S covalent bond, the Asp residue in P1 contributes to about 30% of the interaction energy. The Asp in P4 and the Val in P5 have important non-covalent contributions to stabilization of the complex, 21% and 17%, respectively. Each of the other two residues (Ala in P2 and Val in P3) contributes to about 12% of the interaction energy. (b) Structure of TRP601. (c) Representative dose–response curve of human recombinant Casp2 and Casp3 inhibition by TRP601. Initial velocities were determined from standard colorimetric microplate assays. (d) Kinetic off rate (k3/Ki) parameters of irreversible caspase inhibitors on Casp2 and Casp3. (e) TRP601 inhibits neuronal caspase activities and prevents serum deprivation (SD)-induced cell death. High-density E14 cortical neuron cultures were subjected to 24 h SD in the presence or absence of 50 μM TRP601. Histograms indicate the means (±S.D.) of 15 independent experiments. (f) Representative pharmacokinetic of TRP601 after intravenous (i.v.) administration in adult rats, through liquid chromatography-mass spectrometry (LC-MS/MS) detection in the plasma and brain homogenates. Note that following intraperitoneal (i.p.) administration of the same dose, TRP601 was detected in the brain at 0.25 h (brain Cmax=25 ng/ml) and the Cmax (20 ng/ml) was obtained in the plasma according to a plateau between 0.5 and 2 h. (g–j) TRP601 reduces excitotoxic lesions in neonates. The 5-day-old mice were subjected to intracerebral ibotenate injection and killed at different time points (g=5 days; h and i=24 h; j=8 h) following the excitotoxic challenge to determine the impact of TRP601, TRP801, and TRP901 (1 mg/kg; i.p.) on lesion severity (g), microgliosis (h), astrogliosis (i), and group II caspases activity (j). Histograms show mean lesion volume (g; ▪, vehicle, n=16; TRP601, n=16; TRP801, n=7; TRP901, n=8), cell density (h and i; n=10 per group), or VDVADase activity (j; n=5 per group)±S.E.M. Asterisks indicate differences from control (▪) (*P<0.05, **P<0.01, ***P<0.001 in Kruskal–Wallis post hoc Dunn's for g, Mann–Whitney for h–j). (k) TRP601 does not enhance protection conferred by short interfering RNA (siRNA)-mediated genetic inhibition of Casp2. The 5-day-old mice were subjected to intracerebral injection (as in c) of either an siRNA against Casp2 (si2-a) or a control siRNA (si2Co), as indicated. After 24 h, ibotenate was administered (intracerebroventrally (i.c.v.)), followed immediately by vehicle (□, n=20; ▪, n=20) or TRP601 (▪; n=24) administration (i.p.). See Supplementary Table 1 for exact values and detailed statistical analysis
