Synthesis, Enzymatic Evaluation, and Docking Studies of Fluorogenic Caspase 8 Tetrapeptide Substrates

Abstract

The synthesis, enzymatic evaluation, and molecular modeling studies of new fluorogenic tetrapeptide-based substrates selective for caspase 8, having the general structure Ac-IETD-AXX, are described. Various fluorescent reporter groups (AXX), i.e., 3- and 4-substituted coumarins and quinolin-2(1H)-ones were synthesized by von Pechmann condensation. They were subsequently coupled with the caspase-8-selective tetrapeptide Ac-IETD-OH under newly developed synthetic conditions to give the desired substrates in good yields and in high enantiomeric purity. Based on K(M) and V(max) values, the new compounds proved to be excellent substrates for recombinant human caspase 8. In contrast, the K(M) values for the same compounds as substrates for human caspase 3 were approximately 10-20-fold higher. Molecular modeling studies based on the X-ray crystal structures of both human caspases 3 and 8 revealed that there is sufficient room within both active sites to accommodate substrates with moderately bulky substituents in the 3- and 4-positions of the fluorogenic coumarins and quinolin-2(1H)-ones. Automated docking of the substrates into the active sites of both human caspases 3 and 8 with the program AutoDock 3 gave structures similar to the published crystallographic structures for the same tetrapeptide bound to caspase 8 in the form of an irreversible inhibitor. The calculated binding energies for the new substrates to either caspase 3 or 8 showed little difference between the substrates, consistent with the K(M) data. In addition, the calculated binding energies (DeltaG) to caspase 8 were considerably more negative than those to caspase 3, also consistent with the K(M) data. A possible molecular interaction that might explain the selectivity of the IETD tetrapeptide motif for caspase 8 over caspase 3 is discussed.

Structure based molecular inhibition of Caspase-8 for treatment of multi-neurodegenerative diseases using known natural compounds

“…In the present study, all the compounds were found to interact mainly through ten amino acid residues R258, R260, N261, G262, T263, H317, A359 and C360 (Table 1). The role of Arg 258 is conspicuous in the structure of caspase 8 as the large protrusion that sticks out in the top-middle of the active site cleft [28]. This residue was also very much involved in the positioning of compounds within the active site of caspase-8.…”

Novel 7‐maleimido‐2(1H)‐quinolones as potential fluorescent sensors for the detection of sulphydryl groups

“…Compounds 2a – 2d were synthesised according to the protocols in the literature . Equimolar amounts of 1,3‐phenylenediamine 1 and the corresponding ethyl acetates (Scheme ) were refluxed.…”

“…Equimolar amounts of 1,3‐phenylenediamine 1 and the corresponding ethyl acetates (Scheme ) were refluxed. After cooling the reaction mixture to room temperature the resulting solids were filtered, washed with methanol, dried and finally recrystallised from methanol .…”

“… 2a : yield 45%, mp 278–279 °C (279 °C ), 2b : yield 55%, mp 327–329 °C (328–329 °C ), 2c : yield 44%, mp 265 °C (278–279 °C ), 2d : yield 45%, mp 334–336 °C (334–336 °C ). …”

Structure–activity relationship studies of 4-methylcoumarin derivatives as anticancer agents

“…Compound 24 was synthesized according to the synthetic protocol established by our group (Aggarwal et al, 2012). Compound 25 was synthesized by following the reported multistep procedure and was then acetylated to yield compound 26 (Reszka et al, 2010). To synthesize compound 27, commercially available 4-bromoresorcinol was reacted with ethyl-4-bromoacetoacetate via the literature procedure (Bourbon et al, 2012).…”