Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP

Abstract

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Figure 1

Structures of parent sn-2 indololactone 1 and target sn-1 indololactones.

Figure 2

Comparison of the C1 domain binding sites of PKCδ C1b with bound phorbol (1PTR), shown in light blue, and RasGRP1 (4L9M), shown in black. The original position of residue Gln 568 in the RasGRP1 structure is colored white, and its adjusted position, as used for docking, is in black. Hydrogen bonds formed by this residue to the backbone are shown as dashed green lines.

Figure 3

Model of the RasGRP1 C1 domain relative to the lipid bilayer. The colored background represents the location of bilayer regions: blue – polar region of charged lipid headgroups and water; green – interfacial region with lipid glycerol backbone and ester groups; yellow – hydrophobic regions with lipid acyl chains. A) Docked complex of compound 1. B) Docked complex of compound 4. Hydrogen bonds to the C1 domain backbone are shown as dashed yellow lines.

Scheme 1

Synthesis of indololactone 2. Reagents and conditions: (a) 1. (CH₃)₂CO, LiHMDS, THF, −78 °C, 4 h; 2. (i) CH₃SO₂Cl, Et₃N, 0 °C, 2 h; (ii) DBU, CH₂Cl₂, room temp, 24 h, 62%; (b) BCl₃, CH₂Cl₂, −78 °C, 2 h, 82%; (c) 1-methyl-1H-indole-3-carbonyl chloride, Et₃N, DMAP, room temp, 24 h, 85%; (d) HF.Et₃N, THF, reflux, 24 h, 78%.

Scheme 2

Synthesis of indololactones 3–8. Reagents and conditions: (a) 1. RCHO, LiHMDS, THF, −78 °C, 2–4 h; 2. (i) CH₃SO₂Cl, Et₃N, 0 °C, 5 h; (ii) DBU, CH₂Cl₂, room temp, 24 h; (b) TBAF, THF, room temp, 30 min; (c) CAN, CH₃CN/H₂O, 0 °C, 20 min; (d) 1-methyl-1H-indole-3-carbonyl chloride, Et₃N, DMAP, room temp, 24 h; (e) BCl₃, CH₂Cl₂, −78 °C, 1 h.