Structure activity relationship studies on Amb639752: toward the identification of a common pharmacophoric structure for DGKα inhibitors

Figures & data

Figure 1. Three of the most studied DGKα inhibitors.

Figure 2. Structures of the deaza analogue of R59949 and Amb639752.

Figure 3. First set of compounds tested for their inhibitory activity on DGKα.

Table 1. Inhibitory activity on DGKα (I).

Compound	Residue activity at 100 µM
R59022	27
R59949	28
Amb639752	4
Amb758897	67
Amb37496	114
Amb626577	127
Amb1926062	88
Amb94966	196
Amb730692	45
Amb98433	81
Amb18718	135
Amb726054	126
Amb731111	130
Amb22018852	160
Amb687392	115
Amb758592	150
Amb629700	101
2	135

Scheme 1. The first synthetic route for the compound 8.

Scheme 2. The second synthetic route for the compound 8.

Figure 4. Carboxylic acids used.

Figure 5. Putative DGKα inhibitors synthesised. In brackets the yield of the coupling reaction with the common intermediate 7.

Table 2. Inhibitory activity on DGKα (II).

Compound	Residue activity at 100 µM	IC50 (µM)
8	5	3.2
11	6	1.6
12	6	3.6
13	14	6.9
14	6	3.0
15	89	–
16	27	32.8
17	41	–
18	47	–
19	26	49.7
20	3	1.8
21	39	–
22	48	–

Figure 6. Dose–response curves for novel DGKα inhibitors. Dose–response of the most active compounds along with their IC₅₀ values. Data from at least three independent experiments performed in triplicate.

Figure 7. Isoform specificity of novel DGKα inhibitors. 293T cells were transfected with different DGK isoforms (A – DGKα, B – DGKζ, C – DGKθ, respectively) or empty vectors and homogenised. All the molecules were tested at 100 μM for their capacity to inhibit the DGK activity of the different isoform homogenates. Data are means ± SEM of at least three independent experiments performed in triplicate.

Figure 8. Novel DGKα inhibitors do not affect serotonin signalling. Human monocytes were pre-incubated for 1 h with the indicated drugs in absence or presence of serotonin and then stimulated with PMA 1 µM for 30 min (■ control unstimulated cells,

PMA stimulated cells, ■ PMA and serotonin stimulated cells). Results are expressed as n moles of reduced cytochrome C/10⁶ cells. Data are means ± SEM of 10 independent experiments performed in triplicate.

Figure 9. DGKα inhibitors partially restores apoptosis in SAP deficient lymphocytes. Lymphocytes from normal subjects were transfected with control or SAP specific siRNA (■ control siRNA, ■ SAP siRNA). After 4 days, the cells were restimulated with CD3 agonist OKT3 (10 ng/mL) in presence of respective inhibitor. Vehicle (DMSO) was used as control. Twenty-four hours later, the % of cell loss was evaluated by PI staining. Data are the mean ± SEM of nine independent experiments performed in triplicate.

Figure 10. Novel DGKα inhibitors slow tumour cell migration. MCF7 monolayer was wounded and treated for 15 h with serum in presence of our new DGKα inhibitors (10 μM) or vehicle (DMSO). Results are expressed as the percentage of wound area compared to the initial area. Data are the mean ± SEM of nine independent experiments.

Figure 11. Proposed pharmacophoric model.