Umbelliferone aminoalkyl derivatives as inhibitors of human oxidosqualene-lanosterol cyclase

Figures & data

Figure 1.  Schematic representation of the relevant steps in cholesterol biosynthesis.

Figure 2.  Structures of umbelliferone aminoalkyl derivatives.

Table I.  Inhibitory effects of umbelliferone aminoalkyl derivatives on sterol biosynthesis in 3T3 fibroblasts incubated with radiolabeled acetate.

Compound	% radioactivity incorporated into non-saponifiable lipids^*
	Squalene	Oxidosqualene	Dioxidosqualene	Lanosterol	Cholesterol
Control	3.11	6.45	2.65	4.59	83.30
1
1 μm	3.71	1.77	1.99	4.56	87.97
10 μm	5.67	18.51	29.37	6.14	40.31
2
0.1 μm	4.06	16.33	3.46	5.21	70.94
1 μm	4.59	50.41	17.99	5.13	21.88
10 μm	4.14	79.62	11.15	2.87	2.22
3
0.1 μm	1.58	20.48	4.07	5.64	68.23
1 μm	3.34	58.03	21.97	4.04	12.62
10 μm	6.03	69.59	15.34	3.29	5.75
4
1 μm	3.71	1.78	1.99	4.55	87.97
10 μm	4.61	3.48	1.71	5.38	84.82
5
0.1 μm	3.96	8.05	2.13	7.75	78.11
1 μm	5.24	7.21	2.91	5.38	79.26
10 μm	3.62	10.72	2.99	7.82	74.85
6
0.1 μm	3.37	8.87	3.23	7.04	77.49
1 μm	4.53	8.44	3.02	6.72	77.29
10 μm	8.13	25.73	15.64	11.57	38.93
7
1 μm	10.20	11.02	6.96	6.50	65.40
10 μm	11.94	13.92	3.97	5.60	64.57
8
1 μm	6.50	9.95	4.33	6.01	73.21
10 μm	5.27	26.32	9.57	4.40	54.44
Ro 48-8071
0.1 μm	1.81	61.21	27.66	4.00	4.92
1 μm	2.49	63.09	27.79	2.37	4.26

* Values are the means of two separate experiments, each one carried out in duplicate. Maximal deviations from the mean were less than 10%.

Table II.  Inhibitory effects of umbelliferone aminoalkyl derivatives on sterol biosynthesis in human keratinocytes incubated with radiolabeled acetate.

Compound	% radioactivity incorporated into non-saponifiable lipids^*
	Squalene	Oxidosqualene	Dioxidosqualene	Lanosterol	Cholesterol
Control	3.93	4.40	2.49	8.98	80.20
1
0.1 μm	1.57	20.48	4.08	5.64	68.23
1 μm	1.84	48.14	27.53	4.71	17.78
10 μm	1.39	53.78	31.68	2.33	10.82
2
0.1 μm	3.06	7.71	3.58	8.46	77.19
1 μm	2.56	23.28	18.40	7.34	48.42
3
0.1 μm	6.12	14.30	11.51	7.99	60.08
1 μm	3.11	41.75	30.33	6.20	18.61
4
0.1 μm	6.90	1.51	1.08	7.60	82.91
1 μm	3.32	2.86	2.71	6.83	84.39
10 μm	4.07	8.77	4.13	4.73	78.30
5
1 μm	2.65	5.30	4.17	7.58	80.30
10 μm	5.20	12.58	5.49	8.55	68.18
6
1 μm	3.95	18.82	5.25	6.22	65.76
10 μm	2.42	30.56	27.33	6.87	32.82
7
1 μm	4.55	4.82	1.96	10.34	78.33
10 μm	5.81	16.73	12.57	8.22	56.67
8
1 μm	2.84	7.86	8.23	7.56	73.51
10 μm	2.57	28.26	25.66	8.66	34.85
Ro 48-8071
0.01 μm	5.26	67.21	23.05	0.71	3.76
0.1 μm	6.18	67.84	22.26	1.98	1.74

* Values are the means of two separate experiments, each one carried out in duplicate. Maximal deviations from the mean were less than 10%.

Table III.  Effect of temperature, pH and detergent concentration on the activity of human OSC expressed in yeast.

	OSC ACTIVITY^* (nmol/h mg protein)
Temperature (°C)	A	B	C
30	0.13		0.26
37	0.17		0.95
45	0.44	0.94	1.30
55	0.11		0.64

A) pH 7.2, polidocanol 0.1% B) pH 8, polidocanol 0.1% C) pH 8, polidocanol 0.05%, glycerol 10%

* Homogenates contain 136 mg/mL protein in 100 mM sodium phosphate buffer. Values are the means of two separate experiments with duplicate incubations, each. Maximal deviations from the mean were less than 10%.

Table IV.  Effect of umbelliferone aminoalkyl derivatives on the activity of human OSC expressed in yeast.

Compound	IC50 (μm)^*
1	1.28
2	0.72
3	0.92
4	38.5
Ro 48-8071	0.17

* Values are the means of two separate experiments, each one carried out in duplicate. Maximal deviations from the mean were less than 10%.

Figure 3.  OSC residues interacting with the compound 2.  (in sticks) in the docking model. For comparison, the reference inhibitor Ro 48-8071 in the minimized model, as derived from X-ray [7], is also reported.

Figure 4.  OSC residues interacting with the compound 1.  (in sticks) in the docking model. For comparison, the reference inhibitor Ro 48-8071 in the minimized model, as derived from X-ray [7], is also reported.

Figure 5.  OSC residues interacting with the compound 3.  (in sticks) in the docking model. For comparison, the reference inhibitor Ro 48-8071 in the minimized model, as derived from X-ray [7], is also reported.

Figure 6.  OSC residues interacting with the compound 4.  (in sticks) in the docking model. For comparison, the reference inhibitor Ro 48-8071 in the minimized model, as derived from X-ray [7], is also reported.

R Thoma, T Schulz-Gasch, B D'Arcy, J Benz, J Aebi, H Dehmlow, M Hennig, M Stihle, and A Ruf. (2004). Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase. Nature 432:118–122.

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