Synthesis and structure-activity relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and discovery of a competitive inhibitor

Figures & data

Figure 1. Sulphatide synthesis by CST: galactosylceramide (cerebroside) is converted to sulphatide by CST in the presence of PAPS as sulphate donor.

Figure 2. Chemical structures of aromatic dyes known as CST inhibitors competing with the co-substrate PAPS.

Scheme 1. Reagents and conditions: (a) (i) PMe₃, THF, then H₂O; (ii) appropriate RCOOH, EDC, CH₂Cl₂; (b) H₂, Pd black; (c) (i) PMe₃, THF, then H₂O; (ii) (Boc)₂O, Et₃N, CH₂Cl₂; (d) (i) HCl, AcOH, H₂O; (ii) nervonic acid, oxalyl chloride, reflux; the acyl chloride is added to crude amine in THF/aq. NaOAc.

Table 1. Investigation of analogues of galactocerebroside as substrates of CST^a.

Compound	Structure	Percent conversion^a at 100 µM	Km^a (µM)	Vmax^a (µmol/min/mg protein)	kcat/Km^a
β-Glycosides
3 Galactosylceramide^b		100%^c	60.3^18	0.0738^18	123^18
8 Psychosine		n.d.	103^18	0.105^18	102^18
9 Glucosylceramide^b		19%	n.d.	n.d.	n.d.
10 β-KRN7000		42%	550	0.113	20.5
α-Glycosides
11		40%	907	0.1080	11.9
12		30%	358	0.0449	12.5
13		22%	392	0.0584	15.0
14		32%	493	0.0590	12.0
15		20%	n.d.	n.d.	n.d.
16		9%	n.d.	n.d.	n.d.
17		25%	521	0.0519	10.0
18 KRN7000		51%	438	0.0758	17.3
19		32%	751	0.0834	11.1
20		15%	n.d.	n.d.	n.d.
21		16%	n.d.	n.d.	n.d.
22		19%	n.d.	n.d.	n.d.

^aStandard errors were typically below 30% of the reported mean values.

^bR: saturated or unsaturated alkyl or hydroxyalkyl residue (C16–C27).

^cConversion of physiological substrate was set at 100%.

K_m and V_max values are shown in bold.

Figure 3. Enzyme kinetics of galactosylceramide sulphotransferase for selected substrates. For K_m and V_max values see Table 1.

Table 2. CST inhibitory activity of galactocerebroside analogues

Compound	Structure	Ki ± SEM (µM) versus galactosylceramide (% inhibition at 100 µM)	Ki ± SEM (µM) versus KRN7000 (% inhibition at 100 µM)
β-Glycosides
9 Glucosylceramide	R = saturated or unsaturated alkyl or hydroxyalkyl residue	(9%)	(−5%)
α-Glycosides
16		127 ± 12	159 ± 55
20		(4%)	(1%)
21		(4%)	(−30%)
22		(−7%)	(1%)

Figure 4. Concentration–inhibition curve of the galactosylceramide sulphotransferase (CST) inhibitor 16. The curve had to be extrapolated due to limited solubility of 16. A K_i value of 127 ± 12 µM versus galactosylceramide as a substrate was determined.