Deciphering the enzymatic target of a new family of antischistosomal agents bearing a quinazoline scaffold using complementary computational tools

Figures & data

Figure 1. In vitro findings for NPD-1246 (1) previously reported¹⁴.

Table 1. In vitro metabolic stability of NPD-1246: percentage of parent compound remaining over time in the presence of mouse liver microsomes.

% Parent compound remaining upon incubation
Phase I/II	Time (min)	NPD-1246		Diclofenac
		Average	SD	Average	SD
CYP450-NADPH	0	100	−	100	−
	15	66	5	87	2
	30	27	9	70	6
	60	8	2	48	1
		(n = 2)		(n = 2)
UGT enzymes	0	100	−	100	−
	15	111	4	41	1
	30	111	2	44	8
	60	106	5	34	2
		(n = 2)		(n = 2)

Figure 2. Effect of NPD-1246 alone (in a dose of 20 or 10 mg/kg/day) or in combination with PZQ (10 mg/kg/day each) for 5 days treatment on (A) worm burden, (B) tissue egg load and (C) oogram pattern in S. mansoni-infected mice sacrificed 10 days post end of treatment. *Significantly different from infected control at p < 0.05. ^#Significantly different from PZQ group at p < 0.05. Numbers above columns and between parentheses represent percentage change from infected control group.

Figure 3. Metabolic site prediction using SMARTCyp web server for NPD-1246. Top-ranked sites (red circles) and minor sites (blue circles) predicted to be metabolised by (A) CYP_2C9, (B) CYP_2D6 and (C) CYP_3A4.

Scheme 1. Synthesis of 3-benzyl-1-(4-(trifluoromethyl)benzyl)quinazolin-2,4(1H,3H)-diones 1–13.

Table 2. Mature worm killing and ovipositing at 100 µM and 50 µM of new quinazolines (2–13) in comparison with previously reported data for NPD-1246 (1)¹⁴.

			Worm killing^a (% of total)		Uncoupling		Reduction in number of eggs (%)
R^1	R^2		100 µM	50 µM	100 µM	50 µM	100 µM	50 µM
H	H	NPD-1246 (1)	100	53	Yes	Yes	100	100
H	F	2	100	0	Yes	No	100	100
H	OMe	3	75	0	Yes	Yes	100	100
6-Br	F	4	100	13	Yes	Yes	100	100
6-Br	OMe	5	100	0	Yes	No	100	31
7-Br	F	6	88	0	Yes	Yes	100	100
7-Br	OMe	7	0	0	No	No	50	35
6-Cl	F	8	100	0	Yes	Yes	100	100
6-Cl	OMe	9	100	29	Yes	Yes	100	100
6,7-diOMe	F	10	100	93	Yes	Yes	100	100
6,7-diOMe	OMe	11	86	0	Yes	Yes	100	100
6-Me,8-Br	F	12	0	0	Yes	No	100	50
6-Me,8-Br	OMe	13	33	0	Yes	Yes	100	100

^aThe final recording of worm killing was determined on day 5 (end of the observation period).

Table 3. Mature (6 weeks old) worm killing and ovipositing under different concentrations of selected compounds in comparison with NPD-1246 (1) data¹⁴ previously reported.

	Worm killing^a (% of total)					EC50	Uncoupling					Reduction in number of eggs (%)
	100 µM	50 µM	25 µM	10 µM	5 µM	(µM)	100 µM	50 µM	25 µM	10 µM	5 µM	100 µM	50 µM	25 µM	10 µM	5 µM
NPD-1246 (1)	100	53	0	0	0	50	Yes	Yes	Yes	No	No	100	100	100	20	10
9	100	88	0	0	0	47	Yes	Yes	Yes	No	No	100	100	100	73	67
10	100	100	50	8	0	25	Yes	Yes	Yes	No	No	100	100	100	80	77

^aThe final recording of worm killing was determined on day 5 (end of the observation period).

Table 4. Mature (male & female) worm killing under different concentrations of selected compounds in comparison with NPD-1246 (1) data¹⁴ previously reported.

	Worm killing^a (male worms)					EC50	Worm killing^a (female worms)					EC50
	100 µM	50 µM	25 µM	10 µM	5 µM	(µM)	100 µM	50 µM	25 µM	10 µM	5 µM	(µM)
NPD-1246 (1)	100	50	0	0	0	50	100	57	0	0	0	50
9	100	100	0	0	0	41	100	75	0	0	0	49
10	100	100	63	14	0	20	100	100	33	0	0	26

^aThe final recording of worm killing was determined on day 5 (end of the observation period).

Table 5. In vitro metabolic stability of 9 and 10: percentage of parent compound remaining over time in the presence of mouse and human liver microsomes.

			% Parent compound remaining upon incubation
Microsomes	Phase I/II	Time (min)	9		10		Diclofenac
			Average	SD	Average	SD	Average	SD
Mouse	CYP450-NADPH	0	100	–	100	–	100	–
		15	91	3	85	5.3	61
		30	51	3.3	39	0.55	34
		60	44	0.45	32	0.43	27
			(n = 2)				(n = 1)
	UGT enzymes	0	100	–	100	–	100
		15	101	2.6	89	8.3	34
		30	103	3.6	83	8.7	32
		60	105	0.8	91	18.3	30
			(n = 2)		(n = 2)		(n = 1)
Human	CYP450-NADPH	0	100	–	100	–	100	–
		15	104	4.9	108	3.7	27
		30	61	2.4	83	18.8	5
		60	67	3.2	65	1.3	1
			(n = 2)		(n = 2)		(n = 1)
	UGT enzymes	0	100	–	100		100
		15	94	27.0	102	2.4	11
		30	95	12.9	106	4.5	9
		60	97	14.1	98	1.8	9
			(n = 2)		(n = 2)		(n = 1)

Figure 4. Quinazoline-related structures crystallised with different target proteins according to the scaffold search in the PDB (access codes included).

Figure 5. Superposition of the crystal structures of human aldose reductase 1IEI, depicted in cyan, S. japonicum aldose reductase 4HBK in magenta and the homology model of S. mansoni aldose reductase in purple, (A) front view and (B) back view.

Figure 6. (A) Superimposition of zenarestat in the crystal structure 1IEI depicted in cyan and validation docking results depicted in purple show the high similarity between both poses in the human enzyme. (B) Detail of the zenarestat binding mode together with the main interactions found in the catalytic site of aldose reductase.

Figure 7. Detail of the binding mode of NPD-01246 in the S. mansoni aldose reductase binding site.

Botros SS, William S, Sabra AA, et al. Screening of a PDE-focused library identifies imidazoles with in vitro and in vivo antischistosomal activity. Int J Parasitol Drugs Drug Resist 2019;9:35–43.

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