Inhibition of PrP^Sc formation in scrapie infected N2a cells by 5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine derivatives

Figures & data

Figure 1. Structures of the tested 5,7,8-trimethyl-1,4-benzoxazine derivatives.

Table 1. Efficacy and cellular toxicity screen of 1,4-benzoxazine compounds for PrPSc inhibition in ScN2a cells

Code	MW	EC50 μM	LD50 μM
TC121	283.16	1.17	10.00
TC145	256.14	-	4.17
TC 158	211.69	20.00	50.00
TC128	283.36	-	>5.00
TC143	283.36	-	>15.00
TC161	283.16	-	>60.00

Figure 2. 22L-ScN2a cells. Effect of 5,7,8-trimethyl-1,4-benzoxazine derivatives on PrP^Sc levels by western blot analysis. (A) inhibitors of PrP^Sc formation. (B) compound TC145 with no inhibitory effects on PrP^Sc accumulation. Following incubation with each compound, the cells were treated (+) or not treated (-) with PK. All treatments were performed in duplicates (C_D1, C_D2 for the control, untreated cells and B_D1, B_D2 for the 5,7,8-trimethyl-1,4-benzoxazines -treated cells). Two representative blots for each active compound, corresponding to different cell passages, are presented in order to show the reproducibility of the acquired results. For PrP-immunostaining, 6H4 was used, whereas for signal normalization, blots were probed with β-actin (C4) antibody.

Figure 3. Dose response and toxicity curves. EC₅₀ value, were 50% of the pathological isoform PrP^Sc is reduced, is 1.17 μM for TC121 (A) and 20 μM for TC158 (C). LD₅₀ value, as derived from the representative toxicity curves, is 10 μM for TC121 (B) and 50 μM for TC158 (D).

Figure 4. N2a cells. Effect of 5,7,8-trimethyl-1,4-benzoxazine derivatives on PrP^C levels by western blot analysis. All treatments were performed in duplicates (C_D1, C_D2 for the control, untreated cells and B_D1, B_D2 for the 5,7,8-trimethyl-1,4-benzoxazines-treated cells).

Figure 5. (A) Relative expression of the PRNP gene in untreated “control Sc N2a” cells and cells treated with different 5,7,8,-trimethyl-1,4-benzoxazine derivatives. (B) Relative expression of the PRNP gene in untreated “control N2a” cells and cells treated with TC121 and TC158. As expected, there is no signal in negative control samples without cDNA. (C) 2% agarose gel showing representatively the amplified fragments of the tested genes. M: relative molecular mass markers, 1, 3, 5, 7: PCR products amplified with the β-actin, PrP, RPL32 and GAPDH primers in the presence of cDNA and 2, 4, 6, 8: in the absence of cDNA.

Table 2. Primer sequences used for the amplification of fragments from genes encoding murine PrP (PRNP) and three reference proteins: β-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), ribosomal protein L32 (Rpl32)

Primer	Gene	Reference number^a	Sequence (5’-3’)	Amplified fragment (bp)
mPrP_103F	PRNP	M13685.1	AATCAGTGGAACAAGCCCAG	103
mPrP_103R	PRNP	M13685.1	CCAGCATGTAGCCACCAAG	103
moACT_139F	Actb	NM_007393.3	TGTTACCAACTGGGACGACA	139
moACT_139R	Actb	NM_007393.3	CTGGGTCATCTTTTCACGGT	139
mGAPDH_132F	Gapdh	NM_008084.2	AACTTTGGCATTGTGGAAGG	132
mGAPDH_132R	Gapdh	NM_008084.2	GGATGCAGGGATGATGTTCT	132
mRPL32_111F	Rpl32	NM_172086.2	AGGCACCAGTCAGACCGATA	111
mRPL32_111R	Rpl32	NM_172086.2	GATGTTGGGCATCAGGATCT	111

^a Reference number of the gene sequence as deposited in Genbank.