Discovery of small molecule inhibitors of Plasmodium falciparum apicoplast DNA polymerase

Figures & data

Figure 1. High-throughput screening. (A) The fluorescent-based DNA polymerase assay used to screen the compound libraries. The DNA template strand is a hairpin loop with a primer annealed to the loop. The 3’ end of the primer is 11 bases away from the 6 bp duplex. Strand displacement activity of the DNA polymerase separates the Cy3 dye from the Black hole quencher leading to an increase in fluorescent signal. (B) The high-throughput assay workflow. The DNA polymerase is incubated with 10 mM compound for 10 min prior to addition of the hairpin substrate. The reaction is quenched at 3 different time points and the fluorescent signal is measured and % inhibition is determined using the provided equation.

Figure 2. A scattergram of the large-scale screen of 11 compound libraries (listed at right). The overall hit rate for the screen was 0.59%.

Table 1. Results from high-throughput screening to identify inhibitors of P. falciparum apicoplast DNA polymerase.

Library	Total # of Compounds	# of Hits	Hit Rate	Dose-response reconfirmation (# of compounds)	Reconfirmation rate, %
Chembridge Diversity	43736	250	0.57	205	82
ChemDiv Diversity	56232	293	0.52	267	91
CMLD	5208	22	0.42	21	96
Life Diversity	15040	64	0.43	54	84
GreenPharma Natural Products	480	23	4.79	20	87
Life chemicals Nature Product-like	8128	114	1.4	71	62
ChemDiv CNS	26136	120	0.46	102	85
ChemDiv BFL/PPL	33864	61	0.18	53	87
Sigma Lopac	1280	35	2.73	27	77
Maybridge Hit finder 2016	14400	217	1.51	148	68
Maybridge MinHitFinder 2016	2000	18	0.9	11	61
Total	206504	1217	0.59	979	80
Average Hit Rate (%)

Figure 3. A plot of FragFp similarity for the non-toxic, specific apPOL inhibitors (102 compounds). Chemical similarity was calculated using the DataWarrior²² software package. The core structure for the two largest clusters are given. Each compound is labelled with a number corresponding to the data in Supplemental file 1. The markers are coloured according to IC₅₀ values and the number of Lipinski violations are indicated by marker shape.

Figure 4. Potential as lead compounds. A plot of the synthetic accessibility versus leadlikeness violations for the non-toxic, specific apPOL inhibitors is shown. Each compound is labelled with a number corresponding to the data in Supplemental file 1. The markers are coloured according to their IC₅₀ values and the number of Lipinski violations are indicated by marker shape. A description of synthetic accessibility and leadlikeness can be found in the main text.

Figure 5. Compounds active against cultured Plasmodium falciparum. A plot of IC₅₀ values versus EC₅₀ values is shown. The markers are coloured according to their Druglinkess values as calculated by the DataWarrior²² software package. The number of Lipinski violations are indicated by marker shape.

Table 2. Inhibition data for compounds found to be active against cultured Plasmodium falciparum.

Molecule	KUID	IC50 Pf-apPOL (µM)	IC50 Pb-apPOL (µM)	IC50 Bb-apPOL (µM)	EC50 (µM)	EC50 + IPP (µM)
1	KU0263501	5.9 ± 0.9	7.1 ± 0.6	3.7 ± 0.2	8.6 ± 0.4	9.3 ± 0.5
21	KU0036696	8 ± 1	9.7 ± 1.6	8 ± 2	3.1 ± 0.1	3.4 ± 0.1
27	KU0260920	15 ± 1	ND	ND	10 ± 1	9 ± 1
31	KU0007309	15 ± 1	ND	15 ± 5	8.9 ± 0.5	8.7 ± 0.6
34	KU0241474	7.2 ± 0.9	ND	ND	11.3 ± 0.9	10.0 ± 0.7
55	KU0177470	10 ± 2	51.9 ± 3.5	1.5 ± 1.0	3.5 ± 0.3	4.3 ± 0.2
94	KU0261556	7.6 ± 0.6	13.9 ± 1.6	30 ± 3	4.6 ± 0.2	3.7 ± 0.5
95	KU0001071	7.4 ± 0.4	8.6 ± 0.7	2.5 ± 0.2	7.5 ± 0.5	7.4 ± 0.4
98	KU0271653	8 ± 2	ND	ND	16 ± 1	11 ± 1

Sander T, Freyss J, von Korff M, Rufener C. DataWarrior: an open-source program for chemistry aware data visualization and analysis. J Chem Inf Model 2015;55:460–73.

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