The MicroSource Spectrum and Killer Collections (referred to hereafter as the MicroSource collection) make up a library of 2,160 known drugs, bioactive compounds and natural products. We screened the entire MicroSource collection against two representative laboratory strains of P. falciparum: 3D7 (drug-sensitive (8
)) and W2 (drug-resistant (9
)). The growth inhibition assay we use consists of a three-day incubation of each compound with P. falciparum cultured in human erythrocytes, followed by detection and quantification of parasite growth via flow cytometry, as detailed in the Methods & Materials Section (6
). Industrial high throughput screening efforts normally use compound concentrations of 1 to 10 μM for cell-based assays. We performed our initial screen at 10 μM final concentration. The quality of the results was found to be quite high, with a median Z-prime factor of 0.75 (Z-prime is a measure of assay quality, with a maximum of 1.0 indicating an ideal assay, and > 0.5 indicating a good, reliable assay(10
)). More than 50% of the compounds in this library exhibited some amount of malaria growth inhibition, presumably due to the nature of the library.
Compounds with Z-score greater than or equal to 1.65, as determined by comparison to negative controls (corresponding to a p-value less than or equal to 0.05), are considered statistically significant hits in biological screening assays. We initially used this cutoff to determine the hit list for active P. falciparum inhibitors in our screen. However, this cutoff included only compounds with 100% inhibition activity against 3D7 and W2. We therefore relaxed the cutoff to a Z-score of 1.50 (corresponding to a p-value less than or equal to 0.067), which then included 126 compounds with inhibition activity greater than 97.5% against 3D7 and 228 compounds with inhibition activity greater than 93.7% against W2. 103 compounds inhibited both 3D7 and W2 P. falciparum strains, making a total of 251 compounds for further investigation. This subset of compounds was screened again at lower concentrations of 1 μM and 100 nM.
72 compounds exhibited significant activity (defined here as greater than 70% growth inhibition relative to control) at 1 μM and 19 compounds exhibited significant activity at 100 nM. The 72 compounds with greater than 70% growth inhibition relative to control at 1 μM were flagged for further investigation (see Supplemental ). Literature searches were conducted to determine current therapeutic uses, toxicity information, and any prior identification of antimalarial activity. Compounds within this set of 72 that lacked literature evidence of anti-P. falciparum activity were then investigated for dose response behavior.
Supplemental Table 1
Compounds with greater than 70% inhibition activity against Plasmodium falciparum at 1 μM.
13 of the 72 active inhibitors of P. falciparum were previously known antimalarial therapeutic agents. These compounds served as an excellent internal validation of our screening assay, as we were blinded to the identities of the compounds. The known antimalarial therapeutics we identified were all active at 100 nM and included: quinine (11
), quinidine (12
), chloroquine and hydroxychloroquine (13
), amodiaquine (14
), cinchonine (15
), cinchonidine (16
), mefloquine (17
), bebeerine, atovaquone (18
), pyrimethamine (13
), dihydroartemisinin (19
) and quinacrine (20
). Six more members of the MicroSource Collection inhibited P. falciparum growth at a concentration of 100 nM. These compounds have been studied previously and confirmed to have antimalarial activity: tetrandrine (21
), actinomycin D (23
), anisomycin (24
), puromycin (24
), methotrexate (25
), and pentamidine (26
). 17 additional known inhibitors of P. falciparum were identified with significant activity at a concentration of 1 μM.
To our knowledge, 36 compounds have not been studied previously for antimalarial activity and inhibited P. falciparum growth at 1 μM. 19 of these compounds were known therapeutics. This set included known antibiotics, topical anti-infectives, natural products, a known anti-trypanasomal, anti-neoplastics, vasodilators and anti-psychotics. Effective 50% inhibitory concentration values (EC50’s) of these novel antimalarial inhibitors are listed in
Representative EC50 values and distribution of MicroSource antimalarial inhibitors with optical, intraenous and oral delivery routes.