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14th June 2015 @ 21:46

Stephan Meister from Elizabeth Winzeler's laboratory at UCSD tested two Series 1 Compounds in a liver stage assay to provide some greater context for the first Open Source Malaria Paper.

The aryl pyrrole OSM-S-5 and near neighbour OSM-S-38 were both blood-stage active compounds from Series 1 and were both evaluated in this assay. OSM-S-5 showed an IC50 of 23 uM, whilst OSM-S-38 had an activity of 19 nM with no HepG2 cytotoxicity. These data may provide some evidence that these two series of compounds may have distinct mechanisms of action.

Previously OSM-S-111, another of the near neighbours (analogous to OSM-S-38) had shown moderate potency in the same assay:

Liver stage malaria activities of OSM-S-106 and OSM-S-111 in Plasmodium berghei.

 

Data:

OSM_results.xlsx

General assay principle:

This assay is based on the murine Plasmodium berghei species transformed with Luciferase. Hepatic human transformed cells (HepG2), pretreated for two hours with the compound to investigate, are infected with freshly dissected P. berghei Luciferase sporozoites. After 48 hours of incubation with the compound to investigate, the viability of P. berghei exoerythrocytic forms (EEF) is measured by bioluminescence.

This assay allows us to identify compounds with an eventual activity against sporozoite infection of liver cell as well the viability of liver schizonts. 

Update Sept 10th 2015

The compounds were re-tested by Stephan and Jenya Antonova, giving broadly similar results: OSM-S-5 showed an IC50 of 14 uM, whilst OSM-S-38 had an activity of 13 nM.

Pberghei results_2015_08_OSDD.xlsx

This raises the question of whether OSM-S-38 should be tested in the Pc liver stage assay at the BPRC in the Netherlands.

 

 

This post originally authored by Alice Williamson. Edited by Mat Todd. Updated with new data by Mat Todd.

SOP :

Parasites.

Plasmodium berghei Luciferase sporozoites were obtained by dissection of infected A. stephensi mosquito salivary glands supplied by the New York University Insectary. Dissected salivary glands were homogenized in a glass tissue grinder and filtered twice through Nylon cell strainers (40 μm pore size, BD Falcon) and counted using a hemocytometer. The sporozoites were kept on ice until needed. 

Strings:

OSM-S-5: TCMDC-123812 CC(N1C2=CC=C(F)C=C2)=C(C(OCC(N)=O)=O)C=C1C InChI=1S/C15H15FN2O3/c1-9-7-13(15(20)21-8-14(17)19)10(2)18(9)12-5-3-11(16)4-6-12/h3-7H,8H2,1-2H3,(H2,17,19) YSUCFIZUNLQZDX-UHFFFAOYSA-N

OSM-S-38: CC1=CC(/C=C(C(N/2)=O)\SC2=N/C3=CC=CC=C3)=C(C)N1C(C=C4)=CC=C4C(F)(F)F InChI=1S/C23H18F3N3OS/c1-14-12-16(15(2)29(14)19-10-8-17(9-11-19)23(24,25)26)13-20-21(30)28-22(31-20)27-18-6-4-3-5-7-18/h3-13H,1-2H3,(H,27,28,30)/b20-13- YBBWTVGRVHTTDD-MOSHPQCFSA-N

OSM-S-111: O=C(/C(S/1)=C/C2=C(C)N(C3=CC=C(OC)C=C3)C(C)=C2)NC1=N\C4=CC=CC=C4 InChI=1S/C23H21N3O2S/c1-15-13-17(16(2)26(15)19-9-11-20(28-3)12-10-19)14-21-22(27)25-23(29-21)24-18-7-5-4-6-8-18/h4-14H,1-3H3,(H,24,25,27)/b21-14- KXIVXNPEYYNDHE-STZFKDTASA-N


Attached Files
14th June 2015 @ 21:26

Compounds 1, 2, 3, 5, 8, 9 and 10 from the top ten compounds were sent to have their efficacy evaluated against Plasmodium falciparum in-vitro at Syngene.

All values in nanoMolar.

They were repeated twice owing to poor quality statistics in the first assay:

 The results didn't reveal any 'killer' compounds but provided the team with some important SAR information.

  • Aliphatic groups in top right of the molecule kill activity.
  • Unsubstituted aromatic group in top right of the molecule kill activity.
  • Substituted pyridines more potent than unsubstituted.
  • Replacing alcohol side chain with methanol kills activity.
  • Polar group in benzylic position of side chain improves activity but mono-methylation of benzylic amine kills activity.

 

The team are currently digesting the latest data and a new set of target molecules will be proposed later today and discussed in an online meeting to be arranged next week. 

General assay principle:
"This protocol assesses compound efficacy against Plasmodium falciparum in-vitro. This assay is using [3H]-hypoxanthine incorporation or DNA labeling by SYBR Green as a markers of parasite growth. 
This procedure is designed for use with culture adapted P. falciparum strains or clones only. On one 96-well plate typically 03 drugs are tested in duplicate. Standard strains: Plasmodium falciparum, NF54 (sensitive to all known drugs), Plasmodium falciparum, K1 (chloroquine and pyrimethamine resistant). The assay can be performed in dose response mode (12 concentrations in duplicate, 24 data points) which allows determining IC50, or in single concentration mode (one concentration in triplicate, 3 data points) which allows determining the percentage of growth inhibition.
For more information, see Desjardins et al. (Antimicrob. Agents Chemother., 16(6), 710, 1979)."

 

(Post originally authored by Alice Williamson)

Attached Files
2nd June 2015 @ 12:56

A further set of Series 4 Triazolopyrazine compounds have been sent to have their efficacy evaluated against Plasmodium falciparum in-vitro at Syngene.

These compounds were 1, 2, 3, 5, 8, 9 and 10 of the top ten compounds:

May Submission Syngene.png


General assay principle:
"This protocol assesses compound efficacy against Plasmodium falciparum in-vitro. This assay is using [3H]-hypoxanthine incorporation or DNA labeling by SYBR Green as a markers of parasite growth. 
This procedure is designed for use with culture adapted P. falciparum strains or clones only. On one 96-well plate typically 03 drugs are tested in duplicate. Standard strains: Plasmodium falciparum, NF54 (sensitive to all known drugs), Plasmodium falciparum, K1 (chloroquine and pyrimethamine resistant). The assay can be performed in dose response mode (12 concentrations in duplicate, 24 data points) which allows determining IC50, or in single concentration mode (one concentration in triplicate, 3 data points) which allows determining the percentage of growth inhibition.
For more information, see Desjardins et al. (Antimicrob. Agents Chemother., 16(6), 710, 1979)."

 

(Post originally authored by Alice Williamson)


Attached Files