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14th June 2015 @ 22:46

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. 

Post originally authored by Alice Williamson. Edited 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 @ 22:26

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 @ 13:56

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
19th May 2015 @ 12:42

Compounds synthesised by Tom Foley and Jamie Scott, masters research students in Edinburgh, from November 2014 - March 2015.

 

EDIreport07052015.pdf

 

 

Lot 4 series 3.png

Lot 4 series 4.png
Attached Files
22nd January 2015 @ 23:47

Melanie Ridgeway in Kiaran Kirk’s laboratory completed some cross-resistance studies, looking at growth-inhibition by several of the OSM Series 4 compounds of several Kirk ATP4 mutant strains (generated by long-term exposure to increasing concentrations of three Malaria Box hits, as documented in their recent paper).

The data are attached to this post. Each graph is averaged from three independent experiments. Upshot: Data are supportive of Series 4 targeting ATP4.

Series 4 growth assays[1].pdf


Each of the six panels shows the response of four strains (the Dd2 parent and three different PfATP4 mutants) to a different compound.  Panels A-C show growth inhibition by three different Series 4 compounds. Panel D shows growth inhibition by NITD/KAE 609. Panels E and F show growth inhibition by chloroquine and artemisinin.

The three mutant PfATP4 strains show resistance to all three of the Series 4 compounds (Panels A-C) and to NITD609 (Panel D), providing further evidence of a common mechanism of action.

(Note: For two of the three mutant strains there is no significant shift of the chloroquine or artemisinin dose-response curves. But for one of the three mutants there is what appears to be small shifts in the artemisinin and chloroquine dose-response curves, with the artemisinin and chloroquine curves shifted in opposite directions.  The meaning of this is currently unclear.)

Attribution for these data: Kiaran Kirk, Adele Lehane, Melanie Ridgeway. Received by email to Mat Todd, 20th October 2014.

Compound Structures:

Compounds used to generate ATP4 mutants


Compound Strings:


NITD609 KAE609 C[C@@H](C1)N[C@@]2(C(NC3=C2C=C(Cl)C=C3)=O)C4=C1C5=CC(F)=C(Cl)C=C5N4 InChI=1S/C19H14Cl2FN3O/c1-8-4-11-10-6-14(22)13(21)7-16(10)23-17(11)19(25-8)12-5-9(20)2-3-15(12)24-18(19)26/h2-3,5-8,23,25H,4H2,1H3,(H,24,26)/t8-,19+/m0/s1 CKLPLPZSUQEDRT-WPCRTTGESA-N

MMV011567 O=C(C1=CC(Cl)=C(OC)C=C1)NC2=NON=C2C3=CC=C(OC)C(OC)=C3 InChI=1S/C18H16ClN3O5/c1-24-13-6-5-11(8-12(13)19)18(23)20-17-16(21-27-22-17)10-4-7-14(25-2)15(9-10)26-3/h4-9H,1-3H3,(H,20,22,23) RUPCNQRICRCGRU-UHFFFAOYSA-N

MMV007275 ClC(C=C1C(NC2=C(C)C=CC(F)=C2)=O)=CC=C1NC3=CC=CC=C3 InChI=1S/C20H16ClFN2O/c1-13-7-9-15(22)12-19(13)24-20(25)17-11-14(21)8-10-18(17)23-16-5-3-2-4-6-16/h2-12,23H,1H3,(H,24,25) YDYIMBIBJGKUCE-UHFFFAOYSA-N

MMV670944 O=C(NC1=CC=NC(C(F)(F)F)=C1)C2=CN=CC3=NN=C(C4=CC=C(OC(F)F)C=C4)N32 InChI=1S/C19H11F5N6O2/c20-18(21)32-12-3-1-10(2-4-12)16-29-28-15-9-25-8-13(30(15)16)17(31)27-11-5-6-26-14(7-11)19(22,23)24/h1-9,18H,(H,26,27,31) UEOZBGRWGZEYED-UHFFFAOYSA-N

MMV670767 O=C(NC1=CC=C(F)C(Cl)=C1)C2=CN=CC3=NN=C(C4=CC=C(OC(F)F)C=C4)N32 InChI=1S/C19H11ClF3N5O2/c20-13-7-11(3-6-14(13)21)25-18(29)15-8-24-9-16-26-27-17(28(15)16)10-1-4-12(5-2-10)30-19(22)23/h1-9,19H,(H,25,29) WFJPUMPZNOYIKX-UHFFFAOYSA-N

MMV671677 O=C(NCC1=C(Cl)C(C(F)(F)F)=CC=C1)C2=CN=CC3=NN=C(C4=CC=C(OC(F)F)C=C4)N32 InChI=1S/C21H13ClF5N5O2/c22-17-12(2-1-3-14(17)21(25,26)27)8-29-19(33)15-9-28-10-16-30-31-18(32(15)16)11-4-6-13(7-5-11)34-20(23)24/h1-7,9-10,20H,8H2,(H,29,33) YLYCMIJACQNNRW-UHFFFAOYSA-N




Attached Files