Biological Evaluation of Compounds

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.

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:

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

The following compounds have been evaluated by Kiaran Kirk and Adele Lehane in their ion regulation assay, and will now be evaluated vs ATP4-resistant mutants in Kiaran's lab. The same samples are being sent to David Fidock's lab at Columbia to testing vs his ATP4 resistant mutants, for comparison. Compounds being shipped 28th August 2014. Results will be posted to this lab notebook when obtained.

Adelaide Dennis, from Kiaran Kirk's lab at the ANU, sent data on June 9th by email:

"I have attached my raw data from experiments testing the effect of compounds OSM-S-4/5/35/51/106/111 at 150 µM and 1 µM on [Na+]i of saponin-isolated SBFI-loaded trophozoite stage 3D7 parasites.

Originally sent: Compounds sent for parasiste ion regulation assays at ANU

These compounds were tested at 150 µM and 1 µM. At 150 µM OSM-S-5 appeared to increase [Na+]i within the parasite. At 150 µM OSM-S-4 did not cause an increase in [Na+]i, and the remaining four compounds (35, 51, 106, 111) caused an 'optical effect'. In this assay, excitation wavelengths are 340 nm and 380 nm, while emission is measured at 520 nm. An 'optical effect' occurs when the addition of a compound causes a disturbance at one of these wavelengths.

At 1 µM OSM-S-4, OSM-S-5 and OSM-S-111 showed no effect on [Na+]i within the parasite, and OSM-S-35, OSM-S-51 and OSM-S-106 showed slight 'optical effects'.

Fluorescence intensity (FI) was calibrated to [Na+]i for the traces showing compounds tested at 150 µM. Fluorescence intensity was not calibrated for traces showing compounds tested at 1 µM."

This assay is used to suggest whether bioactives are targeting PfATP4. This screen was carried out to investigate whether OSM Series 1 or 3 might have such a mechanism of action, as Series 4 appears to. The data suggest they do not.

Compounds assayed:

Preparation of OSM-S-4

Preparation of OSM-S-5

Preparation of OSM-S-35

Preparation of OSM-S-51

Preparation of OSM-S-106

Preparation of OSM-S-111

Adelaide made her original lab book entry available - the team uses Labarchives:

https://mynotebook.labarchives.com/share/Adelaide%2520Dennis/MTIwLjl8MjYyODgvOTMvVHJlZU5vZGUvNTI1ODM5NDMwfDMwNi45

Professor Kiaran Kirk's laboratory at the Australian National University have kindly agreed to test some OSM compounds in a their 'parasite ion regulation assay'.

The compounds below have been sent for evaluation:

InChi (in order OSM-S-4, 5, 35, 51, 111, 106)

InChI=1S/C13H12FNO2/c1-8-7-12(13(16)17)9(2)15(8)11-5-3-10(14)4-6-11/h3-7H,1-2H3,(H,16,17)

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)

InChI=1S/C22H19N3OS/c1-15-13-17(16(2)25(15)19-11-7-4-8-12-19)14-20-21(26)24-22(27-20)23-18-9-5-3-6-10-18/h3-14H,1-2H3,(H,23,24,26)/b20-14-

InChI=1S/C21H18N4OS/c1-14-12-16(15(2)25(14)19-10-6-7-11-22-19)13-18-20(26)24-21(27-18)23-17-8-4-3-5-9-17/h3-13H,1-2H3,(H,23,24,26)/b18-13-

InChI=1S/C22H19N3OS/c1-15-13-17(16(2)25(15)19-11-7-4-8-12-19)14-20-21(26)24-22(27-20)23-18-9-5-3-6-10-18/h3-14H,1-2H3,(H,23,24,26)/b20-14-

InChI=1S/C12H10N4O2S2/c13-12-11-9(15-6-16-12)5-10(19-11)7-2-1-3-8(4-7)20(14,17)18/h1-6H,(H2,13,15,16)(H2,14,17,18)

Excel File:

ChemDraw:

Ion regulation assays to be performed by Kiaran Kirk on representative compounds from early series of OSM project. Relevant consultation:

https://github.com/OpenSourceMalaria/OSM_To_Do_List/issues/77

Draft Chemdraw of structures posted here. Require addition of potencies and molecular weights and then re-posting, partly as reference for Kirk Lab when doing testing.