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28th August 2013 @ 04:22

Part of launch of Series 4. Five compounds were originally evaluated in ion regulation assays by Kiaran Kirk since this could indicate the compounds are inhibiting PfATP4.

Data sheet from MMV on the compounds is attached:

Initial PfATP4data.pdf

Structures of the compounds:

Original PfATP4 Activities.jpg


Ion regulation activity (vs potency):

MMV669000: no (potency: inactive)

MMV669304: yes (potency: 280 nM)

MMV669360: yes (potency: 356 nM)

MMV669542: yes (potency: 185 nM)

MMV669848: yes (potency: 114 nM)

(Note the correlation: compound inactive in ion regulation assay is the inactive Pf analog)

Raw ion regulation assay data (older codes used - correlations below)
Compound screen - Summary for Mat.pdf
MMV669000 (labelled PCCBTAK-0083) did not dissipate the plasma membrane Na+ gradient or increase the plasma membrane pH gradient consistent with it not inhibiting PfATP4 at the concentration tested.
The other compounds (MMV669304 (PCCBTAK-0127), MMV669360 (PCCBTAK-0156), MMV669542 (PCCBTAK-0160) and MMV669848 (PCCBTAK-0194) dissipated the plasma membrane Na+ gradient and increased the plasma membrane pH gradient at a concentration of 2 μM, consistent with them being PfATP4 inhibitors.
The ion measurements were performed with saponin-isolated P. falciparum parasites (3D7 trophozoites) loaded with fluorescent ion indicator:  either SBFI (Na+ measurements) or BCECF (pH measurements).
In the pH experiments following the addition of the test compound we then added Concanamycin A (Con A, a specific inhibitor of the parasite’s plasma membrane H+ pump) to dissipate the pH gradient.  This was to verify that the phenotype was the same as that seen for the spiroindolones (see Spillman et al. Cell Host & Microbe paper).

The experiments were done by Adelaide Dennis, then a Research Assistant and now a PhD student in the Kirk lab


Compound Strings
MMV669000: O=C(N1CC(C=CC=C2)=C2C1)C3=CN=CC4=NN=C(C5=CC=C(OC(F)F)C=C5)N43; InChI=1S/C21H15F2N5O2/c22-21(23)30-16-7-5-13(6-8-16)19-26-25-18-10-24-9-17(28(18)19)20(29)27-11-14-3-1-2-4-15(14)12-27/h1-10,21H,11-12H2
MMV669304: FC(F)OC(C=C1)=CC=C1C2=NN=C3C=NC=C(CCCC4=CC=CC=C4)N32; InChI=1S/C21H18F2N4O/c22-21(23)28-18-11-9-16(10-12-18)20-26-25-19-14-24-13-17(27(19)20)8-4-7-15-5-2-1-3-6-15/h1-3,5-6,9-14,21H,4,7-8H2
MMV669360: FC(F)OC(C=C1)=CC=C1C2=NN=C3C=NC=C(COCC4=CC=C(F)C(F)=C4)N32; InChI=1S/C20H14F4N4O2/c21-16-6-1-12(7-17(16)22)10-29-11-14-8-25-9-18-26-27-19(28(14)18)13-2-4-15(5-3-13)30-20(23)24/h1-9,20H,10-11H2
MMV669542: FC(F)OC(C=C1)=CC=C1C2=NN=C3C=NC=C(C(NC4=CC=CC(Cl)=C4)=O)N32; InChI=1S/C19H12ClF2N5O2/c20-12-2-1-3-13(8-12)24-18(28)15-9-23-10-16-25-26-17(27(15)16)11-4-6-14(7-5-11)29-19(21)22/h1-10,19H,(H,24,28)
MMV669848: FC(F)OC(C=C1)=CC=C1C2=NN=C3C=NC=C(CN4CC(C=CC=C5)=C5C4)N32; InChI=1S/C21H17F2N5O/c22-21(23)29-18-7-5-14(6-8-18)20-26-25-19-10-24-9-17(28(19)20)13-27-11-15-3-1-2-4-16(15)12-27/h1-10,21H,11-13H2

Attached Files
1st August 2013 @ 05:06


OSM-S-106 had a liver stage IC50 of 2.348 μM and OSM-S-111 had an IC50 of 345.4 nM. OSM-S-106 had no effect on the HepG2 host cells at the 10uM maximal concentration, while OSM-S-111 displayed some cytotoxicity with an IC50 of 4.386 μM.


OSM-S-106 and 111.xlsx

IC50 graphs.pdf

Materials & Methods

Parasites. P. berghei Luciferase sporozoites are 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), spun down at 10,000xg, resuspended in media and counted using a hemocytometer. The sporozoites are diluted to a final concentration of 200 sporozoites per μl and kept on ice until needed.

Cell lines. HepG2-A16-CD81EGFP cells stably transformed to express a GFP-CD81 fusion protein (Silvie, O. 2006), are cultured at 37°C in 5% CO2 in DMEM (Invitrogen, Carlsbad, USA) supplemented with 10% FCS, 0.29 mg/ml glutamine, 100 units penicillin and 100 μg/ml streptomycin.

Sporozoite invasion assay. 3x103 HepG2-A16-CD81EGFP cells in 5μl of medium (2x105 cells/ml, 5%FBS, 5xPen/Strep/Glu) are seeded in 1536-well plates 20-26 hours prior to the actual infection. 18 hours prior to infection, 50nl of compound in DMSO (0.5% final DMSO concentration per well) are transferred with a PinTool (GNF Systems) into the assay plates (10 μM final concentration). Atovaquone and 0.5% DMSO are used as positive and negative controls, respectively. Penicillin and streptomycin are added to the sporozoite preparation for a final 5x-fold increased concentration in the well. The HepG2-A16-CD81EGFP cells were then infected with 103 sporozoites per well (5 μl) with a single tip Bottle Valve liquid handler (GNF), and the plates spun down at 37°C for 3 minutes in an Eppendorf 5810 R centrifuge with a centrifugal force of 330x on lowest acceleration and brake setting. After incubation at 37°C for 48 hours the EEF growth was quantified by bioluminescence.

Bioluminescence quantification of exo-erythrocytic forms (EEFs). Media is removed by spinning the inverted plates at 150xg for 30 seconds. 2 μl BrightGlo (Promega) are dispensed with the MicroFlo (BioTek) liquid handler. Immediately after addition of the luminescence reagent, plates are vortexed at the median intensity setting for 10 seconds and read by the Envision Multilabel Reader (PerkinElmer). IC50 values are obtained using measured bioluminescence intensity and a non-linear variable slope four parameter regression curve fitting model in Prism 6 (GraphPad Software Inc). 

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