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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.
 
Method:
 
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
Original PfATP4 Activities.jpg
Initial PfATP4data.pdf
Compound screen - Summary for Mat.pdf