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27th August 2014 @ 01:56

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.

 

Kirk and Fidock ATP4 Resistance Assay

Attached Files
14th August 2014 @ 22:58

An assay was performed on several Series 1 compounds in July 2013 by GSK to determine activity vs P. falciparum Dihydroorotate dehydrogenase (DHODH). Assay carried out by María José Lafuente. Reports/Data attached.

Compounds tested:

PMY 12-5 OSM-S-8 Preparation of OSM-S-8

PMY 27-2 OSM-S-16 Preparation of OSM-S-16

PMY 31-5 OSM-S-19 Preparation of OSM-S-19

PMY 34-1 OSM-S-21 Preparation of OSM-S-21

ZYH 3-1 OSM-S-35 Preparation of OSM-S-35

ZYH 5-1 OSM-S-37 Preparation of OSM-S-37

ZYH 6-1/6-2 OSM-S-38 Preparation of OSM-S-38

ZYH 7-2 OSM-S-39 Preparation of OSM-S-39

ZYH 10-2 A OSM-S-42 Preparation of OSM-S-42

ZYH 10-2 B OSM-S-43 Preparation of OSM-S-43

ZYH 12-1/12-2 OSM-S-45 Preparation of OSM-S-45

ZYH 15-1 OSM-S-48 Preparation of OSM-S-48
ZYH 16-1 OSM-S-49 Preparation of OSM-S-49
ZYH 17-1 OSM-S-50 Preparation of OSM-S-50
ZYH 18-1 OSM-S-51 Preparation of OSM-S-51
ZYH 19-1 OSM-S-52 Preparation of OSM-S-52
ZYH 22-3 OSM-S-54 Preparation of OSM-S-54
ZYH 23-1 OSM-S-55 Preparation of OSM-S-55

PMY12-1 OSM-S-7 Preparation of OSM-S-7
PMY 2-4 OSM-S-2 Preparation of OSM-S-2
PMY 6-1 OSM-S-3 Preparation of OSM-S-3
PMY 8-2 OSM-S-4 Preparation of OSM-S-4
PMY 11-2 (TCMDC-123794) OSM-S-6 Preparation of OSM-S-6

PMY 10-2 (TCMDC-123812) OSM-S-5 Preparation of OSM-S-5

PMY 14-1 OSM-S-9 Preparation of OSM-S-9

Result: No activity

 

(This post authored by Mat Todd)

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31st July 2014 @ 12:54

Update 13th Aug 2014 (original post below)

Final compound list is this, with some reasons (following input from Paul Willis):

Aldehyde Oxidase Shortlist 2

Codes of compounds to be sent:

MMV668956
MMV669784
MMV669844
MMV669846
MMV670246
MMV670250
MMV670944
MMV670946
MMV671927
MMV672727
MMV672939

Plus ONE of these two, whichever has the most sample available:

MMV670947
MMV672942

 

Original Post:

Exciting news. We have a lab willing and able to run the aldehyde oxidase assay, and not just any lab but one of the experts in this field - Scott Obach’s lab at Pfizer, Groton.

(Interestingly Pfizer are the originators of the currently most active series in OSM, Series 4, on which this assay will be deployed).

Scott has previously published on the increasing profile of AO in drug discovery/development. Several Series 4 compounds have shown a susceptibility to metabolic clearance:

Metabolism ID Study for Three Triazolopyrazines

Physiochemical Evaluation and Metabolism of 8 Test Compounds in Human and Mouse Liver Microsomes

There’s a summary on the wiki of all the current metabolic data for this series.

One of the possible problems is that these molecules contain aza-aromatic structures able to be oxidized adjacent to the nitrogen atoms - that is, I believe, a known substrate for AO. If that’s happening the it’s something we can deal with - and was probably something that was briefly looked at in the original work, explaining the synthesis and evaluation of these analogs.

But before we worry too much, we should see if these compounds are substrates for this enzyme. Following our appeal for help, I contacted Scott to see if he’d be interested, and he is. Pfizer have agreed to this, and to be named as contributing to OSM in this way, which is fantastic.

The aim is to run the assay on a set of 6-12 molecules at the outset in a human cytosolic lability assay vs. positive controls to test for AO-mediated consumption (here's the general method). If we see activity and want to examine several compounds further, we could then look at a rodent AO assay, since there are known to be species differences. One step at a time.

Experimental data would allow a comparison with the predictive work performed by Chris Swain.

Scott took a look at the Series 4 wiki and chose the long list of molecules below. Only some of these are available: the list of current stocks is here. Paul Willis added MMV669784, which is available.

AO First Shortlist

So, question. We have space for up to 6 more structures. What should we include, and why? Scott needs 1-2 mg of each. Again, the full list of what’s known about Series 4 metabolism is here, and we’re already including/addressing the most interesting compounds I think.

Some other possibilities from Series 4 are these:

AO Assay Second Set of Possibles

The first two, MMV669846 and MMV670250 seem like obvious choices given their variation of the core heterocycle. MMV670946 is a surrogate for MMV670945 which was on the original list but is no longer available. MMV672727 might be interesting as an ether resistant to benzylic oxidation. We have no good structures containing aliphatic amines, as far as I can tell.

Are we light on the regular ethers, i.e. should we include one more compound analogous to MMV639565 or MMV670437? Maybe we provide one from newly-synthesised stocks in Sydney? Are there other interesting structures available that people feel might ask/answer interesting AO-related questions, particularly alterations in the core triazolopyrazine core?

On the other hand, could we also ship compounds from previous series, to answer questions there too, for publication purposes? (The Series 1 paper is an an advanced state of write-up).

To summarize: which other compounds should go into this assay? We have room for up to 6 more.

Deadline for decision: Morning of Wednesday August 6th 2014. Then we’ll ship.

Places for comments/suggestions: Below (login with Google account to comment) or Github or G+ or Twitter using @O_S_M or (last resort) email (opensourcemalaria@gmail.com) but please make it clear you’re happy to have comments relayed publicly with attribution.

(This post authored by Mat)

Attached Files
9th July 2014 @ 06:36

Compounds in OSM Series 4 and Series 3, as well as some compounds synthesised at the start of an open source TB project, were submitted to GSK for potency evaluation: Compounds Submitted for Testing at GSK Tres Cantos for anti-malarial and anti-TB activity

Single shot data was received from GSK (Laura Maria Sanz-Alonso) on June 27th 2014 and can be found here: Single Shot Potencies for Compounds Submitted to GSKCompounds found to have percentage of inhibition  50% (along with some controls) were progressed to IC50 determination assays.

Initial Comments:

Full data is here:

20140631_whole cell_IC50 determinatio_IC50 values.xlsx

Phenethyl ether compounds OSM-S-187 and OSM-S-189 look promising and have ClogP <5.

Striking difference in potency of amides OSM-S-201 and OSM-S-202 that differ only in the presence of an ortho-methyl group. Perhaps more notable was the low inhibition of OSM-S-204, bearing an ortho-fluoro group. OSM-S-204 wasn't subject to the IC50 assay but showed only 24% inhibition in the single shot assay, this might suggest that the nature of the ortho substituent, rather than steric bulk influences activity.

OSM-S-206 has a promising IC50 but ClogP is too high.

MMV669844, (OSM-S-218) an enantioenriched compound was inherited at the start of the series and was found to have an EC50 of 0.040 uM in an erythrocyte assay (NF54 with 3H-Hypoxanthine at 72hr time point). OSM-S-208 is the racemic form of this compound and was also found to have a high potency in the IC50 assay described.

Strings

OSM-S-106 Preparation of OSM-S-106 O=S(C1=CC=CC(C2=CC3=NC=NC(N)=C3S2)=C1)(N)=O 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)

OSM-S-142 Preparation of OSM-S-142 NC1=C2C(C=C(C3=CC(C(N4CCN(C)CC4)=O)=CC=C3)S2)=NC=N1 InChI=1S/C18H19N5OS/c1-22-5-7-23(8-6-22)18(24)13-4-2-3-12(9-13)15-10-14-16(25-15)17(19)21-11-20-14/h2-4,9-11H,5-8H2,1H3,(H2,19,20,21)

OSM-S-187 Preparation of OSM-S-187 N#CC(C=C1)=CC=C1C2=NN=C3C=NC=C(OCCC4=CC=CC=C4)N32 InChI=1S/C20H15N5O/c21-12-16-6-8-17(9-7-16)20-24-23-18-13-22-14-19(25(18)20)26-11-10-15-4-2-1-3-5-15/h1-9,13-14H,10-11H2

OSM-S-189 Preparation of OSM-S-189 ClC1=CC=CC=C1CCOC2=CN=CC3=NN=C(C4=CC=C(C#N)C=C4)N32 InChI=1S/C20H14ClN5O/c21-17-4-2-1-3-15(17)9-10-27-19-13-23-12-18-24-25-20(26(18)19)16-7-5-14(11-22)6-8-16/h1-8,12-13H,9-10H2

OSM-S-194 Preparation of OSM-S-194 C12=CC=CC=C1C3=C(C4(CCN(CC5=CC=CC=C5)CC4)OCC3)N2 InChI=1S/C22H24N2O/c1-2-6-17(7-3-1)16-24-13-11-22(12-14-24)21-19(10-15-25-22)18-8-4-5-9-20(18)23-21/h1-9,23H,10-16H2

OSM-S-201 Preparation of OSM-S-201 O=C(NC1=CC=CC(Cl)=C1C)C2=CN=CC(N23)=NN=C3C4=CC=C(OC(F)F)C=C4 InChI=1S/C20H14ClF2N5O2/c1-11-14(21)3-2-4-15(11)25-19(29)16-9-24-10-17-26-27-18(28(16)17)12-5-7-13(8-6-12)30-20(22)23/h2-10,20H,1H3,(H,25,29)

OSM-S-202 OSM-S-202 O=C(NC1=CC=CC(Cl)=C1)C2=CN=CC(N23)=NN=C3C4=CC=C(OC(F)F)C=C4 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)

OSM-S-204 OSM-S-204 O=C(NC1=CC=CC(Cl)=C1F)C2=CN=CC(N23)=NN=C3C4=CC=C(OC(F)F)C=C4 InChI=1S/C19H11ClF3N5O2/c20-12-2-1-3-13(16(12)21)25-18(29)14-8-24-9-15-26-27-17(28(14)15)10-4-6-11(7-5-10)30-19(22)23/h1-9,19H,(H,25,29)

OSM-S-206 Preparation of OSM-S-206 O=C(NC1=CC(C(F)(F)F)=CC(C(F)(F)F)=C1)C2=CN=CC3=NN=C(C4=CC=C(OC(F)F)C=C4)N32 InChI=1S/C21H11F8N5O2/c22-19(23)36-14-3-1-10(2-4-14)17-33-32-16-9-30-8-15(34(16)17)18(35)31-13-6-11(20(24,25)26)5-12(7-13)21(27,28)29/h1-9,19H,(H,31,35)

OSM-S-208 OSM-S-208 FC1=C(F)C=CC(C(OC)COC2=CN=CC3=NN=C(C4=CC=C(C#N)C=C4)N32)=C1 InChI=1S/C21H15F2N5O2/c1-29-18(15-6-7-16(22)17(23)8-15)12-30-20-11-25-10-19-26-27-21(28(19)20)14-4-2-13(9-24)3-5-14/h2-8,10-11,18H,12H2,1H3

Attached Files
1st July 2014 @ 01:23

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

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