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14th July 2015 @ 00:41

What are the best strategies for minimizing metabolic clearance in OSM Series 4? This has been, and remains, a key scientific issue for us, along with maintaining potency and improving solubility.

We're devising the next set of compounds to make (also GHI301) and we're meeting next week (GHI317), so this is a good time to re-engage with metabolic clearance.

Separate Issues will be created for each relevant To Do item on Github, but here are all the background data:

1) All the current clearance data are in the wiki. Some data inherited, others obtained more recently. For the moment we are deprioritising the amide series (top row of data in the main wiki scheme) because of a probable hERG liability. So let's focus mostly on the non-amides.

2) Chris Swain predicted metabolic hotspots (in red, below) for three Series 4 compounds, MMV669844, MMV669848 and MMV670936.

Swain Predictions and MetID

When Chris ran the same compounds through a Cyp predictor the same sites came up minus those on the ring, suggesting the ring could be a substrate for another enzyme such as aldehyde oxidase. However, measured AO clearance values suggest MMV669844, for example, is not an AO substrate (see below).

3) Met and MetID work on these same three compounds above from Sue Charman's lab suggested that oxidation on the triazolopyrazine-aromatic system was occurring in most cases, with other liabilities shown above. The analog of MMV669844 with a CHF2 group in place of the CH3 (MMV670652, 17 nM) was tested in microsomal studies, giving CLint HLM<8, RLM 30.

CH3 vs CHF2 met data

4) Aldehyde Oxidase Screen. Performed, with summary data. Amides appeared to be more susceptible to AO oxidation, but in general several potent members of the ether class were found not to be substrates for AO. We should both not worry about AO for this series and yet periodically re-assess Series 4 compounds in the assay.

5) The nature of the core heterocyclic ring has a major effect on clearance - compare the parent triazolopyrazine MMV639565 with the imidazopyrazines MMV669846 and MMV670250.

Core Triazole Modifications

The potency and reasonable clearance of MMV669846 make it interesting to look at again. The very high clearance of MMV670250 suggests...what?

6) We know that blocking the pyrazine ring in the 8-position reduces potency and that the N-oxide in the pyrazine ring is inactive (below), and we also now know that replacement of the pyrazine southwest N with CH lowers potency. Is there any reason to investigate these further, e.g. for metabolic clearance rate on these compounds?

Pyrazine Mods



6) To Do/Questions

i) Benzylic Blocking: Are there blocking groups in the northwest benzylic position we ought to include that might improve metabolic stability? Is it possible to run some of these through predictive software to see if such structures might help?

ii) Acquiring New Metabolic Data on Existing Compounds: MMV670652 (above) has not yet been studied in hepatocytes, and has not had MetID performed. Is there justification for this, to confirm that we see improvements over MMV669844, or can this be assumed?

iii) Acquiring New Metabolic Data on New Compounds: Of the newer compounds synthesised (1, 2, 3), are there any that ought to be evaluated at the Charman lab for microsomal stability (GHI213) (and solubility: GHI260)? Should we re-run Chris Swain's analysis on any new target compounds?

iv) Trends in the Data: In general the newer Monash data (in green in the wiki) shows that a number of the analogs with blocking groups in the northwest benzylic position and OCHF2 in the northeast have higher than expected clearance rates vs. what might be expected given the data for the analogs with similar blocking groups in the benzylic position and a cyano group in the northeast. Is it possible that the northeast Ar substituent is playing a role in metabolic liability of the aromatic system?

v) Triazole Mods: We should resynthesise the imidazopyrazine MMV669846 shown above to verify potency and microsomal stability. Underway. Are there other groups that could be entertained on the triazole, or in place of it? What is known of good mimics of such a ring system?

vi) Pinning Down Site of Oxidation: Is it worth establishing where on the triazolopyrazine-Ar structure the oxidation is occurring, given the prediction that Cyp will not oxidise on the TP ring and given the low AO clearance experimentally determined? (Related to GHI124).


Post originally authored by Mat Todd

Attached Files
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