Potential Candidates for in vivo screening

[fantasy121]

Hi @OpenSourceMycetoma/corecontrib, I saw in my SAR some interesting fenarimol analogues that are promising for in vivo. I wonder if @wendy you are already testing some of these, I think these will be quite good in terms of rationalising the SAR, and potentially furthering our LogD vs in vivo potency theory.

These compounds I highlighted with a red nt (not tested) in the tables below, they all have in vitro <20 and they all have logD <2.5

(More "+" equals higher priority, in my opinion)

• MYOS00014 EPL-BS0115 (++)
• MYOS00042 EPL-BS0374 (+++)
• MYOS00106 EPL-BS0613 (++++)
• MYOS00133 HPD37-1 (+)
• MYOS00057 EPL-BS0729 (+)
• MYOS00072 EPL-BS0572 (++++)
• MYOS00067 EPL-BS0248 (+)
• MYOS00073 EPL-BS0595 (++)
• MYOS00074 EPL-BS0609 (+++)

Update:

• MYOS00051 EPL-BS0566 (++)

(Draft tabulated data for paper)

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[wwjvdsande]

@fantasy121 So far we did not start testing any of the compounds in vivo. However if there is a good rationale of course we can do that. @MA-Jjingyi as a first control we will test if they are still having the same potency after storage. If so than we could take them along in case there are not too many compounds to be tested from the new series. When we know the in vivo activity of the new compounds we will make up a definitive list to test. When we have data on these compounds earlier (in terms if they are still active) we might be able to screen them first. Do you have a ranking in case we cannot screen all?

[fantasy121]

@fantasy121 So far we did not start testing any of the compounds in vivo. However if there is a good rationale of course we can do that. @MA-Jjingyi as a first control we will test if they are still having the same potency after storage. If so than we could take them along in case there are not too many compounds to be tested from the new series. When we know the in vivo activity of the new compounds we will make up a definitive list to test. When we have data on these compounds earlier (in terms if they are still active) we might be able to screen them first. Do you have a ranking in case we cannot screen all?

I added a scale of priority. More +++ more priority. Hope this helps : )

[wwjvdsande]

@fantasy121 @mattodd @bendndi we checked the list and retested the compounds against M. mycetomatis to make sure they are still active after prolonged storage. Unfortunately after retesting they don't seem to be active anymore. Can the compounds selected by hung be sent over one more time so that we can test them in vitro and in vivo?

[MA-Jjingyi]

@kym834 Hi, Kymberly. thanks for your compounds. we received them last Friday. there is an issue that two compounds have different weight number from the bottle to the excel. please have a check with those two compounds weight called MYOS_00173_00_01(HPD053-1P5) and MYOS_00174_00_01(HPD044-1P5) .

[mattodd]

@wwjvdsande it's a significant worry that compounds are not retaining potency and may be deteriorating. 1) How old are those samples you tested, roughly? 2) How are they stored? In DMSO at room temp? 3) Is the problem affecting all the compounds tested? 4) Could a chemistry team re-examine a batch of one of these compounds (stored in Sydney or UCL) by NMR to see if the compounds are degrading?

[dmitrij176]

@mattodd @wwjvdsande I recently did an NMR re-check of compounds which are at least 7-8 months old (eg: DM29-1, DM30-1, DM31-1) and no stability issues were detected. Previously, here at UCL we investigated the effect of DMSO solvent on one of the fenarimols for 2 months and so far the structure was stable. I can recall only one compound that failed to fully replicate in vitro results- DM16-1 positive control and it might be due to the same problem.

[fantasy121]

@wwjvdsande @mattodd The compounds recommended by me above are all from the epichem library, except for MYOS_00133, which was synthesised by me. I can re-characterise MYOS_00133 to see if it's still stable, and will let you know. But Sydney is currently in lockdown so there will be a delay getting back to you.

[fantasy121]

@kym834 Hi, Kymberly. thanks for your compounds. we received them last Friday. there is an issue that two compounds have different weight number from the bottle to the excel. please have a check with those two compounds weight called MYOS_00173_00_01(HPD053-1P5) and MYOS_00174_00_01(HPD044-1P5) .

@MA-Jjingyi Where there is a discrepancy, can you please you the values supplied in the cover letter MYOS_00173_00_01(HPD053-1P5) 2.90mg and MYOS_00174_00_01(HPD044-1P5) . 2.66mg

[fantasy121]

@OpenSourceMycetoma/corecontrib For the latest batch of shipped compounds from me, I have re-checked for stability (with NMR) before shipping them over as well, due to the lag time between me making them (Q4 2020) and shipping them (now). So to the best of my knowledge, all of my re-checked compounds are viable thus far.

[fantasy121]

@wwjvdsande it's a significant worry that compounds are not retaining potency and may be deteriorating.

1. How old are those samples you tested, roughly?
2. How are they stored? In DMSO at room temp?
3. Is the problem affecting all the compounds tested?
4. Could a chemistry team re-examine a batch of one of these compounds (stored in Sydney or UCL) by NMR to see if the compounds are degrading?

From memories, I think the epichem compounds are stored refrigerated in DMSO. All synthesised compounds are shipped as is, then similarly stored once testing is undertaken.

[MA-Jjingyi]

@kym834 MYOS_00174_00_01(HPD044-1P5) . 2.90mg and MYOS_00173_00_01(HPD053-1P5).2.66mg are in the glass bottles stickers. this is the different with the excel. so I should trust the number in the excel not the sticker in the glass bottles?

[kym834]

@MA-Jjingyi Glad the compounds made it!

From what you are saying that the masses are switched for these two compounds. The sticker on the glass bottle for MYOS_00173_00_01 has a mass of 2.66 mg but on the excel it has 2.90 mg. And for MYOS_00174_00_01 the glass bottle has 2.90 mg but the excel has 2.66 mg. Is this right?

These are @fantasy121 compounds but I think he wants you to use the numbers in the excel (table) that we provided in the cover letter. So use these below

MYOS_00173_00_01 (HPD053-1P5) has a mass of 2.90 mg MYOS_00174_00_01 (HPD044-1P5) has a mass of 2.66 mg

Thanks :)

[MA-Jjingyi]

@kym834 ok. i will follow this number: MYOS_00173_00_01 (HPD053-1P5) has a mass of 2.90 mg MYOS_00174_00_01 (HPD044-1P5) has a mass of 2.66 mg. thank you.

[wwjvdsande]

@wwjvdsande it's a significant worry that compounds are not retaining potency and may be deteriorating.

1. How old are those samples you tested, roughly?
2. How are they stored? In DMSO at room temp?
3. Is the problem affecting all the compounds tested?
4. Could a chemistry team re-examine a batch of one of these compounds (stored in Sydney or UCL) by NMR to see if the compounds are degrading?

From memories, I think the epichem compounds are stored refrigerated in DMSO. All synthesised compounds are shipped as is, then similarly stored once testing is undertaken.

@fantasy121 the compounds were dissovled in DMSO upon arrivel and stored at -20. We retested all of them since it was a while since we last used them and it is not that much more work to test all in the first screening or to test only a few. The freezers are on a monitoring system and nothing happened to the temperature since they were frozen in.

[wwjvdsande]

The samples tested are from 2016. We also included itraconazole as a control and that still worked. We repeated the assay 3 times now to make sure. A chemistry re-examine might be done however is it not most practicle to see if these compounds can be resend and retested and hopefully to make it by the end of this larvae season. We don't have to do the full in vitro analyses for those before the larvae, we can simply do a high low screening and move further. After the larvae season we than repeat the rest of the in vitro screenings to make the dataset complete.

[mattodd]

As per the meeting just now, yes, let's ask Epichem if they have any more of these compounds destined for in vivo testing, since we're in doubt about whether our stocks at Erasmus MC are still OK. In vitro testing of any Epichem compounds should tell us whether those stocks are still OK!

[wwjvdsande]

@fantasy121 I checked the list you sent

MYOS00014 EPL-BS0115 (++) --> has an IC50 of 8,0 will be tested MYOS00042 EPL-BS0374 (+++) --> does not inhibit growth at 25 uM --> cannot reach that concentration in the larvae MYOS00106 EPL-BS0613 (++++) --> does not inhibit growth at 25 uM --> cannot reach that concentration in the larvae MYOS00133 HPD37-1 (+) --> IC50>16. We will retest this to see what happens MYOS00057 EPL-BS0729 (+) --> does not inhibit growth at 25 uM --> cannot reach that concentration in the larvae MYOS00072 EPL-BS0572 (++++) --> does not inhibit growth at 25 uM --> cannot reach that concentration in the larvae MYOS00067 EPL-BS0248 (+) --> does not inhibit growth at 25 uM --> cannot reach that concentration in the larvae MYOS00073 EPL-BS0595 (++) --> does not inhibit growth at 25 uM --> cannot reach that concentration in the larvae MYOS00074 EPL-BS0609 (+++) --> does not inhibit growth at 25 uM --> cannot reach that concentration in the larvae

Seeing these activities I thing it is good to test MYOS00014 in the larvae and reanalyse HPD37-1 to check what the inhibitory concentration in MM55 is. However since the other compounds did not inhibit growth at a concentration of 25 uM I doubt that they will be effective in vivo because we cannot get a high enough concentration within the larvae. Any other ideas or suggestions?

[MA-Jjingyi]

@wwjvdsande @mattodd @kym834 @fantasy121 @dmitrij176. Today the first in vitro results for the new compounds arrived. These are the results of only one testrun and we are currently running the duplicate experiments. So-far it seems that from series 1 (the fenarimols) MYOS_00147_00_01, MYOS_00148_00_01, MYOS_00150_00_01, MYOS_00191_00_01, MYOS_00192_00_01, myos_00166_00_01, myos_00167_00_01, myos_00168_00_01, myos_00170_00_01, myos_00171_00_01 and myos_00174_00_01 were able to inhibit M. mycetomatis at both 100 uM and 25 uM. Those will be tested for IC50 and MIC as well. MYOS_00149_00_01, MYOS_00151_00_01, myos_00165_00_01, myos_00172_00_01, myos_00173_00_01, myos_00175_00_01 and myos_00189_00_01 inhibited M. mycetomatis only at 100 uM. The only fenarimol which also had potent activity against F. senegalensis was MYOS_00191_00_01. This compound inhibited F. senegalensis at 100 uM and 25 uM.

For series 2 (the aminothiazoles), myos_00176_00_01 was able to inhibit M. mycetomatis and F. senegalensis at 100 uM and 25 uM. The only other compound which demonstrated some activity was myos_00186_00_01, which was able to inhibit M. mycetomatis growth at 100 uM but not at 25 uM.

For series 6 MYOS_00193_00_01 was able to inhibit both M. mycetomatis and F. senegalensis at 100 uM and 25uM. MYOS_00194_00_01 inhibited both species at 100 uM only, not at 25 uM.

[dmitrij176]

@wwjvdsande @mattodd @kym834 @fantasy121 @dmitrij176. Today the first in vitro results for the new compounds arrived. These are the results of only one testrun and we are currently running the duplicate experiments. So-far it seems that from series 1 (the fenarimols) MYOS_00147_00_01, MYOS_00148_00_01, MYOS_00150_00_01, MYOS_00191_00_01, MYOS_00192_00_01, myos_00166_00_01, myos_00167_00_01, myos_00168_00_01, myos_00170_00_01, myos_00171_00_01 and myos_00174_00_01 were able to inhibit M. mycetomatis at both 100 uM and 25 uM. Those will be tested for IC50 and MIC as well. MYOS_00149_00_01, MYOS_00151_00_01, myos_00165_00_01, myos_00172_00_01, myos_00173_00_01, myos_00175_00_01 and myos_00189_00_01 inhibited M. mycetomatis only at 100 uM. The only fenarimol which also had potent activity against F. senegalensis was MYOS_00191_00_01. This compound inhibited F. senegalensis at 100 uM and 25 uM.

For series 2 (the aminothiazoles), myos_00176_00_01 was able to inhibit M. mycetomatis and F. senegalensis at 100 uM and 25 uM. The only other compound which demonstrated some activity was myos_00186_00_01, which was able to inhibit M. mycetomatis growth at 100 uM but not at 25 uM.

For series 6 MYOS_00193_00_01 was able to inhibit both M. mycetomatis and F. senegalensis at 100 uM and 25uM. MYOS_00194_00_01 inhibited both species at 100 uM only, not at 25 uM.

Great news. Will be waiting for full results. Thank you @MA-Jjingyi .

[MA-Jjingyi]

@wwjvdsande @mattodd @kym834 @fantasy121 @dmitrij176. Today the first in vitro results for the new compounds arrived. These are the results of only one testrun and we are currently running the duplicate experiments. So-far it seems that from series 1 (the fenarimols) MYOS_00147_00_01, MYOS_00148_00_01, MYOS_00150_00_01, MYOS_00191_00_01, MYOS_00192_00_01, myos_00166_00_01, myos_00167_00_01, myos_00168_00_01, myos_00170_00_01, myos_00171_00_01 and myos_00174_00_01 were able to inhibit M. mycetomatis at both 100 uM and 25 uM. Those will be tested for IC50 and MIC as well. MYOS_00149_00_01, MYOS_00151_00_01, myos_00165_00_01, myos_00172_00_01, myos_00173_00_01, myos_00175_00_01 and myos_00189_00_01 inhibited M. mycetomatis only at 100 uM. The only fenarimol which also had potent activity against F. senegalensis was MYOS_00191_00_01. This compound inhibited F. senegalensis at 100 uM and 25 uM. For series 2 (the aminothiazoles), myos_00176_00_01 was able to inhibit M. mycetomatis and F. senegalensis at 100 uM and 25 uM. The only other compound which demonstrated some activity was myos_00186_00_01, which was able to inhibit M. mycetomatis growth at 100 uM but not at 25 uM. For series 6 MYOS_00193_00_01 was able to inhibit both M. mycetomatis and F. senegalensis at 100 uM and 25uM. MYOS_00194_00_01 inhibited both species at 100 uM only, not at 25 uM.

Great news. Will be waiting for full results. Thank you @MA-Jjingyi .

it won't be long. 😁 I will post the results as soon as I get them.

[mattodd]

@dmitrij176 it'd be very useful if, when the results are confirmed, you could add in the structures and colour code according to activity, then post here directly? Also highlight any structures that are the structurally closest to those we have evaluated, thereby allowing us to see whether any of the activities are surprising - that will assist with the SAR analysis you're doing with @fantasy121

[dmitrij176]

@dmitrij176 it'd be very useful if, when the results are confirmed, you could add in the structures and colour code according to activity, then post here directly? Also highlight any structures that are the structurally closest to those we have evaluated, thereby allowing us to see whether any of the activities are surprising - that will assist with the SAR analysis you're doing with @fantasy121

@mattodd already started a draft version.