series 5: benzimidazoles

[wwjvdsande]

@mattodd @bendndi @kym834 @fantasy121 @dmitrij176 @MA-Jjingyi @Wilson-Lm As mentioned in our last meeting we found some interesting hits with the benzimidazoles. After screening the pandemic response box, two benzimidazoles were able to prolong larval survival in Madurella mycetomatis infected larvae. Furthermore all three also showed some acivity against Falciformispora senegalensis, another prominent mycetoma causative agent.

Benzimidazoles have been developed for numorous diseases

For fungal infections in plants, some benzimidazoles are currently in use. They act on betatubulin:

Due to the large availability of benzimidazoles as anti-infectives several benimidazoles were included in the MMV boxes. The activity against Madurella mycetomatis is shown below

Compound	MMVnumber	Growth inhibition at 100 uM	Growth inhibition at 25 uM	IC50	MIC50	% Larval survival	Significant?
Membendazole	MMV003152	110,90
Fenbendazole	MMV003143	7,16	2,38	2,66	0.5	24,4	yes
Carbendazim	MMV344625	8,48	3,52	3,52	0.5	6,7	no
MMV1782387	MMV1782387	15,57	4,41	0,26	4	28,9	yes
MMV687730	MMV687730	7,67	-3,36	11,55
MMV687248	MMV687248	-2,72	64,71	40,7
MMV676474	MMV676474	2,84	76,60	52,15
MMV687703	MMV687703	-0,73	60,96	37,35
MMV024311	MMV024311	0,36	76,40	52,05
MMV676445	MMV676445	91,10
MMV020081	MMV020081	117,66
MMV011903	MMV011903	146,74
MMV676449	MMV676449	93,74
Maribavir	MMV1593521	81,75	93,63
Veliparib		87,56	129,58

The structure of these molecules is as follows:

Is there any correlation between the structure of the molecule and the activity in M. mycetomatis? Are there compound collections we should and can try?

[MFernflower]

There are two other easily available compounds: @wwjvdsande Flubendazole Egaten

A unrelated but interesting compound from the agricultural market that might be worth trying is Toltrazuril

[MFernflower]

@wwjvdsande another possible compound to try is Oxibendazole

all four of the molecules I have mentioned are somewhat common agrochemicals - but as such since I do not what the chemical suppliers near you carry - I assume it will be a bunch of phonecall work to see what they have!

[MFernflower]

I also found an interesting paper: https://innovareacademics.in/journal/ijpps/Vol6Issue5/9426.pdf

[kym834]

Hi @OpenSourceMycetoma/corecontrib

I had a look through the pandemic box and found two other interesting compounds which can add some value to this discussion. MMV394033 and MMV000725 - both these compounds are benzimidazole imines with good values at 25 uM.

Based on these and the carbamates that you shared at the meeting, I think that having a nitrogen bonded to the C2 will be important for activity, possible related to its ability to hydrogen bond.

@Wilson-Lm, let me know once you've finished that table and I'll explore a little further :)

[MFernflower]

@Wilson-Lm @wwjvdsande Have these two agrochemicals been tested vs eumycetoma?

[wwjvdsande]

@MFernflower @MA-Jjingyi as far as i know they are not tested (if they are not in the masterlist than they are not tested). I noticed they are available from Sigma but expensive. Is there an other way where we can obtain them to test?

[MFernflower]

@wwjvdsande have you checked any local biochem suppliers? Sigma is always 💰💰

[MFernflower]

@bendndi could you check if you have the two drugs mentioned possibly to donate to Wendy's lab?

[MFernflower]

Also @wwjvdsande egaten is fda approved so if it's potent could allow a speed lined deployment to actual patients

[MFernflower]

@bendndi Oxfendazole is being worked on by DNDI for river blindness if I'm not mistaken - would it be possible for @wwjvdsande to get some for screening vs fungi? @mattodd

[MFernflower]

@OpenSourceMycetoma/corecontrib I just saw the latest paper and added it to the front page of this github repo - and Oxfendazole, Oxibendazole and Mebendazole are inactive - my attention now turns to trying to get Wendy's lab some egaten (triclabendazole)

Anybody have some possible connections for sourcing this molecule?

[bebi78]

@OpenSourceMycetoma/corecontrib Triclabendazole is commercially available from ABCR or TCI (1g-5g, 46 to 70 Euros). I can order it and send it to Wendy if you like.

[bebi78]

@MFernflower @wwjvdsande Do you have access to triclabendazole? If not, I can order it paid by our institute budget and send it to Wendy.

[bebi78]

@mattodd @MFernflower @wwjvdsande You mentioned above that carbendazim is not significant in larvae. Do you think it would be a good idea to prepare further derivatives of carbendazim in order to see if there is some improvement in the in vivo model? Because most of these benzimidazoles are targeting the beta-tubulin of fungi, do you think molecular docking using fungal tubulin might lead to new interesting antifungal compounds? Are there tubulin structures known for the eumycetoma causing fungi?

[wwjvdsande]

@bebi78 If you look at the survival curve and also in our just recently published paper on this you see that carbendazim does not signficantly prolong larval survival at day 10. However at day 4 it does. Typically we treat the larvae only in the first 3 days after infection. So directly after treatment stopped carbendazim did still result in prolonged larval survival however this effect was diminished in the periode in which no treatment was received. unfortunately due to injuries to the larvae we cannot treat the whole 10 day periode. So I think it would certainly be worthwile to prepare further derivatives of carbendazim.

With respect to the tubulin structures. For Madurella mycetomatis there are no cristal structures prepared for any of the proteins however with modelling, models of Madurella proteins were made and placed in Uniprot (eg. betatubulin: https://www.uniprot.org/uniprot/A0A175W1F8)

[bebi78]

@wwjvdsande Thanks. Congratulations to the accepted paper! It is very inspiring and useful to know all the compounds already tested by you. Because I added trimethoprim to the package for you and I've seen now it was already tested. But maybe you can use it for other research projects instead. I have a question about the results for 8-p-tosyl-aminoquinoline (line 179 in the supporting excel file). Has this compound an IC50 value between 25 and 100 µM?

[wwjvdsande]

Yes. For this compound we noted that in the triplicates with 100 uM there was already some variation and the same was seen for 25 uM. The IC50 was at least above 25 uM but since we did not determine values above that (except 100 uM) I will not be able to pinpoint where it will be.

[mattodd]

@bebi78 is trimethoprim not in the MycetOS Master List? We are trying to maintain an up-to-date list of compounds that have been tested, but I guess those published compounds may not be in the list yet? @MA-Jjingyi @wwjvdsande ?

[wwjvdsande]

@mattodd @bebi78 all data on the pandemic box is also in the mastersheet under the tab MMV boxes screened

[bebi78]

@mattodd Yes, trimethoprim is part of the DNDi pandemic box. I did not know that. Maybe Wendy or her colleagues can keep or use it for other projects.

[fantasy121]

@OpenSourceMycetoma/corecontrib I also have some series 5 analogues I wish to synthesise for testing in the coming months. See #2

[MFernflower]

@bebi78 I have made a new repository for the products you have sent and possible future directions:

https://github.com/OpenSourceMycetoma/Adjuvant-Screen

[wwjvdsande]

@mattodd @AndreaOtago at the moment @MA-Jjingyi is starting to prepare a manuscript on the compounds we tested for series 5 so far. She needs this for her PhD thesis. However, series 5 are including benzimidazoles and benzoxazoles. Central will be MYOS326, MYOS333 and MYOS334 for which MYOS333 and MYOS334 prolonged larval survival and MYOS326 did not. They were created by Imperial College Students. Can you help us with the chemical analysis? Which compounds belong to which group and which ones are structurally closely related??? They also have a DNDi number, where they all from a same project???

[AndreaOtago]

@wwjvdsande @MA-Jjingyi RE you comment above, Yes all the Imperial and Otago benzoxazoles were made for the same DNDi project - Open Synthesis Network "Project 5" , you can download all that data off this website. https://dndi.org/research-development/portfolio/open-synthesis-network/ under "P5". This is also where the DNDi code is from and the compounds in the biological data sheet also list a DNDi code. In terms of chemical analysis - ask Charlotte at Imperial, sorry I don't know her user name on here off hand. I assume Charlotte/the students who worked on the project have all the NMR, MS, HPLC data on the final compounds. I could help with this for any Otago generated compounds only.

[MA-Jjingyi]

25032024 MyctOS meeting -MA.pptx Hi, @AndreaOtago @mattodd . this is the slide of series 5 compounds summary. with Andrea's suggestion, we did find the chemical structures and LogP from OSN P5 database(shown in slides). Would you mind helping with further chemical analysis? or anyone would like to help us with that?

[AndreaOtago]

Hi @MA-Jjingyi , sorry I missed a great meeting and presentation! I saw the meeting text title of UK noon time and then ignored all subsequent meeting posts thinking this was 1am and NZ time but I think you met earlier? Anyway I will know for next time :) I am happy to help but unsure what you need doing, what do you mean by 'chemical analysis' - do you structure-activity-relationship thoughts? Or just chemical analysis, eg clogP, clogD(7.4), PSA, other physicochemical metrics. If it is SAR then I don't know enough about the biology/what the numbers mean to have a good go at this, happy to try but would need help from someone who knew about the biology. RE 'benzoxazoles or benzimidazoles' - all on slide 15 and benzoxazole - the N and O in the Ar system = benzoxazole, N/NH is the named benzimidazole.

[wwjvdsande]

@AndreaOtago, @MA-Jjingyi and I are sitting together and looked at your comment. What we want to know if there were chemical differences which could explain the activity. So a SAR. logP, logD etc were generated but SAR. We understand the biology so what would you need to know for the SAR? In the previous paper we did a SAR on in vitro activities and on in vivo. Of course only 2 were active in vivo. In vitro we have more results. We also have a scaling there so we know the IC50 and the MIC. Those are mentioned in the spreadsheet but we can also make a small summary in a table here with the data you need.

[AndreaOtago]

@MA-Jjingyi @wwjvdsande it's more that from the tables of data I don't know what number is 'significantly' different from another in terms of the activity. Perhaps if you annotate this slide, screen shot pasted below, with the MYOS compound codes at each stage (for everything other than the starting 33 compounds) then this will summarise what is 'active' and I can comment on if there are any chemical trends across this same slide too and a good SAR summary.

[AndreaOtago]

[MA-Jjingyi]

Hi @AndreaOtago , sorry for reply late. I worked on the database of series 5 to make it easier to read. please check. and if you have any doubts about this database, please let me know. Thanks for your kind help. series5 data base.xlsx

[AndreaOtago]

Hi @MA-Jjingyi , it would be better if you could please annotate that powerpoint slide with what compound is mentioned where, then I can just refer to the excel sheet for details if I need to. e.g. "3 compounds active against both strains " - what 3 compounds? I don't want/can't look for this myself in the data in the excel file. So can you please reproduce this slide but with compounds codes in each part of the flow chart.

[MA-Jjingyi]

Hi @AndreaOtago , good morning. here you are.

[AndreaOtago]

Great thanks! Yes this was what I was after, now I can take a look at the chemical structures at each stage in the flow chart and see if there are any patterns in terms of chemical properties etc. I'll get back to you over the next week or so regarding my thoughts about this.

[AndreaOtago]

Hi @MA-Jjingyi , I am just coming to do this, however, sorry I didn't realise when I first commented on your image, I actually can't see the codes well enough (resolution wise) to be sure what are '3' and '9' - it looks grainy, I do not want to get the wrong compound. Can you please post as higher res. image or a link to a powerpoint file? Thanks.

[MA-Jjingyi]

Hi @AndreaOtago , here you go. sereis 5 data summary21042024.pptx

[AndreaOtago]

Hi @MA-Jjingyi , great thanks, I am starting to look at this. I am just wondering - the series5data base excel sheet you have a few posts above - is there a reason the other series 5 compounds are not included, is this because the other/more recent compounds have not undergone as much testing? I am referring to MYOS_00521_00_01 MYOS_00522_00_01 MYOS_00523_00_01 MYOS_00524_00_01 MYOS_00525_00_01 MYOS_00526_00_01 MYOS_00527_00_01 MYOS_00528_00_01 MYOS_00529_00_01 MYOS_00530_00_01 MYOS_00531_00_01 Which are also all benzoxazoles. I just thought I would ask as if looking at SAR across a compound series - better to include everything. If the compounds above didn't progress in testing as were inactive, this is also very useful SAR info.

[wwjvdsande]

@AndreaOtago these compounds were tested in vitro too. Of these only MYOS_00527 showed activity at 25uM. MYOS_00521, MYOS_00522, MYOS_00523 and MYOS_00524 did inhibit growth at 100 uM but not at 25 uM. The IC50 of MYOS_00527 was 3.1 uM and the MIC against or animal strain was 4 uM. This means that this compound is considered an in vitro hit. It was not tested in vivo (yet). We also did not test it against Falciformispora yet. The updated powerpoint is attached sereis.5.data.summary23042024.pptx

[AndreaOtago]

@AndreaOtago these compounds were tested in vitro too. Of these only MYOS_00527 showed activity at 25uM. MYOS_00521, MYOS_00522, MYOS_00523 and MYOS_00524 did inhibit growth at 100 uM but not at 25 uM. The IC50 of MYOS_00527 was 3.1 uM and the MIC against or animal strain was 4 uM. This means that this compound is considered an in vitro hit. It was not tested in vivo (yet). We also did not test it against Falciformispora yet. The updated powerpoint is attached sereis.5.data.summary23042024.pptx

@MA-Jjingyi @wwjvdsande - great thanks for the update. @MA-Jjingyi - can you please add these compounds in the correct places on that summary sheet? I can't quickly see what species/assay the activity for the newer compounds is for.

[AndreaOtago]

@AndreaOtago these compounds were tested in vitro too. Of these only MYOS_00527 showed activity at 25uM. MYOS_00521, MYOS_00522, MYOS_00523 and MYOS_00524 did inhibit growth at 100 uM but not at 25 uM. The IC50 of MYOS_00527 was 3.1 uM and the MIC against or animal strain was 4 uM. This means that this compound is considered an in vitro hit. It was not tested in vivo (yet). We also did not test it against Falciformispora yet. The updated powerpoint is attached sereis.5.data.summary23042024.pptx

@MA-Jjingyi @wwjvdsande - great thanks for the update. @MA-Jjingyi - can you please add these compounds in the correct places on that summary sheet? I can't quickly see what species/assay the activity for the newer compounds is for.

sorry! I just realised you have already updated the summary :) I should have read to the end...... Great, I will take a look at this this week and be in touch

[AndreaOtago]

@wwjvdsande CHEM 305 students at the University of Otago have made 8 benzoxazoles in their undergrad lab this year. Structures and photos below. Do you want these 8 compounds to test? I am happy to post over. We made these for/are sending into the DNDi open synthesis network too, for their parasite panel screen. If you want the compounds sent in, then I will enter/assign a MYOS code and also the structures etc in a dwar file.

[wwjvdsande]

@AndreaOtago yes please.

[AndreaOtago]

@wwjvdsande great! I will add into the spreadsheet and give a MYOS code etc. @mattodd - advice on how to list a mixture of endo and exo norbornene isomers? We have one compound pure 'exo' isomer (so I'll list this as one, single diastereoisomer) and another compound that is a 80:20 endo:exo mix - how to enter this? I assume the Mycetoma project would still be keen to test this, as other than the endo:exo isomer mix the compound is 'pure'. Thoughts before I enter compounds into the sheet?

[mattodd]

@AndreaOtago tricky. Yes definitely interested to test. Can you insert two Smiles in the relevant cell? If not is there a comment column where you could add a note about the minor, while giving the Myos code for the major?

[AndreaOtago]

thanks @mattodd sounds good, I'll input and compound code today. @wwjvdsande can you please confirm compound shipping address and who it is attn of. The international couriers also like a phone # (whether it is an office phone or dept admin/reception phone). Thanks.

[MA-Jjingyi]

@AndreaOtago these compounds were tested in vitro too. Of these only MYOS_00527 showed activity at 25uM. MYOS_00521, MYOS_00522, MYOS_00523 and MYOS_00524 did inhibit growth at 100 uM but not at 25 uM. The IC50 of MYOS_00527 was 3.1 uM and the MIC against or animal strain was 4 uM. This means that this compound is considered an in vitro hit. It was not tested in vivo (yet). We also did not test it against Falciformispora yet. The updated powerpoint is attached sereis.5.data.summary23042024.pptx

@MA-Jjingyi @wwjvdsande - great thanks for the update. @MA-Jjingyi - can you please add these compounds in the correct places on that summary sheet? I can't quickly see what species/assay the activity for the newer compounds is for.

sorry! I just realised you have already updated the summary :) I should have read to the end...... Great, I will take a look at this this week and be in touch

Hi @AndreaOtago , do you have any other doubts about the data summary we sent before? if so, please let me know. we are currently working on this manuscript.

[AndreaOtago]

@AndreaOtago these compounds were tested in vitro too. Of these only MYOS_00527 showed activity at 25uM. MYOS_00521, MYOS_00522, MYOS_00523 and MYOS_00524 did inhibit growth at 100 uM but not at 25 uM. The IC50 of MYOS_00527 was 3.1 uM and the MIC against or animal strain was 4 uM. This means that this compound is considered an in vitro hit. It was not tested in vivo (yet). We also did not test it against Falciformispora yet. The updated powerpoint is attached sereis.5.data.summary23042024.pptx

@MA-Jjingyi @wwjvdsande - great thanks for the update. @MA-Jjingyi - can you please add these compounds in the correct places on that summary sheet? I can't quickly see what species/assay the activity for the newer compounds is for.

sorry! I just realised you have already updated the summary :) I should have read to the end...... Great, I will take a look at this this week and be in touch

Hi @AndreaOtago , do you have any other doubts about the data summary we sent before? if so, please let me know. we are currently working on this manuscript.

Hi @MA-Jjingyi I got as far as redoing the summary to this, taking away some of the arrows and including the whole compound tally, but unfortunately I have not then had a chance to actually have a look at compound structure. Hopefully in a week or so, it is a very busy teaching time currently :)

[MA-Jjingyi]

thank you for your quick reply, @AndreaOtago. enjoy the teaching and keep in touch.

[MA-Jjingyi]

@wwjvdsande CHEM 305 students at the University of Otago have made 8 benzoxazoles in their undergrad lab this year. Structures and photos below. Do you want these 8 compounds to test? I am happy to post over. We made these for/are sending into the DNDi open synthesis network too, for their parasite panel screen. If you want the compounds sent in, then I will enter/assign a MYOS code and also the structures etc in a dwar file.

Hi, @AndreaOtago , We get the parcel with 8 compounds of UO-compounds. Hope your teaching goes well. Would you mind giving us an update on chemical analysis?

[AndreaOtago]

@wwjvdsande great the compounds have arrived! Yes, the SAR analysis is now 2nd to top of my list :) behind marking some lab reports. I'll be in touch in the next few days.

[AndreaOtago]

@MA-Jjingyi @wwjvdsande I made a start on updating that excel sheet you sent, by pasting from the main master sheet (for the last 11 year 2023 compounds), but I realised the column headings / data breakdown in conc's etc is not exactly the same in this sheet and the master one, so could one you please update the one species of in vitro data for the 2023 otago compounds? I can then look at everything all together, thanks series5.data.base_2805_AV.xlsx