Mycetoma monthly meeting 24 January 2023

[wwjvdsande]

Time: January 24th (12 noon UK, 1pm EU, 11pm Aus) Timezones.

The Outlook invite can be found here

Location: https://uni-sydney.zoom.us/j/99281549497. In case of failure, use https://ucl.zoom.us/j/99789829423 and in case of double failure use MycetOS meeting teams link Chair: UCL Minute taker: ErasmusMC Previous meeting: OpenSourceMycetoma/Series-1-Fenarimols#83 and recording at https://youtu.be/gRVsCUZWUWEv Recording of this meeting:

MINUTES

1. Outstanding items from previous meeting on the fenarimols a) Paper writing
   • [ ] @dmitrij176 @fantasy121 SAR gap analysis and the Epichem library. A refinement of this diagram is needed so that we know whether to request compounds from Epichem for the current paper, or not. i.e. which matched pairs would be instructive in answering questions we have for the current SAR.
   • [ ] @fantasy121 @dmitrij176 Is the chemical experimental section complete (in main paper and in SI document)? Looking at the current draft it looks like there are still remaining issues on the experimental section on the chemistry. If @fantasy121, @dmitrij176 @kym834 could take a look at it and go through it to check the issues highlighted and remove comments when solved. The ppt looks fine.
   • [ ] Supplemental files for the manuscript: A combined labarchive was suggested as a supplemental file. In that way all needed supplemental data could be obtained from an downloadable offline copy in labarchive. Can @fantasy121 @dmitrij176 take action on this? For the biological supplemental data, which is not in labarchive, an EXCELL sheet can be generated from our current spreadsheet displaying all data on the fenarimols tested. @MA-Jjingyi and @wwjvdsande will take care of that.
   • [ ] @mattodd @fantasy121 @dmitrij176 @MA-Jjingyi @wwjvdsande need to double check that all fenarimols made are tested in vitro/vivo. If @fantasy121 @dmitrij176 can check if all their molecules are entered in the spreadsheet and sent than @MA-Jjingyi and @wwjvdsande will check if they are screened.
   • [ ] @fantasy121 has suggested molecules from EPICHEM to fill the SAR gap. However the reasoning was not provided. Can @fantasy121 add that. Need the molecules be obtained and tested?
   • [ ] @mattodd to review current draft

b) Statistics for in vivo data --> @wwjvdsande will have to contact the statistical department of ErasmusMC

2. Actions on other compound series: Series 2: aminothiazoles:
   • [ ] Rohan's contribution of compounds from Griffith University QLD: Numbers of compounds, screening capacity, costs and motifs of interest, to be discussed.
   • [ ] Crowdsourcing update - incoming contributions from e.g. @Whitgift, @JuliaSarju, @PaulMcKeatingSevenoaks, Hampton School. Others @kym834 (e.g. Barker College (@katieterrett))? Update on meeting held is here. Actions include a check on compound novelty, and the provision of targets for @Whitgift and @PaulMcKeatingSevenoaks.
   • [ ] Meeting is needed to make sure every school has a different target @mattodd @kym834
   • [x] Compounds shown here have been evaluated. Wiki will be updated
   • [x] ELN needed for @JuliaSarju team. Action on @mattodd. Done: https://tinyurl.com/YorkChemELN

Series 5: Actions based on potencies observed for most recent compound batches_ - e.g. [oxazoles](https://github.com/OpenSourceMycetoma/Series-1-Fenarimols/issues/82#issuecomment-1325150305) oxazoles were selected because they resembled the benzimidazoles which showed potent in vivo efficacy. The oxazoles were validated _in vitro against Madurella mycetomatis and appeared to be very potent. THey will be tested against Falciformispora senegalensis too. This series will be evaluated in vivo in the new larval season.

• [x] @bebi78 are there more oxazoles out there? Could you check with DNDi

Series 6: ketoximes --> 19 compounds designed. Most of them are synthesized by UCL.

• [x] @dmitrij176 could you put a figure of the compounds which will be shipped out on the ketoxime wiki so that everyone can take a look on the molecules made.

Series natural products.

• [ ] Compounds tested in vitro. @bebi78 to go over them to see if there might be interesting leads
• [x] naphthazarin manuscript bb.naphthazarin.mycetoma.ChemBiodiv.Template.docx ready for @mattodd to look at

3. Updates: USYD update: no updates, USYD was not present during the meeting UCL update: UCL gave an update on series 6 compounds mentioned above. Next stage may be hybrids should be thought of as well. Erasmus update: spreadsheet was updated with the screenings performed. MIC50 will be next on the agenda.

4. Any other business? Was there a discussion needed around the DNDi project described in this paper?

[wwjvdsande]

@OpenSourceMycetoma/corecontrib I made a new agenda for the next meeting. Based on the minutes from previous meeting I simply rotated the chair and the minute taker. If it needs to be different, please correct. Hope to see you all next Tuesday!

[wwjvdsande]

@OpenSourceMycetoma/corecontrib: @MA-Jjingyi and @wwjvdsande have updated the spreadsheet with the in vitro susceptibility data.

[bebi78]

@wwjvdsande @MA-Jjingyi @MFernflower Two new niclosamide derivatives 4SF5-Closamide and 4SCF3-Closamide are quite promising. Might be candidates for toxicity testing in the next summer. Maybe you can test the missing fragrances (maritima, nootkatone) and metal compounds until spring. I could write a paper about the fragrances then. @wwjvdsande mentioned that methylene blue was investigated as part of a photodynamic therapy before. Is there a publication about that?

[wwjvdsande]

@bebi78 no we never published the methylene blue study. It was quite tricky with the light we used. it did seem to work though but only under the special light

[bebi78]

@wwjvdsande Yes, the absorption maximum of MB is 664 nm, so lasers emitting in that range should be applied to get maximum phototoxic activity. Because MB in the ´´dark´´ or daylight already has an IC50 value of 2.1 µM, I think that submicromolar activities can be reached upon irradiation with a proper laser.

[MFernflower]

@bebi78 niclosamide seems promising - I guess it would be up to DNDI to work on clinical trials for it for mycetoma

seeing the 4SF5-Closamide gave me this idea for a molecule though: 4-Ethyl-Closamide

[bebi78]

@MFernflower Thanks for the suggestion. This compound was published in 2011 in a Czech journal I don't have access to (see below). Its abstract tells that it was tested for antituberculosis activity. But its butyl analog was mentioned as the most active derivative of this compound series. Our university compound database says that the 4-ethyl and 4-butylaniline precursors are available at our local Macromolecules department. So I will try to get them and prepare these compounds.

The antimycobacterial salicylanilides, potential inhibitors of ATP synthesis By: Waisser, Karel; Petrlikova, Eva; Kunes, Jiri; Vrabcova, Petra; Kolar, Karel; Stolarikova, Jirina Folia Pharmaceutica Universitatis Carolinae (2011), 39, 17-23

[bebi78]

@wwjvdsande @MFernflower Dr. Wainwright from the University of Liverpool has developed a promising lipophilic MB derivative (S137, see link below to a recent paper using G. mellonella fungal infection model). I think we should ask him for a sample of S137. https://link.springer.com/article/10.1007/s43630-022-00258-4

[MFernflower]

"Our university compound database says that the 4-ethyl and 4-butylaniline precursors are available at our local Macromolecules department. So I will try to get them and prepare these compounds."

Sounds good!

[fantasy121]

Addn'l Epichem Compounds Refined List.zip

[fantasy121]

I refined the list of gap compounds to 13, please see above.

Re: draft, my writing is completed.

[fantasy121]

Sorry I won't make it to today's meeting. Please send my apologies. I will catch up afterwards, just tag me if there's any action item for me thanks.

[wwjvdsande]

@fantasy121 will someone from USyd join? If not that we should use the link from UCL.

[mattodd]

Hi @fantasy121 for the additional Epichem compounds you've selected above, can you please just walk us through 1) Why those, i.e. what SAR questions will they help us answer, and 2) Whether there are any important SAR gaps that you think remain unanswered, and whether we need to make any compounds to answer them. This helps us justify any extra request to Epichem (which comes with a resources cost) as well as plan any remaining synthesis that we think is unavoidable.

[mattodd]

@fantasy121 will someone from USyd join? If not that we should use the link from UCL.

...and we'd need an alternative chair unless @kym834 or @alintheopen can join. If not, I can do this, given that Erasmus is minute-taker.

[mattodd]

OK, let's use UCL link https://ucl.zoom.us/j/99789829423

[wwjvdsande]

@mattodd could you add the recording to the minutes displayed above?

[fantasy121]

1. Why those, i.e. what SAR questions will they help us answer, and

• Match-pairs of tested compounds to show the potency of the 2-substituted position on the phenyl ring. What kind of impacts does removing 2-F substitution have?
• 3-Substitution is not well represented in our current analysis (a gap in SAR), so I grabbed some with 3-substitution to match-pair with current data of tested compounds.
• Extending the scope of SAR on modification of pyridyl ring
• Some of these compounds address more than one of the above points

2. Whether there are any important SAR gaps that you think remain unanswered, and whether we need to make any compounds to answer them.

• From the angle of 2-F or 2-substitution being important for potency, and 4-substitution being less important, there's an opportunity to modify 4-substitution further to see if activity can be improved (credits: to @kym834 for suggesting this). I'll be working on Suzuki cross-coupling to extend the 4-substituted phenyl ring. I made a new GHI for this, here

[dmitrij176]

Here is my edited SAR list. Blue: Matched with USYD suggestions compounds Red: Additional suggestions by UCL

Pyridyl ring: the heme coordinating region has not been well explored, so including at least 4 entries would be high priority. The top 3 molecules have the preferred 2F,4Cl substitution on ring 2, while the CF3 on ring 3 remains conserved. Having only pyridyl ring modifications as the only variable can allow to draw conclusions on their importance. Alkylation seems useful as well. (question asked: how that might affect coordination, potency etc.) Core modifications: in the paper we have concluded that modifications at the central carbon atom are essential for potency, especially the -OH group which is involved in hydrogen bonding with the target CYP51. Fluorine can H-bond as well and many recently approved antifungals have 2F linkers in their structure. Previously we have explored the -OH, -NH2 and -MeO motifs at C1. 3-Substiutution is indeed unexplored, so I agree on including these molecules (like 15839) Fluorine substitutions on ring 3: I added another 3 molecules (red: 5914, 2845, 2846) to the list. It has been long established that fluoro substitutions enhance safety profiles of the drugs which ultimately resulted in many marketed antifungals (most notable example-Fluconazole; Voriconazole, Posaconazole,Ravuconazole and many others). Again, here we have a set of 3 molecules with conserved structures except the fluoro substitutions (2,4; 3,4 and 2,5 positions). Definitely an SAR gap worth exploring. Ring 2 substitutions with CN and F groups: In the in vivo RD section (for the G1 scaffold) we conclude that the highest 10 day larval survival was achieved with scaffold bearing 2-CN on ring 2 and 4-Cl on ring 3, while 2-F,4-Cl on ring 2 generated similar results. For example, compound (ending with) 4842 would explore similar configuration, but with 2-F, 4-CN, whereas 0083 would show the importance 2-F group. Lastly, N,N-dimethyl aniline motif has been unexplored and has a different configuration to classical phenyl ring system which might give interesting assay results.

[mattodd]

@fantasy121 @dmitrij176 In advance of the next meeting (#3):

1. The Matched Pairs

For matched pairs, it helps to have an active compound already, as the comparator. Otherwise, if both compounds are inactive you don't really gain any information about the importance of the change. So in the cases we're discussing above can we just be 100% sure that there is already an active we're comparing against, and if so, what is the structure/code? Please can we draw these pairs side by side, to be sure - the known active (with potency) and the suggested matched pair compound? If we don't have an existing active, then we could consider just throwing the dice and getting a new pair tested, e.g. SCYX0001294212 and SCYX0001332845 where there is only a 3-F change. But let's be clear

I) 2-substitution on phenyl ring. What's the matched pair, i.e. what's the compound that has already been tested, and what's the new suggested test compound? Can either remove the F or replace it or both.

ii) 3-substitution. Ditto

iii) Pyridyl ring - definitely some useful options, but what's the matched compound? The first three compounds SCYX0001335912, SCYX0001574841 and SCYX0001574838 look good, but something needs to be active (these or something matched) in case all three are inactive.

So I think we need to test between 3 and 6 compounds here if we already have actives as comparators.

2. SAR gaps

4-substitution, as per https://github.com/OpenSourceMycetoma/Series-1-Fenarimols/issues/86. Just be careful of logD. N-alkyl rings might be a better solution as mentioned. But are any of the compounds you're proposing suitable as matches to things we already have in the paper? SCYX0001325593 suggested by @dmitrij176 has this kind of motif and would be useful to check too.

Obviously I'm assuming that none of these structures are already present in the Master List!

[MFernflower]

Found this really neat article - the drug used is super cheap might be worth screening against mycetoma

https://www.science.org/content/article/repurposed-drug-battles-brain-eating-amoeba

@bebi78

[bebi78]

@MFernflower @wwjvdsande Has nitroxoline already been tested as part of the pandemic response box?

[MFernflower]

I do not know would be a question for Wendy and her students

I may not attend this meeting however will have to see how it goes

@bebi78 @mattodd

[bebi78]

@MFernflower Haven't found nitroxoline in Wendy's pandemic box paper/supplement. So I suppose it has not been tested until now.

[MFernflower]

@MFernflower Haven't found nitroxoline in Wendy's pandemic box paper/supplement. So I suppose it has not been tested until now.

Seems like a cheap drug to get some for testing @bebi78 @mattodd

[bebi78]

@MFernflower I tried to prepare it from 8-hydroxyquinoline and have obtained a yellow powder now. Analysis will be done asap.

[MFernflower]

@bebi78 seems good

[bebi78]

@MFernflower Nitroxoline and 4-Ethyl-Closamide successfully prepared.