Open Source Mycetoma Meeting 25 Jan 2022

[mattodd]

Open Source Mycetoma (MycetOS) Meeting

Recurring Meeting Zoom link: https://uni-sydney.zoom.us/j/99281549497

Time: New York 7 AM - UTC 12 noon - London 12 noon - Rotterdam 1 PM - Sydney 11 PM - (2022 schedule discussed below)

Chair: Erasmus

Minute taker: UCL

Recording can be found [here]

Minutes can found below

Link to previous meeting #66

---

AGENDA

1. Outstanding items from previous meeting

• [ ] Dmitrij and Hung to agree on common grounds for colour-coding visual summaries (threshold or criteria for green/amber/red/black). We agreed to keep the visual summary concise and free of clutters such as compounds IDs, potency values etc (reserving these for tabulation of data). This conversation should continue at GHI Structural Activity Relationship Analysis of biological evaluation For Fenarimol Analogues/Epichem compounds #51
• [ ] Manuscript needs urgent updates of Chemistry data (Dmitrij ready, Hung to do) @dmitrij176 @fantasy121 #55
• [ ] Literature reading is recommended to see if our SAR agree with exisiting SAR somewhere else. (See Keenan’s SAR papers of fenarimol for Chagas, and other papers). Point of interest for lit reading: Azoles vs fungal cytochrome P450 (CYP51 protein) @dmitrij176 @fantasy121

Potential starting points (Keenan's papers on Fenarimols vs Chagas, SAR, CYP51 etc.) DOI: 10.1021/jm2015809 DOI: 10.1016/j.bmc.2013.01.050 DOI: 10.1074/jbc.M113.497990 DOI: 10.1021/jm401610c

https://doi.org/10.1016/j.bioorg.2020.104240 (History of the development of antifungal azoles: A review on structures, SAR, and mechanism of action)

2. Discussion points for this meeting

1) 2022 schedule will be:

4th Tuesday of each month:

JAN 1pm EU FEB 10pm EU MAR 1 EU APR 10 EU MAY 1 EU JUNE 10 EU JULY 1 EU AUG 10 EU SEPT 1 EU OCT 10 EU NOV 1 EU DEC 10 EU (but likely no meeting)

• [x] @kym834 to send out these invitations to the core team using outlook.

• [ ] @mattodd to consider how to set up a shared sheet of email addresses for participants and as a means of having people sign up for MycetOS newsletters as we did for OSA.

• USYD update: @fantasy121 described his work on the SAR for the paper. Everyone is encouraged to read and suggest edits directly.

• Link to paper writing #55 .
• Recap of work at #68.
• Further ongoing discussions of SAR at #51

• [ ] @fantasy121 to forward paper to @alintheopen and Peter Rutledge (can someone please remind me of Peter's Github handle?) and ask for edits.
• [ ] @fantasy121 to create a list of targets that arise from this analysis for potential future consideration. e.g. combination of L1 amide linker region with quaternary centre OH.

Discussion of Mechanism of Action: @fantasy121 presented some reading on mechanism. There are no crystal structures of madurella proteins (correct?) and no structures of proteins from other organisms in which are bound fenarimols (correct?). @wwjvdsande has models created previously for madurella proteins. It is possible we could ask SSGCID to express and solve a madurella protein and even soak in a known binder (@mattodd to reach out to @bartrum about this possibility). Knowledge of how to express other madurella proteins would be v useful (or we could pursue something easier with high homology e.g, Albicans). Ultimately it would be nice to demonstrate target ID. (@wwjvdsande this did not come up, but in theory could you generate a conditional knockdown for Cyp51 and see whether the IC50 for a known active is reduced? This is quite common in malaria.

• [ ] @fantasy121 to post notes on MoA below. Please can the info also be added to the MoA page, which is very out of date.

• [ ] @wwjvdsande to dig up and share previous computational models created for Cyp51s

• [ ] @mattodd to investigate possible inputs from SSGCID

• [ ] @wwjvdsande to post any useful technical details of madurella protein expression that might help SSGCID.

• UCL update

@dmitrij176 spoke about SAR analysis and the design of 3 compounds including dioxolane functionality. All those can be made and shipped along with 5 ketoxime analogs in next batch. The latter will be evaluated as mixtures of E and Z isomers, with the possibility of separation being examined if activity is observed (note new repo for these compounds https://github.com/OpenSourceMycetoma/Series-1B-Ketoximes)

• [ ] @dmitrij176 to post powerpoint update slides below.

• Erasmus update

Ran out of time!

There was a discussion about the importance of pursuing combination therapies. That after this paper is complete we should prioritise new targets as a means to develop molecules that can complement each other. @wwjvdsande mentioned several lines of enquiry that look promising e.g. olorofim (vs DHODH) and the benzimidazoles.

@kym834 mentioned that there was interest at Sydney for high school students to synthesise compounds in the benzimidazole series. @mattodd is also in discussion with @AndyBishopSevenoaks about exactly this and the students are keen to get going.

• [ ] @AndyBishopSevenoaks to advise if he needs any further inputs in advance of lab work starting.

3. Any other businesses?

Did not come up, but do we have any compound requests currently with Martine at Epichem, or were they all sent?

World NTD Day is on Jan 30th. Here is a picture we can share on social media to highlight MycetOS, and I'd suggest we link back to this issue (please @fantasy121 and @dmitrij176 can you post your slide decks for this meeting below before then).

4. Action Items for Aug 2021 Meeting

Action items are those with checkboxes above.

[fantasy121]

@wwjvdsande To confirm.

I am missing IC/MIC values for MYOS 00170, 00171, 00175 and in vivo for MYOS 00174.

• MYOS 00170 is not tested for IC/MIC because @25 uM activity is 38%. So this one is cleared.
• MYOS 00174 has excellent in vitro all the way to MIC50. However, the larval season has ended before in vivo can be tested on this compound. I want to double check I have the facts correct so I can explain this in the paper. (So it's not that this one has been tested in vivo but results are not updated, for instance)
• That leaves MYOS 00171 and 00175 still missing IC/MIC values. I need an update whether these were tested for IC/MIC (but not yet updated) or these are not tested for IC/MIC (which needs explaining in the paper because these guys have excellent activity @ 25 uM.)

I am particularly interested in 00175 result because this would clarify the SAR further.

[MFernflower]

@mattodd I may be missing something but CYP51F is a core eukaryotic enzyme so complete knockout of both copies of the gene will cause the cells to stop working

Maybe knocking out one copy of the gene is possible?

[MFernflower]

@mattodd @wwjvdsande @kym834 With regard to the benzimidazole type tubulin inhibitors

There are two commercial compounds we have not tested vs eumycetoma:

[MFernflower]

@dmitrij176 Just sort of randomly came up with this idea - should be a single step from the chlorinated common precursor and Dimethylethanolamine

[mattodd]

Predictions of Madurella protein structures (all of them, I think?) https://alphafold.ebi.ac.uk/search/organismScientificName/Madurella%20mycetomatis

[wwjvdsande]

The two CYP51 variants of Madurella mycetomatis. The heme group is indicated in ball and stick.

Between the two variants only one aminoacid difference is found. In the picture above this difference is indicated with the arrow.

[mattodd]

@wwjvdsande What's the lighter coloured spaghetti in the middle? There's nothing bound in there, right? Just amino acid side chains? @fantasy121 Where would an azole drug be expected to bind, based on the xstal structures you've been looking at?

[wwjvdsande]

@mattodd there was nothing bound. The ball and stick is just the heme group. Similar to the improved Aspergillus fumigatus CYP51A model, the M. mycetomatis CYP51A structure is predicted to be accessible from the left cleft beside the heme structure.

[wwjvdsande]

The fastafiles of the two variants are found here:

CYP51A protein sequence 499S variant MGLVQDIASPLAEGFSRFGLASQIGIAFGVFLFVAVLLNVLQQVLFRNPNEPPLVFHWFPLIGSTITYGMDPPRFFKENRAKYGECFTFVLLGKKTTVYLGTQGNDFILNGKIRDVCAEEIYTVLTTPVFGKDVVYDCPNSKLMEQKKFMKVALTTDAFRSYVPIISDEVTSYFKRTSDFKGQSGIVNICPKMAQITIFTASHALQGKEIRSKFDESLADLYHDLDMGFSPINFKLHWAPLPWNQRRDHAQRTIAKIYMDTIKSRRARGETDAKDIMWHLMNSEYKNGVKVPDHEIAHMMIALLMAGQHSSSSTSSWIMLRLASRPDIMEELYQEQVKNLGADLPPLKYEDLAKLPLNQAIVKETLRLHAPIHSIMRAVKQPMPIPGTKYVIPTNHVLLAAPGVSASDPQYFPEPDLWEPHRWEKESPLAPSIVRNEAAEEDEEKVDYGYGLVSKGAGSPYLPFGAGRHRCIGEQFANVQLQTIVAMTVRLFKFRNVDSSNKVIGTDYASLFSRPLEPANIYWERRDKE CYP51A protein sequence 499G variant MGLVQDIASPLAEGFSRFGLASQIGIAFGVFLFVAVLLNVLQQVLFRNPNEPPLVFHWFPLIGSTITYGMDPPRFFKENRAKYGECFTFVLLGKKTTVYLGTQGNDFILNGKIRDVCAEEIYTVLTTPVFGKDVVYDCPNSKLMEQKKFMKVALTTDAFRSYVPIISDEVTSYFKRTSDFKGQSGIVNICPKMAQITIFTASHALQGKEIRSKFDESLADLYHDLDMGFSPINFKLHWAPLPWNQRRDHAQRTIAKIYMDTIKSRRARGETDAKDIMWHLMNSEYKNGVKVPDHEIAHMMIALLMAGQHSSSSTSSWIMLRLASRPDIMEELYQEQVKNLGADLPPLKYEDLAKLPLNQAIVKETLRLHAPIHSIMRAVKQPMPIPGTKYVIPTNHVLLAAPGVSASDPQYFPEPDLWEPHRWEKESPLAPSIVRNEAAEEDEEKVDYGYGLVSKGAGSPYLPFGAGRHRCIGEQFANVQLQTIVAMTVRLFKFRNVDGSNKVIGTDYASLFSRPLEPANIYWERRDKE

[MA-Jjingyi]

@fantasy121 Hi, Hung, sorry to reply a little late. we already have known that MYOS170,171,175 didn't work below 25uM. and the final tests will be finished next week.
as for MYOS174, it was tested in vivo test just one time in last larvae season. and it looks weird, because all larvae died in the 3rd day. it will be the priority in the upcoming larvae season. i will update the data about IC50s and IC90s as soon as i finish them.

[fantasy121]

@wwjvdsande What's the lighter coloured spaghetti in the middle? There's nothing bound in there, right? Just amino acid side chains? @fantasy121 Where would an azole drug be expected to bind, based on the xstal structures you've been looking at?

Sorry for the late reply, I was sick for a few days. My understanding is that the nitrogen atom on the aromatic ring of the Azole co-ordinates to the iron of the haeme, this is the main interaction. The H-donor/acceptor (like a hydroxy group) and the disubstituted phenyl group interacts with nearby residues (usually Tyrosines, the positions of these vary slightly between CYP51 of different species) which disrupt these residues from stabilising (D-rings of) the haeme and instead strengthen the H-bond network of the drug with the haeme. Usually the active pocket can only accommodate these bits of the azoles and the remainder of the drugs (if present) will sit outside of the pocket forming more interaction with the residues around the entry to the active site, for more stabilising effects.

I am not experienced in reading off the crystal structures directly and will seek help from friendly biochemists in my building if more detailed clarification is required but the above points are what I gathered from reading the papers that publish these structures.

[mattodd]

@kym834 Hello, sorry to hassle, but are you happy to send out the Outlook invites for the next meeting? I almost double booked myself just now - I think we're due to meet 10pm EU time on Feb 22nd?

[kym834]

@mattodd. All done. @OpenSourceMycetoma/corecontrib please forward the two outlook invitations on. I've not get everyones email address

[kym834]

Also @mattodd, we already made a sign up form for the newsletter - #42

I don't remember what email address I used to create it though as it is not within the Drive of any of those that I use regularly. Do we have a gmail account for MycetOS that maybe I used?

[mattodd]

Hi @kym834 @fantasy121 should have the sign-in deets for the MycetOS gmail address. If not, I can dig them out somewhere. Yes, it would be useful to manage all interested contacts using that.

[MA-Jjingyi]

@wwjvdsande To confirm.

I am missing IC/MIC values for MYOS 00170, 00171, 00175 and in vivo for MYOS 00174.

• MYOS 00170 is not tested for IC/MIC because @25 uM activity is 38%. So this one is cleared.
• MYOS 00174 has excellent in vitro all the way to MIC50. However, the larval season has ended before in vivo can be tested on this compound. I want to double check I have the facts correct so I can explain this in the paper. (So it's not that this one has been tested in vivo but results are not updated, for instance)
• That leaves MYOS 00171 and 00175 still missing IC/MIC values. I need an update whether these were tested for IC/MIC (but not yet updated) or these are not tested for IC/MIC (which needs explaining in the paper because these guys have excellent activity @ 25 uM.)

I am particularly interested in 00175 result because this would clarify the SAR further.

Hi, @fantasy121 Hung. the IC50, IC90 and MIC50 data about 00171 and 00175 are updated. please check.