Project Launch. 3. Are There Other Sources of Molecules?

[mattodd]

MycetOS needs to identify other potential sources of molecules (actual samples) for Series 1 (the fenarimols, and subject of the preprint) but also as possible starting points for future series.

Does anyone know of a group in academia or industry that might be interested in donating a few milligrams of potential antifungal compounds for evaluation? These samples can be sent to Wendy van de Sande's lab in Rotterdam.

The compounds screened to date are in the preprint (MMV's Pathogen and Stasis boxes), and contained in the Master List. An appeal for which other compounds from the compound library in Epichem is discussed in #1. In addition, Wendy and others have in the past screened a number of "usual suspect" antifungals described in the following Word file (also archived here). See also this review for some of these data.

In vitro susceptibility of Madurella mycetomatis.docx

So the question remains, which other groups should we reach out to? Please post your suggestions below, or on Twitter, or if the suggestion is sensitive in some way please email the project (opensourcemycetoma@gmail.com)

[MFernflower]

@mattodd @wwjvdsande I think it's worth screening a handful of S4 malaria compounds against Madurella just to see if there is any ion channel cross reactivity

[MFernflower]

@wwjvdsande would you be able to screen paclobutrazol against the fungus? https://www.sigmaaldrich.com/catalog/product/sial/46046?lang=en&region=US

[wwjvdsande]

I could but what is the rationale behind it. Why this compound?

[MFernflower]

It's a pretty lipophillic azole drug so should be able to penetrate grain

[MFernflower]

But I feel that pouring all our effort into making a YACI (Yet Another Cytochrome Inhbitor) seems to be less than optimal

[wwjvdsande]

May be. But by using different proporties in the molecules we can also find out what characteristics are needed to penetrate the grain. We know of the cyp51 gene that when inhibited the fungus is hampered in growth in vitro but in vivo it is less efficiently inhibited due to the poor drug penetration. If a lipophilic azole would do the trick better this leads to insighs to proporties a drug needs to be able to penetrate the drug and cure it. So it indeed might be worth a try

[MFernflower]

@wwjvdsande I also wonder if current treatment protocol for the infection is flawed in some way - I.E if injecting fluconazole directly into the growths would be better than the current protocol of buckets upon buckets of sporanox given I.V? It would not be a pleasant treatment for the patient but it still beats out amputation by a long shot ! For example: in C. Neoformans brain infections the fungicides are given in the spinal fluid as opposed to IM or IV

[MFernflower]

@wwjvdsande Just curious. how long does it take to run a moth larva based screen?

[mattodd]

If we could try to also focus on sources of molecules, in addition to their identity, that'd be very good. I'm thinking of existing molecules AND where they might be. In the case of paclobutrazol (good discussion above) we'd not want to buy 250 mg, so we're then looking for someone willing to ship, or some other source?

Wendy, for an in vitro and in vivo evaluation you need what, 5 mg dry mass?

Further to the discussion over at OpenSourceMycetoma/General-Start-Here#5 for example, I'm wondering whether there are good sources of samples of aminothiazoles. Such requests may get more traction coming from our co-workers at DNDi or MMV, clearly.

[MFernflower]

(sorry for the repost - I clicked the wrong button) @mattodd @mattodowd25 I suggest that USyd indeed buy the 250mg of paclobutrazol from Sigma as we could send 20mg to Wendy and keep the rest for derivation experiments (see image below)

The end goal of all these shenanigans is to end up producing and sending a small library of paclo derivatives (4 or so compounds including the parent compound!) to @wwjvdsande for screening

[wwjvdsande]

The Moth model takes a month usually per run. I culture thet fungus, infect, treatment and monitor for 10 days. I repeat the experiment three times usually. WitHet any New drug i have to determine toxicityou firstaat. Which takes 20 days

[wwjvdsande]

Sorry. The last sentence is: with any New drug i have to determine toxicity first. Phone overwrites me with its spelling controle.

[wwjvdsande]

In vitro screening takes about the same time. It is a Slow Growing fungus.

[wwjvdsande]

In vitro screening takes about the same time. It is a Slow Growing fungus.

[bendndi]

@mattodd I'm looking into accessing the aminothozoles at the moment. Not sure if we still have access to the samples (we don't have our own compound storage, instead everything sits with partners. The aminothiazole work was done in collab with Advinus, and i should have more info on this middle of this week.

[mattodowd25]

@mattodd A paper I found just recently with a list of compounds that showed antifungal activity. Some similar to the fenarimol compounds in the EpiChem library. Maybe worth investigating as another source of molecules.

[MFernflower]

Can you post a few molecules from the paper so I can get a grasp on them? @mattodowd25 I don't have access to that paper

[mattodd]

Here are some representative structures from that paper @mattodowd25 found:

They are a little PAINS like, and the growth inhibition observed is remarkably similar across the structures, but I will email the corresponding author to ask if samples exist, and report back if anything emerges.

[MFernflower]

Thiazolidine-2,4-diones are known to be rather sketchy when it comes to whole cell screens - see the odd side effects and off targets of the glitazone class of diabetes drugs! @mattodd

[MFernflower]

The commercial wood preservative Cyproconazole also seems to bear resemblance to our S1 compounds

https://upload.wikimedia.org/wikipedia/commons/thumb/7/75/Cyproconazole.svg/200px-Cyproconazole.svg.png

@mattodd would it be possible for someone to ship this molecule to wendy?

[mattodd]

I had a fascinating day at Syngenta the other day. I was introduced by Dr Ed Emmett to useful resources summarising compounds used for herbicides, fungicides and insecticides (these posters are public domain but are all, I believe, copyrighted). It made me wonder whether we have, in our screens, covered this space (mainly herbicides and fungicides) @wwjvdsande and @bendndi ? Do you think there are commercial herbicides and fungicides we've not adequately covered? Or potential mechanisms of action we've not screened for that we ought? In essence, is there value in our requesting, of a company, samples of known molecules to ensure the space is covered?

There would obviously be value in our asking companies for samples of molecules that are not public domain/commercial, but that's a more difficult IP situation even though we're evaluating vs something that is presumably not directly related to their bottom line. I might come to you @bendndi for help with that if it looks like we could make headway, though those negotiations might have to go closed a little.

[MFernflower]

Apologies for the repost but my phone decided it would be a good idea to eat the formatting!!!!

Some agrochemicals that I think could be effective against M. mycetomatis at least in vitro @mattodd @wwjvdsande

Kresoxim-methyl [Mitochondrial poison] [May have already been tested!] Azoxystrobin [Mitochondrial poison] [May have already been tested!] Paclobutrazol [CYP51] Diclocymet [targets melanin synthesis] Tolprocarb [targets melanin synthesis] Flusilazole [CYP51] Metrafenone [Actin filament targeting] Procymidone [DAF1 kinase inhibitor] Tebuconazole [CYP51] Fluopicolide [Spectrin inhibitor] Cyproconazole [similar to fenarimol/CYP51] Iprodione [DAF1 kinase inhibitor] N-phenylsuccinimide [DAF1 kinase inhibitor] Synthetic protocol is super easy and can be done by highschoolers https://pubs.acs.org/doi/abs/10.1021/ed100983x

[MFernflower]

@mattodd @bendndi Think we can broker some sort of agreement to get a few API samples of Syngenta's goodies to be sent to Wendy for screening?

[wwjvdsande]

@MFernflower @mattodd In the pathogenbox we tested azoxystrobin and M. mycetomatis was potently inhibited in vitro. However in vivo no enhanced survival was obtained. The number of grains and the size of the grains in vivo did decrease. We received the pandemic box last week and we are planning to screen it this week and upcomming week. Fluopicolide is included, the rest of the above list not. Next to fluopicolide in the pandemic box the following antifungal agents are included which we did not screen in the past. Eberconazole (Seems promising - AMS) Ciclopirox (most likely too toxic - AMS) fenbendazole (Seems promising - AMS) OSU-03012 (Seems promising - AMS) pafuramidine (most likely too toxic - AMS) deferasirox buenafine (Seems promising - AMS) AN-2718 Metipirox NSC527092 TSC-INH Flutolanil (Seems promising - AMS) N-epoxymethyl-1,8-naphthalimide Abafungin (Seems promising - AMS) NSC 527094 FRurvina Oxfendazole Carbendazim (Seems promising - AMS) Alexidine (most likely too toxic - AMS) Amorolfine (Seems promising - AMS) Cispentacin (Seems promising - AMS) Lomefloxaxin

I m trying to hire a Bachelor student to help us with the in vitro testing. The assay I use is to difficult for a highschool student

[MFernflower]

This class of drugs also seems promising @wwjvdsande @mattodd @bendndi

https://www.frontiersin.org/articles/10.3389/fmicb.2016.02014/full

[wwjvdsande]

We could make a panel from fungicides and herbicides to complement the screening of the pandemic box. Hopefully I will have the screening data of these boxes by the end of this month. M. mycetomatis does grow slowly unfortunate. But if interested to see which molecules are in the pandemic box you can find out here: https://www.mmv.org/sites/default/files/uploads/docs/mmv_open/Pandemic_Response_Box_list_of%20compounds_March2019.xlsx

[bendndi]

We received the pandemic box last week and we are planning to screen it this week and upcomming week. Fluopicolide is included, the rest of the above list not. Next to fluopicolide in the pandemic box the following antifungal agents are included which we did not screen in the past

@wwjvdsande great that you recieved the PRB! I was the lead at DNDi for designing the content of the PRB on the Anti Bacterial and Antiviral side but the Antifungal contribution came exclusively from the team at MMV. If you want to know how they made the selection I can ask the MMV team.

[bendndi]

@mattodd @MFernflower Regarding accessing industry collections or contributions (e.g. Syngenta), I'd have to ask my screening colleagues at DNDi how to go about this. I'd imagine that Syngenta have a pre-prepared panel of diverse fungicides which they screen as standard for their internal projects. They may be prepared to send out a copy of that plate, perhaps blinded....

[MFernflower]

I edited your comment to highlight the ones in the box I think may be useful @wwjvdsande

Very good score with the pathogen box!

[MFernflower]

@mattodd @wwjvdsande @drc007 I figured I might share this - These are a few derivatives of terbinafine I was playing with a bit in silico as a possible killer of Balamuthia mandrillaris (a slime mold like protist known to cause fatal infections in humans) - this project never materialized so here's the three compounds:

Thoughts everyone on getting these made?

[wwjvdsande]

@MFernflower in our larval model, terbinafine was able to prolong larval survival, however to a lesser extent as some of the fenarimols. However, we only tested terbinafin itself. There was no analogue library tested so may be they could be more potent. It would be interesting to test some more.

[MFernflower]

@wwjvdsande During some free time I did some digging through the IRAC insecticide library (like I did with the fungicide library) and found a few chemicals that appear to mess with chitin synthesis - maybe they will work to bust cell wall in fungi?

Clofentezine, Diflovidazin, Hexythiazox, Etoxazole, Novaluron

[wwjvdsande]

@MFernflower they might work. For medical fungi nikkomycinZ is also in development and it is inhibiting chitin synthesis as well. In the past for aspergillus we also tested polyoxin D. However, from my experience with aspergillus the chitin inhibitors work better in combination with betaglucan inhibitors such as caspofungin. The main constituents of the fungal cell wall are chitin and betaglucan and they together are responsible for the strength of the fungal cell wall. In a previous study with aspergillus we (and others as well) demonstrated that if you inhibit betaglucan synthesis, chitin synthesis goes up to remain turgor in the fungal cell. When chitin was inhibited the opposite occurs, betaglucan is up. When you combine the cell wall inhibitors you get swollen cells, hardly any hyphae.

[MFernflower]

@wwjvdsande I wonder if inhibiting chitin production in vivo would allow for remission as the increase in beta glucan would activate the immune system - kinda like cancer immunotherapy

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762275/

[wwjvdsande]

@MFernflower for aspergillosis it has been demonstrated that interfering with either betaglucan or chitin can influence the immune response. So I don't see why it should not be so for M. mycetomatis

[MFernflower]

@wwjvdsande @mattodd Finished updateing my list of interesting agrochemicals - @bendndi do you have any list members that are not strobilurins in stock?

[mattodd]

FYI New Pandemic Response Box data posted by @wwjvdsande over at OpenSourceMycetoma/What-other-molecules-to-screen#1.