Kinome

[mattodd]

Some things arising from the interesting Mycetoma ISNTD session the other day.

1) Really quite striking when the current clinical trial regimen (Eisai/DNDI) was shown by Ed - a whole year of treatment with surgery in the middle. We meed new medicines!

2) Niclosamide as a possible drug. This is @wwjvdsande 's 2021 paper. Also MMV665807. Is this project being handled separately, i.e. no inputs needed from MycetOS?

(CC-BY image)

3) Below's a screenshot of a nice slide from Ed. We need this kind of "pipeline status" on our Mycetoma Github web pages somewhere in a way that can be maintained. Maybe a separate repo detailing currently-available meds, or those in development. Volunteers? @dmitrij176 @fantasy121 @kym834 if you're ever writing stuff like this for reports/theses, please duplicate on Github, or vice versa. @AndyBishopSevenoaks let us know if any of your students might be interested in doing some writing like this.

(Hope it's OK to share this Ed).

4) Olorofim seems v promising @wwjvdsande . We're not looking at it as part of MycetOS. As for niclosamide, Wendy, this project's proceeding just fine away from MyectOS? Or should we be doing something?

5) @dmitrij176 is looking at the MoA of fenarimols and of the (probably not related) ketoxime series. We need to keep this in mind so that we can be more aware of the influence this might have on what we make - i.e. are there known aspects of SAR against other pathogens that we can learn from. This info needs to go on the wiki describing current candidates too.

6) Are there any obvious chemical libraries that have not yet been screened in vitro, e.g. PKIS?

7) The DepMap provides a genetic resource for essentiality in cancer - which mutations cause exploitable vulnerabilities. Is there an equivalent for e.g. Madurella, to allow for rational prediction of which unexplored targets could be pursued?

[wwjvdsande]

@mattodd @Wilson-Lm @MFernflower @dmitrij176 @fantasy121

2. Niclosamide and MMV665807 was tested by two PhD students from the laboratory of Samy Khalid and Pascal Mayer. I helped them with setting up the MIC testing. May be they would like to join. Might be a nice avenue and also good to attract more collaborators for MycetOS
3. As part of a Dutch thesis, the PhD studeten also has to write a review and publish that. Wilson wrote a nice review on the compounds he tested during his PhD and discussed the pro's and con's. It is a bit more extensive than Ed's review over here.
4. For now I would consider olorofim in a different light. It is already in clinical trials and the gap between synthesizing new drugs and testing them in vitro and in vivo and the clinic is very big. Olorofim is already in clinical trials for other fungal infections and might be able to bridge this gap. It seems to work fine and could potentially be the next candidate for clinical investigation in mycetoma patients. It has a different mode of action so combination therapy might also be interesting (although in vitro we only found indifference), but we all learned from mycetOS that in vitro is not always predictive for in vivo
5. With regards to chemical libraries which we did not screen. We did not screen a true repruposing library. @MA-Jjingyi has been very busy adapting our assay so that we could also screen larger libraries. We can now screen in 384 wells plates, which means that we could do more molecules at the same time. However most libraries are commercial libraries and simply out of our reach financially. Most of my grants run out by the first half of 2022 so that means that money becomes thight and I can't afford to buy such libraries.
6. There is no DepMap for Madurella. CDC now sequenced the genome in more depth so that we might have a better genome. We are also going to sequence Falciformispora senegalensis and some of the actinomycetes. May be that helps.

[mattodd]

OK re 2) @wwjvdsande would you like to invite those students to join? Maybe they could come to the next MycetOS meeting, or have a conversation with you or me?

3) Any existing writing from any students about the status of medicines for mycetoma - would be very good to convert to something on the wiki.

4) I'll reach out to see if PKIS is available.

7) But beyond the genome you need to know which targets are essential, right? Through knockouts etc?

[wwjvdsande]

@mattodd

2. I will do so.
3. We can make M. mycetomatis fluorescent now by randomly integrating GFP into the genome. Our post-doc Saskia du Pre was able to pull this off. She is trying to make a targeted knock-out but M. mycetomatis prefers the non-homologous pathway so it is very difficult. Unfortunately her post-doc with us will end by the end of this year. I would love to have her longer on the project but I simply lack the funding. With a bit more time she would certainly be able to pull this off. She is very good!

[mattodd]

Hi @wwjvdsande I reached out to the SGC folks at UNC Chapel Hill about the kinase inhibitor library:

"Mat, PKIS does not exist anymore. It has been replaced by KCGS. KCGS is accessible through Ximbio. https://ximbio.com/reagent/157681/the-kinase-chemogenomic-set-kcgs KCGS is only 184 compounds – the set is smaller than PKIS because it was optimized for human kinase diversity not chemical diversity. Tim"

So would someone (e.g. @Wilson-Lm or @MA-Jjingyi) like to see if the set can be ordered for free, for academic use? I got to the screen where you have to make an account with the supplier, and then stopped...

Kinase inhibitors for fungal targets is not, it seems, a bad idea?

https://www.mdpi.com/2079-6382/10/10/1223

[MFernflower]

@mattodd @wwjvdsande A few of the enzymes targeted in that 184 molecule set are quite ancient and have homologs in fungi

[MFernflower]

@MOUSEY007 Didn't a few kinase inhibitors show good efficacy vs PFLA'S? could be interesting to see if anything overlaps provided Wendy can get the kinase box and screen it.

[MOUSEY007]

Yes @MFernflower there were several kinase inhibitors identified in our drug repurposing paper here; https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0008353

A better and in depth analysis of the kinome is needed for these organisms.

[wwjvdsande]

@mattodd @MFernflower @MOUSEY007 I contacted ximbio with the question if they could donate the library for this project. I will let you know when we get a response

[AndyBishopSevenoaks]

@mattodd yes even though the synthesis is the more exciting part of the project this sounds like something that would work really well for our students. The students would need some direction on what the content needed to look like but they are a bright group and are passionate about the open source pharma model. Last year the group produced a little video in lock down, to give you an idea as to what they are capable of producing in the space of a few hours! https://www.youtube.com/watch?v=fFEuHvsb-YA

[mattodd]

I think @MFernflower highlighted this recent paper and motif: https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01299

[bebi78]

@MFernflower @mattodd @wwjvdsande Very nice. Has there been any progress with the kinase inhibitors? I published two papers about new kinase inhibitors and new dual HDAC/kinase inhibitors as anticancer agents based on an older work from 2010. Both projects are ongoing and the published compounds are in stock. 103.3ClQuin-SAHA.IJMS.pdf @MFernflower @mattodd @mattodd Very nice. Has there been any progress with the kinase inhibitors? 12.EJMECH3991.tyrphos.2010.pdf 97.ThioIva.IJMS.pdf

[MFernflower]

I don't think the kinome of this fungus is fully known - you did already include a selection of covalent type inhibitors (e.g curcumin analogues) in your shipment so I guess we will see if something hits @bebi78

On Fri, Feb 11, 2022, 2:05 PM bebi78 @.***> wrote:

@MFernflower https://github.com/MFernflower @mattodd https://github.com/mattodd @wwjvdsande https://github.com/wwjvdsande Very nice. Has there been any progress with the kinase inhibitors? I published two papers about new kinase inhibitors and new dual HDAC/kinase inhibitors as anticancer agents based on an older work from 2010. Both projects are ongoing and the published compounds are in stock. 103.3ClQuin-SAHA.IJMS.pdf https://github.com/OpenSourceMycetoma/What-other-molecules-to-screen/files/8051019/103.3ClQuin-SAHA.IJMS.pdf @MFernflower https://github.com/MFernflower @mattodd https://github.com/mattodd @mattodd https://github.com/mattodd Very nice. Has there been any progress with the kinase inhibitors? 12.EJMECH3991.tyrphos.2010.pdf https://github.com/OpenSourceMycetoma/What-other-molecules-to-screen/files/8051018/12.EJMECH3991.tyrphos.2010.pdf 97.ThioIva.IJMS.pdf https://github.com/OpenSourceMycetoma/What-other-molecules-to-screen/files/8051017/97.ThioIva.IJMS.pdf

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[bebi78]

@mattodd @MFernflower I should also have some of the known commercially available kinase inhibitors such as sorafenib and others in reasonable amounts (min. 2 mg) we use as control compounds. I can make a list next week if you like.

[bebi78]

@MFernflower Yes, but the curcuminoids and cinnamides don't inhibit kinases.

[MFernflower]

Is the MOA of those shipped covalent inhibitor compounds known? If I had to predict I'd say maybe they target metabolism like maybe mitochondrial enzymes? @bebi78

[bebi78]

@mattodd @MFernflower From time to time I order known kinase inhibitors for anticancer research from LC Laboratories because of their prices (see link below).

Concerning the curcuminoids, we have not tested these compounds for kinase inhibition because kinase inhibition is rather uncommon for these compounds or rarely investigated as anticancer mode of action as far as I know (while there are several examples for substituted acrylonitriles including in vivo data). MePip-SF5 showed cell death and apoptosis induction, ROS formation and effects on stem cells (see attached file). More recent results show that it is also well tolerated and suppresses melanoma metastasis formation in mice (upon i.p. treatment with non-toxic doses). A paper is in preparation with my colleagues of the University Hospital Mainz about that in vivo effects.

https://www.lclabs.com/ 101.SF5.biomolecules.earlyview.pdf

[bebi78]

@mattodd @MFernflower In terms of the niclosamide compounds, the exchange of the CF3 group of MMV665807 by a SF5 group (often dubbed as super trifluoromethyl group) might lead to an interesting compound.

[MFernflower]

"MePip-SF5 showed cell death and apoptosis induction, ROS formation and effects on stem cells" @bebi78 if I am not mistaken isn't that similar to what niclosamide does?

the kinome exploration is ultimately up to @wwjvdsande and her students however

[bebi78]

Yes, you are right. I suppose the nitro-arene of niclosamide contributes to ROS formation. I'm not sure if the CF3 or SF5 groups have a similar mechanism or if, as you mentioned before, rather mitochondrial damage is the source of higher ROS levels for them. BTW, the quinazolines (dual HDAC/kinase inhibitors) of the paper 103.3ClQuin-SAHA have no Michael system and belong to the class of gefitinib related compounds. I just was happy to see that you plan to test kinase inhibitors and if I can do something in this regard let me know since I'm working on such compounds anyway. @MFernflower @mattodd @wwjvdsande

[MFernflower]

@bebi78 It was fascinating talking to you

I'm not sure if the mycetoma kinome has even been mapped!!!! I'm certain Wendy would know more than I do about that!

[wwjvdsande]

@MFernflower @bebi78 @mattodd @Mickey-Konings so far I did not receive any reply from ximbio for the kinase library. So we did not screen those yet. However, CDC just released the new genome of Madurella mycetomatis and we have transcriptomic data on grain development in the Galleria mellonella larvae model which we are currently writing up. We might be able to do an in silico analysis of this first to determine at least the kinome in silico. Would that be of any help? These data are all there we simply did not use it in this way yet. Might also be of use for any other target we think of. For the transcriptome I have collaborated with Imad Abugessaisa of RIKEN in Japan. I'm sure he would love to be part of such an endavour. Any ideas where to get other kinase inhibitor libraris or would the compounds from @bebi78 library be good to test out to make it a more complete story?

[mattodd]

I think pursuing the ximbio library is a sound idea, since that's an excellent collection. Possibly reach out again? Then @bebi78's inhibitors would obviously also be v interesting, if there is no overlap with the ximbio compounds. How many compounds are we talking about, all told?

[wwjvdsande]

@mattodd I approached them again. Fingers crossed!

[bebi78]

@mattodd @mattodd Yes, try to get the ximbio library first. In case they don't want to cooperate we have a plan B.

[wwjvdsande]

@mattodd @bebi78 Ximbio got back to me. Because they are a non-profit organisation, they only can offer their tools for a fee. The fee was £2400 plus shipping costs. Are there other kinase libraries which we could try to obtain?

[mattodd]

How many compounds is it again, and would they be able to ship enough for the assay and follow-up? That many not be too much money, actually, and we could search around for potential funds to purchase.

[wwjvdsande]

The Kinase Chemogenomic Set (KCGS) v1.0 is a collection of 187 potent and selective small molecule ATP-competitive inhibitors that were produced in drug discovery programs targeting protein kinases. Each inhibitor has been profiled for binding against >200 human kinases at DiscoverX. Each inhibitor has a Kd < 100 nM for its target kinase with a selectivity index S10 < 0.04 at 1 µM. In total, KCGS indexes 215 human kinases including many poorly studied (dark) kinases. KCGS is available from the SGC in aliquots of 1 µL of a 10 mM solution in DMSO. This means that we will not be able to screen it at 100 uM but we have to screen lower (especially if we will screen in duplo or triplo which we normally do). It will not be enough to screen also in the larvae. We usually treat with 20 uM in the larvae. However, the latter we might be able to resolve by either remaking the compounds or obtaining single compounds in a larger quantity of course.

[bebi78]

@wwjvdsande @mattodd Sounds good anyway. If there is a hit from the in vitro assay we can buy more of the hit compound from Sigma-Aldrich or other providers for the larvae assay.

[wwjvdsande]

@mattodd @MFernflower @bebi78 I just sent an application to the fungal infection trustfond to try to obtain funding for purchasing the ximbio kinase chemogenomic set. Keep our fingers crossed!

[wwjvdsande]

@MFernflower @bebi78 @mattodd hereby the preliminary data presented. We will make a sepperate tab in the spreadsheet to upload all.

The hits were: <html xmlns:m="http://schemas.microsoft.com/office/2004/12/omml" xmlns="http://www.w3.org/TR/REC-html40">

Hits (Duplo) | compound | alternate name | KCGS 2.0 | member of original KCGS | member of KCGS Expansion | SMILES string -- | -- | -- | -- | -- | -- | -- A10 | GW855857 | yes | yes | no | Cc1cccc(n1)-c1nc(Nc2ccncc2)c2ccccc2n1 B18 | GSK2606414 | yes | yes | no | FC(C1=CC(CC(N2CCC3=C2C=CC(C4=CN(C)C5=NC=NC(N)=C54)=C3)=O)=CC=C1)(F)F M11 | GW569293E | yes | yes | no | OC(=O)\C=C\C(O)=O.OCCCNc1nccc(n1)-c1c(nn2cc(ccc12)C(F)(F)F)-c1ccc(F)cc1 M15 | THZ531 |   | yes | yes | no | CN(C)C\C=C\C(=O)NC1=CC=C(C=C1)C(=O)N1CCC[C@H](C1)NC1=NC=C(Cl)C(=N1)C1=CNC2=CC=CC=C12 N2 | AKI-063a |   | yes | no | yes | N1(=C(N=CC=C1C=3(N2(C(C=NC=C2)=NC=3C4(=CC(=CC=C4)C))))NC5(CCCCC5)) O6 | PA-16-0081B | yes | no | yes | O=C(NC)C1=NN(C2=C1C(C)(CC3=CN=C(N=C23)N([H])C4=C(C=C(C=C4)N5CCOCC5)OC)C)C

[bebi78]

@OpenSourceMycetoma/corecontrib Because staurosporine was active but many other kinase inhibitors were inactive, I checked the Covid Response Box results again for kinase inhibitors. Indeed, the close staurosporine analog midostaurin was inactive, like many other well-established kinase inhibitors. However, the EGFR inhibitor osimertinib and the AXL inhibitor bemcentinib showed promising activity against M. m. and F. s., both drugs might be available for clinical tests from the providers. In addition, osimertinib has shown antifungal activity (see link). https://www.translationalres.com/article/S1931-5244(22)00073-1/fulltext

[wwjvdsande]

@OpenSourceMycetoma/corecontrib This paper recently appeared on pubmed: https://pubmed.ncbi.nlm.nih.gov/37398159/ It is on the woronin body, which is a fungal specific organelle. Apparantly the DYRK kinase YakA plays a role in the blocking of the septal pore by the Woronin body under stress. Inhibiting YakA with 1-ethoxycarbonyl-beta-carboline (1-ECBC) prevents stress mediated septal pore blocking and synergises with the azoles. So this might be a good compound (series) to explore further. Has anyone access to 1-ECBC or analogues thereof. i did not see them at Sigma.

[MFernflower]

It almost looks like the series 3 compounds - especially the curaxin ones!

Message ID: <OpenSourceMycetoma/What-other-molecules-to-screen/issues/7/1835995822@ github.com>

[bebi78]

@MFernflower @wwjvdsande I checked it on Scifinder and Biosynth (UK) offers 1 mg 1-ECBC for 135 USD as the cheapest provider.

[bebi78]

@MFernflower @wwjvdsande Quite interesting, tyrphostin A9 was very active against Mm. Very simple compound with in vivo activity in various cancer models.

[bebi78]

@MFernflower @wwjvdsande Harmine and INDY were also described as DYRK1 inhibitors.

https://aacrjournals.org/mcr/article/15/4/371/89773/A-Systematic-Analysis-of-Negative-Growth-Control

[bebi78]

@OpenSourceMycetoma/corecontrib Here are the structures and summary of the active kinase inhibitors I sent.