Hypha Results (Jan 2021)

[danaklug]

We have received results from the Hypha screen of OSA_000821 against their standard PolyCYP and AOX/FMO3 panel (more information here; raw data here; and notebook page here).

Summary

The compound of interest (OSA_000821) is incubated with panel enzymes, which carry out metabolic reactions. The resulting mixture is analyzed for metabolites of interest. Output is mass, retention time, and TIC (I believe) of metabolites.

The major species detected are two monohydroxylated metabolites (+16 Da, 0.88 min and 0.95 min) produced by the PolyCYP platform and one glucuronide (+176 Da, 0.90 min) produced by human liver S9 enzymes.

Follow-up and outstanding questions

• The next step is to scale up, isolate, and identify the metabolites of interest and discussions regarding this are ongoing.

• Can we get confirmation from Hypha that the data provided in the spreadsheet is indeed the TIC of each species?

[MFernflower]

@danaklug I'm guessing since the glucuronide will be difficult - the next step would be to incubate a bunch of OSA_000821 with lots of PolyCYP 483 and try to determine the two sites of hydroxylation - @mattodd will hypha do this for us or will it be done in UCL?

[mattodd]

Yes, the next steps for this very useful initial work is to check the conversion of the PolyCYP 483 oxidation of OSA_000821 and then scale it up, which Hypha have offered to do (probably in February). Then handover of the material to us here at UCL to purify the target metabolites and elucidate the structures with NMR. I'll post any developments received by email.

[MFernflower]

Thanks @mattodd See you friday!

[mattodd]

Update from Julia at Hypha by email Feb 15th.

"The dose escalation study is complete and Emily will be undertaking the scale-up so that, all being well, we will be able to send you the extract for purification week commencing 1st March.

We are dosing the substrate in the scale-up at 300mg/L which should give a higher absolute yield of the +16s, although the overall % conversion will be lower than that observed at a lower dose.

One of the +16s is very minor and Richard thinks this may not be related to any enzyme reaction, but the other +16 is definitely a metabolite of PolyCYP 483. Structure elucidation of the products should reveal all!"

Thank you Hypha colleagues, we're looking forward to receiving this and isolating the metabolite.

[mattodd]

Scale-up material has been shipped from Hypha and should arrive at UCL tomorrow.

[danaklug]

Hypha HPLC chromatograms have been uploaded and attached to the notebook page above.

[edwintse]

UPDATE: Hypha metabolite purification has been completed. I've got ~1-2 mg (balance accuracy questionable...) but I've managed to get 1H, 13C, COSY, HSQC and HMBC on it and have confirmed the structure as hydroxylated on the southern methylene carbon.

[MFernflower]

@edwintse perhaps it might be worth taking some OSA_000821 and mixing it with MCPBA in DCM to see if you can abiotically make more of the hypa metabolite?

[MFernflower]

@edwintse @danaklug The paper referenced from above:

https://applbiolchem.springeropen.com/track/pdf/10.1007/s13765-018-0404-2.pdf

[edwintse]

Characterisation data for the isolated metabolite (1D and 2D NMRs and the mnova file) have been uploaded here.