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Everything to do with Open Source Malaria Series 3
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CRISPR editing #19

Closed mbhebhe closed 1 year ago

mbhebhe commented 4 years ago

Our collaborators at the Wellcome Sanger Institute did some CRISPR editing to confirm that the Asn-tRNA ligase mutation is causal for resistance. The mutant parasite was generated by using CRISPR gene editing. They introduced the R487S mutation that came up in the selection with the most resistant clone (#12 ). Then, the isogenic mutant parasite underwent a drug response assay with OSM-S-106 to determine IC50 value in parallel with the wild-type Asn-tRNA ligase parasite (in this case they used both Dd2 and 3D7 wild type strains). Their most recent work can be found here

MFernflower commented 4 years ago

If the T-RNA synthease isn't the target but allows for resistance perhaps our drug targets the synthesis of the amino acid itself? @mattodd

MFernflower commented 4 years ago

@mattodd perhaps the molecule behaves as a competitive asparagine synthetase inhibitor at the ATP binding site? @mbhebhe

drc007 commented 4 years ago

@mbhebhe Do you know if the mutated enzyme is still functional?

holeung commented 4 years ago

We know for sure that Asn-ligase is not the target?

mbhebhe commented 4 years ago

@MFernflower if 106 binds to the ATP binding site then that would affect translation. We have seen that the molecule does not inhibit translation. @drc007 I will ask them. @holeung not completely. It could still be the target.

MFernflower commented 4 years ago

@mbhebhe @drc007 I have a hunch that while it doesn't bind to the enzyme that loads empty transfer RNA's with asparagine - it might bind to the enzyme that converts aspartic acid to asparagine, thus lowering the protist's pool of asparagine to fatal levels - somebody could confirm this by measuring levels of an ASN rich protein - if after drugging the levels fall sharply, then we may have our MOA!

mbhebhe commented 4 years ago

They don't know if the mutated enzyme is still functional. They would need to engineer it as a recombinant protein and then have it tested in an in vitro assay. If they assume the protein is essential, then it presumably is still functional.

MFernflower commented 4 years ago

@holeung The sequence of Plasmodium Vivax ASNS is known - Would you be able to try to model and dock 106 to it?

MCGILAIFHSSIERHRLRRKALELSKKLRHRGPDWNGIVVEENEDGTTNVLAHERLAIVD VLSGHQPLYDDAQEVCLTINGEIYNHMELRKLLPEDTIKKLTSHSDCAVIPNLYKNFGEK TPSMLNGIFSGVISDHKKNTFFAFRDPIGVCPLYIGYAADGSIWFASEFKALKDSCIRYV NFPPGHYYINCKNKGEFVRYFNPNWWDLSAAIPNNRADLEQIRVHLERAVVKRLMGDVPF GVLLSGGLDSSIVAAIVARHLKKIQGEGSGGGSGAAAAHQAAHEASPPNGEDNCGNPPAD SCHLKSFSIGLRNSPDLKAAKEVANFLGSHHTEFHFTVEEGVDSLHDVIYHIETYDITTI RASTPMYILSRLIKSSCVKMVLSGEGADEIFGGYLYFHKAPNREEFHRELQRKVHDLHLY DCLRANKSTMAFGIEARVPFLDLHLLDVVMNIDPKEKMCSEGHIEKDILRRAFAGYLPDH ILYRQKEQFSDGVGYNWIDGLKEYAESKVSDIQFSRAPFLFPYNTPKTKEAYLYRCIFSE CFPEQCAQESVPEGSSIACSSSKAVEWDASFKQNPDQSGRSVLGVHHSAKQFADVKPVQL PDDESRALLTRAS

mattodd commented 1 year ago

Closing. This page is linked on the wiki. The data linked at the top of the page supported Asn-tRNA synthetase as the target.