AaRS assay at SGC-UNICAMP

[mbhebhe]

Sub-issue of #12 as that issue is getting long

OSM-S-106 (hit) and OSM-S-137 (negative control) have been sent to SGC-UNICAMP (Brazil). They will be expressing (or have expressed) the asparagine-tRNA ligase and running our compounds in their assay. Hopefully this is the target!

[MFernflower]

Really exciting stuff!

[mbhebhe]

AsnRS assay at SGC - UNICAMP:

They say they tried several approaches and they saw no inhibition or interaction between OSM-S-106 and the enzyme. They tried Isothermal Titration Calorimetry (ITC) and DSF.

"ITC can detect binding (10s of microM to low nM). Using ITC we detected binding between the protein and the amino acid. We could not detect binding between the protein and the inhibitor (we did not try with the negative control)." "I have performed the DSF (differential Scanning Fluorimetry) using the enzyme in the presence of OSM-S-106 and OSM-S-137, and varying the concentration of amino acid (from 25 uM to 10 mM of L-asparagine), with and without MgCl2 (sometimes act as co-factor for these enzymes); for all curves, I could not observe any increase in the thermostability (which could suggest the binding of the compounds to the enzyme). For now, I don't have further idea to assess if the binding is happening."

We are waiting on them to send us data and a write up on what they did. Once I have that I will post it one here.

I came across a paper (https://doi.org/10.1073/pnas.1405994111) that had a compound that inhibited our target. Unfortunately they don't have the compound anymore, we wanted to use it as a positive control for the AsnRS assay.

Plasmodium falciparum encodes for two copies of asparaginyl-tRNA synthetases; cytosolic and one located in the apicoplast. The guys at SGC - UNICAMP expressed the cytosolic AsnRS, the same as what MalDA reported to be the target. So the two sets of results are in disagreement. Since the cell free plasmodium translation assays also shows no inhibiton #15 , should we move on to the other suggested candidates reported by MalDA? Nucleoside transporter 4 and Glutamate Dehydgrogenase #12 . I will start looking into who runs assays for these two targets.

Any suggestions on what to do next?

[MFernflower]

I'd vote for the glutamate dehydrogenase assay @mbhebhe

[drc007]

@mbhebhe in the PNAS you reference they describe Borrelidin, a natural compound that inhibits threonyl-tRNA synthetase. Did I miss mention of AsnRS inhibition?

[mbhebhe]

In Table 1 they list some analogues that inhibit other Pf tRNA synthetases and one of them inhibit AsnRS. Most of the paper they talk about Borrelidin. From what I have read, I think the reason they did the inhibition assay was because the analogue (CHEMBL1163087) had been previously identified as an antimalarial drug targeting aminoacyl-tRNA synthetases and it lacked potency data. So they ran it in their assay and their data suggests that it inhibits the cytosolic asparaginyl-tRNA synthetase.

[drc007]

OK I see, but the inhibitor of AsnRS is not a Borrelidin analogue.

[drc007]

Perhaps a step back and reassess all the data to date?

How strong is the evidence that either Nucleoside transporter 4 or Glutamate Dehydgrogenase is the molecular target?

[mbhebhe]

We are waiting for SGC-UNICAMP to send us data.

As for the other targets, this is what MalDA had sent us last year:

"Results for OSM-S-106 are a little more complicated but there is some consistency across the 3D7 and Dd2 results.

In the 3D7 line, PF3D7_0103200 (nucleoside transporter 4) has a His26Gln mutation and PF3D7_0802000 (glutamate dehydrogenase, putative) has a Lys64Gln mutation in all clones. There may also be slight amplification across PF3D7_1107800 (AP2 domain transcription factor, putative). I’ve noted that the resistant clones all showed a ~3x shift so the consistency of these mutations makes sense.

In the Dd2 line, there are also mutations in PF3D7_0103200 (nucleoside transporter 4), but they are different across flasks 2 and 3 – His320Leu in flask 2 clones and Ser22Cys in flask 3 clones. An Asp200Tyr mutation in PF3D7_0802000 (glutamate dehydrogenase, putative) is in both clones from 3 but not 2. Interestingly, both clones from flask 2 have an Arg487Ser mutation in PF3D7_0211800 (asparagine--tRNA ligase), while the clones from flask 3 have an amplification across this region but not the SNV. There is a much more significant difference in the fold-shifts for the resistant lines (~9x in flask 2 clones vs. ~2-3x in flask 3 clones) so the SNV vs. CNV may account for that discrepancy. I think the mostly likely explanation of these results is that asparagine--tRNA ligase is the target and nucleoside transporter 4 and glutamate dehydrogenase mutations are resistance/compensatory mechanisms (though glutamate dehydrogenase is also a possible Pf target)."

People are currently working on validating the three targets

[mbhebhe]

The guys from SGC-UNICAMP have sent us the report.

CONCLUSION: no activity could be detected for both the positive and negative control on PfAsnRS

[mbhebhe]

@rafacounago we have been working with Prof Shozeb Haider from UCL and we told him about the conflicting results we have been receiving. He suggested that co-crystallization of the active compound with the asparagine-tRNA ligase should work. He suggested that maybe chop off the loops (as seen in #20 ) in the Pf ligase and make it like the Hs one and it should crystalize like the Pf one. Is that something you guys could do?

[rafacounago]

@mbhebe before we commit to pursuing crystallization efforts, we´d like to have a positive result on the protein-ligand interaction from a biochemical/biophysical assay. We just introduced a new biochemical assay for tRNA synthases in the lab. I will post what we find here as soon as the results are in. Best wishes.

[mbhebhe]

@rafacounago thanks for letting us know. Fingers crossed

[mbhebhe]

Since OSM-S-106 wasn't inhibiting the tRNA ligase in the AaRS assay, we asked our other collaborators working on the genetics side of the project to have a look at the data and results from the AaRS assay. They had a few questions, comments and suggestions for the guys at SGC-UNICAMP. This is a summary of the discussion:

1. PlasmoDB gene ID for the enzyme they are expressing: PlasmoDB PF3D7_0211800
2. They expressed the full length protein with 610 residues with the N terminal his tag.
3. Q: Did their reaction buffer include PPiase? For some aminoacylation reaction, the PPi released could be inhibiting so adding PPiase to break it down into 2 molecules of Pi could help with increasing the signal. A: They didn’t use PPiase in the buffer reaction.
4. It was suggested that they repeat the inhibition studies with an alternative activity assay where S/B is higher and high variance lower. They have started another assay, a luciferase-based assay. They will also be increasing the concentration of the compounds on their assays.”

[mattodd]

Closing. This page is linked from the wiki, and the report is available here and on Github. Paper to be published shortly will likely explain the lack of activity seen in the biochemical assay.