Closed mbhebhe closed 1 year ago
Really interesting result. As you mention crystal structures of an asparagine-tRNA ligase are available e.g. 1X56. Perhaps we can build a homology model?
I can work on a homology model and try to identify an OSM-S-106 binding site. I can also produce Pf asparagine-tRNA ligase in the lab, verify binding to OSM-S-106, and pursue a co-crystal structure.
Excellent
Looks like the structure of Pf asparagine-tRNA ligase has already been done, PDB 5JLD. The paper was published in 2016, Dimerization of Arginyl-tRNA Synthetase by Free Heme Drives Its Inactivation in Plasmodium falciparum.
@holeung Would you be able to dock the drug to the PDB? I'd bet our drug is binding to the ATP intake site on the protein possibly causing a conformation change that deactivates the enzyme!
@holeung Just realized the PDB entry is for the malarial arginine ligase - perhaps it could still be useful for binding study as it could have the same atp intake site?
Right you are. Thanks for pointing that out. I need to use my reading glasses more.
Just updated the wiki with a link back to this GHI @mbhebhe @mattodd
A homology model would be great. @holeung how easy/difficult will it be to produce Pf asparagine-tRNA ligase? @MFernflower I had the same thought about OSM-S-106 binding to the ATP binding site. Thanks for updating the wiki.
Does not seem too difficult no glycosylation - just order an arabad plasmid with the pfal sequence from plasmid builder service and insert into TOP10 E. Coli
Purification and co crystal formation is what takes time
@mbhebhe
Also @mbhebhe if it is a mimic of ATP the purine analogue of OSM-S-106 should be somewhat more potent yes?
Purification and co crystal formation is what takes time
and we should not under estimate the length of time it can take.
Attached is a homology model of Pf AsnRS docked to OSM-S-106. The homology model was created with I-TASSER. ATP was then included, and the complex was subject to energy minimization in solvent using Yasara, with the Yasara2 knowledge-based force field. OSM-S-106 was docked into the ATP binding site region with smina, using the Vinardo scoring function. The score for the best pose was -6.8, which is very poor (more negative is better). The initial homology model might not be very good given that there are no crystal structures of AsnRS closely conserved with that from Pf. The zipped file contains the PDB and .mol2 files.
I conclude that the homology model supports the possibility of OSM-S-106 acting as an ATP mimic for AsnRS but cannot be considered strong evidence. The homology model may be useful for SAR analysis.
ATP is in gray.
I will look into the logistics of producing the Pf AsnRS protein. It shouldn't be too difficult, but it requires time, money, and labor, which are always challenges. My immediate goal is to get biochemical/biophysical evidence of binding, to provide preliminary data for a grant application, hopefully with @mattodd and Prof. Winzeler.
@holeung thanks for producing the homology model! Should we wait for the target validation studies and conditional knock down experiments to be done first before trying to produce the Pf AsnRS protein? I will be sending them more compound to them by end of this week
@mbhebhe , sounds good. What kind of validation studies will they perform?
Would it be possible to dock 3-(4,2-diaminothieno[3,2-d]pyrimidin-6-yl)benzenesulfonamide to the homology model when you have time? @holeung
NC1=NC(N)=C2SC(=CC2=N1)C1=CC=CC(=C1)S(N)(=O)=O
@holeung Validation studies through CRISPR/Cas9
@mbhebhe, great. I will contact them directly so we can coordinate and avoid duplication of efforts.
Its actually people from the Wellcome Sanger Institute in England. I will ask them to get a GH account so that we can easily communicate on here
I apologize for assuming it was the Winzeler laboratory! @mbhebhe @holeung
@mbhebhe , thank you for the clarification. Nice that we have so many contributors!
@holeung Was just wondering if you have access to poseview to create a 2d image of the docking study?
@MFernflower , 2D diagram attached. Made using Schrodinger Maestro.
^^ Gives me an idea:
NC=1N=CC=C2C1SC(=C2)C=2C=C(C=CC2)S(=O)(=O)N IPIRNZZMWQZXIW-UHFFFAOYSA-N
Still looks like replacing the sulphonamide with carboxylic acid should work.
^^^^ @drc007 @mbhebhe perhaps the reason the acid was not potent was because it could not cross the cell membrane - (M)ethyl ester perhaps?
Sigma stocks the required boronic acids in Australia but seems slightly pricey: https://www.sigmaaldrich.com/catalog/product/aldrich/591130?lang=en®ion=AU https://www.sigmaaldrich.com/catalog/product/aldrich/527548?lang=en®ion=AU
Hi all, just to connect you all to the MalDA consortium and explaining who is doing what. I am the Program Manager overseeing all the projects and coordinating the multiple groups (14 in total) in their efforts. We received your compound OSM-S-106 last year and I asked Eva Istvan from the Goldberg lab to perform the in vitro selections. Once she had isolated resistant clones the Winzeler lab performed the sequence analysis and identified the potential target. We have now reached out to Marcus Lee to validate the target by CRISPR/Cas9 and Jacquin Niles for conditional knockdown studies. We are all excited to be part of this group and working with you. Please let us know how we can help. Best, Sabine You can also reach me by email: sottilie2@gmail.com
@sottilie , thanks for the intro post! After the validation tests, I am interested in following up by biophysics and crystallography. Or do you already have that covered?
No I don't thing we will be able to do biophysics and crystallography - so wonderful if you can follow up with this. @holeung
Great thread based on a fascinating result. My guess is that you'd want to wait on the knockdowns before investing any experimental time @holeung ?
@mbhebhe have we not made the CH analog @MFernflower suggests? The thieno[2,3‐c]pyridine?
@holeung the pose of the ATP in the active site - how confident are you of the pose? I'd thought that in kinases (at least) the methionine residue (which you have) would be pointing at the free NH2? i.e. flipped over from what you have. Which is not to say that that applies here.
Shortest path to validation could be to find someone running assays for this enzyme in vitro? Try the authors of https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868750/ and https://www.ncbi.nlm.nih.gov/pubmed/24196969
Charise in Jacquin Niles' lab has received some of your compound and is already starting experiments on her cKD cell line.
@mbhebhe @mattodd I'd also be curious what effect the thieno[2,3‐c]pyridine system has on real world water solubility - IIRC the S3 hit had poorer than expected water solubility due to strong hydrogen bonds between molecules of OSM-S-106
We have not made the thieno[2,3‐c]pyridine analogue. The precursor is expensive though.
There are many synthetic methods reported @mbhebhe @mattodd https://doi.org/10.1016/j.bmcl.2006.08.041 https://pubs.acs.org/doi/abs/10.1021/jo016171j https://patents.google.com/patent/WO2014186035A1/en https://pubs.acs.org/doi/abs/10.1021/acs.joc.8b01037
Please excuse my mistake regarding the Sigma Aldrich product
There are a couple of other straightforward ways of gathering validation data that I can think of. First is that if asparagine tRNA synthetase is the target, the compound ought to block protein synthesis. We could test that relatively easily by radioisotope labelling of amino acids in the presence and absence of the inhibitor. Second is that the parasites may be less susceptible to the inhibitor in the presence of higher concentrations of asparagine, as the tRNA synthetase may be more efficient in the presence of higher asparagine levels. This may not be as pronounced if the inhibitor is indeed binding only to the ATP binding pocket, but is nonetheless a very easy experiment to conduct. Our lab would be happy to help out with either of those experiments if you are interested in going ahead with them. Stuart.
I think the 14C-asparagine experiment would be super useful - Thoughts @mattodd and @mbhebhe and @drc007 ???
It wouldn't have to be 14C-asparagine - although this seems more direct, if you get inhibition of protein synthesis, it would likely impact all amino acids, so you could use whatever radio-labeled amino acid you have handy
The Winzeler lab is also able to do a protein synthesis inhibitor assay since we have another aa RS target for a different compound. Only problem we are out of your compound OSM-106.
Sabine Ottilie, Ph.D. Project Manager Malaria Drug Accelerator (MalDA) Consortium Yeast Drug Target Discovery Lab of Prof. E. Winzeler | UC San Diego Division of Host-Microbe Systems & Therapeutics Biomedical Research Facility 2 | 5220D 9500 Gilman Dr | MC 0760 | La Jolla, CA 92093-0760 http://winzeler.ucsd.edu/
On Tue, Jan 15, 2019 at 6:01 PM MFernflower notifications@github.com wrote:
I think the 14C-asparagine experiment would be super useful - Thoughts @mattodd https://github.com/mattodd and @mbhebhe https://github.com/mbhebhe and @drc007 https://github.com/drc007 ???
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@StuartRalph This might be an obvious question but what happens when the ribosome runs out of a certain trna - does translation just lock up on that codon or does the chain prematurely terminate?
Great thread based on a fascinating result. My guess is that you'd want to wait on the knockdowns before investing any experimental time @holeung ?
Not 100% clear on what you are expecting from the knockdown experiments. This is almost certainly an essential protein, and the piggybac data indicate it can't be disrupted. The parasite will have reduced protein synthesis and may be more susceptible to the inhibitor, but that might also apply to knockdown of other off-target proteins required for protein synthesis. (eg if you knockdown an off target ribosomal component, protein translation will be compromised, and those parasites may also be more sensitive to a second (chemical) insult to translation. I would have thought that induced or constitutive over-expression of the target (with or without the SNPs) would be a more direct and more conclusive genetic experiment.
You would see a shift in IC50 if the compound indeed hits the target. It's a quick validation study we do for all our putative 'targets' that we find by in vitro selections.
Sabine Ottilie, Ph.D. Project Manager Malaria Drug Accelerator (MalDA) Consortium Yeast Drug Target Discovery Lab of Prof. E. Winzeler | UC San Diego Division of Host-Microbe Systems & Therapeutics Biomedical Research Facility 2 | 5220D 9500 Gilman Dr | MC 0760 | La Jolla, CA 92093-0760 http://winzeler.ucsd.edu/
On Tue, Jan 15, 2019 at 6:10 PM Stuart Ralph notifications@github.com wrote:
Great thread based on a fascinating result. My guess is that you'd want to wait on the knockdowns before investing any experimental time @holeung https://github.com/holeung ?
Not 100% clear on what you are expecting from the knockdown experiments. This is almost certainly an essential protein, and the piggybac data indicate it can't be disrupted. The parasite will have reduced protein synthesis and may be more susceptible to the inhibitor, but that might also apply to knockdown of other off-target proteins required for protein synthesis. (eg if you knockdown an off target ribosomal component, protein translation will be compromised, and those parasites may also be more sensitive to a second (chemical) insult to translation. I would have thought that induced or constitutive over-expression of the target (with or without the SNPs) would be a more direct and more conclusive genetic experiment.
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@sottilie But would it be possible to obtain reliable results when the target is an essential piece of machinery for protein synthesis? I'd be worried about abnormally early cell mortality causing test interference
Jacquin Niles has done this for quite a few aaRS and it seems to work well.
On Jan 15, 2019, at 6:18 PM, MFernflower notifications@github.com wrote:
@sottilie But would it be possible to obtain reliable results when the target is an essential piece of machinery for protein synthesis?
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^^ Extremely interesting....
@StuartRalph I can send some compound to you guys. Is 5mg enough?
There are a couple of other straightforward ways of gathering validation data that I can think of. First is that if asparagine tRNA synthetase is the target, the compound ought to block protein synthesis. We could test that relatively easily by radioisotope labelling of amino acids in the presence and absence of the inhibitor. Second is that the parasites may be less susceptible to the inhibitor in the presence of higher concentrations of asparagine, as the tRNA synthetase may be more efficient in the presence of higher asparagine levels. This may not be as pronounced if the inhibitor is indeed binding only to the ATP binding pocket, but is nonetheless a very easy experiment to conduct. Our lab would be happy to help out with either of those experiments if you are interested in going ahead with them. Stuart.
@sottilie I will send some more compound tomorrow
The Winzeler lab is also able to do a protein synthesis inhibitor assay since we have another aa RS target for a different compound. Only problem we are out of your compound OSM-106. Sabine Ottilie, Ph.D. Project Manager Malaria Drug Accelerator (MalDA) Consortium Yeast Drug Target Discovery Lab of Prof. E. Winzeler | UC San Diego Division of Host-Microbe Systems & Therapeutics Biomedical Research Facility 2 | 5220D 9500 Gilman Dr | MC 0760 | La Jolla, CA 92093-0760 http://winzeler.ucsd.edu/
JFTR , assuming https://pubchem.ncbi.nlm.nih.gov/compound/44528665, MQMXDJVOZKMSNT-UHFFFAOYSA-N and OSM-S-106 are one and the same :) PubChem provides a useful list of 33 analogues https://www.ncbi.nlm.nih.gov/pccompound?cmd=Link&LinkName=pccompound_pccompound&from_uid=44528665. This represents the 90% Tanimoto shell as computed internaly. Note that other OSMs are also in there (via ChEMBL) and that 18 should be available via credit card (OK, I know OSM doesn't have one). It's a slight shame the internal synonym did not make it in (i.e. OSM-S-79 is PubChem-positive but S-106 is not) but this is fixable.
Last year, we sent OSM-S-106 to the Winzeler Lab (UCSD) for mechanism of action studies via the generation of resistant mutants. Last week they emailed us the results! Exciting!! Raw data is available here.
This was they said in the email:
"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)."
In short, asparagine-tRNA ligase is the target.
I have been doing a bit of reading on asparagine tRNA ligase/synthetase (AsnRs). From what I have read this is novel Pf target and I could not find any inhibitors for this target. We asked the Winzeler Lab if they knew of other molecules that hit this enzyme. They don't know of any other molecules but they offered to do more target validation through CRISPR/Cas9 and conditional knock down experiments. (Is this a good idea?).
Since this is a novel target, it seems that this Pf enzyme has only been expressed/cloned once (Ref 1). Please let us know if you know of anyone that has worked on Pf AsnRs or has it. We would like to do some binding studies on this enzyme. Conducting binding studies on Homo sapien AsnRs, would also be great . On the protein data bank website there are 8 AsnRs structures from 4 organisms (one human and no Pf).
Asparagine tRNA synthetase catalyses the attachment of Asn to its cognate tRNA to produce tRNAAsn, a substrate for translation. This is done via a 2 step reaction. An ATP molecule and Asn molecule enter the active site, forming an aminoacyl-adenylate intermediate followed by the esterification of the Asn to the 30 end of the tRNA, forming the final tRNAAsn (Ref 2).
The papers I have read so far talk about Pf proteome having a lot of asparagine rich regions but yet there is only one gene that encodes tRNAAsn. They conducted studies to see if there is any correlation between these asparagine rich regions and tRNAAsn. AsnRS is highly expressed in the cell and it is not the limiting 'reagent' in the first step of the asparaginylation reaction (Ref 1&3).
Stuart Ralph and colleagues from UniMelb wrote a review on Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites. In this review they say that disrupting tRNA synthetases will poison the process of protein translation and this will affect the parasite. Parasites have bacteria like protein translation pathways and are different to those of human pathways. We can target the binding site for ATP, amino acid and the fold for tRNAamino acid recognition and binding (Ref 2). In summary, aminoacyl-tRNA synthetases are promising drug targets and if OSM-S-106 truly does target the novel, Pf AsnRs then we are have a promising drug.
Ref 1: doi: 10.1074/jbc.M113.522896 Ref 2: http://dx.doi.org/10.1016/j.ijpddr.2013.10.001 Ref 3: doi: 10.1093/femsre/fux046
Sorry for the text overload! Not even a single diagram :'(