Seeking advice about SPR for a new approach to inhibitors

I should first say that I know very little about SPR as it is not my field. I have been reading up about the basics for the moment.
The reason I have joined the forum is to try to obtain advice from people with much more knowledge of this field than I.
The background is as follows - our target is Discoidin domain receptor 2 (DDR2) and we have been working on a new approach towards inhibiting this target for nearly two years now. This approach is to create inhibitors that work on the extracellular part of DDR2 and not the kinase domain that is intracellular.
After nearly two years we have a selection of molecules, some more drug-like than others that work in our cellular assay (inhibition of DDR2 activation by collagens). Some of the inhibitors reach IC50s of around 2 to 3uM and have moderate solubilities (15 to 100uM). However, although we had a binding assay at the start that seemed to give us information about affinity, this binding assay turned out to be a bit of a red herring and so we were forced to abandon it.
This means that we need something to replace the binding assay and so my thoughts turned to SPR. The problem is that there is almost no information published about SPR with the extracellular portions of the DDR receptors (DDR1 & DDR2). I have found some info in patents about SPR experiments with DDR1 and antibodies but patents contain very little experimental detail.
So my questions are as follows;
1) How to attached DDR2 ECD domain to a chip? I know that we can buy DDR2 ECD with a 6xHisTag - we have used this in our binding assay. Can this be attached to a chip? I know that the NTA chips are suitable for polyhistidine tag attachment - at least that is what I have read. is this true in practice? Here is an example:
www.novoprolabs.com/p/human-ddr2-cd167b-his-tag-503180.html

Can other tags be of use for attachment to chips? Here is another commercially available DDR2 ECD but his time Fc tagged:

www.creativebiomart.net/description_5076_12.htm

Why am I considering only commercially available DDR2 ECD - because of cost and time.

2) How important is a known ligand when setting up SPR experiment for ligand-protein binding? There are no known small molecules to use as standards - our molecules work in cellular assays but we do not yet know how. The only other known ligands that are better characterised are collagens and collagen peptides (these collagen peptides are a bit lower in molecular weight than collagen itself - they are around 4kDa)

For the moment I will stop writing questions (I do have more) and will wait to see if anyone can help me.

Hi,
Welcome on the forum of the SPRPages.

1)
The fastest way to immobilize a protein to a sensor chip is using the amine coupling and a dextran based sensor chip. This method has some drawbacks (random orientation of the protein for instance; coupling is permanent) but to get a first impression it can be good enough.
Using a 6xHIS-protein will give a oriented immobilization (I would say capture) of the protein to the sensor chip. For this method a special prepared NTA-sensor chip is needed. Second benefit is that the surface can be regenerated and used again. Sometimes the strength of the capture is low, resulting in some dissociation of the protein (decaying surface).
Capturing using a Fc-TAG is also a good option since most of these systems are fairly stable and you can regenerate and use the surface again. For this the sensor surface has to be modified with an antibody recognising the Fc-TAG.

2)
I would be nice to have a positive control to confirm that the protein on the sensor surface is intact and functional. The benefit of SPR is that you will see if the analyte (the compound flowed over the immobilized protein) will bind or not.

Which type of SPR instrument do you have access to?
There are alternative methods to look for interaction between molecules ( www.biapages.nl ).

Kind regards
Arnoud

Arnoud - thank you for taking the time to answer my questions.

What instrument do I have access to? At the moment none. I am trying to inform myself as much as possible before starting to look for someone that can do the experiments for us (as a paying service or as a collaboration).

As for attaching the His-tag DDR2 ECD, I understand the chemistry behind this (Nickel chelation) and the His-tag is in the correct place (C-terminal that would be towards the transmembrane part of the DDR2 receptor) so a DDR2 His-tag ECD attached to a chip should align itself in the correct way. The compounds we wish to test would not be good chelators at all so no problem there, although I understand that proteins can sometimes attach to NTA chips through histidine side amino acids that are not part of the His-tag.

As for the Fc-tag, what is the mode of attachment?

As for checking if the DDR2 ECD is attached, do you think we could use collagen peptides? There are known binding motifs and known non-binding motifs for DDR2 (see link below). Professor Farndale has determined not only which peptides activate the DDR2 receptor but also which bind and activate and which do not bind and do not activate and which do not bind at all, so this at least is quite clear.

collagentoolkit.bio.cam.ac.uk/toolkits/sequences

We already have some of these as we were using them for the now redundant binding assay. Can these be used in the same type of SPR instrument as would be used for small molecules? As I said before, these are not huge (MW around 4kDa) but they are at least 10 times bigger than the small molecules. Is this still in the range of an SPR instrument for use with small molecules?

I have considered other techniques such as Thermal Shift Assays but the concern here is that the ECD has two domains, discoidin and discoidin-like and this can make interpretation rather complicated.

As for the Fc-tag, what is the mode of attachment?

The protein you looked up has the Fc-part of human IgG1. Ideally you start with immobilizing anti IgG1 antibody to a sensor chip and capture the Fc-DDR2 ECD on the chip. So this is an affinity interaction.

The collagen peptides would be nice to verify the immobilization of the DDR2 ECD and to check if the protein is still active. As for analyte size cut-off, it depends on the instrument sensitivity but most commercial instruments can routinely measure down to 500 Da and high end even lower.

I have considered other techniques such as Thermal Shift Assays but the concern here is that the ECD has two domains, discoidin and discoidin-like and this can make interpretation rather complicated.

Surface plasmon resonance measurement will also not distinguish between the two domains. Depending on the strength of interaction one of the interactions may be more pronounced. However, SPR cannot tell you which of the two binds more without special experimental procedures such as making mutations in the DDR2 ECD interaction domains.

Kind regards
Arnoud

Thanks again Arnoud.

From your experience or knowledge, which attachment method would you try first?
As for the two domains, I am not worried about not being able to distinguish to which domain the small molecules bind. That can come later with a bit of mutational work. First I want to see an interaction with the ECD of DDR2 that correlates in some sort of way with the cellular inhibitory activity observed.
As for the thermal shift assay, again not my field, but I have been told that it can be difficult to interpret thermal shift assay of proteins with more than one domain and on top of this, from my basic understanding of SPR, SPR should give me more information than I could gain from TSAs.
I have to choose one technique as trying to get both done could be too energy and above all money consuming.

For you I think the NTA chip will be the easiest. Buy the 6xHIS protein and the NTA chip and find an SPR instrument.