Protocol Synthesis of 124I-labeled epichaperome probes and assessment in visualizing pathologic protein-protein interaction networks

I'm going to discuss today our work or an overview first of our approach to treatment, which is to target 1313 interaction, rather than a protein or an Interact or interaction of 2 proteins as generally or at least historically we think about therapy. And the paper I was invited to discuss is it's a protocol we published recently. In cancer cells SAR protocols, and it's called Synthesis of Labeled Epichaperome Probes and assessment in visualizing pathologic protein interaction networks in tumor-bearing mice.

Understanding Epichaperome Probes

I mentioned the context perhaps of cancer treatment chemotherapy, as we know, it's been historically the way of treatment. And as we have started to understand more about the drivers of cancer, we move to the targeted approach. Initially, we knew how to inhibit. So, there has been an explosion in kinase inhibitor. So even the first successful inhibitor, the BCR inhibitor. Yeah. It's kind of inhibitor. As we moved again, to understand more about the proteins involved and how they interact we moved into disrupting protein interaction, and now the big way is to actually degrade proteins. It's a very fashionable way to try to develop new treatments. But we actually know, although perhaps our human mind, it thinks more as one protein or one individual. We are not the one individual that just has proteins are not one protein. We exist in a, sorry, we exist in a society.

Epichaperome Researcher at Memorial Sloan Kettering Cancer Center

We exist as a group of interactions, whether those are a group of our colleagues, our family society as a whole, who is, are shaping us and we are shaping them. And it's this interaction network. That's really defining our word, our cellular functions our it's developing who we are and. Become, and that's exactly the same for proteins.

It's becoming more and more understand that also in disease and of course in cancer as well proteins are shaped and are in their significance and in their oncogenic behavior. They're interactive partners and these interactive partners are can be contact and tissue dependent, meaning one protein pathways such as Raf Kinase Inhibitor (RKIP) different interactions and different contexts.

And it's really this really the network of these interactions that will defining how to target the protein pathway or how the protein's really influencing the cell death or the phenotype as a whole in disease. So, in this context, disease is a really a severity of perturbation of these complex networks or molecular interaction in cell lines.

How to Target Epichaperome Probes?

So, how can you target such complexity? It's not much discussed because it's very difficult to really approach something like that, therapeutically and what we believe. So, in this context, you can think as disease is really. As I said a dysfunction in this protein networks study it's created not by one driver.

We like to think in cancer is genetics, but it's actually, as we will know it's a combination of factors, whether it's from aging environment, whether it's like a cell environment or the environment as a whole. What. Other diseases or comorbidities that we may have with the genetics and other factors that combine shape or negatively impact the cell is an environment creating disease or phenotype by restructuring this big networks of the proteins in How we can really address this complexity is for discovery. We published not long ago. First in in nature is that. The restructuring of protein pathways in disease has a helper. And this helper comes from a restructuring of a large protein family which is called the chaperone or molecular chaperones.

And we all know about chaperones that they fold. They interact dynamically with all sorts of proteins to help them fold or they work in aggregation, disaggregation. This is what we are talking about. What we're talking about is a restructuring of these members of the chaperone family into scaffolding platforms, we term Epichaperome Probes.

What are the Epichaperome Probes platforms?

Epichaperome Probes platforms do not fault. What they do is the reorganize protein interactions at the protein level and disease to regulate. The need of the cell under the pathologic process. Whatever that is. So, we have shown these appearing cancer, the Alzheimer disease of Parkinson. So, there's a mechanism of pathologic transformation through buying of protein networks through the formation of these scaffolding platforms called epichaperome. So why is this important? It's important because now you can think about a way to actually address targeting protein interaction.

You can target protein interaction by actually targeting the mediators of these alteration in protein pathways, destruction, meaning Epichaperome Probes. So, if there is a way that you can actually. Target these, detect these you can create both therapeutics and diagnostics for treatment and emerging or detection of these transformations in disease.

How to Detect Epichaperome?

The paper that we are in in discussing is really detailing how we created not only drugs to target this dysfunction, but also small molecules that we can use to detect these dysfunctions in protein pathways interaction networks. That, excuse me. I'm mediated by these Epichaperome Probes. And the paper describes the synthesis of these radio labeled agents.

There are small molecules that have attached, and I done 124. Which can be used in positive and emission tomography to visualize tissues tumors that are afflicted by these changes that are driven by epichaperome formation. And we have shown the use of these probes in a variety of settings from obviously to clinical, but also to all the way to clinical level.

Epichaperome Probe Application

And here, I'm going to show you this, an example in the application of these probes are detecting tumor cells that have this transformation mechanism. So, the alteration and protein networks that are driven by these Epichaperome Probes this is patient. This is an Alzheimer patient. You can clearly see the presence, the anatomical presence. And quantification through these imaging probes of specific lesion in the brain of this patient that very well aligned to disease known to manifest. You can also see another example here of a lung cancer patient is by the way, was a metastatic, triple negative breast cancer patient.

And these be published as you can see in the journals that I'm mentioning here. And these 2 probes add to another probe we develop, which is from hematologic malignancies. So, this is a as these are imaging agent, these are flow cytometry based agent that can detect these changes in subpopulations they're specific to the specific hematological malignancy.

And again, this was Also published and also its application to clinic published recently in the MPJ position, oncology paper in 2021. You asked me Stephanie what are the questions people are asking is us. And one of the major one that we are receiving is how can you actually differentiate?

Can You Create These Agents that can Differentiate Epichaperome Probes

How can you create these agents that can differentiate these Epichaperome Probes, which are only affection in the human body and are specific to these tissues undergoing the disease process. From this very abundant family of Epichaperome Probes. Epichaperome Probes, if you might know as I'm showing here, the one of the most abundant proteins in humans, and there are 10% of tall (inaudible) mass in human patients.

Which, and if you count for one of them, for example, HSP90, that really amounts to hundreds of grams of protein in any human. So how can you make these small molecules that can differentiate one from the chap ones from the Epichaperome Probes. So, you only want target the epi chaplains. And one of the issues here is also, and I think the question is coming because as you may know, Epichaperome inhibitors, such as HS dose have been tried extensively on oncology.

And have had issues with perhaps lack of efficacy or toxicity. So, there was a bias in the field or in, in the oncology field on this topic. So how can you differentiate an Epichaperome binding agent from a chap binding agent? And we have shoulders in an extensive study, which was published last year in nature communications that you can create a molecule that can differentiate this really abundant family from the small faction Epichaperome by modulating the kinetic activity of these agents.

Kinetic Activity, What Does it Mean in Epichaperome?

So, what does kinetic activity mean means that if this firm binds here, it exists very rapidly. And has no effect. However, if it binds to the epi gets stuck and it stay there. So that's why you can actually really use them to emerge these lesions. And here we are showing one of these strokes that I just mentioned, how it can detect only, you can see the red only in this brain tumor, but not in the normal brain anywhere that's around, which has, as I said, grams of the Epichaperome. So, the study really takes you from synthesis to testing in animals, to proof of principle in human patients, obviously in a clinical study setting. So, another question we are receiving is how can you implement or why do you wanna implement these agents to a clinic?

For several reason one is you can detect this tumor. So, what does, what is good that tells you. Well, it's, it really tells you that level of therapeutic vulnerability to the agents that target Epichaperome, because we have shown and published extensively that the higher the app Epichaperome levels, the higher, the number of proteins hired a number of proteins that are being negatively.

How therefore the higher, the number of a band protein food interaction less was the more vulnerable the cancer cell lines to the, a Epichaperome therapy. And we have in addition to extensive preclinical evidence, we have shown in a in a pilot study. Where this is a metastatic breast cancer study which patients at baseline.

Epichaperome Therapy Combination

So, before they ran on Epichaperome therapy combination with the bra vaccine, they were image with these agents and emission tomography agents. And you can see some cancer patients. The tumor lit up, meaning they're really high in this protein for detection network dysfunctions that are modulated by Epichaperome. You can see this patient went on therapy.

Clearly see how the tumor went down, both by cat scan and FDG patients who had. Negative Epichaperome level at the baseline, right? So, these tumors have lot of Epichaperome, but not Epichaperome, not these dysfunction protein for intervention networks that are mediated by Epichaperome and clear no signal on, on this post emission tomography, clearly mission demography, clearly, no response.

So, we could actually show in the small study that those cancer patients who actually. To stay longer therapy benefited longer from this therapy were those that had higher Epichaperome level, higher dysfunction in this protein pathway network. So, it's a way of stratifying selecting patients for this therapy.

Epichaperome Uses:

What other uses are for this? Obviously in addition to that you can actually derive target engagement measurements so you can understand what is the dose and the schedule you need to treat specific patient. Using this assay, we could actually derive that those that you need to give to these patients for treatment.

So, you don't, so it's an approach, not by maximum to. But maximum saturated those up to target, then you can gain from using these emerging agents and lastly what you can also we have learned from this is that using these app Epichaperome Probes problems, you can control how 1313 networks behave and manifest in the context of tumors.

So, we have recently published that you can modulate with these inhibitors the protein pathway interaction networks in a way that you make them very vulnerable to current therapy. So, in this paper, what we show is that by using these Epichaperome Probes inhibitors, we can bring the tumor to a state that is highly vulnerable, to already known therapy.

And we show this through a principle in pancreatic cancer. These cancer cells were there, as we all know, don't respond to, for example, MAC inhibitors, although RAS Kinase it's a known known driver activated pathway in these tumors. However, if these tumors were in MAC inhibitors were applied to these tumor, After we drove them into this epichaperome mediated state of vulnerability, they became very responsive to the MAC inhibitor.

Epichaperome Probes Synthesis of 124I-labeled Details

Epichaperomes are pathologic scaffolding linked with disease that are made up of firmly bonded chaperones and co-chaperones. They enable precision therapy by detecting and targeting faulty protein pathways interaction networks rather than a single protein. This technique discusses the manufacture and characterization of two 124I-labeled epichaperome probes, [124I]-PU-H71 and [124I]-PU-AD, which have both been used in clinical trials. It details how to utilize these reagents to visualize and quantify epichaperome-positivity in tumor-bearing mice using positron emission tomography.

Author Credentials and Affliations

Gabriela Chiosis, Ph.D., Researcher at Memorial Sloan Kettering Cancer Center. In this video, she speaks about the Synthesis of 124I-labeled epichaperome probes and assessment in visualizing pathologic protein-protein interaction networks in tumor-bearing mice.