Identifying new molecular targets for application to other less known diseases has been just as much a drug discovery process. The BLT1 is an immune/inflammatory regulator within the leukotriene signalling pathway. This knowledge about how it functioned and whether it could be a candidate for treatment is what made space for new drugs – especially new imaging.

Figure 1. Structure and binding site of hBLT1. (Michaelian N, et al.; 2021)

I will explain in this blog post why BLT1 is important for drug discovery, why disease is rooted in BLT1, and how new strategies are being conceived to target the receptor and snipe at it for therapy.

The Leukotriene B4 Channel: An Overview

Leukotrienes are lipid messengers formed from arachidonic acid by 5-lipoxygenase (5-LO) enzyme. But the most important of all these leukotrienes is the anti-inflammatory molecule leukotriene B4 (LTB4). LTB4 is a protein produced by many immune cells, to beckon and direct inflammatory cells to an injury or infection.

LTB4 works by attaching to two G-protein-coupled receptors – mostly BLT1 (or LTB4R1) and BLT2. Particularly, BLT1 shows up on every inflammatory cell: neutrophils, eosinophils, T lymphocytes. LTB4 stimulation of BLT1 initiates downstream signalling responses (phospholipase C, intracellular calcium increase, inflammatory cytokines and chemokines). This kind of signalling mediates leukocyte chemotaxis, adhesion and infiltration of the inflammatory process.

This inflammation function meant BLT1 triggered asthma, rheumatoid arthritis, inflammatory bowel disease, psoriasis and atherosclerosis. That's why BLT1 has become an attractive model for the design of anti-inflammatory medicines to dampen leukotriene signalling.

BLT1 Pathogenesis of Disease

BLT1 is a causal agent of several chronic inflammatory conditions, and overexpression contributes to disease progression and symptom severity. Voici a few diseases for which BLT1 is implicated:

• Asthma: In asthma, BLT1-mediated signaling draws inflammatory cells like eosinophils and neutrophils to the airways, where they block the airway, secrete mucus and hyperresponsiveness. Blocking BLT1 signalling has already been effective for alleviating asthma symptoms and avoiding exacerbations.
• Rheumatoid Arthritis (RA): In RA, an autoimmune disease with a chronic joint inflammation, BLT1 recruits immune cells to the synovium. The results are pro-inflammatory cytokines and damage to the joint tissues. BLT1 antagonists can prevent inflammation and protect the joint.
• Inflammatory Bowel Disease (IBD): In IBD (such as Crohn's disease and ulcerative colitis), BLT1 signaling kickstarts the inflammatory response to damage the gut. Tilting BLT1 could lower inflammation and re-repair mucosal repair in IBD patients.
• Atherosclerosis: BLT1 is responsible for attracting inflammatory cells into atherosclerotic plaques where it makes plaques unstable and contributes to cardiovascular disease. BLT1 antagonists could even help with inflammation of the plaque and cardiovascular health.

Because BLT1 is so implicated in these diseases, agents that target and inhibit BLT1 signalling are particularly promising as therapies for these disorders. But they would have to know a lot more about BLT1's function and how it causes disease before such treatments can be made and put on the market. Here is where imaging tools step in.

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Leukotriene B4 Receptor1 Imaging Analysis

Why Imaging Is Crucial for Drug Discovery

Imaging has been a must-have in drug discovery, particularly when finding and validating new targets. For BLT1, imaging can allow scientists to monitor receptor expression, see how receptors activate and how drugs candidates affect in real-time. Imaging can also be extremely valuable to the pharmacokinetics and pharmacodynamics of BLT1 inhibitors to optimise their efficacy and reduce their side-effects.

Some newer imaging techniques are being applied to research BLT1 and its disease activity:

1. Radiolabeled Ligand Binding Studies

This is a common approach to characterise the binding of receptors such as BLT1 using radiolabelled ligands. If they pair a radioactive isotope with a ligand binding specifically to BLT1, they could monitor how the ligand moved around and bound in real time. This protocol enables BLT1 activity in tissues and organs to be plotted and offers key information on receptor abundance and tissue distribution. We can imaging the spread of radiolabeled BLT1 ligands in animal models, for instance, or even in human trials, by positron emission tomography (PET).

2. Fluorescence Imaging and Confocal Microscopy

Fluorescence imaging like confocal microscopy allows for high-resolution, real-time monitoring of BLT1 expression and receptor signalling in the cell. By labelling BLT1 or its signalling molecules with fluorescent tags, scientists could monitor receptors in living cells and tissues. It is possible to use this method to dynamically monitor BLT1 activity during drug treatment, providing useful information about how BLT1 antagonists or agonists work. Fluorescence imaging can also be used to investigate receptor internalisation, which is also crucial to BLT1 signalling. Once activated, BLT1 endocytoses and this is what terminates signaling. If you track receptor internalisation, scientists can assess whether a drug is able to control this process and stop the signals.

3. Vivo Imaging of Disease Models in Vivo Imaging of Disease Models

To make inferences about the function of BLT1 in disease, scientists use animal models of inflammatory diseases very much like human ones.

In a model of rheumatoid arthritis, for instance, the imaging with MRI could show joint inflammatory activity, and calculate the impact of BLT1 inhibition on progression of joint damage. The same applies to animal asthma models: NIRF can be used to monitor the movement of inflammatory cells into the airways and measure anti-inflammatory activity of BLT1-targeting medications.

4. Single-Cell RNA Sequencing and Imaging

We can now learn more about the cell heterogeneity that underlies BLT1-induced inflammation with new technologies for single-cell RNA sequencing. With the combination of single-cell RNA sequencing and imaging, they can identify where BLT1 is expressed within individual cells and what role it plays in the inflammatory process at the molecular level. This allows us to find biomarkers that may lead to more specific therapies for BLT1 disease.

Problems with BLT1 Imaging and Drug Discovery

Although imaging is much better now, there are still some issues with BLT1-targeting drugs and imaging tools:

• No Specificity: There's still a big hurdle to overcome when it comes to designing highly specific BLT1 ligands for imaging. Some of the study ligands also bind BLT2 or similar receptors, so we cannot fully separate BLT1-based effects. There is currently a race to find more selective ligands that can be used to precisely target BLT1.
• Plasticity of the Receptor: BLT1 can be modulated by many things, such as cytokines, environmental signals and tissue conditions. This receptor plasticity makes imaging studies difficult because receptor expression and activity may be variable with disease status or therapeutic intervention.
• Technical Limitations: Imaging techniques like PET, MRI and fluorescence microscopy are promising, but still have spatial resolution, sensitivity, and penetration. To break through these restrictions will need more imaging technology.

Conclusion

BLT1 is an inflammation- and immune-responsive receptor that's a target of interest for drugs targeting various chronic inflammatory disorders. Radiolabeled ligand binding, fluorescence and in vivo imaging technologies are giving researchers novel imaging tools to examine BLT1 expression, activation and activity. Such imaging techniques are not only validating BLT1 as a target but are also being used to develop more powerful and targeted therapies. More targeted therapies that deliver better outcomes with fewer side effects for inflammatory and autoimmune disease patients will become available as BLT1 imaging and drug development advance.

1. Michaelian N, et al.; Structural insights on ligand recognition at the human leukotriene B4 receptor 1. Nat Commun. 2021, 12(1):2971.

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