Illuminating C5aR Biology: The Role of Fluorescent Ligands in GPCR Research

GPCRs are one of the most important families of therapeutic targets in the pharmaceutical industry. They play a role in various pathologies, including neurological, oncological, degenerative, metabolic, and immunological conditions. Approximately one-third of the drugs currently in clinical use are GPCR ligands.

The Impact of Twist Bioscience

Twist Bioscience serves life science researchers who are dedicated to improving the world. These scientists come from diverse fields such as medicine, agriculture, industrial chemicals, and data storage. They utilize synthetic genes, oligo pools, and NGS target enrichment to enhance lives and promote sustainability. Twist Bioscience's technology addresses inefficiencies and enables cost-effective, rapid, precise, high-throughput DNA synthesis and sequencing.

However, researchers faced a challenge with the target C5aR, as they lacked the appropriate tools to study it in depth. The C5a anaphylatoxin chemotactic receptor 1, also known as CD88, is part of the rhodopsin family of GPCRs. Interest in this receptor has surged recently due to its involvement in several inflammatory pathologies, including asthma, arthritis, sepsis, and more recently, Alzheimer's disease and cancer.

Its activation triggers immunological responses, such as chemotaxis, activation, and inflammatory signaling. Understanding the molecular binding mechanism behind C5a and C5aR interaction is crucial for developing novel immunological therapeutics.

To accelerate ligand development for C5aR, new tools must be developed. Fluorescence-based assays, such as flow cytometry or fluorescence polarization, can be used for medium or high-throughput screening. However, there is a notable lack of fluorescent probes available in the market for this receptor.

Celtarys Conjugation Technology

Figure 2. General structure of ligands architecture obtained by Celtarys Technology.

At Celtarys, we employ various conjugation techniques, including our proprietary semi-combinatorial approach. This method has been validated for developing fluorescent ligands with optimal properties for different assays and has been applied to several GPCRs.

1. A bibliographic search accompanied by in silico modelling is essential to determine the appropriate pharmacophore. A deep understanding of the structure-activity relationship helps identify a suitable location for the linker. The final pharmacophore is derived from a set of at least 3-5 different chemical scaffolds.

   
   

2. The pharmacophore is then functionalized in the best position for introducing a linker. Various spacers and hinges are utilized at this stage, and the biological evaluation of these compounds enables us to identify the most effective linker for the target.

   
   

3. The final step involves introducing fluorophores suitable for the desired assays. The activity of the final molecules is measured in binding or functional assays, allowing us to select the best candidate.

   
   

Figure 3. Development process of fluorescent probes using Celtarys technology and its stages.

C5aR Fluorescent Ligand Development

Initially, a detailed analysis of the published ligands for C5aR is performed. For competition-based screening, antagonists are preferred as they exhibit the same affinity for both active and inactive receptor conformations and do not trigger internalization.

Three scaffolds were selected (P1, P2, and P3), considering their activity range, structure-activity relationship, available information, chemical scaffold, and synthetic accessibility.

The C5aR has been crystallized with the cyclopeptidic antagonist PMX53. This provides valuable information regarding the potential fitting of our three pharmacophores through computational methods, along with the SAR studies conducted after the chemical functionalization of the scaffolds.

During Stage 1 of the project, four promising functionalized structures of P1 demonstrated a K*B** of less than 100nM* in a Calcium flux assay (Ready-to-AssayTM, C5aR Anaphylotoxin Receptor Frozen Cells from Eurofins). These four candidates were selected for the next step, which involved the introduction of linkers (Table 1, Stage 2).

Table 1. Biological activity of the most representative compounds synthesized in C5aR fluorescent ligand development project. * In addition to the W-54011, the unmodified pharmacophore 1 was used as internal control for further assay validation.

Using our proprietary technology, several linkers were assembled, combining the suitable functionalized pharmacophores with different hinges and spacers. The linker structures are filtered based on the desired physicochemical properties.

An initial set of compounds based on functionalized P1 combined with different linkers was synthesized. However, none of these compounds exhibited a K*B** of less than 100nM*, unlike the functionalized P1 scaffold. Consequently, different combinations of P1+linkers and a P3 functionalized scaffold + linker were also tested, with the P3+linker (MFLV50) emerging as the highlight (Table 1, blue).

The most promising scaffolds were labeled with a red-emitting fluorophore, Cy5. While the activity was not optimal, there was a discrepancy between biological results and expected activity based on the docking studies using the crystal structure. For instance, MFLV18 (Table 1, blue) was anticipated to establish an intramolecular hydrogen bond, simulating the fold present in PMX53.

Neither fluorescent P1 nor P3 showed good activity in calcium functional assays. The best compound identified was CELT-58, which was obtained by combining MFLV18 with Cy5, showing a K*B** of 5788nM* (Table 1, red).

Further assays were conducted in a more extensive manner. Seven compounds based on P1 and P3 were tested by Twist Bioscience. A Flow Cytometry C5aR binding assay was performed in both C5aR-HEK (Multispan) and C5aR-Chem1 (DiscoverX) transfected cell lines, along with the cAMP HunterTM eXpress C5aR CHO-K1 GPCR Assay.

cAMP Functional Assays

Only P1 (MFLV59) and the P3+linker conjugates MFLV50 and MFLV66, as well as the fluorescent compound CELT-68 (based on P3), demonstrated activity in cAMP assays (Figure 4, Table 1).

Figure 4. cAMP functional assays performed on representative precursors and final fluorescent probes.

Flow Cytometry Binding Assays

The best saturation curves of the seven fluorescent ligands were obtained in C5aR Chem-1 transfected cells, indicating high specific binding (Figure 5).

Figure 5. Specific binding of the most promising fluorescent antagonists in Chem 1 cell lines. The signal in C5aR transfected Chem-1 is compared with the untransfected parent cell line to study fluorescent probe specific binding.

Both CELT-58 and SG65 exhibited strong binding properties across different cell lines.

Figure 6. EC50 affinities obtained by flow cytometry saturation binding experiments in C5aR transfected Chem-1 cell line. For those curves which did not reach plateau the EC50 was not reported since the data may not be accurate.*

Subsequently, CELT-58 and CELT-68 were utilized in competition assays at EC50 concentration, against the endogenous peptidic ligand C5a (Figure 7). CELT-58 achieved a remarkable EC*50** of 30.38nM*, while CELT-68 demonstrated high activity in cAMP.

Figure 7. C5aR competition binding of CELT-58 and CELT-68 with the endogenous ligand C5a by flow cytometry.

Discussion

The efficiency, versatility, and convergence of our proprietary conjugation technology enabled the design and synthesis of numerous exploratory compounds in a short time. Over 50 different molecules were synthesized following the established three-stage process, leading to two optimal fluorescent tools for C5aR screening.

Good biological activity was observed in the Calcium Flux Assay for the functionalized ligands based on P1 (low nanomolar range). However, this activity diminished in Stage 2 after the linkers were attached. Consequently, Stage 3 labeling was performed with moderate activity conjugates.

Seven fluorescent ligands with P1 and P3 pharmacophores were characterized biologically in greater depth. CELT-58 and CELT-68 were identified as valuable tools for conducting competition binding assays by flow cytometry.

These results underscore how the type of assay can yield different results and how critical information may be lost by not conducting sufficient studies.

Conclusions

By applying our proprietary technology, we have designed and synthesized two optimal fluorescent probes for C5aR: CELT-58 and CELT-68. Both ligands exhibit high specific binding to C5aR in saturation binding assays (Figure 5) and demonstrate good competition with the endogenous ligand C5a by flow cytometry (Figure 7). Both are orthosteric ligands with antagonistic activity in Calcium and cAMP assays (Table 1).

These two fluorescent probes have proven to be optimal tools to perform fluorescence-based assays to unlock the therapeutic potential of this important receptor.

References

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