C5aR Fluorescent Ligands: Need for new Research Tools

GPCRs are one of the most important families of therapeutic targets in the pharmaceutical industry. They are involved in several pathologies, ranging from neurological, oncological, degenerative, metabolic, immunological… around a third of the drugs in clinical use are GPCR ligands.¹

Twist Bioscience serves Life Science researchers who are changing the world for the better. Coming from diverse fields of medicine, agriculture, industrial chemicals and data storage, scientists use their synthetic genes, oligo pools, and NGS target enrichment to better lives and improve the sustainability of the planet. Twist Bioscience technology overcomes inefficiencies and enables cost-effective, rapid, precise, high-throughput DNA synthesis and sequencing.

They found themselves with a target, C5aR, lacking the appropriate tools to study it in depth. This receptor, the C5a anaphylatoxin chemotactic receptor 1 (also known as CD88), is part of the rhodopsin family of GPCRs. Interest in this receptor has recently increased as it participates in several inflammatory pathologies, such as asthma, arthritis, sepsis, and more recently has been found to participate in Alzheimer’s disease and cancer.²

Its activation triggers immunological responses, such as chemotaxis, activation and inflammatory signaling.³ Improving the understanding of the molecular binding mechanism behind C5a and C5aR interaction is of high interest for the development of novel immunological therapeutics

Figure 1. C5aR intracellular signalling. C5aR interacts directly or indirectly with kinases (purple), GTP binding/regulatory proteins (red), transcription factors (pink), other signalling enzymes (blue) or structural proteins (grey). Internalization of C5aR is mediated by clathrin, which associates with receptor-bound b-arrestin (Ar) and the actin cytoskeleton. Proteins, such as hsp27, phosphorylated by MAP kinase-activated protein kinase 2 (MAPKAP-K2), may regulate the actin cytoskeleton. MAPKAP-K2 is itself activated by the mitogen-activated kinase (MAPK/ERK/ JNK) cascade, in turn activated by kinase Akt (also known as PK-B) or by p21-associated protein kinase (PAK) complexed with Rac/Cdc42 guanine nucleotide exchange factor PIXa, cdc42 and G-protein-coupled receptor kinase-interactor 2 (GIT2). G-protein a-subunits are deactivated by regulator of G-protein signalling 1 (RGS1) that stimulates GTP conversion to GDP; in the GDP-bound state, a-subunits can bind to and modulate the activity of the NADPH-oxidase component p67phox. bg-subunits directly activate PAK and indirectly activate PK-Cb by increasing diacylglycerol and intracellular Ca2 þ ([Ca2 þ ]i) through phospholipase Cb (PLCb). bg may be sequestered by G-protein-coupled receptor kinase (GRK), which also phosphorylates C5aR along with PK-Cb. Transcription factors signal transducer and activator of transcription 3 (STAT3), cAMP responsive element binding protein (CREB) and nuclear factor (NF)-kB are activated at the convergence of the kinase pathways, and apoptosis inhibited by phosphorylation of Bcl-associated death promoter (BAD) and upregulation of caspase degradation. JNK, c-Jun N-terminal kinase; NADPH, nicotinamide adenine dinucleotide phosphate.³

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

Celtarys Conjugation Technology

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

At Celtarys, we work with different conjugation techniques, among them is our proprietary semi-combinatorial approach. It has been validated for the development of fluorescent ligands with optimal properties for different assays and applied to several GPCRs.^4–6

1. A bibliographic search accompanied by in silico modelling is needed to determine the appropriate pharmacophore and a deep understanding of the structure-activity relationship is used to find a good location for the linker. The final pharmacophore is detected from a set of at least 3-5 different chemical scaffolds.
   

2. The pharmacophore is then functionalized in the best position for the introduction of a linker. Different spacers and hinges are used at this step, and the biological evaluation of these compounds allows us to identify the best linker for the target.
   

3. The last step is to introduce fluorophores suitable for the desired assays. The activity of the final molecules measured in binding or functional assays allows us to select the best one.

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

C5aR Fluorescent Ligand Development

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

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

The C5aR has been crystalized with the cyclopeptidic antagonist PMX53. Thus, information on the possible fitting of our 3 pharmacophores was obtained by computational methods, as well as the SAR studies performed after the chemical functionalization of the scaffolds.

During Stage 1 of the project, four promising functionalized structures of P1 showed a K_B of less than 100nM in a Calcium flux assay (Ready-to-Assay^TM, C5aR Anaphylotoxin Receptor Frozen Cells from Eurofins). These four were selected for the next step, 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 and combined with different linkers were synthesized, but none of them showed a K_B of less than 100nM, unlike the functionalized P1 scaffold. Thus, different combinations of P1+linkers and a P3 functionalized scaffold + linker was also tested – with the P3+linker (MFLV50) being the highlight (Table 1, blue).

The most promising scaffolds were labeled with a red-emitting fluorophore, Cy5. While the activity was not great, there was a discrepancy between biological results and expected activity based on the docking studies using the crystal structure.⁷ For example, MFLV18 (Table 1, blue) was predicted to establish an intramolecular hydrogen bond, simulated the fold present on PMX53.

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

Other assays were performed, in a more extensive manner. 7 compounds based on P1 and P3 were tested by Twist Bioscience. Flow Cytometry C5aR binding assay was performed in both C5aR-HEK (Multispan) and C5aR-Chem1 (DiscoverX) transfected cell lines and cAMP Hunter^TM 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) showed 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 7 fluorescent ligands were obtained in C5aR Chem-1 transfected cells, showing 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 showed strong binding properties in 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.

Afterwards, CELT-58 and CELT-68 were used in competition assays at EC50 concentration, against the endogenous peptidic ligand C5a (Figure 7). CELT-58 had a remarkable EC₅₀ of 30.38nM, and CELT-68 showed a 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 made it possible to design and synthesize many exploratory compounds in a shot time. Indeed, 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 seen in the Calcium Flux Assay for the functionalized ligands based on P1 (low nanomolar range), but this activity was lower in Stage 2 after attaching the linkers. Thus, the Stage 3 labelling was performed with moderate activity conjugates.

Seven fluorescent ligands with P1 and P3 pharmacophores were characterized biologically in more depth. CELT-58 and CELT-68 were identified as tools to perform competition binding assays by flow cytometry.

These results highlight how the type of assay can lead to different results, and how information may be lost by not performing enough studies.

Conclusions

Applying our proprietary technology, two optimal fluorescent probes for C5aR have been designed and synthesized, CELT-58 and CELT-68. Both ligands show high specific binding to C5aR in saturation binding assays (Figure 5) and 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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