Label-free binding kinetics, affinity, and specificity on Biacore 8K
Direct binding analysis for small molecules, fragments, antibodies, peptides, and nucleic acids against active enzymes and non-catalytic proteins. Assays performed in Malvern, PA, USA.
Surface Plasmon Resonance (SPR) is a highly sensitive technique for accurate analysis of the interactions of two biomolecules with respect to binding kinetics and affinity as well as binding specificity.
Reaction Biology performs high-throughput fragment screening, kinetics and affinity determination, binding specificity profiling, and antibody characterization. Our experts have extensive expertise in analyzing and resolving surface plasmon resonance challenges with ‘difficult to test’ proteins.
| Step | Description |
|---|---|
| Sensor chip preparation | Attaching the target to the sensor chip. |
| Assay validation | Testing the behavior of the target on the sensor chip. |
| Assay reproducibility | Testing whether the optimized conditions are reproducible. |
| Protein activity and stability | Confirming target activity and stability on the sensor chip. |
| Analyte testing | Binding of the test analyte is measured. Kinetics and affinity use either a 10-concentration multi-cycle or 5-concentration single-cycle measurement, depending on off-rates and regeneration conditions. |
| Analyte ranking | For screening studies, a single concentration measurement can be used for analyte ranking. |
SPR analysis is an optical method measuring changes in the mass of biomolecules immobilized on a gold film. Upon binding an analyte, the refractive index of the metal film changes, resulting in a changed reflection angle of light (the surface plasmon resonance phenomenon). SPR binding assays are widely used in drug discovery, from fragment screening through analyte ranking and in-depth kinetic characterization.
Low molecular weight fragment compounds (100 to 300 Da) tend to demonstrate low binding affinity; thus, the compounds require screening at a high concentration. This is better tolerated in the SPR platform than in many biochemical assays.
Promiscuous binders, which appear as false positives in biochemical inhibitor assays, can be identified by SPR when used as the secondary screening technology.
The kinetics of drug binding and unbinding, especially the residence time, play a crucial role in a drug’s in vivo efficacy. SPR can rank the kinetic selectivity of drug analogs for the selection of the best drug candidates.
SPR measures the direct binding between analyte and target, which is why the target does not need to be an enzyme and no substrate is needed. This suits non-active proteins and targets with unknown substrates.
The impact of various co-factors on analyte-target interaction can be tested. Established example: a substrate competitive inhibitor of PRMT5/MEP50 that binds its target only in the presence of SAM or SAM analogs such as MTA and SAH.
SPR can be used for antibody affinity determination, determination of kinetic parameters, epitope mapping, binding specificity, and cross-reactivity. Established assays include FcRn/FCGRT and Fc gamma receptors FCGR1A, FCGR2A, FCGR2B, FCGR3A, and FCGR3B.
SPR-based assays are sensitive, high-throughput options to accurately measure plasma protein binding of analytes.
Combining kinetic information with affinity and potency data early in the drug discovery process ensures that promising compounds are not being discarded.
Kinetic profile of an analyte-target binding reaction.
SPR detects changes in the refractive index at the surface of a sensor chip as a result of molecular mass changes of a target upon binding of the analyte. The target (also referred to as the ligand) is immobilized to the surface of the sensor chip. During the association phase, the analyte flows over the surface, and binding to the target is monitored. The flow then switches to the running buffer, and the dissociation of the analyte from the target is monitored.
Example of a co-factor analysis by SPR.
In this example study, we investigated a substrate-competitive inhibitor that binds to its target enzyme, PRMT5/MEP50, only in the presence of SAM or SAM analogs such as MTA and SAH. No dose-dependent responses were observed for analyte binding to apoprotein (left figure). The binding to the MTA-bound target is relatively weak (KD~20 µM) with fast kinetics (on/off). The binding affinity increased by ~10x for the SAH-bound target (Kd ~2µM). While the on-rates are similar for the MTA- and SAH-bound conditions, the off-rates are approximately 100X slower. The highest affinity (Kd ~3nM) and slowest off-rate (100x less than SAH-bound) was observed for analyte binding to the SAM-bound target. Single-cycle kinetics mode, which does not require dissociation to baseline in between doses, was used due to the slow off-rate observed for this condition. A slower off-rate indicates longer occupancy of the analyte on the target.
Example of binding affinity determination of an inhibitor to two bromodomains.
The BD1 or BD2 domain of BRD4 were immobilized to the sensor chip surface. The analyte was applied to the chip surface in increasing concentrations approaching saturation.
Right graph: The relative response at equilibrium for each dose was plotted against the analyte concentration to determine the equilibrium dissociation constant, Kd. The analyte is 10-fold more selective for BD2 than for BD1.
SPR measures direct binding between an analyte and an immobilized target. Deliverables are the association rate constant ka, the dissociation rate constant koff, and the binding affinity Kd.
No. SPR measures direct binding, so the target does not need to be an enzyme and no substrate is required. This makes SPR suitable for non-catalytic proteins and targets with unknown substrates.
Fragments, small molecules, peptides, proteins, antibodies, and nucleic acids.
Approximately two weeks for development of an assay against a new target, and approximately two weeks for screening with an established assay. Projects run on a first-come, first-serve basis.
Malvern, Pennsylvania, USA.
A purity of 90% or greater is recommended. Targets can be client-supplied, selected from our off-the-shelf proteins, or custom-made.
Yes. Established assays include VHL/EloB/EloC and the Cereblon CULT domain, supporting molecular glue and PROTAC programs.
Yes. Applications include affinity determination, kinetic parameters, epitope binning, binding specificity, and cross-reactivity. FcγR and FcRn assays are established.
SPR is commonly used as a secondary screen to identify promiscuous binders that appear as false positives in biochemical inhibitor assays.
| Target | HGNC Symbol | Synonyms | Target Class | Assay Type | Data Sheet |
|---|---|---|---|---|---|
| B-cell lymphoma-2 | BCL2 | BCL-2 | Apoptosis Regulators | Small molecule | Data Sheet |
| Bcl-2-like protein 1 isoform L | BCL2L1 | BCL-XL | Apoptosis Regulators | Small molecule | Data sheet |
| Bcl-2-like protein 2 | BCL2L2 | BCLW, KIAA0271 | Apoptosis Regulators | Small molecule | Data sheet |
| BRD2-1 | BRD2 | O27.1.1, Really interesting new gene 3 protein | Bromodomains | Small molecule | Data sheet |
| BRD2-2 | BRD2 | O27.1.1, Really interesting new gene 3 protein | Bromodomains | Small molecule | Data sheet |
| BRD3-1 | BRD3 | RING3-like protein | Bromodomains | Small molecule | Data sheet |
| BRD3-2 | BRD3 | RING3-like protein | Bromodomains | Small molecule | Data sheet |
| BRD4-1 | BRD4 | Bromodomain containing 4, domain 1 | Bromodomains | Small molecule | Data sheet |
| BRD4-2 | BRD4 | Bromodomain containing 4, domain 2 | Bromodomains | Small molecule | Data sheet |
| BRDT-1 | BRDT | Cancer/testis antigen 9, RING3-like protein | Bromodomains | Small molecule | Data sheet |
| CREB-binding protein | CREBBP | Histone lysine acetyltransferase CREBBP, Protein-lysine acetyltransferase CREBBP | Bromodomains | Small molecule | Data sheet |
| Histone acetyltransferase p300 | EP300 | EP300, p300 | Bromodomains | Small molecule | Data sheet |
| SMARCA2b | SMARCA2 | BAF190B, BRM, SNF2L2 | Bromodomains | Small molecule | Data sheet |
| Cereblon CULT Domain | CRBN | MRT2, MRT2A | E3 Ligases and Degrader Components | Small molecule | Data sheet |
| VHL/EloB/EloC | VHL/ELOB/ELOC | von Hippel-Lindau tumor suppressor/Elongin B/Elongin C | E3 Ligases and Degrader Components | Small molecule | Ternary complex | Data sheet |
| FCGR1A | FCGR1A | Fc gamma receptor Ia; CD64; CD64A; FcgammaRI; FcgammaRIa; FCG1; FCGR1 | Fc Receptors | Antibody-related | Data sheet |
| FCGR2A | FCGR2A | Fc gamma receptor IIa; CD32; CD32A; IGFR2; CDw32; Fc-gamma-RIIa; FcgammaRIIa | Fc Receptors | Antibody-related | Data sheet |
| FCGR2B | FCGR2B | Fc gamma receptor IIb; CD32; CD32B; FcgammaRIIb; FcGRIIB | Fc Receptors | Antibody-related | Data sheet |
| FCGR3A | FCGR3A | Fc gamma receptor IIIa; CD16; CD16a; FcgammaRIIIa; FcGRIII | Fc Receptors | Antibody-related | Data sheet |
| FCGR3B | FCGR3B | Fc gamma receptor IIIb; CD16; CD16b; FcgammaRIIIb; FcRIIIb | Fc Receptors | Antibody-related | Data sheet |
| FCGRT | FCGRT | Fc gamma receptor and transporter; FcgammaRn; FcRn | Fc Receptors | Antibody-related | Data sheet |
| EGFR: wildtype (aa 671-998) | EGFR | Epidermal growth factor receptor, Proto-oncogene c-ErbB-1, Receptor tyrosine-protein kinase erbB-1 | Kinases | Small molecule | Data sheet |
| Mitogen-activated protein kinase 1 | MAPK1 | MAPK1, ERT1, ERK2, MAPK 2 | Kinases | Small molecule | Data sheet |
| cGAS | CGAS | Cyclic GMP-AMP synthase, fragment | Nucleotidyltransferases | Small molecule | Data sheet |
| cGAS (aa 157-522) | CGAS | Cyclic GMP-AMP synthase, fragment | Nucleotidyltransferases | Small molecule | Data sheet |
| ACE2 | ACE2 | Angiotensin I converting enzyme 2 | Other | Small molecule | Data sheet |
| Cyclophilin A | PPIA | Peptidylprolyl isomerase A | Other | Small molecule | Peptide | Data Sheet |
| Poly(ADP-ribose) glycohydrolase | PARG | hPARG111 | Other | Small molecule | Data sheet |
| SARS-CoV-2 S protein – ACE2 interaction | n.a. | Spike glycoprotein, receptor binding domain; Angiotensin I converting enzyme 2 | Other | Protein-protein | S protein webpage |
| SHP2 | PTPN11 | Tyrosine-protein phosphatase non-receptor type 11, Protein-tyrosine phosphatase 1D (PTP-1D), Protein-tyrosine phosphatase 2C (PTP-2C), SH-PTP2, SH-PTP3 | Other | Small molecule | Data sheet |
| G9a | EHMT2 | Histone-lysine N-methyltransferase EHMT2, Euchromatic histone-lysine N-methyltransferase 2, HLA-B-associated transcript 8, Histone H3-K9 methyltransferase 3 (H3-K9-HMTase 3), Lysine N-methyltransferase 1C | Protein Methyltransferases | Small molecule | Data sheet |
| Histone-lysine N-methyltransferase SETD2 | SETD2 | SET2 | Protein Methyltransferases | Small molecule | Data sheet |
| NSD2-SET | NSD2 | SET domain of NSD2 | Protein Methyltransferases | Small molecule | Data sheet |
| PRMT1 | PRMT1 | Protein arginine N-methyltransferase 1 | Protein Methyltransferases | Small molecule | Data sheet |
| PRMT5/MEP50 | PRMT5/WDR77 complex | Protein arginine methyltransferase 5, WD repeat domain 77 | Protein Methyltransferases | Small molecule | Data sheet |
| H-Ras | HRAS | HRAS, HRAS1 | RAS Pathway | Small molecule | Data sheet |
| K-Ras | KRAS | C-K-RAS, CFC2, K-RAS2A, K-RAS2B, K-RAS4A, K-RAS4B, KI-RAS, KRAS1, KRAS2, NS, NS3, RASK2 | RAS Pathway | Small molecule | Data sheet |
| N-Ras | NRAS | NRAS, NRAS1 | RAS Pathway | Small molecule | Data sheet |
| SOS1 | SOS1 | SOS Ras/Rac guanine nucleotide exchange factor 1, son of sevenless homolog 1 | RAS Pathway | Small molecule | Data sheet |
| GNMT | GNMT | Glycine N-methyltransferase | Small Molecule Methyltransferases | Small molecule | Data sheet |
| HNMT | HNMT | Histamine N-methyltransferase | Small Molecule Methyltransferases | Small molecule | Data sheet |
| INMT | INMT | Indolethylamine N-methyltransferase | Small Molecule Methyltransferases | Small molecule | Data sheet |
| NNMT | NNMT | Nicotinamide N-methyltransferase | Small Molecule Methyltransferases | Small molecule | Data sheet |
| PNMT | PNMT | Phenylethanolamine N-methyltransferase | Small Molecule Methyltransferases | Small molecule | Data sheet |
| TPMT | TPMT | Thiopurine S-methyltransferase | Small Molecule Methyltransferases | Small molecule | Data sheet |
