Stroke is the second leading cause of death on the planet. But only Tissue plasminogen activator (tPA) is currently available through the U.S. Food and Drug Administration (FDA) for acute ischemic stroke. Hence, new treatments for stroke are needed as quickly as possible. But discovery and development of drugs are very laborious and expensive. There are many stages of this: target discovery, verification, hit screening, lead optimization, preclinical evaluation, and formulation. The subject used to be driven by natural resources but has migrated towards synthesis with high performance and combinatorial chemistry. Modern practices adopt ever more advanced technologies such as ultra-high-throughput screening and artificial intelligence to solve cost and time issues and continue to strive to cut costs and make resources work better.

Surface plasmon resonance (SPR) is one of the most useful tools in contemporary targeted drug discovery and development. The technique has multiple use cases in stroke drug discovery and development such as, but not limited to, target-directed drug discovery, high-throughput lead compound screening, affinity, binding kinetic analysis, hit validation and optimization, mode of action studies, biosensor construction, and fragment-based drug discovery strategies. SPR could shorten the time to development for stroke drugs, boost candidate drug pharmacokinetics, and significantly lower the cost of current screening technologies.

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What is Surface Plasmon Resonance?

SPR is a biophysical effect in which polarized light passes through a thin film of metal (usually gold or silver) at the edge of a dielectric. The interaction energizes collective vibratory oscillations of conducting electrons, surface plasmons, producing resonant coupling that changes depending on the refractive index of the medium in which it's interacting. SPR can be used to investigate molecular dynamics as they can be used to directly observe molecular adsorption and kinetics instantaneously, label-free, and quantitatively by studying the reflection or transmission of light at different angles or wavelengths.

Fig. 1. The operational mechanism of an SPR sensor involves the detection of light reflection on a detector, whereby the process entails the binding of antibodies with a certain protein. (Acharya, et al., 2024)

What Are the Benefits of Surface Plasmon Resonance?

Each biophysical technique has unique capabilities for studying biological systems, but SPR stands out in drug discovery due to its superior attributes.

• No labeling required: This feature simplifies sample preparation and eliminates concerns that tags might interfere with reactions.
• Real-time analysis: SPR allows detailed monitoring of binding events, providing insights into association and dissociation kinetics, which is crucial for structure/activity relationship (SAR) studies.
• Higher throughput: Compared to other label-free technologies like titration or scanning calorimetry, SPR biosensors require less sample volume and enable more rapid analysis.

Applications of Surface Plasmon Resonance in Stroke Drug Discovery and Development

SPR has made significant contributions to drug discovery and development by evaluating binding kinetics and affinity, screening and identifying hits, studying mechanisms of action, characterizing and developing antibodies, assessing off-target binding and safety, and facilitating fragment-based drug discovery. This technique plays a vital role across various stages of stroke drug discovery and development, showcasing its diverse applications in the field.

Target Identification Using SPR in Stroke Drug Discovery

Target identification is a critical step in drug discovery, previously slow but now accelerated by advanced techniques such as genetic, affinity-based, chemical proteomics, and computational methods. SPR, a label-free optical technique, facilitates target characterization for stroke.

Binding Affinity Determination and Kinetic Analysis

SPR assays can measure binding affinities between drug candidates and target proteins through concentration-dependent experiments, determining the dissociation constant (K_d). A lower K_d indicates stronger binding affinity. Key kinetic parameters, including on and off rates, influence the interaction's stability and strength, providing insights into potential in vivo behavior.

Epitope Mapping

Epitope mapping through SPR helps identify antigenic determinants on targets that interact with antibodies or other molecules. This analysis reveals the specificity and selectivity of interactions, enabling the assessment of potential off-target effects and guiding improvements in drug potency and efficacy.

Stability and Aggregation Analysis

SPR can also assess the stability and aggregation propensity of target proteins by monitoring binding responses under varying conditions. This data aids in identifying stable drug targets and optimizing formulations.

Target Validation Using SPR in Stroke Drug Discovery

SPR, being a protein-protein interaction interrogation technique, provides insights into the binding mechanism and pertinent kinetic parameters. The advancement in the application of the technique in the form of SPR imaging and microscopy and its merge-up with exploratory-based assays could be used in drug target validation and further steps of stroke drug discovery.

HIT Identification Using SPR in Stroke Drug Discovery

After initial screening, lead optimization involves assessing numerous hits to identify the most promising candidates for further development. SPR biosensors are highly valued in this phase for their ability to measure the association rate constant (K_on) and dissociation rate constant (K_off), which can be used to calculate additional parameters such as binding affinity (K_D) and free energy changes.

Enhancing Drug Delivery Through SPR-guided Optimization

SPR is a powerful analytical technique used in the formulation development stage of drug development. It can evaluate the stability of drug formulations affected by factors such as temperature, pH, and humidity. By immobilizing the drug formulation or its components onto a sensor chip, researchers can monitor changes in binding interactions to assess how these factors impact the formulation's stability.

Applications of SPR in Preclinical Pharmacokinetics and Toxicity Studies

Pharmacokinetics Assessment with SPR

The ability of SPR to monitor serum protein binding interactions in real-time represents an advancement over traditional methods like ultracentrifugation and dialysis. By immobilizing target molecules such as transporters or plasma proteins on a biosensor surface, researchers can assess binding kinetics and selectivity.

Metabolism Studies

Understanding drug metabolism is vital for predicting the fate and efficacy of drugs. SPR-based biosensors have been employed to study interactions between drugs and metabolic enzymes, particularly cytochrome P450 enzymes.

Drug Excretion Analysis

SPR can be used to assess drug excretion by immobilizing efflux transporters on sensor surfaces. This allows researchers to analyze the binding kinetics of substrates to multidrug resistance proteins, aiding in drug design to combat resistance.

Mechanistic Pathway Investigations

In addition to the above, SPR has been used for the investigation of mechanistic pathways of drugs. Through receptor profiling, competition assays, kinetic analysis, and ligand-receptor binding mapping, one can rule out the mechanism through which the drug molecule shows activity.

Applications of SPR in Stroke-related Biomarker Discovery and Validation

SPR technology has been applied in clinical trials through the development of SPR-based immunosensors for analyzing clinical samples. A functionalized gold SPR chip was developed for the specific detection of stroke biomarkers using an SPR POC device. This was the first time SPR technology had been utilized as a POC device for the detection of blood-based biomarkers.

Fig. 2. Point-of-Care surface plasmon resonance biosensor for stroke biomarkers NT-proBNP and S100β using a functionalized gold chip with specific antibody. (Harpaz, et al., 2019)

1. Acharya, B., et al. (2024). Optimizing drug discovery: Surface plasmon resonance techniques and their multifaceted applications. Chemical Physics Impact, 8, 100414.
2. Harpaz, D., et al. (2019). Point-of-Care surface plasmon resonance biosensor for stroke biomarkers NT-proBNP and S100β using a functionalized gold chip with specific antibody. Sensors, 19(11), 2533.