Fragment-based drug design (FBDD) is an increasingly popular method to devise novel molecules with medicinal properties.

A typical FBDD workflow begins with screening large libraries of fragments, to select those which interact with the chosen drug target. Each fragment in the library is about half the size of the final drug molecule and once selected, it can be grown or merged with another fragment to improve its potency.

The physicochemical properties of successful fragment hits are usually consistent with ‘the rule of three’ proposed by the Astex Technology researchers in Drug Discovery Today, 2003:

  • Molecular weight < 300 Da
  • Number of hydrogen bond donors ≤ 3
  • Number of hydrogen bond acceptors ≤ 3
  • ClogP (computed partition coefficient of a compound) ≤ 3
  • Number of rotatable bonds ≤ 3
  • Polar surface area (PSA) ≤ 60 A˚2

The identification and characterization of promising fragments in such libraries requires sensitive biophysical methods, capable of detecting weak affinity interactions of low molecular weight compounds.

The core approaches to detect low-affinity fragment compounds today are nuclear magnetic resonance (NMR), X-ray crystallography, differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR).

The SPR-based biosensors are particularly well suited for this task as they provide sufficient sensitivity and throughput to complete fragment screening on libraries of several thousand compounds in just a few weeks per target. 

Learn more about SPR and SPR-based biosensor system Pioneer FE on Pall ForteBio website.

How SPR works

The figure below shows the principle of the SPR method implemented in systems such as Pioneer and Pioneer FE, recently acquired by Pall ForteBio from SensiQ Technologies.

Fig 1 SPR surface plasmon wave

Figure 1. SPR surface plasmon wave

In simple terms, SPR is a technique for detecting changes in refractive index at the surface of a sensor. The sensor is comprised of a glass substrate and thin gold coating.

Light passes through the substrate and is reflected off of the gold coating. At certain angles of incidence, a portion of the light energy transfers through the gold coating and creates a surface plasmon wave at the sample and gold surface interface.

The angle of incident light required to sustain the surface plasmon wave is very sensitive to changes in refractive index at the surface (due to mass change), and it is these changes that are used to monitor the association and dissociation of biomolecules.

However, limited solubility of some fragments presents an analytical challenge for characterizing weak affinity and low MW hit compounds. Often fragment test concentrations will be limited to a range below KD due to solubility concerns. High sensitivity of instrumentation and accuracy of sample preparation is required to characterize fragment hits for affinity to the protein target.

The manual sample preparation also takes time and introduces an additional error into the measurement process.

Learn more about how Pioneer FE system deals with this challenge on Pall ForteBio website.

OneStep® – the Pioneer solution to the challenge 

The Pioneer and Pioneer FE systems are sensitive, 3-channel, fully-automated surface plasmon resonance (SPR) instruments, with a unique OneStep® gradient injection technology. 

Increasing sample throughput and data content through the generation of gradients, while decreasing sample preparation time, OneStep® Injections can dramatically improve SPR-based fragment screening.

A single injection performed with OneStep® allows to acquire kinetics and affinity for a full analyte titration up to 3 orders of magnitude – offering unmatched speed compared to other SPR-based systems.

The technique generates a continuous concentration gradient using the sample and running buffer, allowing to streamline binding analysis by testing a full concentration series in a single injection. This not only saves sample preparation time and materials, but also reduces human error by eliminating the preparation of multiple sample dilutions. 

Figure 2. A work-flow for Fragment Screening on the Pioneer Systems.

Figure 2. A work-flow for Fragment Screening on the Pioneer Systems.

The Pioneer FE system is also capable of performing standard injections (FCI), creating a flow of uniform analyte concentration.

The figure below shows how OneStep® Injection technology compares to the standard FCI method.

Figure 3. 1000 fragments tested on the Pioneer FE system using OneStep Injection (left) and the Biacore S51 (right). Data scaled according to fractional binding occupancy. There is less apparent scatter in the Pioneer system experiment suggesting a more stable target environment.

Figure 3. 1000 fragments tested on the Pioneer FE system using OneStep Injection (left) and the Biacore S51 (right). Data scaled according to fractional binding occupancy. There is less apparent scatter in the Pioneer system experiment suggesting a more stable target environment.

In a typical fragment screen, both conventional FCI and OneStep® Injections confirm similar hits (Figures 3 and 4). However, active fragment candidates are identified directly from the primary screen in data generated using the OneStep® method. 

Also, less scatter is readily visible in data generated using OneStep® Injections, thus making the decision of which fragments to work with further downstream much easier.

Figure 4. Similar sets of confirmed hits (colored squares) would be selected by both the Biacore S51 and the Pioneer FE system, but OneStep data gave KD information directly from the screen.

Figure 4. Similar sets of confirmed hits (colored squares) would be selected by both the Biacore S51 and the Pioneer FE system, but OneStep data gave KD information directly from the screen.

The Pioneer FE system is also equipped with the NeXtStepTM Injection technology, which allows two sample components to be injected over the biosensor surface to perform a full kinetic analysis with site-specific competition.

As stated previously, secondary screening can either be reduced or completely avoided as fragment candidate selection is optimized during primary screening (Figure 5).

Figure 5. A) Optimized workflow using dynamic injection SPR (diSPR). Initial compound screening is followed by a specificity test analysis that leads to full characterization of the identified hits. The selected compounds can then be used in various applications in medicinal chemistry. B) Conventional fragment screening workflow. An initial screening process is followed by a secondary screening process where samples have to be prepared at different concentrations and analyzed separately to allow for affinity characterization and fragment hit confirmation.

Figure 5. A) Optimized workflow using dynamic injection SPR (diSPR). Initial compound screening is followed by a specificity test analysis that leads to full characterization of the identified hits. The selected compounds can then be used in various applications in medicinal chemistry. B) Conventional fragment screening workflow. An initial screening process is followed by a secondary screening process where samples have to be prepared at different concentrations and analyzed separately to allow for affinity characterization and fragment hit confirmation.

The Pioneer systems are enabling technologies – providing more information faster than traditional methods and helping our customers in their quest to develop drugs in a more rapid and cost-effective manner,” says Dominik Arnold, General Manager at Pall ForteBio.

To learn more about Pioneer FE system and what it can do, visit Pall ForteBio website or get in touch with a Pall ForteBio representative.