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Fragment-Based Drug Discovery

Drug discovery using libraries of small molecule fragments with molecular weights of typically ≤300 Da offer an innovative approach to hit identification for a range of biological targets.  Sygnature Discovery offers FBDD as a cost-effective alternative to other methods of hit identification.

Fragment-based drug discovery (FBDD) utilises a range of biophysical techniques, such as surface plasmon resonance (SPR), differential scanning fluorimetry (DSF) (also termed fluorescent thermal shift assay; FTSA), X-Ray crystallography and nuclear magnetic resonance (NMR), to screen fragment libraries for specific binding to a biological target.

The low complexity of these fragments increases the chance of complementarity to any protein surface; so it may only be necessary to screen several hundred molecular fragments, rather than the hundreds of thousands of compounds often tested during high-throughput screening (HTS) to obtain hits.  This fragment-based hit-finding screen is often followed with X-ray crystallography to provide detailed knowledge of exactly how and where the fragments bind to a specific biological target.  This structural information then facilitates the optimisation of these low affinity hits – either by careful growing off the fragments, or by combining different fragments together into one molecule.

The small size of the fragments makes subsequent expansion of the hits in an atom efficient manner, through the addition of well-matched functionality, more feasible.  This contrasts with the slower, iterative cycles of piece-by-piece optimisation of more complex, larger molecular weight hits (commonly identified by HTS).  At Sygnature, this fragment hit optimisation process is particularly effective as our fragment library is proprietary and the novel fragments are well-designed for subsequent medicinal chemistry-driven expansion.

Sygnature’s Unique, Proprietary Fragment Library

Sygnature Discovery has designed and synthesised a unique, proprietary fragment-based library to aid clients in their drug discovery activities.  Sygnature’s fragment library consists of >1,000 (50% non-commercial) high purity (≥95% by LC-MS and NMR) compounds that have been rigorously selected for their suitability for fragment screening applications:

  • Conformation to the ‘Rule of 3’ (Ro3) criteria, e.g. <300 MW with, clogP, rotatable bonds, H-bond donors, and acceptors all <3.
  • Maximal chemical diversity to generate high-quality hits.
  • Broad coverage of chemical space.
  • Easier elaboration into drug-like molecules.

 ♦ Sygnature Library | ♦ Commercially Available Libraries 

Figure 1. Sygnature Discovery’s excellent coverage of the soluble fragment chemical space, demonstrated via Principal Component Analysis.

 

Our novel fragment library has been designed to be readily amenable to medicinal chemistry optimisation to rapidly establish any structure-activity relationship (SAR) around each core fragment. Each fragment contains at least one point of diversity which is amenable to further derivatisation.  A small number of analogues around each functional group are also included within our fragment library to provide an early indication of SAR to guide subsequent hit expansion libraries utilising the technology and expertise that Sygnature has built whilst being a key member of the European Lead factory.  These follow-on molecular analogues could be quickly designed in close collaboration with the client and subsequently synthesised at Sygnature in our high throughput synthesis labs via a single synthetic step from the diversity point(s) in the chosen fragment core.  The ability to rapidly increase affinity to the level at which functional biochemical or cellular activity is measurable is especially important when addressing novel protein targets or seeking selective allosteric modulators.

Surface Plasmon Resonance (SPR) based Screening

Sygnature utilises high-sensitivity Biacore 8K and Biacore T200 SPR instruments that are able to efficiently screen our proprietary fragment library collection at high concentration.  A counter-screen against an unrelated control protein can also enable us to identify hits that may be more selective towards the client’s target.

The Biacore 8K is the new ‘gold standard’ SPR instrument for fragment-based drug discovery.  Its 8 channels and 16 flow surfaces offers the greatest flexibility of set-up, alongside unparalleled throughput and sensitivity:

  • Up to 2,300 compounds in a day.
  • Full kinetic characterisation of up to 64 fragment hits in only 4 hours.
  • Fully-automated compound handling, including robotic and ECHO acoustic dispensing to ensure accuracy down to 2.5nl.
  • Automated, validated analysis packages to turn around the client’s data as soon as possible.

Fragment hits identified via SPR are characterised to accurately measure their affinity (KD).  These hits are subsequently confirmed using an array of orthogonal biophysical techniques, which are also available at Sygnature.  Upon characterisation, follow-on compounds are synthesised which may also be screened in the various biophysical assays.

X-ray Crystallography and NMR-based Fragment Screening

Our affiliate company, Peak Proteins, works closely with Sygnature Discovery to provide a commercial fragment screening service.  Peak Proteins applies its extensive expertise in protein production and crystallisation to generate appropriate constructs for co-crystallisation, or soak protocols for the biological target of interest in order to screen fragment libraries or determine binding mode of hits using X-ray crystallography.  Constant review of binding interactions observed in such X-ray structures enables ligand efficient optimisation to provide potent and selective leads for advancement towards development.

With regard to NMR-based fragment screening, Sygnature utilises several cryoprobe-equipped Bruker 500 to 800 MHz NMR spectrometers, which offer the excellent resolution and throughput.  These instruments also have auto-samplers for high-throughput screening capabilities, facilitating screening of the fragment library in a matter of hours.  Sygnature Discovery has experience with fragment-based NMR screening methods using this instrumentation, including saturation transfer difference (STD) and waterLOGSY, and can apply these approaches to either a fragment library screen or a hit profiling study.

Additional Biophysical Technologies

Sygnature Discovery utilises a range of orthogonal biophysical technologies that best address the client’s target and stage of drug discovery.

 

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