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Fragment Screen on BRD4 Using NMR Spectroscopy

Fragment-based drug discovery (FBDD) has become widely adopted approach in lead discovery bestowing on it all the advantages of starting small. In its essence, the idea of FBDD is simple:

Objective

  • After acquiring the fragment library of desired features, the key to a successful screen is the use of different techniques to find orthogonalized weak binders.
  • Our objective in this study was to test the Saturation Transfer Difference (STD) NMR as a screening method on hand-picked Bromodomain-containing protein 4 (BRD4) binders and non-binders.
  • The results were analysed and the degree of agreement between NMR (Fidelta) and SPR and X-ray (BioFocus) data assessed.

 

STD NMR

  • The STD NMR experiments are based on saturation of protein resonances, magnetization transfer to bound ligand and detection of the free ligand relaxation following the dissociation.
  • The selective saturation of BRD4 was performed using Gauss G3 cascade pulse in duration of 3 s and on-resonance frequency of -1 ppm. The excitation sculpting scheme was used to suppress the HDO signal while relaxation filtering was employed to suppress protein resonances.
  • A known binder I-BET762 was used to test the experimental conditions for the screen.
  • 18 chosen fragments (Table 1) were screened, the results interpreted as answers to a yes-no question. Figure 1 shows examples of STD NMR spectra belonging to a yes-binder (fragment 9) and no-binder (fragment 17).

 

Figure 1. A binder (fragment 9) vs. non-binder (fragment 17): a) 1H spectra; b) STD NMR spectra with BRD4; c) STD NMR spectra in absence of BRD4; all in TRIS buffer at 25 °C

Table 1. Comparison of STD NMR results (Fidelta) with SPR and X-ray data (BioFocus)

         *low solubility under experimental conditions
         **not soluble under experimental conditions
         NT=not tested

Summary

  • Structure integrity and solubility check was performed under screening conditions prior to the screen, resulting in four discarded fragments.
  • STD NMR was tested as a fragment screening method against BRD4. A known binder I-BET762 was used to adjust the experimental conditions of the screen. The result of the screen were five unambiguous and two possible binders.
  • Comparison with SPR data revealed a good correlation between the techniques identifying three fragments which qualified as orthogonalized binders: fragments 3, 9 and 10.
  • Experimentally determined protein-ligand crystal data was obtained for two out of four NMR hits.
  • STD NMR screen revealed an additional two weak binders (fragments 12 and 13) undetected by other techniques that warrant further investigation in fragment-to-lead chemistry.

References

  • A. Čikoš, Fragment Screen on BRD4 using NMR Spectroscopy, Fidelta poster at FBLD 2014, Basel, Switzerland

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