Crystallography in Fragment-based drug discovery

Fragment-based drug discovery (FBDD) serves as a starting point for the development of drug candidates to generate high affinity ligands. X-ray crystallography, a structural method, can be used to map the interactions of small molecules with proteins, rapidly and efficiently increasing the development of drug discovery. Early FBDD projects utilizing crystallography method as primary screening methods, can directly discover truly positive fragments, although crystal structures of protein should have a high diffraction value.

The process of fragment screening based on crystallography is illustrated in Fig.1.[1, 2] The purpose of this method is to expose protein to fragments and solve the crystal structures of the complexes. It, in most cases, involves growing crystals of the target protein and soaking them in solutions of the fragments, either as single compounds or as cocktails of compounds.[2]



Figure 1.Typical flow chart for high-throughput ligand screening using crystallography. [1, 2]

Fragments are followed by “rule of three” [3], i.e. molecular weight < 300 Da; clogP ≤ 3; number of hydrogen-bond donors ≤ 3; number of hydrogen-bond acceptors ≤ 3. Compared with small molecules, fragments have a lower binding affinity, but a higher hit rate to the target protein. Because of its low binding affinity feature to the target protein, a relatively high concentration of compounds are used in crystallography, in the region of 25–100 mM.[2]

The crystal structure of a protein with a high resolution is important for this method. It must be robust, stable under soaking conditions and diffract to beyond about 2.5 Å resolution—sufficient to place fragments unambiguously in electron density.[4] Besides, it is necessary to generate crystals of a similar size and quality on a large scale for the fragment screening.

From previous researches, crystallography for fragment-based screening has been successfully used to discover inhibitors, especially some difficult targets such as b-secretase[5], as well as inhibitors of a wide range of other enzymes: hPNMT, Phosphodiesterase 4A, Hsp90, Bromodomain, Adenosine A2 receptor, etc.. (Table1) One classic example is the discovery of inhibitors of β-secretase.

Murray and co-workers[5] screened a library containing 347 fragments in cocktails containing six compounds. Two hits found to have nearly identical interactions with Beta secretase-1(BACE-1), forming hydrogen bonds with catalytic aspartate residues D32 and D228. Then, they identified further fragments to access to two important regions which were important for substrate peptide binding by molecular docking and crystallography for fragment-based screening.

Article title Target protein Primary/secondary
FBDD screening
Application of Fragment Screening by X-ray
Crystallography to the Discovery of
Aminopyridines as Inhibitors of -Secretase
β-Secretase X-ray crystallography Fluorescence-based
activity assay
Missing fragments: detecting cooperative
binding in fragment-based drug design
hPNMT X-ray crystallography ITC/Molecular
dynamics free energy
Fragment-based screening for inhibitors of
PDE4A using enthalpy arrays and X-ray
Fragment-Based Drug Discovery Applied to
Hsp90. Discovery of Two Lead Series with
High Ligand Efficiency
Hsp90 NMR/X-ray
Fragment-Based Discovery of Bromodomain
Inhibitors Part 1: Inhibitor binding modes and implications for lead discovery
Bromodomain Fluorescence anisotropy
anisotropy assay
Fragment-Based Discovery of Bromodomain
Inhibitors Part 2: Optimization of
Phenylisoxazole Sulfonamide
AcK pocket
Fluorescence anisotropy
assay/Modelling X-ray
SPR/Thermal shift
Structure-based design of potent and
ligand-efficient inhibitors of CTX-M class A
based bioassays/
Antibacterial activity
Discovery of 1,2,4-triaine derivatives as
adenosine A2A antagonists using structure
based drug design
Adenosine A2
Discovery and Optimization of New
Benzimidazole- and Benzoxazole-Pyrimidone
Selective PI3Kβ Inhibitors for the Treatment
of Phosphatase and TENsin homologue
(PTEN)-Deficient Cancers
PI3K In vitro enzyme
assay/Cell based assay
X-ray crystallography
In vitro enzyme
Synthesis, Structure–Activity Relationship
Studies, and X-ray Crystallographic Analysis
of Arylsulfonamides as Potent Carbonic
Anhydrase Inhibitor
Stopped-flow kinetic
Implications of Promiscuous Pim-1 Kinase
Fragment Inhibitor Hydrophobic Interactions
for Fragment-Based Drug Design
Pim-1 Kinase Docking/X-ray
Mobility shift assay

Table 1. Some examples of fragment-based screening.[4]

Blog written by Xiangrong Chen


[1] I. Tickle, A. Sharff, M. Vinkovic, J. Yon, H. Jhoti, High-throughput protein crystallography and drug discovery, Chemical Society Reviews, 33 (2004) 558-565.

[2] H. Jhoti, A. Cleasby, M. Verdonk, G. Williams, Fragment-based screening using X-ray crystallography and NMR spectroscopy, Current Opinion in Chemical Biology, 11 (2007) 485-493.

[3] M. Congreve, R. Carr, C. Murray, H. Jhoti, A ‘Rule of Three’ for fragment-based lead discovery?, Drug Discovery Today, 8 (2003) 876-877.

[4] Z. Chilingaryan, Z. Yin, A.J. Oakley, Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls, International Journal of Molecular Sciences, 13 (2012) 12857-12879.

[5] M. Congreve, D. Aharony, J. Albert, O. Callaghan, J. Campbell, R.A.E. Carr, G. Chessari, S. Cowan, P.D. Edwards, M. Frederickson, R. McMenamin, C.W. Murray, S. Patel, N. Wallis, Application of Fragment Screening by X-ray Crystallography to the Discovery of Aminopyridines as Inhibitors of β-Secretase, Journal of Medicinal Chemistry, 50 (2007) 1124-1132.


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