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High-throughput crystallography for lead discovery in drug design

Key Points

  • Knowledge of the three-dimensional structures of target proteins provides a starting point for structure-based approaches to drug design by defining the topographies of the complementary surfaces of ligands and their protein targets.

  • X-ray crystallography is the most widely used technique for protein structure determination, but technical challenges and time constraints have traditionally limited its use primarily to lead optimization.

  • Recent advances in molecular biology, biochemistry, crystallography, chemoinformatics and bioinformatics have faciliated the development of high-throughput X-ray crystallography. Consequently, the use of crystallographic techniques is now being extended beyond structure determination, into new approaches for lead discovery.

  • Once the structure of the target has been solved, virtual screening, coupled with high-throughput X-ray crystallography, can be used to identify one or more weakly binding small-molecule fragments from compound libraries that consist of hundreds of small-molecule fragments.

  • High-resolution definition of these binding interactions provides an information-rich starting point for medicinal chemistry. X-ray crystallography can then be used to rapidly guide the elaboration of the fragments into larger molecular-weight lead compounds.

Abstract

Knowledge of the three-dimensional structures of protein targets now emerging from genomic data has the potential to accelerate drug discovery greatly. X-ray crystallography is the most widely used technique for protein structure determination, but technical challenges and time constraints have traditionally limited its use primarily to lead optimization. Here, we describe how significant advances in process automation and informatics have aided the development of high-throughput X-ray crystallography, and discuss the use of this technique for structure-based lead discovery.

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Figure 1: Examples of structure-based drug design.
Figure 2: Close-up of the three-dimensional structure of the active site of renin in complex with a peptidic inhibitor.
Figure 3: Fragment-based screening.
Figure 4: Examples of small-molecule fragments bound into a pocket of trypsin.
Figure 5: Structural screening.
Figure 6: Targeting protein–protein interactions.

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Acknowledgements

We would like to acknowledge the help of A. Cleasby, M. Hartshorn, E. Southern, I. Tickle, A. Scharff, M. Verdonk, J. Yon and N. Wallace in the preparation of this manuscript.

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Correspondence to Tom L. Blundell.

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DATABASES

Medscape DrugInfo

amprenavir

imatinib

nelfinavir

zanamivir

Protein Data Bank

c-ABL

FGF1–FGFR2–heparin

HIV protease

neuraminidase

pepsin

renin

SWISS-PROT

GFP

FURTHER INFORMATION

Cambridge Crystallographic Data Centre

Stanford Synchrotron Radiation Laboratory

Astex Technology Ltd

Integrative Proteomics, Inc.

Plexxikon, Inc.

Structural GenomiX, Inc.

Syrrx, Inc.

TRIAD Therapeutics

Accelrys

Nature

Glossary

CPK COLOURING

The CPK colour scheme for elements is based on the colours of the popular plastic space-filling models developed by Corey, Pauling and Kultun, and is conventionally used by chemists. In this scheme, carbon is represented in light grey, oxygen in red, nitrogen in blue and sulphur in yellow.

SYNCHROTRON

A synchrotron accelerates charged particles in a circular orbit. This produces very intense X-rays, which allows the use of smaller and more easily obtained crystals than can be used with conventional X-ray crystallography, and also boosts relevant signals while minimizing noise. The wavelength of synchrotron X-radiation can be varied to perform multiwavelength anomalous diffraction (MAD) experiments.

GEL ELECTROPHORESIS

A method that separates macromolecules on the basis of size, electric charge and other physical properties.

INCLUSION BODIES

Protein overexpression often leads to the production of insoluble aggregates of misfolded protein, which are known as inclusion bodies.

GREEN FLUORESCENT PROTEIN

Autofluorescent protein originally identified in the jellyfish Aequorea victoria.

MOSAICITY

Measure of the degree of order of a crystal. Lower mosaicity indicates better-ordered crystals and hence better diffraction.

STRUCTURE-FACTOR AMPLITUDES

Structure factors are related to the electron density by a mathematical operation called a Fourier transform. Structure-factor amplitudes are determinable from the measured intensities in an X-ray diffraction experiment, but the phases of the diffracted beams, which are needed to reconstitute the electron density, cannot be determined directly.

VAN DER WAALS SURFACE

The van der Waals radius is that which defines the normal contact distance with another non-covalently bound atom. The van der Waals surface is defined by the radii of all such atoms in the molecule.

sp3 CARBON

An sp3 carbon has four substituents.

PHARMACOPHORE

The ensemble of steric and electronic features that is necessary to ensure optimal interactions with a specific biological target structure and to trigger (or to block) its biological response.

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Blundell, T., Jhoti, H. & Abell, C. High-throughput crystallography for lead discovery in drug design. Nat Rev Drug Discov 1, 45–54 (2002). https://doi.org/10.1038/nrd706

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