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. 2019 Jun 17;377(2147):20180422.
doi: 10.1098/rsta.2018.0422.

Structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping

Sherine E Thomas  1 , Patrick Collins  2 , Rory Hennell James  1   3 , Vitor Mendes  1 , Sitthivut Charoensutthivarakul  4 , Chris Radoux  5 , Chris Abell  4 , Anthony G Coyne  4 , R Andres Floto  6   7 , Frank von Delft  2   3   8   9 , Tom L Blundell  1
Affiliations

Structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping

Sherine E Thomas et al. Philos Trans A Math Phys Eng Sci. .

Abstract

Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 1040 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as 'warm spots' for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue 'Fifty years of synchrotron science: achievements and opportunities'.

Keywords: Mycobacterium abscessus; SAICAR synthetase (PurC); fragment-based drug discovery; structure-guided; synchrotron.

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Conflict of interest statement

We declare we have no competing interests.

Figures

Figure 1.
Figure 1.
(a) Schematic depiction of the enzyme reaction catalysed by PurC (SAICAR synthetase) in the bacterial purine biosynthesis pathway. (b) Crystal structure of apo form of M. abscessus PurC refined at 1.5 Å resolution, coloured by secondary structure.
Figure 2.
Figure 2.
M. abscessus PurC in complex with natural ligands and fragment hits. (a) Crystal structure of Mab PurC in complex with ATP (blue) showing the position of fragments 1 (pink) and 2 (green) with respect to ATP adenine ring; (b) crystal structure of Mab PurC in complex with ATP (blue) and the inferred position of substrate CAIR (green) in MabPurC derived by superposition with E. coli PurC (PDB code 2GQS); (c) fragment 1 and (d) fragment 2, showing interaction of fragment hits (yellow stick) with residues at the ATP site (grey stick). Hydrogen bonding interactions are depicted in blue, π-interactions in black and hydrophobic contacts in red dotted lines, respectively. The corresponding two-dimensional structures of fragments and biophysical data are shown below.
Figure 3.
Figure 3.
Hotspot mapping of PurC. Crystal structure of MabPurC protein (white), shown in surface representation, with hotspot maps showing hydrogen bond donor (blue), acceptor (red) and hydrophobic (yellow) regions. ATP (blue stick) and CAIR (green stick) are also shown. (a) Three hotspot regions are observed at the active site cleft at the front, when the maps are contoured at 17. (b) A fifth hotspot consisting of an acceptor region is seen at the rear of the protein. (c) A warm spot (warm spot 2) can also be seen in addition to the three hotspots, when the maps are contoured at 14 and the hydrophobic patches (yellow) at all the hotspot regions become more prominent at this contour. (d) The fifth hotspot at the rear of the protein when observed at contour 14. The hotspot maps were generated as described in [22].
Figure 4.
Figure 4.
Fragment binding modes identified from screening of diverse fragment libraries by crystallography and analysis using PanDDA program. Hotspot maps are contoured at 14 and MabPurC protein is shown as surface electrostatic representation. (a) Front view of the protein with fragment binding sites 1–5 and representative hits. (b) Rear view of the protein having fragments binding sites 6–8 with representative hits. The average occupancies and B-factors corresponding to each site are also illustrated in the blue box.
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