Advantages of crystallographic fragment screening: functional and mechanistic insights from a powerful platform for efficient drug discovery

Abstract

X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this "chemical interrogation" of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility.

Keywords: Fragment screening; HIV; Influenza; X-ray crystallography.

Figure 1

Crystal structure of HIV-1 reverse transcriptase-rilpivirine complex soaked with 20% (v/v) d₆-DMSO. Color-coding: p66 subdomains as fingers (blue), palm (red), thumb (green), connection (yellow), and RNase H (orange), and the p51 subunit (cyan). Rilpivirine is shown as yellow and blue spheres, ordered waters are shown as blue dots, and d₆-DMSO molecules are shown as green, yellow, and red spheres. Crystal structure revealed a total of 16 d₆-DMSO binding sites. This crystal form was very robust to fragment soaking leading to ~90% of crystals yielding high-resolution datasets.

Figure 2

Importance of quality control of compounds used for crystallographic screening. A.) Electron density from a difference map (contoured at 3.0σ) observed upon soaking of 1 into crystals of HIV-1 RT-rilpivirine complex. Electron density envelope does not accommodate the ligand in question. B.) Electron density with metal ions and coordinating water molecules modeled in. Anomalous density indicating probable metal binding is also shown (cyan mesh contoured at 3.0σ). The metal atoms were unexpectedly present in a “pure” commercial sample; apparent high inhibition (RNase H) led to excitement that was mitigated when fractionation and purification of the compound resulted only in loss of activity.

Figure 3

Crystal structure of RT-rilpivirine with a fragment bound at the Knuckles site, revealing a novel allosteric inhibitory site for HIV-1 RT that is potentially druggable. This pocket is not observed to be open in any of hundreds of published RT structures, nor in the many hundreds of structures solved during our fragment screening campaign, unless occupied by similar fragments. Residues that shift in presence of bound ligand are shown in cyan and in their non-bound positions as green. Y115 and F116 form part of the substrate dNTP-binding site, suggesting that these conformational changes may account for the observed inhibition by fragments binding to this site.

Figure 4

Cartoon representation of apo PA_N active site from a 100 mM CaCl₂ soak (inspired by observation of a third Mn²⁺ during fragment screening) showing electron density (light blue mesh contoured at 1.5σ) for the metal ions and coordinating waters. The locations of the metal ions from a fragment soak are displayed in green whereas the locations of the calcium cations from a 100 mM soak are in yellow. The third metal had not been observed in previously reported structures and may have functional significance in stabilization of the reaction products.

Figure 5

Crystal structure (1.51 Å resolution) of HIV-1 RT-rilpivirine complex (RT52A construct), showing two water molecules making hydrogen-bonding interactions (dashed lines) with the bound drug molecule. The previously undetected water molecule (upper left corner) may account for some of the increased inhibitory potency of rilpivirine (in yellow) conferred by the cyanovinyl substituent vs. related diarylpyrimidine analogs. The crystal used for data collection was treated with 6% (w/v) TMAO in the cryoprotective solution; without TMAO this crystal form usually yields 1.8 Å resolution datasets.

Figure 6

Structure-guided lead development of a fragment hit, 5-chloro-2,3-dihydroxypyridine, identified from a screening campaign targeting influenza endonuclease. Electrostatic surface (APBS) is shown for each ligand bound structure. The availability of numerous crystal structures allowed for molecular modeling to be used, resulting in not only an increase in the specificity of binding to the active site but also an improvement in the inhibitory activity of the compounds. Merger of substitutions at the 5- and 6-positions yielded compounds with greater than a thousand-fold increase in potency compared to the initial hit.