Toward rational fragment-based lead design without 3D structures

Abstract

Fragment-based lead discovery (FBLD) has become a prime component of the armamentarium of modern drug design programs. FBLD identifies low molecular weight ligands that weakly bind to important biological targets. Three-dimensional structural information about the binding mode is provided by X-ray crystallography or NMR spectroscopy and is subsequently used to improve the lead compounds. Despite tremendous success rates, FBLD relies on the availability of high-resolution structural information, still a bottleneck in drug discovery programs. To overcome these limitations, we recently demonstrated that the meta-structure approach provides an alternative route to rational lead identification in cases where no 3D structure information about the biological target is available. Combined with information-rich NMR data, this strategy provides valuable information for lead development programs. We demonstrate with several examples the feasibility of the combined NMR and meta-structure approach to devise a rational strategy for fragment evolution without resorting to highly resolved protein complex structures.

Figure 1

The individual stages of fragment-based lead (drug) design (FBLD). Starting from a suitable chosen small molecule fragment library, biophysical techniques (SPR, NMR, or X-ray) are used to identify weak binders. (A) Structure-based FBLD exploits 3D structural information about ligand binding modes to rationally evolve starting fragments in iterative rounds of optimizations. (B) Fragment evolution is performed by either merging individual fragments binding to different interaction sites or by ligand extension using medicinal chemistry substitution. (C, D) Meta-structure-based fragment-based lead (drug) design strategies for ligand merging (C) and extension (D). (C) Meta-structure homologies are used to discern putative binding modes based on available 3D structure information of the homologue. (D) Suitable sites for ligand derivatization are identified using ligand-based NMR spectroscopy (AFP-NOESY). In this experiment, intraligand ¹H–¹H cross relaxation is monitored as a function of spin lock power. Protons exposed to the solvent exhibit a sign inversion with increasing spin lock power (red). In contrast, protons embedded in hydrophobic clusters (i.e., being part of a dense proton network) display a markedly different behavior (blue) due to spin diffusion. This differential behavior can be used to identify suitable sites for ligand derivatization.

Figure 2

Selection of meta-structure-derived DRUGBANK homologues for the lipocalin protein Q83. (A) Streptavidin, (B) fatty-acid binding protein (FABP), and (C) chorismate lyase. The lipocalin Q83 is shown in green, the DRUGBANK homologues in blue. Regions of structural similarity are indicated in red (Q83) and orange (homologues). Chemical formulas of small molecule ligands for chorismate lyase (D, vanillic acid, VA), and Q83 (E, bacterial siderophore enterobactin). The similar location of enterobactin and vanillic acid in the respective bound states are also indicated in F and G. Figures were prepared using the programs TopMatch (Sippl) and PyMOL (graphics).

Figure 3

NMR verification of vanillic acid binding to Q83. (A) 2D ¹H–¹⁵N HSQC spectra of unbound Q83 (blue) and bound to vanillic acid (red). (B) Location of observed chemical shift changes mapped onto the 3D solution structure of Q83. Most of the affected residues are located in the calyx, where also the bacterial siderophore enterobactin binds. (C) Nonlinear chemical shift changes are observed as a function of increasing vanillic acid (VA) concentration, indicating the formation of a ternary VA:Q83 (2:1) complex. (D) Binding isotherm of VA probed by isothermal calorimetry (ITC). Raw data (top) and fitted data (bottom) are shown. The experimental ITC data could only be reliably fitted assuming two binding events (K_D1 = 0.4 mM; K_D2 = 70 mM). The extracted thermodynamic parameters are given in Table 1.

Figure 4

Experimental verification of improved ligand binding to Q83. (A) Location of observed chemical shift changes induced by the improved ligand; 4-amino-1,1′-azobenzene-3,4′-disulfonic acid mapped onto the 3D solution structure of Q83. (B) Fluorescence quench-based measurement of binding affinity. The dissociation constant K_D* (K_D* ∼ K_D1*K_D2) K_D2 was determined to K_D* = 25 μM.

Figure 5

Chemical structures of meta-structure-derived ligands (ligand scaffolds) for β-catenin. The identified ligands using the meta-structure approach (bottom) are compared with known β-catenin inhibitors (top). To illustrate the feasibility of the method to identify relevant chemical fragments, the corresponding moieties of known β-catenin inhibitors are shown in bold.

Figure 6

1D ¹H-STD NMR spectra of 30 μM β-catenin in association with 1 mM aqueous solutions of the meta-structure-derived ligands fluorescein and eosin Y. The corresponding negative control in the absence of the protein is shown below each spectrum.

Figure 7

Pharmacophore mapping of the β-catenin fluorescein complex using adiabatic fast passage (AFP) NOESY. Experimental AFP cross-relaxation rates are shown as a function of tilt angle (e.g., increasing RF spin lock amplitude). The following protons were selectively inverted and acted as sources of magnetization transfer: (A) H₁, H₂, H₇, and H₈ from the biphenylic ether fragment and (B) H_4′, H_5′, and H_6′ from the attached aromatic ring system. Protons of H₄ and H₅ of the biphenylic ether scaffold were the detected spins in A while protons of H₁, H₂, H₇, and H₈ were the detected spins in B. The lack of zero passage in both experiments is indicative of the prevalence of spin diffusion effects and thus suggests that the biphenylic ether moiety is embedded in a largely hydrophobic binding cleft.

Figure 8

NMR monitoring of fragment extension. 1D ¹H-STD NMR is used to observe ligand binding of individual fragments (A, B) and the covalently linked ligand (C). 1D ¹H-STD NMR spectra of 30 μM solution of β-catenin in association with 0.5 mM solutions of sodium 2-phenoxybenzoate (A), 4-chloro-2-methylaniline (B), and the merged ligand fused via an imine bond (C). The molecular formulas of the individual compounds are indicated. The experimental data also convincingly confirm the ligand binding deduced by the AFP-NOESY spectrum, as shown in Figure 6. The considerably higher binding affinity of the merged ligand is indicated by the increased average STD amplification factor: 3.64 of (C) vs 0.63 for the sodium 2-phenoxybenzoate (A) at saturation time 1 s.