On scaffold hopping: challenges in the discovery of sulfated small molecules as mimetics of glycosaminoglycans

Abstract

The design of sulfated, small, nonsaccharide molecules as modulators of proteins is still in its infancy as standard drug discovery tools such as library of diverse sulfated molecules and in silico docking and scoring protocol have not been firmly established. Databases, such as ZINC, contain too few sulfate-containing nonsaccharide molecules, which severely limits the identification of new hits. Lack of a generally applicable protocol for scaffold hopping limits the development of sulfated small molecules as synthetic mimetics of the highly sulfated glycosaminoglycans. We explored a sequential ligand-based (LBVS) and structure-based virtual screening (SBVS) approach starting from our initial discovery of monosulfated benzofurans to discover alternative scaffolds as allosteric modulators of thrombin, a key coagulation enzyme. Screening the ZINC database containing nearly 1 million nonsulfated small molecules using a pharmacophore developed from the parent sulfated benzofurans followed by a genetic algorithm-based dual-filter docking and scoring screening identified a group of 10 promising hits, of which three top-scoring hits were synthesized. Each was found to selectively inhibit human alpha-thrombin suggesting the possibility of this approach for scaffold hopping. Michaelis-Menten kinetics showed allosteric inhibition mechanism for the best molecule and human plasma studies confirmed good anticoagulation potential as expected. Our simple sequential LBVS and SBVS approach is likely to be useful as a general strategy for identification of sulfated small molecules hits as modulators of glycosaminoglycan-protein interactions.

Figure 1

Structures of some monosulfated benzofuran dimers discovered as first small molecular allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem. 2011, 54, 5522). Key units of this scaffold include: two aromatic rings, hydrophobic substituents at 3 positions and monosulfate at the 5 position. Pharmacophore query designed on the basis of this SAR is as shown in Fig. 2.

Figure 2

Three-dimensional (3D) pharmacophore query developed on the basis of SAR study with monosulfated benzofuran dimers (see Fig. 1).^, The atom in red represents an oxygen of a hydroxyl group. See text for details.

Figure 3

Virtual screening algorithm (combination of LBVS and SBVS) used in the discovery of sulfated allosteric thrombin modulators (A) and the final 10 molecules proposed by the algorithm as the top 10 hits (B).

Figure 4

Direct inhibition of human α-thrombin by VS hits 1, 2 and 3. The inhibition of thrombin was determined spectrophotometrically through Spectrozyme TH hydrolysis assay at pH 7.4 and 25 °C. Solid lines represent sigmoidal fits to the data to obtain IC₅₀, as described in the experimental section.

Figure 5

Michaelis – Menten kinetics of Spectrozyme TH hydrolysis by human α-thrombin in the presence of 0 (○), 20 (▲), 70 (□) and 185 (◆) μM 1 (see Supplementary Information). Solid lines represent non-linear regression fits to the data to yield K_M and V_MAX.

Figure 6

Residual activity of factors VIIa, IXa, Xa, XIa and XIIa at a fixed concentration of 365 μM of inhibitors 1 (grey bars), 2 (black bars) and 3 (white bars). Residual activity was measured in a spectrophotometric assay as described in Supplementary Information. See text for details.

Figure 7

Prolongation of clotting time as a function of concentration of 1 in either APTT (A) or PT (B) assay. Solid lines are trend lines from which the concentration of 1 needed to double clotting times, i.e., 2×APTT and 2×PT, was derived as shown by the dotted lines. See Supplementary Information for detailed protocol.