Figure 1.
Framework of CoreSiMMap-based screening method.
In Step 1, GEMDOCK was used to generate docked poses for HpSK and MtSK by screening compound libraries (Maybridge and NCI). For each target (HpSK or MtSK), the protein-compound interacting profile was derived by the top 2% (∼6,000) of compounds ranked by docking energy. In Step 3, conserved interactions of the target protein and chemical moieties of ligands are identified to deduce the anchors of HpSK and MtSK. The CoreSiMMap is constructed based on the features that are conserved between orthologous target site-moiety maps, which will be used to select candidate compounds for the enzymatic assay. Finally, the model is refined based on the bioassay of candidate compounds.
Figure 2.
(A) Superimposed apo-form anchors of HpSK and MtSK. (B) Superimposed closed-form anchors of HpSK and MtSK. (C) The apo-form CoreSiMMap and (D) the closed-form CoreSiMMap include six consensus anchors derived from consensus anchors (A) and (B), respectively. Each consensus anchor shares conserved residues between HpSK and MtSK and has the same interaction type of binding environment. (E) Features of the six consensus anchors of the apo-form CoreSiMMap. Each of the T groups (T1–T4) represents a given chemical moiety and T-O* indicates other chemical groups. H1, V1, and H2 are situated at the ATP-binding site, while H3, V2, and E1 are at the shikimate-binding site. Each consensus anchor includes conserved interacting residues (•) and the major chemical moieties of the compound candidates.
Figure 3.
Interaction profiles between selected anchor residues and 27 tested compounds.
(A) Anchor profile of tested compounds on shikimate kinases. (B) Group I: NCI compounds (orange). (C) Group II: Maybridge compounds (yellow). (D) Group III: kinase inhibitors (cyan). The NCI compounds consistently occupy anchors E1 and V2 at both ATP and shikimate sites. The NCI compounds except NSC45174 are competitive inhibitors with both ATP and shikimate. For the Maybridge compounds, none form electrostatic interactions with R57 and R132 on the consensus anchor E1. The two kinase compounds are located at the ATP site, which fact is consistent with the kinetic results that reveal that these compounds exhibited competitive inhibition with ATP and noncompetitive inhibition with shikimate.
Figure 4.
Ranks of active compounds using CoreSiMMap, energy-based, and combined scoring methods for apo and closed forms of HpSK and MtSK.
Figure 5.
Properties of some potent inhibitors of HpSK and MtSK.
Docked poses of NSC45611, NSC162535, and NSC45612 are located at the ATP site (H1, H2, and V1) and the shikimate site (H3, V2, and E1), and these inhibitors are competitive inhibitors of ATP and shikimate (SKM). AG538 is a competitive inhibitor of ATP, in agreement with its docked pose.
Figure 6.
Characterization of shikimate kinase inhibitors by enzyme assay, CoreSiMMaps, site-mutagenesis studies, and analogues.
(A–C) Structures of three inhibitors, NSC162535 (2), NSC45611 (1), and NSC45612 (3). (D–F) Relationships between anchors and docked mode of each inhibitor for HpSK. These compounds consistently include two negative charge moieties (SO3− or CO2−) that form hydrogen bonds with conserved interacting residues of anchors E1 and H1. (G) Comparison of relative activities of HpSK mutants. The conserved interacting residues for each anchor were mutated. R57, R132, R116, and F48 located in the shikimate site were critical for the enzymatic functions. (H) Potency of NSC162535 analogues. The substitution moieties of analogues are indicated in black. Those that lack the E1 moiety greatly lost the inhibitory effects (IC50>100 µM).
Figure 7.
Performance of CoreSiMMap method on apo-form HpSK and MtSK.
(A) True-hit rates of energy-based and CoreSiMMap scoring approaches. The CoreSiMMap scores (solid line) of adaptive inhibitors are significantly better than the energy-based scores (dashed line) for the 6000 top-ranked compounds by combining the Maybridge and NCI databases. (B) Distribution of number of polar atoms. (C) Molecular weights of top 100 compounds from CoreSiMMap scores and energy-based scores.