Selective Inhibitors of Helicobacter pylori Methylthioadenosine Nucleosidase and Human Methylthioadenosine Phosphorylase

Abstract

Bacterial 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes adenine from its substrates to form S-methyl-5-thioribose and S-ribosyl-l-homocysteine. MTANs are involved in quorum sensing, menaquinone synthesis, and 5'-methylthioadenosine recycling to S-adenosylmethionine. Helicobacter pylori uses MTAN in its unusual menaquinone pathway, making H. pylori MTAN a target for antibiotic development. Human 5'-methylthioadenosine phosphorylase (MTAP), a reported anticancer target, catalyzes phosphorolysis of 5'-methylthioadenosine to salvage S-adenosylmethionine. Transition-state analogues designed for HpMTAN and MTAP show significant overlap in specificity. Fifteen unique transition-state analogues are described here and are used to explore inhibitor specificity. Several analogues of HpMTAN bind in the picomolar range while inhibiting human MTAP with orders of magnitude weaker affinity. Structural analysis of HpMTAN shows inhibitors extending through a hydrophobic channel to the protein surface. The more enclosed catalytic sites of human MTAP require the inhibitors to adopt a folded structure, displacing the phosphate nucleophile from the catalytic site.

Figure 1.

Transition states and reactions catalyzed by MTAP and HpMTAN. (A) MTAP catalyzes the reaction via a ribocationic transition state and phosphate as the nucleophile. Adenine and methylthio-α-d-ribose 1-phosphate are the products. Bonds to the leaving group and the attacking nucleophile are weak, less than 0.1 Pauling bond order, making the reaction more S_N1 than S_N2 in character. (B) HpMTAN also catalyzes its reaction via a ribocationic transition state. Water acts as the nucleophile. Adenine and methylthio-D-ribose are the products.

Figure 2.

Transition-state analogue inhibitors of MTAP and HpMTAN. Inhibitors were ranked based on their K_d value for MTAP. ND means inhibition not detected at a 5 μM inhibitor concentration. The specificity ratio (K_d Hs/Hp) is the affinity for human MTAP relative to HpMTAN.

Figure 3.

Stereoview of the binding sites of MTAP in complex with transition-state analogue inhibitors. The inhibitor complexes of 15, 16, 30, and 32 are shown in panels A, B, C, and D, respectively. The residues interacting with inhibitors from monomer-A are shown in green and from the neighboring subunit are shown in light blue. Selected hydrogen bond interactions are shown in orange dotted lines.

Figure 4.

Stereoview of the binding sites of HpMTAN in complex with transition-state analogue inhibitors. The inhibitor complexes of 15, 16, 30, and 32 are shown in panels A, B, C, and D, respectively. The residues interacting with inhibitors from monomer-A are shown in yellow and from monomer-B are shown in light blue. Selected hydrogen bond interactions are shown in orange dotted lines.

Figure 5.

Catalytic site conformations of apo- and ligand-bound MTAP and HpMTAN. (A) Stereoview superposition of unliganded MTAP (PDB ID: 3OZE; green) with four inhibitor-bound structures, including MTAP-15 (PDB ID: 6DYZ; cyan), MTAP-16 (PDB ID: 6DZ0; yellow), MTAP-30 (PDB ID: 6DZ3; blue), and MTAP-32 (PDB ID: 6DZ2; light pink), are overlapped. The β1−β2 loop (highlighted with a red star) is altered substantially on the binding of 30 and 32. (B) Superposition of an unliganded MTAN binding site (E. coli MTAN PDB ID: 1Z5P; blue) with four inhibitor-bound complexes of HpMTAN, including MTAN-15 (PDB ID: 6DYU; brick red), MTAN-16 (PDB ID: 6DYV; cyan), MTAN-30 (6DYY; yellow), and MTAN-32 (6DYW; red). Helix 6 and associated loop of the unliganded MTAN change conformation to elongate helix 6 as a result of inhibitor binding (highlighted with a red star).

Scheme 1

Scheme 2.

Reagents: (i) tBuOCH(NMe₂)₂, DMF, 100 °C;(ii) Zn, HOAc; (iii) POCl₃ 100 °C; (iv) aq NH₃, CuCl, 120 °C; and (v) HCHO, aq EtOH, 80−100 °C

Scheme 3.

Reagents: (i) Picoline Borane, 7, MeOH; (ii) 7 N NH₃/MeOH, 120 °C; and (iii) aq HCl, MeOH

Scheme 4.

HCl (conc.) 3:1 v/v; (iii) 9-Deazaadenine, Formaldehyde, EtOH/Water, 70–100 °C (Microwave), 2–6 h; (iv) MeI, Allyl Bromide, nBuBr or BnBr, NaN₃, CuI, MeOH; and (v) NaOMe, MeOH, and then 2-Chloropyrimidine