Functional AdoMet Isosteres Resistant to Classical AdoMet Degradation Pathways

Abstract

S-adenosyl-l-methionine (AdoMet) is an essential enzyme cosubstrate in fundamental biology with an expanding range of biocatalytic and therapeutic applications. We report the design, synthesis, and evaluation of stable, functional AdoMet isosteres that are resistant to the primary contributors to AdoMet degradation (depurination, intramolecular cyclization, and sulfonium epimerization). Corresponding biochemical and structural studies demonstrate the AdoMet surrogates to serve as competent enzyme cosubstrates and to bind a prototypical class I model methyltransferase (DnrK) in a manner nearly identical to AdoMet. Given this conservation in function and molecular recognition, the isosteres presented are anticipated to serve as useful surrogates in other AdoMet-dependent processes and may also be resistant to, and/or potentially even inhibit, other therapeutically relevant AdoMet-dependent metabolic transformations (such as the validated drug target AdoMet decarboxylase). This work also highlights the ability of the prototypical class I model methyltransferase DnrK to accept non-native surrogate acceptors as an enabling feature of a new high-throughput methyltransferase assay.

Figure 1

Utilization and degradation of AdoMet. General scheme highlighting methyltransferase (MT)-catalyzed use of AdoMet in methylation reactions (upper) and major chemical degradation pathways of AdoMet (lower). AdoMet, S-adenosyl-l-methionine (also referred to as SAM); AdoHcy, S-adenosyl-l-homocysteine (also referred to as SAH).

Figure 2

Synthesis of AdoMet analogs and corresponding relevant degradation pathways. (A) Synthesis of l^tMet: (i) (Boc)₂O, pyridine, NH₄HCO₃, rt, 5 h, 97%; (ii) (TFA)₂O/pyridine (1:1), THF, 0 °C, 3 h, 80%; (iii) NaN₃, ZnBr₂, H₂O/2-propanol (2:1), 80 °C, 16 h, 65%; (iv) Et₂NH, CH₂Cl₂, rt, 0.5 h, 82%. (B) General scheme for hMAT2A-catalyzed synthesis of AdoMet, Ado^tMet, ^7dzAdoMet, and ^7dzAdo^tMet. (C) Degradative pathways for Ado^tMet, ^7dzAdoMet, and ^7dzAdo^tMet. Fmoc, fluorenylmethyloxycarbonyl.

Figure 3

DnrK colorimetric assay. (A) General scheme for a high-throughput DnrK colorimetric assay enabled by the surrogate acceptor 2-chloro-4-nitrophenol (ClNP). (B) Representative assay results demonstrating DnrK-catalyzed alkylation of ClNP diminishes classical ClNP color (A₄₁₀) over time [30 μM DnrK; 500 μM ClNP; AdoMet: (i) 100 μM, (ii) 500 μM, (iii) 800 μM, (iv) 1,000 μM, (v) 1,600 μM, and (vi) 2,000 μM; 25 mM Tris· HCl; pH 8.0].

Figure 4

DnrK ligand-bound structures. (A) DnrK-Ado^tHcy (gray) and DnrK-AdoHcy-carminomycin (green) ligand-bound structures. Polar contacts between Ado^tHcy and nearby residues (stick models) and water molecules (spheres) are highlighted by dashed lines. In this model, the distance between carminomycin O-4 and the AdoHcy or Ado^tHcy sulfur is 4.2 and 3.8 Å, respectively. (B) DnrK-AdoHcy-ClNP (purple) and DnrK-AdoHcy-carminomycin (green) ligand-bound structures with ligands represented as stick models. In this model, the distance between carminomycin O-4 or ClNP O-1 and the AdoHcy sulfur is the same (4.2 Å).