Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli

Abstract

Thiolactomycin inhibits bacterial cell growth through inhibition of the beta-ketoacyl-ACP synthase activity of type II fatty acid synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC(50) and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays, but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Cocrystallization of these two inhibitors with the condensing enzyme from Escherichia coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site.

Table 1

5-position TLM derivatives

Table 1

5-position TLM derivatives

Table 2

Analogues of TLM examining structural features of the isoprene side chain

Scheme 1

Synthesis of simple 5-position analogues

Scheme 2

Synthesis of analogues to examine importance of isoprene double bonds in TLM, 32-34^{a a}Reagents and conditions: (a) NH₂NH₂·H₂O (39 equiv), 30% H₂O₂ (aq), EtOH, 0 °C → rt, 1.5 h; (b) NH₂NH₂·H₂O (500 equiv), 30% H₂O₂ (aq), EtOH, 0 °C → rt, overnight; (c) i) 9-BBN, THF, rt, 6 h; ii) 3M NaOH (aq), 30% H₂O₂ (aq), 0 °C → rt, 10 min; (d) NH₂NH₂·H₂O (935 equiv), 30% H₂O₂ (aq), EtOH, 0 °C → rt, overnight; (e) MsCl, Et₃N, CH₂Cl₂, rt, 3 h; (f) NaI, acetone, reflux, 4 h; (g) t-BuOK in THF, CH₂Cl₂, rt, 30 min.

Scheme 3

Syntheses of 5-alkylidene analogues and diolefinic desmethyl-TLM^{a a}Reagents and conditions: (a) i) LiHMDS, THF, 0 °C; ii) paraformaldehyde, 0 °C→ rt; (b) MOMCl, DIPEA, CH₂Cl₂, rt; (c) Dess-Martin periodinane, CH₂Cl₂, rt; (d) AcCH₂P(=O)(OEt)₂, DIPEA, LiCl, CH₃CN, rt; (e) NaBH₄, CeCl₃·7H₂O, MeOH, rt; (f) Burgess reagent, toluene, rt; (g) NaHSO₄·SiO₂, CH₂Cl₂, rt.

Scheme 4

Synthesis of compound 50 ^aReagents and conditions: (a) MOMCl, DIPEA, CH₂Cl₂, rt; (b) Grubbs' II catalyst, trans-2-butene, sealed tube, CH₂Cl₂, 45 °C; (c) silica gel, polymer-bound TsOH, CH₂Cl₂, rt.

Figure 1

(A) IC₅₀ of TLM (open circles) and racemic TLM (closed circles) at E.coli FabB. (B) MIC of TLM (open circles) and racemic TLM (closed circles) in E. coli strain ANS1. (C) IC₅₀ of TLM against M. tuberculosis KasA (open circles) and KasB (closed circles).

Figure 2

Detail of TLM-FabB co-crystal highlighting placement of isoprene side chain. Left panel: TLM binds on the malonyl-ACP side of the active site and forms two strong hydrogen bonds with both active site histidines. The isoprene side chain inserts between the peptide bond of residues 271-272 and 391-392. Right panel: Space filling model of TLM in the same orientation as shown at left. The isoprene chain interacts with the peptide backbone through van der Waals interactions that leave no room for non-planar or bulky substituents at the 5-position.

Figure 3

Compound 47-FabB (left, PDB code 2AQB) and 50-FabB (right, PDB code 2AQ7) co-crystal complexes. Simulated annealing omit maps are shown in red for each ligand, and the docked ligands are shown in cyan. Both compounds bind to the active site in a similar way to TLM, and this is emphasized by preserving the orientation of the active site that is shown for the TLM-FabB complex in Figure 2. The maps are contoured at 2.5 σ.