Antitubercular nucleosides that inhibit siderophore biosynthesis: SAR of the glycosyl domain

Abstract

Tuberculosis is the leading cause of infectious disease mortality in the world by a bacterial pathogen. We previously demonstrated that a bisubstrate inhibitor of the adenylation enzyme MbtA, which is responsible for the second step of mycobactin biosynthesis, exhibited potent antitubercular activity. Here we systematically investigate the structure-activity relationships of the bisubstrate inhibitor glycosyl domain resulting in the identification of a carbocyclic analogue that possesses a KIapp value of 2.3 nM and MIC99 values of 1.56 microM against M. tuberculosis H37Rv. The SAR data suggest the intriguing possibility that the bisubstrate inhibitors utilize a transporter for entry across the mycobacterial cell envelope. Additionally, we report improved conditions for the expression of MbtA and biochemical analysis, demonstrating that MbtA follows a random sequential enzyme mechanism for the adenylation half-reaction.

Figure 1

Biosynthesis of the mycobactins by a mixed NRPS-PKS assembly line initiated by MbtA.

Figure 2

The bisubstrate inhibitor template is comprised of four domains: aryl, linker, glycosyl, and base. The acylphosphate linkage of the acyl adenylate intermediate 2 is mimicked by an acylsulfamate linkage in salicyl-AMS 6. The expanded portion of the figure shows the glycosyl modifications described herein.

Figure 3

Dose-response of fractional initial velocity of γ-[³²P]-ATP formation catalyzed by MbtA as a function of inhibitor 8 concentration. The curve represents the best non-linear fit of the data to the Morrison equation. The data points represent the mean with standard error of duplicate experiments.

Figure 4

A) K_I^app as a function of ATP concentration. B) K_I^app as a function of salicylic acid concentration.

Figure 5

Schematic diagram of key nucleoside/protein interactions for analog 6, based on docking. For comparison with the DhbE X-ray structure, residues are numbered according to PDB entry 1MDB.

Scheme l^a

^aReaction conditions: (a) 2,2′-dimethoxypropane, MeSO₃H, acetone, 84%; (b) NH₂SO₂Cl, NaH, DME, 63%; (c) DBU, DMF, 60%; (d) 80% aq TFA, 66%.

Scheme 2^a

^aReaction conditions: (a) TBSC1, imidazole, cat. DMAP, DMF, 84% (20); (b) 50% aq TFA, 60%; (c) p-TsOH, MeOH, 78% over 2 steps (23); (d) NH₂SO₂C1, NaH, DME, 38% (24), 82% (25); (e) Cs₂CO₃, DMF,; (f) DBU, DMF, 87% (28); (g) Pd/C, H₂, MeOH, 17% over 2 steps (29); (h) TBAF, THF, 50%; (i) 80% aq TFA, 53% over 2 steps.

Scheme 3^a

^aReaction conditions: (a) HC(OMe)₃, pTsOH; (b) 0 Ac₂O, h) 6N HC1, 48% from 14; (c) NH₂SO₂C1, NaH, DME, 55%; (d) 16, DBU, DMF, 81%; (e) 80% aq TFA, 80%; (f) Pd/C, H₂, MeOH, 36%.

Scheme 4^a

^aReaction conditions: (a) NH₂SO₂C1, NaH, DME, 45%; (b) 26, DBU, DMF, 44%; (c) Pd/C, H₂, MeOH, 98%.