Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis

Abstract

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.

Figure 1. Structures of sansanmycins A-C (1–3), synthetic dihydrosansanmycins A–C (4–6) and inhibitory activity against Mtb H37Rv.

MIC values of isolated sansanmycins A–C (1–3) were previously reported by Xie et al. MIC₅₀ values of 4–6 represent the average of two independent experiments, each performed in triplicate; positive controls RIF: MIC₅₀=0.006±0.002 μM; INH: MIC₅₀=0.025±0.005 μM; MIC₅₀ (HEK293) for 4–6>200 μM.

Figure 2. Synthesis of 1st generation sansanmycin analogues 7–17 with inhibitory activities against Mtb H37Rv.

PG=side chain protection necessary for the synthesis of 5, 14, 15 and 16. MIC₅₀ values represent an average of two independent experiments each performed in triplicate; positive controls RIF: 0.006±0.002 μM; INH: MIC₅₀=0.025±0.005 μM; MIC₅₀ (HEK293) for 7–17>200 μM.

Figure 3. Synthesis of 2nd generation sansanmycin analogues 21–37 with inhibitory activities against Mtb H37Rv.

MIC₅₀ values represent average of two independent experiments, each performed in triplicate; positive controls RIF: MIC₅₀=0.006±0.002 μM; INH: MIC₅₀=0.025±0.005 μM; MIC₅₀ (HEK293) for 21–37>200 μM.

Figure 4. Inhibition studies of the synthetic sansanmycin analogues against Mtb MurX.

(a) Reactions catalysed by Mtb MurX to generate lipid I and Mtb MurG to generate lipid II. (b) Exemplar TLC assay from Mtb mc² 6230 membranes for the inhibition of Mtb MurX–MurG by dihydrosansanmycin analogue 37 (IC₅₀=41 nM). Positive control=pacidamycin D and tunicamycin. Ala, alanine; Glu, glutamate; mA₂P, meso-diaminopimelic acid. Product of MurX=decaprenyldiphosphoryl-MurNAc-pentapeptide (lipid I), product of MurG=decaprenyldiphosphoryl-MurNAc-pentapeptide-GlcNAc (lipid II), product of WecA=decaprenyldiphosphoryl-GlcNAc. (c) Inhibition assay of Mtb MurX with dihydrosansanmycin analogue 37 (IC₅₀=16 nM) using dansylated UDP-MurNAc pentapeptide as substrate. K_{m(app) UDP-MurNAc-pentapeptide}=51±4 μM and V_max(app)=69±1.9 μM min⁻¹; K_{m(app) polyisoprenylphosphate}=56±9 μg ml⁻¹ (Supplementary Figs 40 and 41). Positive control tunicamycin: IC₅₀=189 nM (Supplementary Fig. 46).