Mevalonate analogues as substrates of enzymes in the isoprenoid biosynthetic pathway of Streptococcus pneumoniae

Abstract

Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway, which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this pathway appears to be regulated at the mevalonate kinase (MK) step, which is strongly feedback-inhibited by diphosphomevalonate (DPM), the penultimate compound in the pathway. The human mevalonate pathway is not regulated by DPM, making the bacterial pathway an attractive antibiotic target. Since DPM has poor drug characteristics, being highly charged, we propose to use unphosphorylated, cell-permeable prodrugs based on mevalonate that will be phosphorylated in turn by MK and phosphomevalonate kinase (PMK) to generate the active compound in situ. To test the limits of this approach, we synthesized a series of C(3)-substituted mevalonate analogues to probe the steric and electronic requirements of the MK and PMK active sites. MK and PMK accepted substrates with up to two additional carbons, showing a preference for small substituents. This result establishes the feasibility of using a prodrug strategy for DPM-based antibiotics in S. pneumoniae and identified several analogues to be tested as inhibitors of MK. Among the substrates accepted by both enzymes were cyclopropyl, vinyl, and ethynyl mevalonate analogues that, when diphosphorylated, might be mechanism-based inactivators of the next enzyme in the pathway, diphosphomevalonate decarboxylase.

Figure 1

The mevalonate pathway. MK, mevalonate kinase; PMK, phosphomevalonate kinase; DPM-DC, diphosphomevalonate decarboxylase.

Figure 2

Mevalonate analogues as substrates for MK and PMK. Reaction progress curves for mevalonate kinase (A) and phosphomevalonate kinase (B). Color coding is identical in panels A and B. Reaction conditions: MK (Mev, 0.050 µM; 2a, 1 µM; 2b , 1 µM; 2c, 20 µM; 2e, 20 µM; 2f, 20 µM; 2i, 20 µM; 2j, 10 µM) or PMK (Pmev, 0.005 µM; 3a, 0.075 µM; 3b, 0.150 µM; 3c, 1.0 µM; 3e, 2.0 µM; 3f, 0.50 µM; 3i, 2 µM; 3j, 0.10 µM), pyruvate kinase (10.0 units/ml), lactate dehydrogenase (20.0 units/ml), ATP (4.0 mM), MgCl₂ (5.0 mM), NADH (0.82 mM), phosphoenolpyruvate (1.0 mM), KCl (50 mM), β-mercaptoethanol (20.0 mM), compounds 2a–2k (200 µM, racemic mixture) or 3a–c, e, f, i, j (100 µM), Hepes/K⁺ (50 mM, pH 8.0), T = 25 ± 2 °C. The activities of compounds 2d, 2g, 2h and 2k with MK were indistinguishable from background. (C) Mevalonate analogues (R-groups) listed in the order of their reaction rate with MK.

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