Taxodione and arenarone inhibit farnesyl diphosphate synthase by binding to the isopentenyl diphosphate site

Abstract

We used in silico methods to screen a library of 1,013 compounds for possible binding to the allosteric site in farnesyl diphosphate synthase (FPPS). Two of the 50 predicted hits had activity against either human FPPS (HsFPPS) or Trypanosoma brucei FPPS (TbFPPS), the most active being the quinone methide celastrol (IC 50 versus TbFPPS ∼20 μM). Two rounds of similarity searching and activity testing then resulted in three leads that were active against HsFPPS with IC 50 values in the range of ∼1-3 μM (as compared with ∼0.5 μM for the bisphosphonate inhibitor, zoledronate). The three leads were the quinone methides taxodone and taxodione and the quinone arenarone, compounds with known antibacterial and/or antitumor activity. We then obtained X-ray crystal structures of HsFPPS with taxodione+zoledronate, arenarone+zoledronate, and taxodione alone. In the zoledronate-containing structures, taxodione and arenarone bound solely to the homoallylic (isopentenyl diphosphate, IPP) site, not to the allosteric site, whereas zoledronate bound via Mg 2+ to the same site as seen in other bisphosphonate-containing structures. In the taxodione-alone structure, one taxodione bound to the same site as seen in the taxodione+zoledronate structure, but the second located to a more surface-exposed site. In differential scanning calorimetry experiments, taxodione and arenarone broadened the native-to-unfolded thermal transition (T m ), quite different to the large increases in ΔT m seen with biphosphonate inhibitors. The results identify new classes of FPPS inhibitors, diterpenoids and sesquiterpenoids, that bind to the IPP site and may be of interest as anticancer and antiinfective drug leads.prenyl synthase | crystallography | prenylation | molecular dynamics | drug discovery F arnesyl diphosphate synthase (FPPS) catalyzes the condensation of isopentenyl diphosphate (IPP; compound 1 in Fig. 1) with dimethylallyl diphosphate (DMAPP; compound 2 in Fig. 1) to form the C 10 isoprenoid geranyl diphosphate (GPP; compound 3 in Fig. 1), which then condenses with a second IPP to form the C 15 isoprenoid, farnesyl diphosphate (FPP; compound 4 in Fig. 1). FPP then is used in a wide range of reactions including the formation of geranylgeranyl diphosphate (GGPP) (1), squalene (involved in cholesterol and ergosterol biosynthesis), dehydrosqualene (used in formation of the Staphylococcus aureus virulence factor staphyloxanthin) (2), undecaprenyl diphosphate (used in bacterial cell wall biosynthesis), and quinone and in heme a/o biosynthesis. FPP and GGPP also are used in protein (e.g., Ras, Rho, Rac) prenylation, and FPPS is an important target for the bisphosphonate class of drugs (used to treat bone resorption diseases) such as zoledronate (compound 5 in Fig. 1) (3). Bisphosphonates targeting FPPS have activity as antiparasitics (4), act as immunomodulators (activating γδ T cells containing the Vγ2Vδ2 T-cell receptor) (5), and switch macrophages from an M2 (tumorpromoting) to an M1 (tumor-killing) phenotype (6). They also kill tumor cells (7) and...

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen

“…1). On the other hand, the inhibitory constant for prenyltransferases is low, indicating a strong binding already at low concentrations (Jahnke et al, 2010;Lindert et al, 2013;Liu et al, 2014). A slow rate of inhibition of isoprene synthase does not rule out a strong binding once the inhibitor reaches the active site.…”

“…3; Tables I and II), indicating a noncompetitive or irreversible inhibition. Existence of such a direct inhibition could be expected given that, in the case of inhibition of GDP, FDP, and GGDP synthases, alendronate and zoledronate bind to the three-Mg 2+ cluster in the allylbinding (DMADP-binding) pocket of the enzyme active site (Jahnke et al, 2010;No et al, 2012;Lindert et al, 2013;Liu et al, 2014). The active site of isoprene synthase also coordinates the diphosphate tail of DMADP by three Mg 2+ atoms (Köksal et al, 2010), and is thus analogous to the allyl-binding active site in prenyltransferases.…”

“…Illustration of dark-release kinetics of C6 lipoxygenase (LOX) pathway volatiles in control and alendronate-inhibited leaves measured with proton transfer reaction-mass spectrometry (PTR-MS; A), and integrated pools of DMADP converted to isoprene and LOX volatiles during a dark period of 15 min in control and zoledronateinhibited leaves measured by gas chromatography-mass spectrometry (GC-MS; B) in leaves of hybrid aspen. (Jahnke et al, 2010;Lindert et al, 2013;Liu et al, 2014). Steric limitations might explain the slow rate of bisphosphonate inhibition observed for isoprene synthase, and can explain the initial increase of emissions when the effect of enhanced DMADP availability was greater than the reduction of the activity of isoprene synthase (Fig.…”

Physiologic Activity of Bisphosphonates – Recent Advances

“…Furthermore, malaria, which is a serious life-threatening infection caused by five different species of protozoa of the genus Plasmodium, is a huge Protozoan farnesyl pyrophosphate synthase (FPPS) is also a valuable target in the search for drugs against protozoa infections, and consequently, bisphosphonates were quite intensively studied here. The initial efforts had been concentrated on screening the influence of various series of these compounds against groups of parasites, such as Leishmania, [154,155] Plasmodium, [46,154] Trypanosoma, [154,156,157] Toxoplasma, [54,154,158] Schistosoma [159] and Cryptosporidium [160]. Thus, intensive screening of large libraries of structurally diverse bisphosphonates resulted in selection of the most effective structures and determination of the molecular mechanisms of their activities.…”

Farnesyl Diphosphate Synthase Inhibitors With Unique Ligand-Binding Geometries

“…20 Since it is also quite lipophilic (clogP = 4.3), 7 could be a new lead for anticancer therapeutics (e.g., against Ras-bearing tumors). 21 We expressed, purified, and crystallized human FPPS as described previously 19 and obtained crystals with 7 by cocrystallization. Full sample preparation, data acquisition, and data processing details are given in the Supporting Information and Table 1.…”