Using Terpene Synthase Plasticity in Catalysis: On the Enzymatic Conversion of Synthetic Farnesyl Diphosphate Analogues.
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SUBMITTER: Hou A
PROVIDER: S-EPMC9292696 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.
| S-EPMC3055917 | biostudies-literature
Project description:Methylated analogues of isopentenyl diphosphate were synthesised and enzymatically incorporated into methylated terpenes. A detailed stereochemical analysis of the obtained products is presented. The methylated terpene precursors were also used in conjunction with various isotopic labellings to gain insights into the mechanisms of their enzymatic formation.
| S-EPMC7065205 | biostudies-literature
Project description:Terpene synthases catalyse the first step in the conversion of prenyl diphosphates to terpenoids. They act as templates for their substrates to generate a reactive conformation, from which a Mg2+ -dependent reaction creates a carbocation-PPi ion pair that undergoes a series of rearrangements and (de)protonations to give the final terpene product. This tight conformational control was exploited for the (R)-germacrene A synthase- and germacradien-4-ol synthase-catalysed formation of a medium-sized cyclic terpenoid ether from substrates containing nucleophilic functional groups. Farnesyl diphosphate analogues with a 10,11-epoxide or an allylic alcohol were efficiently converted to a 11-membered cyclic terpenoid ether that was characterised by HRMS and NMR spectroscopic analyses. Further experiments showed that other sesquiterpene synthases, including aristolochene synthase, δ-cadinene synthase and amorphadiene synthase, yielded this novel terpenoid from the same substrate analogues. This work illustrates the potential of terpene synthases for the efficient generation of structurally and functionally novel medium-sized terpene ethers.
| S-EPMC6334173 | biostudies-literature
Project description:The relaxed complex scheme is an in silico drug screening method that accounts for receptor flexibility using molecular dynamics simulations. Here, we used this approach combined with similarity searches and experimental inhibition assays to identify several low micromolar, non-bisphosphonate inhibitors, bisamidines, of farnesyl diphosphate synthase (FPPS), an enzyme targeted by some anticancer and antimicrobial agents and for the treatment of bone resorption diseases. This novel class of farnesyl diphosphate synthase inhibitors have more drug-like properties than existing bisphosphonate inhibitors, making them interesting pharmaceutical leads.
| S-EPMC3671582 | biostudies-literature
Project description:Schistosomiasis affects over 200 million people worldwide, with over 200,000 deaths annually. Currently, praziquantel is the only drug available against schistosomiasis. We report here that Schistosoma mansoni farnesyl diphosphate synthase (SmFPPS) and geranylgeranyl diphosphate synthase (SmGGPPS) are potential drug targets for the treatment of schistosomiasis. We expressed active, recombinant SmFPPS and SmGGPPS for subsequent kinetic characterization and testing against a variety of bisphosphonate inhibitors. Recombinant SmFPPS was found to be a soluble 44.2-kDa protein, while SmGGPPS was a soluble 38.3-kDa protein. Characterization of the substrate utilization of the two enzymes indicates that they have overlapping substrate specificities. Against SmFPPS, several bisphosphonates had 50% inhibitory concentrations (IC50s) in the low micromolar to nanomolar range; these inhibitors had significantly less activity against SmGGPPS. Several lipophilic bisphosphonates were active against ex vivo adult worms, with worm death occurring over 4 to 6 days. These results indicate that FPPS and GGPPS could be of interest in the context of the emerging resistance to praziquantel in schistosomiasis therapy.
| S-EPMC3837879 | biostudies-literature
Project description:Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis.
| S-EPMC4360152 | biostudies-literature
Project description:BackgroundIsoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.MethodsThe isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.ResultsThe expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68?±?5 ?M, 7.8?±?1.3 ?M and 2.06?±?0.4 ?M. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.ConclusionsThe rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.
| S-EPMC3679732 | biostudies-literature
Project description: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 (IC50 versus TbFPPS ∼ 20 µM). Two rounds of similarity searching and activity testing then resulted in three leads that were active against HsFPPS with IC50 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 (Tm), quite different to the large increases in ΔTm 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.
| S-EPMC4078810 | biostudies-literature
Project description:Farnesyl diphosphate synthase (FDPS) is a crucial enzyme in isoprenoid and cholesterol biosynthesis and the target for nitrogen-containing bisphosphonate (N-BP) drugs widely used for osteoporosis treatment. Here we show that FDPS inhibition overcomes tumor resistance to immune checkpoint blockade (ICB) therapy by activating endogenous retroviruses (ERVs) and promoting a potent type I interferon response. FDPS inhibition, either by genetic knockout or alendronate, enhances the efficacy of ICB therapy substantially in multiple murine tumors by remodeling the immune microenvironment and enhancing lymphocyte infiltration, which is caused by increased type I interferon upregulation. Mechanistically, FDPS inhibition attenuates mitochondrial membrane potential (MtMP), enabling endonuclease G (EndoG) migration from the mitochondria into the nucleus, increasing DNA double-strand breaks and degradation of the heterochromatin maintenance protein TRIM28(KAP1), promoting enhanced ERV transcription and activation of the MDA5/MAVS pathway. The ensuing type I interferon upregulation is responsible for enhanced antigen presentation and sensitization to ICB therapy.
2024-11-14 | GSE281577 | GEO
Project description:Glioblastoma is a highly malignant tumor that easily acquires resistance to treatment. The stem-cell-like character (stemness) has been thought to be closely associated with the treatment resistance of glioblastoma cells. In this study, we determined that farnesyl diphosphate synthase (FDPS), a key enzyme in isoprenoid biosynthesis, plays an important role in maintaining glioblastoma stemness. A comparison of the mRNA expression in patient-derived glioblastoma sphere cells, which maintain stemness, and their differentiated counterparts, which lose stemness, via RNA sequencing showed that most of the altered genes were networked in the cholesterol biosynthesis pathway. We screened Federal Drug Administration (FDA)-approved drugs targeting specific enzymes in the cholesterol biosynthesis pathway for their ability to inhibit glioblastoma sphere formation. Inhibitors of FDPS, such as alendronate and zoledronate, significantly reduced the formation of glioblastoma spheres, and alendronate was effective at a lower molar concentration than zoledronate. Knockdown of FDPS using short hairpin RNA also completely inhibited the formation of secondary spheres. FDPS mRNA in patients with glioblastoma was associated with malignancy in three independent microarray data sets. RNA sequencing showed that alendronate treatment reduced the embryonic stem cell signature and activated development- and necrosis-related pathways in glioblastoma spheres. These results suggest that FDPS is important for the maintenance of glioblastoma stemness and that alendronate, a drug widely used to treat osteoporosis, can be repositioned to treat glioblastoma.
| S-EPMC6193020 | biostudies-literature