Dataset Information

Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates.

ABSTRACT: Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3?Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca(2+) ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg(2+) ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS-46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

SUBMITTER: Aripirala S

PROVIDER: S-EPMC3949514 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates.

Aripirala Srinivas S Gonzalez-Pacanowska Dolores D Oldfield Eric E Kaiser Marcel M Amzel L Mario LM Gabelli Sandra B SB

Acta crystallographica. Section D, Biological crystallography 20140222 Pt 3

Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray ...[more]

PMID: 24598749

Similar Datasets

Project description:Human farnesyl pyrophosphate synthase (hFPPS) catalyzes the production of the 15-carbon isoprenoid farnesyl pyrophosphate. The enzyme is a key regulator of the mevalonate pathway and a well-established drug target. Notably, it was elucidated as the molecular target of nitrogen-containing bisphosphonates, a class of drugs that have been widely successful against bone resorption disorders. More recently, research has focused on the anticancer effects of these inhibitors. In order to achieve increased non-skeletal tissue exposure, we created phenylaminopyridine bisphosphonates (PNP-BPs) that have bulky hydrophobic side chains through a structure-based approach. Some of these compounds have proven to be more potent than the current clinical drugs in a number of antiproliferation assays using multiple myeloma cell lines. In the present work, we characterized the binding of our most potent PNP-BPs to the target enzyme, hFPPS. Co-crystal structures demonstrate that the molecular interactions designed to elicit tighter binding are indeed established. We carried out thermodynamic studies as well; the newly introduced protein-ligand interactions are clearly reflected in the enthalpy of binding measured, which is more favorable for the new PNP-BPs than for the lead compound. These studies also indicate that the affinity of the PNP-BPs to hFPPS is comparable to that of the current drug risedronate. Risedronate forms additional polar interactions via its hydroxyl functional group and thus exhibits more favorable binding enthalpy; however, the entropy of binding is more favorable for the PNP-BPs, owing to the greater desolvation effects resulting from their large hydrophobic side chains. These results therefore confirm the overall validity of our drug design strategy. With a distinctly different molecular scaffold, the PNP-BPs described in this report represent an interesting new group of future drug candidates. Further investigation should follow to characterize the tissue distribution profile and assess the potential clinical benefits of these compounds.

Project description:BACKGROUND:The enzyme farnesyl diphosphate synthase (FPPS) is positioned in the intersection of different sterol biosynthesis pathways such as those producing isoprenoids, dolichols and ergosterol. FPPS is ubiquitous in eukaryotes and is inhibited by nitrogen-containing bisphosphonates (N-BP). N-BP activity and the mechanisms of cell death as well as damage to the ultrastructure due to N-BP has not yet been investigated in Leishmania infantum and Giardia. Thus, we evaluated the effect of N-BP on cell viability and ultrastructure and then performed structural modelling and phylogenetic analysis on the FPPS enzymes of Leishmania and Giardia. METHODS:We performed multiple sequence alignment with MAFFT, phylogenetic analysis with MEGA7, and 3D structural modelling for FPPS with Modeller 9.18 and on I-Tasser server. We performed concentration curves with N-BP in Leishmania promastigotes and Giardia trophozoites to estimate the IC50via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry. RESULTS:The nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC50 of 13.8 µM, followed by ibandronate and alendronate with IC50 values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC50 of 311 µM for risedronate). Giardia treated with N-BP displayed concentric membranes around the nucleus and nuclear pyknosis. Leishmania had mitochondrial swelling, myelin figures, double membranes, and plasma membrane blebbing. The same population labelled with annexin-V and 7-AAD had a loss of membrane potential (TMRE), indicative of apoptosis. Multiple sequence alignments and structural alignments of FPPS proteins showed that Giardia and Leishmania FPPS display low amino acid identity but possess the conserved aspartate-rich motifs. CONCLUSIONS:Giardia and Leishmania FPPS enzymes are phylogenetically distant but display conserved protein signatures. The N-BPs effect on FPPS was more pronounced in Leishmania than Giardia. This might be due to general differences in metabolism and differences in the FPPS catalytic site.

Dataset Information

Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates.

Publications

Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets