Human Farnesyl Pyrophosphate Synthase Research Articles

Metal-dependent enzyme symmetry guides the biosynthetic flux of terpene precursors

Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential.

Design, synthesis, molecular docking, dynamics and in vitro evaluation of novel 2- substituted-1-hydroxyethane-1, 1-bis(phosphonic acid) derivatives as human farnesyl pyrophosphate synthase inhibitors with expected anticancer activity.

Nitrogenous bisphosphonates (NBPs) are the major class of drugs that are used to treat osteoporosis. Recently, bisphosphonates (BPs) were reported to have an anticancer effect. These agents feature a high affinity that enables them to bind strongly to the human farnesyl pyrophosphate synthase enzyme. The correlation between this affinity and their anticancer effect was confirmed. To date, the use of an oxygen atom as an isosteric replacement for the electronegative nitrogen atom in NBPs has not been reported, and its ability to retain the linker length and bisphosphonate pharmacophore remains unknown. The main aim of this work was to design some isosteric bisphosphonate analogs with oxygen atoms and evaluation of their binding affinity and anticancer activity. The binding mode and stability of the designed compounds were achieved using human farnesyl pyrophosphate synthase (HFPPS) by docking and dynamic simulations. The compounds were synthesized, characterized, and screened for their anticancer activity against the breast cancer MCF-7 cell line and lung cancer A-549 cell line. The inhibitory activity of the tested compounds against HFPPS was evaluated. The compounds under investigation showed potential anticancer activity against the lung cell line with IC50 values of 41.7, 47.4, and 34.8 μg/ml in comparison to that of Risedronic acid (115 μg/ml). However, they do not exhibit potential activity against the breast cancer cell line. Compounds VII and VIII showed in vitro inhibition of human farnesyl pyrophosphate synthase with IC50 values of 82.2 and 98.8 μg/ml, respectively. Further optimization may be required in the future.

Investigation on Controlling Therapy of Bone Skeletal and Marrow Cancer: A Biophysical Chemistry and Molecular Dynamic Study of Bisphosphonates Interaction with Bone Structures

For more than four decades, the bisphosphonates family has been applied for osteoporosis and skeletal metastasis therapy. These drugs decrease the viability of cancer cells that are guided through the HER group of receptor tyrosine kinases. We discussed that bisphosphonates straightly bind to and inhibit HER kinases. In this study for docking a nitrogen-containing bisphosphonate with human FPPS and a few other targets, the iGEMDOCK docking software has been used. Nitrogen-containing bisphosphonates (NBPs) are mostly applied for bone treatment and also for the loss of skeletal disorders. The adsorption, retention, diffusion, and release of (NBPs) in bone are controlled by their affinities to such mineral compounds. Bisphosphonates have a high affinity for Ca2+ and therefore attack bone minerals, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. Nitrogen-containing bisphosphonates (NBPs), including Alendronate, Zolendronate, Risedronic, Ibandronate, and Pamidronate, are functionalized as effective inhibitors of bone resorption diseases. It targets FPPS (osteoclast farnesyl pyrophosphate synthase) to inhibit protein prenylation. Generally, the strong interaction sequence is as follows Alendronate > Risedronic > Pamidronate > Zolendronate > Ibandronate, and this was because of strong electrostatic interactions between amine groups and phosphate ions.

PharmaNet: Pharmaceutical discovery with deep recurrent neural networks.

The discovery and development of novel pharmaceuticals is an area of active research mainly due to the large investments required and long payback times. As of 2016, the development of a novel drug candidate required up to $ USD 2.6 billion in investment for only 10% rate of approval by the FDA. To help decreasing the costs associated with the process, a number of in silico approaches have been developed with relatively low success due to limited predicting performance. Here, we introduced a machine learning-based algorithm as an alternative for a more accurate search of new pharmacological candidates, which takes advantage of Recurrent Neural Networks (RNN) for active molecule prediction within large databases. Our approach, termed PharmaNet was implemented here to search for ligands against specific cell receptors within 102 targets of the DUD-E database, which contains 22886 active molecules. PharmaNet comprises three main phases. First, a SMILES representation of the molecule is converted into a raw molecular image. Second, a convolutional encoder processes the data to obtain a fingerprint molecular image that is finally analyzed by a Recurrent Neural Network (RNN). This approach enables precise predictions of the molecules’ target on the basis of the feature extraction, the sequence analysis and the relevant information filtered out throughout the process. Molecule Target prediction is a highly unbalanced detection problem and therefore, we propose that an adequate evaluation metric of performance is the area under the Normalized Average Precision (NAP) curve. PharmaNet largely surpasses the previous state-of-the-art method with 97.7% in the Receiver Operating Characteristic curve (ROC-AUC) and 65.5% in the NAP curve. We obtained a perfect performance for human farnesyl pyrophosphate synthase (FPPS), which is a potential target for antimicrobial and anticancer treatments. We decided to test PharmaNet for activity prediction against FPPS by searching in the CHEMBL data set. We obtained three (3) potential inhibitors that were further validated through both molecular docking and in silico toxicity prediction. Most importantly, one of this candidates, CHEMBL2007613, was predicted as a potential antiviral due to its involvement on the PCDH17 pathway, which has been reported to be related to viral infections.

Fragment-Based Discovery of Non-bisphosphonate Binders of Trypanosoma brucei Farnesyl Pyrophosphate Synthase.

Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their poor pharmacokinetic properties, they are not well suited for antiparasitic therapy. Recently, an allosteric binding pocket was discovered on human FPPS, but its existence on trypanosomal FPPS was unclear. We applied NMR and X-ray fragment screening to T. brucei FPPS and report herein on four fragments bound to this previously unknown allosteric site. Surprisingly, non-bisphosphonate active-site binders were also identified. Moreover, fragment screening revealed a number of additional binding sites. In an early structure-activity relationship (SAR) study, an analogue of an active-site binder was unexpectedly shown to bind to the allosteric site. Overlaying identified fragment binders of a parallel T. cruzi FPPS fragment screen with the T. brucei FPPS structure, and medicinal chemistry optimisation based on two binders revealed another example of fragment "pocket hopping". The discovery of binders with new chemotypes sets the framework for developing advanced compounds with pharmacokinetic properties suitable for the treatment of parasitic infections by inhibition of FPPS in T. brucei parasites.

Farnesyl Pyrophosphate Synthase as a Target for Drug Development: Discovery of Natural-Product-Derived Inhibitors and Their Activity in Pancreatic Cancer Cells.

Human farnesyl pyrophosphate synthase (Homo sapiens FPPS, HsFPPS) is a target for treating bone resorption diseases and some cancers. HsFPPS is potently inhibited by bisphosphonates, but due to poor cell penetration and distribution in soft tissue, there is currently interest in the development of non-bisphosphonate inhibitors as cancer therapeutics. Here, we report the discovery and development of HsFPPS inhibitors based on the phenolic diterpene carnosic acid (CA), an antimicrobial found in rosemary and sage, which showed better cellular anticancer activities than the bisphosphonate drug zoledronate in pancreatic cancer cell lines, as well as an HsFPPS-dependent mechanism of action. Hit-to-lead optimization of CA improved HsFPPS inhibition by >100-fold. A slow dissociation inhibition pattern and a noncompetitive allosteric binding mode were found, and cellular mechanism-of-action studies showed that these inhibitors inhibit tumor cell growth primarily by inhibiting HsFPPS, leading to downregulation of Ras prenylation and cell apoptosis. The discovery of this series of compounds together with proof-of-mechanism in pancreatic cancer cells may pave the way for targeting HsFPPS in soft tissue cancers using natural-product-derived inhibitors.

Chirality-Driven Mode of Binding of α-Aminophosphonic Acid-Based Allosteric Inhibitors of the Human Farnesyl Pyrophosphate Synthase (hFPPS).

Thienopyrimidine-based allosteric inhibitors of the human farnesyl pyrophosphate synthase (hFPPS), characterized by a chiral α-aminophosphonic acid moiety, were synthesized as enantiomerically enriched pairs, and their binding mode was investigated by X-ray crystallography. A general consensus in the binding orientation of all (R)- and (S)-enantiomers was revealed. This finding is a prerequisite for establishing a reliable structure-activity relationship (SAR) model.

Novel bisphosphonates with antiresorptive effect in bone mineralization and osteoclastogenesis

Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 μM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS showing an IC50 of 0.31 μM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some representative novel bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even better than zoledronic acid as a anti-resorptive agent.

QSAR Models for Nitrogen Containing Monophosphonate and Bisphosphonate Derivatives as Human Farnesyl Pyrophosphate Synthase Inhibitors Based on Monte Carlo Method.

Human farnesyl pyrophosphate synthase (hFPPS) is a well-settled therapeutic target and it is an enzyme of the mevalonate pathway which catalyzes the biosynthesis of the C-15 isoprenoid farnesyl pyrophosphate. QSAR studies by using Monte Carlo method for human farnesyl pyrophosphate synthase inhibitors has been carried out using balance of correlation technique with Index of ideality correlation. For construction of QSAR models, six random splits were prepared from the data of 73 phosphonates and hybrid optimal descriptors procured from graph (HFG) and SMILES based notations were employed. The developed QSAR models have robustness, good fitting ability, generalizability and internal predictive ability. The external predictive ability has been certified by testing various precedents. The values of R2, IIC, Q2 and ∆R2 m for the best model are 0.9304, 0.9614, 0.9061 and 0.0861 respectively. The developed QSAR models met with the specified standards given in OECD guideline and applicability domain. The structural feature promoters for the end point increase and promoters for end point decrease have been extracted. The predicted pIC50 for the new proposed compounds have also been reported.

In Silico-Based Repositioning of Phosphinothricin as a Novel Technetium-99m Imaging Probe with Potential Anti-Cancer Activity.

l-Phosphinothricin (glufosinate or 2-amino-4-((hydroxy(methyl) phosphinyl) butyric acid ammonium salt (AHPB)), which is a structural analog of glutamate, is a recognized herbicide that acts on weeds through inhibition of glutamine synthetase. Due to the structural similarity between phosphinothricin and some bisphosphonates (BPs), this study focuses on investigating the possibility of repurposing phosphinothricin as a bisphosphonate analogue, particularly in two medicine-related activities: image probing and as an anti-cancer drug. As BP is a competitive inhibitor of human farnesyl pyrophosphate synthase (HFPPS), in silico molecular docking and dynamic simulations studies were established to evaluate the binding and stability of phosphinothricin with HFPPS, while the results showed good binding and stability in the active site of the enzyme in relation to alendronate. For the purpose of inspecting bone-tissue accumulation of phosphinothricin, a technetium (99mTc)–phosphinothricin complex was developed and its stability and tissue distribution were scrutinized. The radioactive complex showed rapid, high and sustained uptake into bone tissues. Finally, the cytotoxic activity of phosphinothricin was tested against breast and lung cancer cells, with the results indicating cytotoxic activity in relation to alendronate. All the above results provide support for the use of phosphinothricin as a potential anti-cancer drug and of its technetium complex as an imaging probe.

3D-QSAR, molecular docking, and ONIOM studies on the structure-activity relationships and action mechanism of nitrogen-containing bisphosphonates.

Nitrogen-containing bisphosphonates (N-BPs) have been used widely to treat various bone diseases by inhibiting the key enzyme farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway. Understanding the structure-activity relationships and the action mechanisms of these bisphosphonates is instructive for the design and the development of novel potent inhibitors. Here, a series of N-BPs inhibitors of human FPPS (hFPPS) were investigated using a combination of three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, and three-layer ONIOM studies. The constructed 3D-QSAR model yielded a good correlation between the predicted and experimental activities. Based on the analysis of comparative molecular field analysis (CoMFA) contour maps, a series of novel N-BPs inhibitors were designed and ten novel potent N-BPs inhibitor candidates were screened out. Molecular docking and ONIOM (B3LYP/6-31+G*:PM6:Amber) calculations revealed that the inhibitors bound to the active site of hFPPS via hydrogen-bonding interactions, hydrophobic interactions, and cation-π interactions. Six novel N-BPs inhibitors with better biological activities and higher lipophilicity were further screened out from ten candidates based on the calculated interaction energy. This study will facilitate the discovery of novel N-BPs inhibitors with higher activity and selectivity.

Crystallographic and thermodynamic characterization of phenylaminopyridine bisphosphonates binding to human farnesyl pyrophosphate synthase.

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.

New insights into human farnesyl pyrophosphate synthase inhibition by second-generation bisphosphonate drugs.

Pamidronate, alendronate, APHBP and neridronate are a group of drugs, known as second-generation bisphosphonates (2G-BPs), commonly used in the treatment of bone-resorption disorders, and recently their use has been related to some collateral side effects. The therapeutic activity of 2G-BPs is related to the inhibition of the human Farnesyl Pyrophosphate Synthase (hFPPS). Available inhibitory activity values show that 2G-BPs act time-dependently, showing big differences in their initial inhibitory activities but similar final IC50 values. However, there is a lack of information explaining this similar final inhibitory potency. Although different residues have been identified in the stabilization of the R2 side chain of 2G-BPs into the active site, similar free binding energies were obtained that highlighted a similar stability of the ternary complexes, which in turns justified the similar IC50 values reported. Free binding energy calculations also demonstrated that the union of 2G-BPs to the active site were 38 to 54kcalmol-1 energetically more favourable than the union of the natural substrate, which is the basis of the inhibition potency of the hFPPS activity.

Pharmacophore Mapping of Thienopyrimidine-Based Monophosphonate (ThP-MP) Inhibitors of the Human Farnesyl Pyrophosphate Synthase

The human farnesyl pyrophosphate synthase (hFPPS), a key regulatory enzyme in the mevalonate pathway, catalyzes the biosynthesis of the C-15 isoprenoid farnesyl pyrophosphate (FPP). FPP plays a crucial role in the post-translational prenylation of small GTPases that perform a plethora of cellular functions. Although hFPPS is a well-established therapeutic target for lytic bone diseases, the currently available bisphosphonate drugs exhibit poor cellular uptake and distribution into nonskeletal tissues. Recent drug discovery efforts have focused primarily on allosteric inhibition of hFPPS and the discovery of non-bisphosphonate drugs for potentially treating nonskeletal diseases. Hit-to-lead optimization of a new series of thienopyrimidine-based monosphosphonates (ThP-MPs) led to the identification of analogs with nanomolar potency in inhibiting hFPPS. Their interactions with the allosteric pocket of the enzyme were characterized by crystallography, and the results provide further insight into the pharmacophore requirements for allosteric inhibition.

Human farnesyl pyrophosphate synthase is allosterically inhibited by its own product

Farnesyl pyrophosphate synthase (FPPS) is an enzyme of the mevalonate pathway and a well-established therapeutic target. Recent research has focused around a newly identified druggable pocket near the enzyme’s active site. Pharmacological exploitation of this pocket is deemed promising; however, its natural biological function, if any, is yet unknown. Here we report that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and lock the enzyme in an inactive state. The Kd for this binding is 5–6 μM, within a catalytically relevant range. These results indicate that FPPS activity is sensitive to the product concentration. Kinetic analysis shows that the enzyme is inhibited through FPP accumulation. Having a specific physiological effector, FPPS is a bona fide allosteric enzyme. This allostery offers an exquisite mechanism for controlling prenyl pyrophosphate levels in vivo and thus contributes an additional layer of regulation to the mevalonate pathway.

Theoretical study of the deposition and adsorption of bisphosphonates on the 001 hydroxyapatite surface: Implications in the pathological crystallization inhibition and the bone antiresorptive action

The effect of different side chain groups of bisphosphonates (BPs) on the adsorption on the hydroxyapatite (HAP) is still a controversial issue. In this work, we studied the deposition and adsorption of a set of 26 BPs on the HAP (001) surface by using density functional theory (DFT) in which has been shown that the charge, the length or the presence of different functional groups at R2 side chain can modulate the adsorption energy of the BP. It was observed that negative charged groups at R2 enhanced the favourable electrostatic interactions between the BP and the HAP surface, but also that the length of R2 was important to enable the formation of the favorable electrostatic interactions between the functional group at R2 and the surface. A crossover study between the HAP/BP model (3D-QSAR/DFT) and the inhibition of the human farnesyl pyrophosphate synthase (FPPS) (3D-QSAR) pointed out that the electrostatic character of the R2 side chain provokes contrary effects in the inhibition of pathological crystallization and in the bone antiresorptive action of BPs.

Developing efficient protocols for synthesis, antiosteoarthritic, antiinflammatory assessments and docking studies of nitrogen-containing bisphosphonate derivatives

We report herein, two synthetic approaches to three types of nitrogen-containing bisphosphonates (N-BPs) in moderate to high yields (58–88%). Ester cleavage of selected bisphosphonates was undertaken to obtain their N-BP-acid analogs. Based on the reported bisphosphonate properties and the prospective biological prediction using computer-assisted molecular modeling (CAMM), new compounds were evaluated in a mouse model of antigen-induced arthritis (AIA) and the delayed-type hypersensitivity granuloma reaction (DTH-GRA) for chronic inflammation. Pharmacological results showed that N-BP-acids are more favorable for antiarthritis activity than N-BP-esters. On the other hand, the majority of N-BPs revealed good antiinflammatory potency compared to their acid analogs. The results also showed that the presence of a free thiol group in a molecule enhances the anti-inflammatory activity. Furthermore, bioscreening results were in good agreement with the prediction investigation. A hypothesis of molecular modeling study, including fitting of the synthesized compounds into 3D-pharmacophore using Discovery Studio 2.5 software and their docking into the human farnesyl pyrophosphate synthase (hFPPS, PDB code: 2F8C protein) showed good results consistent with the observed pharmacological properties.

The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins.Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs.In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

Mechanistic insights into protonation state as a critical factor in hFPPS enzyme inhibition.

Zoledronate and risedronate are the most powerful available nitrogen-containing bisphosphonates used in the treatment of bone-resorption disorders. Knowledge about inhibition mechanisms of these molecules is based on available crystallographic structures of human farnesyl pyrophosphate synthase (hFPPS). However, there is a lack of information explaining the inhibition potency of these two molecules compared to the natural substrate, dimethylallyl pyrophosphate. We carried out a molecular dynamics study that shown: (1) that NBPs potency is related to higher electrostatic interactions with the metallic cluster of the active site than to the natural substrate, and (2) the protonation of the R2 side chain is a critical factor to stabilize the NBPs into a closely irreversible ternary complex with the hFPPS.

Probing the molecular and structural elements of ligands binding to the active site versus an allosteric pocket of the human farnesyl pyrophosphate synthase

In order to explore the interactions of bisphosphonate ligands with the active site and an allosteric pocket of the human farnesyl pyrophosphate synthase (hFPPS), substituted indole and azabenzimidazole bisphosphonates were designed as chameleon ligands. NMR and crystallographic studies revealed that these compounds can occupy both sub-pockets of the active site cavity, as well as the allosteric pocket of hFPPS in the presence of the enzyme’s Mg2+ ion cofactor. These results are consistent with the previously proposed hypothesis that the allosteric pocket of hFPPS, located near the active site, plays a feed-back regulatory role for this enzyme.