Joris W. De Schutter

Design and Synthesis of Active Site Inhibitors of the Human Farnesyl Pyrophosphate Synthase: Apoptosis and Inhibition of ERK Phosphorylation in Multiple Myeloma Cells

Human farnesyl pyrophosphate synthase (hFPPS) controls intracellular levels of FPP and post-translational prenylation of small GTPase proteins, which are essential for cell signaling and cell proliferation. Clinical investigations provide evidence that N-BP inhibitors of hFPPS are disease modifying agents that improve survival of multiple myeloma (MM) patients via mechanisms unrelated to their skeletal effects. A new series of N-BPs was designed that interact with a larger portion of the GPP subpocket, as compared to the current therapeutic drugs, and rigidify the (364)KRRK(367) tail of hFPPS in the closed conformation in the absence of IPP. An analogue of this series was used to demonstrate inhibition of the intended biological target, resulting in apoptosis and down-regulation of ERK phosphorylation in human MM cell lines.

Thienopyrimidine Bisphosphonate (ThPBP) Inhibitors of the Human Farnesyl Pyrophosphate Synthase: Optimization and Characterization of the Mode of Inhibition.

Human farnesyl pyrophosphate synthase (hFPPS) controls the post-translational prenylation of small GTPase proteins that are essential for cell signaling, cell proliferation, and osteoclast-mediated bone resorption. Inhibition of hFPPS is a clinically validated mechanism for the treatment of lytic bone diseases, including osteoporosis and cancer related bone metastases. A new series of thienopyrimidine-based bisphosphonates (ThP-BPs) were identified that inhibit hFPPS with low nanomolar potency. Crystallographic evidence revealed binding of ThP-BP inhibitors in the allylic subpocket of hFPPS. Simultaneous binding of inorganic pyrophosphate in the IPP subpocket leads to conformational closing of the active site cavity. The ThP-BP analogues are significantly less hydrophilic yet exhibit higher affinity for the bone mineral hydroxyapatite than the current N-BP drug risedronic acid. The antiproliferation properties of a potent ThB-BP analogue was assessed in a multiple myeloma cell line and found to be equipotent to the best current N-BP drugs. Consequently, these compounds represent a new structural class of hFPPS inhibitors and a novel scaffold for the development of human therapeutics.

Toward a computational tool predicting the stereochemical outcome of asymmetric reactions: development of the molecular mechanics-based program ACE and application to asymmetric epoxidation reactions.

The development and application of ACE, a program that predicts the stereochemical outcome of asymmetric reactions is presented. As major implementations, ACE includes a genetic algorithm to carry out an efficient global conformational search combined with a conjugate gradient minimization routine for local optimization and a corner flap algorithm to search ring conformations. Further improvements have been made that enable ACE to generate Boltzmann populations of conformations, to investigate highly asynchronous reactions, to compute fluctuating partial atomic charges and solvation energy and to automatically construct reactants and products from libraries of catalysts and substrates. Validation on previously investigated reactions (asymmetric Diels Alder cycloadditions and organocatalyzed aldol reactions) followed by application to a number of alkene epoxidation reactions and a comparative study of DFT‐derived and ACE‐derived predictions demonstrate the accuracy and usefulness of ACE in the context of asymmetric catalyst design.

Ternary complex structures of human farnesyl pyrophosphate synthase bound with a novel inhibitor and secondary ligands provide insights into the molecular details of the enzyme's active site closure.

BackgroundHuman farnesyl pyrophosphate synthase (FPPS) controls intracellular levels of farnesyl pyrophosphate, which is essential for various biological processes. Bisphosphonate inhibitors of human FPPS are valuable therapeutics for the treatment of bone-resorption disorders and have also demonstrated efficacy in multiple tumor types. Inhibition of human FPPS by bisphosphonates in vivo is thought to involve closing of the enzyme’s C-terminal tail induced by the binding of the second substrate isopentenyl pyrophosphate (IPP). This conformational change, which occurs through a yet unclear mechanism, seals off the enzyme’s active site from the solvent environment and is essential for catalysis. The crystal structure of human FPPS in complex with a novel bisphosphonate YS0470 and in the absence of a second substrate showed partial ordering of the tail in the closed conformation.ResultsWe have determined crystal structures of human FPPS in ternary complex with YS0470 and the secondary ligands inorganic phosphate (Pi), inorganic pyrophosphate (PPi), and IPP. Binding of PPi or IPP to the enzyme-inhibitor complex, but not that of Pi, resulted in full ordering of the C-terminal tail, which is most notably characterized by the anchoring of the R351 side chain to the main frame of the enzyme. Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering. Structure analysis identified a cascade of conformational changes required for the C-terminal tail rigidification involving Y349, F238, and Q242. The residues K57 and N59 upon PPi/IPP binding undergo subtler conformational changes, which may initiate this cascade.ConclusionsIn human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the “off” position in the absence of bound IPP. Q242 plays the role of a gatekeeper and directly controls the anchoring of R351 side chain. The interactions between the residues K57 and N59 and those upstream and downstream of Y349 are likely responsible for the switch activation. The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores.

Novel bisphosphonate inhibitors of the human farnesyl pyrophosphate synthase.

A structure-based approach was pursued in designing novel bisphosphonate inhibitors of the human farnesyl pyrophosphate synthase (hFPPS). Preliminary SAR and structural evidence for the simultaneous binding of these inhibitors into the isopentenyl pyrophosphate (IPP) and the geranyl pyrophosphate (GPP) substrate sub-pockets of the enzyme are presented.

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 enzymes Mg(2+) 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.

Targeting Bacillosamine Biosynthesis in Bacterial Pathogens: Development of Inhibitors to a Bacterial Amino-Sugar Acetyltransferase from Campylobacter jejuni

The glycoproteins of selected microbial pathogens often include highly modified carbohydrates such as 2,4-diacetamidobacillosamine (diNAcBac). These glycoconjugates are involved in host-cell interactions and may be associated with the virulence of medically significant Gram-negative bacteria. In light of genetic studies demonstrating the attenuated virulence of bacterial strains in which modified carbohydrate biosynthesis enzymes have been knocked out, we are developing small molecule inhibitors of selected enzymes as tools to evaluate whether such compounds modulate virulence. We performed fragment-based and high-throughput screens against an amino-sugar acetyltransferase enzyme, PglD, involved in biosynthesis of UDP-diNAcBac in Campylobacter jejuni. Herein we report optimization of the hits into potent small molecule inhibitors (IC50 < 300 nM). Biophysical characterization shows that the best inhibitors are competitive with acetyl coenzyme A and an X-ray cocrystal structure reveals that binding is biased toward occupation of the adenine subpocket of the AcCoA binding site by an aromatic ring.

Crystal structures of hFPPS in complex with novel anticancer drug leads

Human farnesyl pyrophosphate synthase (hFPPS) produces farnesyl pyrophosphate, an isoprenoid required for a variety of essential cellular processes. Inhibition of hFPPS has been well established as the mechanism of action of the nitrogen-containing bisphosphonate (N-BP) drugs, currently best known for their anti-bone resorptive effects. Recent investigations indicate that hFPPS inhibition also produces potent anticancer effects both in vitro and vivo: N-BPs inhibit proliferation, motility, and viability of tumor cells, and act in synergy with other anticancer agents [1,2]. However, the physicochemical properties of the current N-BP drugs seriously compromise their full anticancer potential in non-skeletal tissues. They show poor membrane permeability and extreme affinity to bone, due mainly to their highly charged bisphosphonate moiety, which mimics the pyrophosphate of the substrates of hFPPS. Both the substrates and N-BPs bind to hFPPS via Mg ion-mediated interactions between their pyrophosphate/bisphosphonate moiety and two aspartate-rich surfaces of the enzyme’s active site cavity. Recently, we took a structure-guided approach to develop bisphosphonates with higher lipophilicity for enhanced uptake into non-skeletal tissues. Surprisingly, some of the new compounds were found to bind to hFPPS even in the absence of Mg ions. Crystal structures of hFPPS in complex with a representative compound revealed that this bisphosphonate binds to the enzyme’s active site in the presence of Mg ions, but also to a nearby allosteric inhibitory site in their absence. Furthermore, removal of a phosphonate group from the bisphosphonate moiety of this compound resulted in an inhibitor that binds exclusively to the allosteric site. Based on the crystal structures with these lead compounds, we generated of a novel class of non-bisphosphonate, allosteric inhibitors of hFPPS with superior physicochemical properties than those of the current N-BP drugs for broader tissue distribution.