Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery

Jahnke, Wolfgang; Rondeau, Jean‐Michel; Cotesta, Simona; Marzinzik, Andreas L.; Pellé, Xavier; Geiser, Martin; Strauss, André; Götte, Marjo; Bitsch, Francis; Hemmig, R.; Henry, Chrystèle; Lehmann, Sylvie; Glickman, J. Fraser; Roddy, Thomas P.; Stout, Steven J.; Green, Jonathan R.

doi:10.1038/nchembio.421

Cited by 117 publications

(192 citation statements)

References 35 publications

Supporting

Mentioning

184

Contrasting

Unclassified

Order By: Relevance

“…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.…”

Section: Bisphosphonate Inhibition Of End Reactions Of the Dxp/mep Pamentioning

confidence: 99%

“…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.…”

Section: Bisphosphonate Inhibition Of End Reactions Of the Dxp/mep Pamentioning

confidence: 99%

“…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.…”

Section: Bisphosphonate Inhibition Of End Reactions Of the Dxp/mep Pamentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2015

View full text Add to dashboard Cite

Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula 3 Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3-to 4-fold, upstream of DMADP in bisphosphonateinhibited leaves, but the DMADP pool was enhanced much less, 1.3-to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

show abstract

Section: Bisphosphonate Inhibition Of End Reactions Of the Dxp/mep Pamentioning

confidence: 99%

Section: Bisphosphonate Inhibition Of End Reactions Of the Dxp/mep Pamentioning

confidence: 99%

Section: Bisphosphonate Inhibition Of End Reactions Of the Dxp/mep Pamentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2015

View full text Add to dashboard Cite

show abstract

“…An enzyme assay confirmed that three of the alternative site ligands were inhibitory. Jahnke et al (23) at Novartis have used NMR fragment screening followed by X-ray crystallography to identify a previously unknown and druggable allosteric pocket on the protein farnesyl pyrophosphate synthase (FPPS). Development of the fragment hits resulted in a number of cell-active submicromolar inhibitors of FPPS.…”

Section: Significancementioning

confidence: 99%

Detection of secondary binding sites in proteins using fragment screening

Ludlow¹,

Verdonk²,

Saini³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

103

106

View full text Add to dashboard Cite

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.protein structure | protein function | X-ray crystallography | fragment-based drug design

show abstract

“…Interestingly, Durrant et al (2011) described a novel screening approach to identify non-bisphosphonate FPP synthase inhibitors which also showed inhibitory properties against UPP synthase enzymes. These compounds were shown to be effective at micromolar concentrations (Durrant et al, 2011), and furthermore they appear to be more amenable to clinical use than their bisphosphonate counterparts, as they are less vulnerable to rapid removal from the circulatory system (Jahnke et al, 2010).…”

mentioning

confidence: 99%