Here we show that in the presence of PtdInsP2 in excised patches, PtdIns, but not PtdIns(4)P, partially inhibited TRPV1 activity at low, but not at high capsaicin concentrations. This is consistent with PtdIns acting as a competitive vanilloid antagonist. However, in the absence of PtdInsP2, PtdIns partially stimulated TRPV1 activity. We computationally identified residues, which are in contact with PtdIns, but not with capsaicin in the vanilloid binding site. The I703A mutant of TRPV1 showed increased sensitivity to capsaicin, as expected when removing the effect of an endogenous competitive antagonist. I703A was not inhibited by PtdIns in the presence of PtdInsP2, but it was still activated by PtdIns in the absence of PtdInsP2 indicating that inhibition, but not activation by PtdIns proceeds via the vanilloid binding site. In molecular dynamics simulations, PtdIns was more stable than PtdInsP2 in this inhibitory site, whereas PtdInsP2 was more stable than PtdIns in a previously identified, nonoverlapping, putative activating binding site. Our data indicate that phosphoinositides regulate channel activity via functionally distinct binding sites, which may explain some of the complexities of the effects of these lipids on TRPV1.Regulation of the heat- and capsaicin-activated transient receptor potential vanilloid 1 (TRPV1) channel by phosphoinositides is complex and controversial. In the most recent TRPV1 cryo-EM structure, endogenous phosphatidylinositol (PtdIns) was detected in the vanilloid binding site, and phosphoinositides were proposed to act as competitive vanilloid antagonists. This model is difficult to reconcile with phosphatidylinositol 4,5-bisphosphate [PtdInsP Its immsP2 , but dissP2 , 9. PtdI2 negatively regulates TRPV1 and that relief from this inhibition plays a major role in sensitization of the channel upon PLC activation by proinflammatory receptor stimulation P, applied directly to excised inside out patches potentiates TRPV1 activity P2 P, demonstrating a direct effect on the channel Pactivity , 15, 16. channel .2 and/or its immediate precursor PtdIns(4)P. Depletion of these lipids by specific inducible phosphoinositide phosphatases resulted in diminished channel activity (2 and PtdIns(4)P are also depleted by Ca2+-induced activation of PLC when TRPV1 is activated by saturating capsaicin concentrations, and this effect plays an important role in desensitization of TRPV1 Pactivity , 14, 18.of TRPV1 , 18, 19.ssion in .2 and PtdIns(4)P being positive regulators of TRPV1 in cellular membranes.The most recent and highest resolution cryo-EM structure of TRPV1 revealed the presence of an endogenous phosphoinositide partially occupying the vanilloid binding site. The lipid was modeled as PtdIns, but it was argued that this site can accommodate a range of phosphoinositide species, and it was proposed that phosphoinositides act as competitive vanilloid antagonist . However2 P2-bound structure of TRPV5 shows an open channel P,5P2 (4). channel , which i channel , 23. A bof TRPV1 . The loc mutants . This puof TRPV5 .2. The inhibitory effect showed an inverse correlation with the concentration of capsaicin, and mutating residues predicted to interact with PtdIns, but not with capsaicin, resulted in higher sensitivity to capsaicin activation and reduced inhibition by PtdIns. These data are compatible with PtdIns acting as a competitive vanilloid antagonist. In the absence of PtdInsP2, on the other hand, PtdIns partially stimulated TRPV1 activity especially in the presence of high capsaicin concentrations, an indication that PtdIns also binds to an activating lipid binding site. We used molecular modeling to characterize the binding of phosphoinositides to both the site partially overlapping with the vanilloid binding site (inhibitory site) and the one located between the S4-S5 linker and the proximal C-terminal TRP-domain (activating site). Theoretical structural models are consistent with the hypothesis that phosphoinositides act primarily on these nonoverlapping binding sites, one with activating and the other with inhibiting effect.Here, we show that in excised inside out patches PtdIns, but not PtdIns(4)P, partially inhibited channel activity in the presence of PtdInsP2 on capsaicin-induced TRPV1 activity, we expressed the channel in Xenopus oocytes and performed excised inside out patch clamp and two electrode voltage clamp experiments to measure TRPV1 currents. We complemented the experimental work with computational modeling, i.e., binding site mapping, molecular docking and molecular dynamics (MD) simulations.To study the effects of PtdIns and PtdInsPA), which is consistent with our earlier findings P2 by phosphatase enzymes in the patch membrane. When we applied the water soluble dioctanoyl (diC8) PtdInsP2 (50 μM), channel activity was restored. Co-application of 50 μM diC8 PtdIns induced a partial, reversible inhibition of TRPV1 activity (A). The effect of PtdIns, was on average ∼50%, and while statistically significant, it was highly variable, ranging from no inhibition to full inhibition (C and D). Next, we tested if the inhibitory effect of PtdIns is decreased at higher and increased at lower capsaicin concentrations, as expected if the lipid acts as a competitive vanilloid antagonist. B–D shows that the inhibitory effect of PtdIns showed an inverse correlation with the concentration of capsaicin, showing essentially no inhibition at 4 μM capsaicin and ∼70% inhibition at 0.2 μM capsaicin. We also tested additional concentrations of PtdIns. We found that 25 μM PtdIns was less effective than 50 μM in the presence of 0.2 μM capsaicin, and 100 μM PtdIns was similarly effective to 50 μM both in the presence of 0.2 μM and 1 μM capsaicin  after patch excision in the presence of 0.5 μM capsaicin in the patch pipette A, which findings . This ruactivity A. The efhibition , C and Dapsaicin .Figure 12. This lipid was shown to also increase channel activity, but with a lower apparent affinity than PtdInsP2 (2 and 0.5 μM capsaicin (A–C). PtdIns (50 μM) on the other hand inhibited TRPV1 when the channel was activated by 50 μM PtdIns(4)P in the presence of 0.5 μM capsaicin (D–F).PtdIns(4)P is the precursor of PtdInsPsP2 , 14. Unlapsaicin , A–C. Ptapsaicin , D–F.Fig8 PtdIns at different capsaicin concentrations in the absence of PtdInsP2. We found that 100 μM diC8 PtdIns had no effect in the majority of patches in the presence of 0.5 μM capsaicin (A), even though in some patches (three out of 10) it induced a small, partial activation, see Figure 3C for summary.Our data support the idea that PtdIns is a competitive channel inhibitor at the binding site overlapping with that for vanilloids. We found earlier that after channel rundown, very high concentrations (500 μM) of long acyl chain (AASt) PtdIns partially reactivated TRPV1 at 0.5 μM capsaicin . Here, wapsaicin A, even t8 PtdIns reliably reactivated TRPV1 (B and C). The amplitudes of the PtdIns-induced currents were 60.4 ± 11.2% (SD) of that induced by 25 μM diC8 PtdInsP2 in the same patches. These data indicate that capsaicin not only increases the apparent affinity for PtdInsP2 in activating TRPV1 . We also found however that PtdIns partially activates TRPV1 in the absence of PtdInsP2, and this effect is more prevalent at higher capsaicin concentrations. These two findings are hard to reconcile with PtdIns binding to a single binding site. We therefore hypothesize that at high capsaicin concentrations, PtdIns activates the channel through a distinct binding site, presumably the same where PtdInsP2 binds to activate the channel .Our data show that PtdIns partially inhibits TRPV1 in the presence of PtdInsP2 binding site, which was proposed to be responsible for the activating effect of PtdInsP2 by the Latorre group P2 added to the purified protein, and the lipid induced a clear conformational change indicating channel opening .Next, we computationally identified residues, which are in contact with PtdIns, but not with capsaicin in the vanilloid binding site (5 Å cutoff): R409 and H410 in pre-S1, D509 and S510 in the S2–S3 loop, K571 and L574 in the S4–S5 segment, and I696, L699, Q700, and I703 in the TRP box A.Figure B and C) yet are seemingly not interacting with capsaicin .In principle, mutation of a residue important for PtdIns binding, but not for capsaicin binding, should reduce inhibition of the channel by endogenous PtdIns, and it would be predicted to increase sensitivity to activation by capsaicin (left shifted concentration response curve), because of weakening inhibition by an endogenous competitive inhibitor. We noticed that R409 and H410 are solvent exposed  and, on bona fide interactions and proceeded with a more computationally intensive characterization of these sidechains. In particular, we calculated the change in PtdIns affinity (ΔΔG) upon mutation of each sidechain into alanine. 2, PtdIns, and capsaicin). We noticed that I696, K571, and L574 also form tight interactions with PtdInsP2 in the putative activating site (B), and therefore, their mutations are likely to result in complex phenotypes. The same is true for Q700, which is involved in an indirect interaction with capsaicin through an intervening hydrogen-bonded water molecule observed in a large fraction of the simulation frames (C and A). Of the remaining list of amino acids, L699A had been reported to show a capsaicin sensitivity very similar to that of WT channels  and D509A (A) mutants showed left-shifted concentration dependence for capsaicin activation. Maximal current amplitudes were similar to WT TRPV1 for both mutants (A). We also transfected HEK293 cells with GFP-tagged WT TRPV1 and GFP-tagged I703A mutant and performed total internal reflection fluorescence (TIRF) imaging experiments. In TIRF microscopy, the excitation light hits the glass–cell interface at a narrow critical angle, so that it is totally reflected. The generated evanescent wave penetrates the cell to less than 100 nm, predominantly exciting the plasma membrane. Both constructs showed punctate distribution in the TIRF mode, which is consistent with earlier reports (B–F).For these reasons, we focused on residues D509 and I703. We found that both the I703A , A–C andnd D509A A mutants mutants A. We als reports . In the  reports , 29. Whi reports , B–F.FigWe also found that the D509A and I703A mutants became more sensitive to activation by low pH . This laD–F shows that 50 μM PtdIns did not inhibit the I703A mutant in excised patches in the presence of 50 μM PtdInsP2 and either 0.5 μM or 4 μM capsaicin. We also found that the I703A mutant was activated by 100 μM diC8 PtdIns in the presence of 4 μM capsaicin and absence of PtdInsP2 (H and I), indicating that the mutation did not affect the activating binding site.Next, we tested if these mutations also altered inhibition of TRPV1 by PtdIns in excised patches. sP2 , H and IC and D). We also found that the D509A mutant had reduced rectification compared with WT TRPV1 and the I703 mutant when activated by capsaicin (E) or by low pH (E), especially at low stimulation strengths. D509A also had significantly slower deactivation compared with WT and I703 after removal of capsaicin combined with application of the TRPV1 antagonist capsazepine (A and B and B and F). Therefore, the D509A mutant, unlike I703A altered several characteristics of the channel, thus the change in the capsaicin concentration response relationship could be because of secondary effects unrelated to PtdIns.The D509A mutant on the other hand was clearly inhibited by PtdIns , C and Dapsaicin E or by ly low pH E, especiA). This residue however may also interact with PtdInsP2 in the activating binding site (B); therefore, its mutation may result in complex effects. Nevertheless, as the L574A mutation is predicted to have a large effect on the binding of PtdIns (A), we also tested the effect of this mutation on PtdIns inhibition. Application of 50 μM PtdIns induced a small and variable inhibition of the L574A mutant in the presence of 0.5 μM capsaicin and 50 μM PtdInsP2 P2 , but thivia a binding site overlapping with that for vanilloid compounds such as capsaicin.Overall, our data with the I703A and L574A mutations give support to PtdIns inhibiting the channel 2 in the putative inhibiting binding site. We compared the root-mean-square deviation (RMSD) of atomic positions of each phosphoinositide along the two MD trajectories . We found that PtdIns is more stable than PtdInsP2 , with RMSD values in the order of 1 to 3 Å for each individual subunit  for PtdIns versus RMSD values in the high 5 Å range (one subunit reaching up to 9 Å RMSD) for PtdInsP2. The high structural stability and binding pose similarity of PtdIns at the inhibiting site is in strong agreement with our experimental evidence suggesting its role as a competitive vanilloid antagonist.First, we assessed the binding site overlapping with the vanilloid site in the lipid nanodisc structure of TRPV1 . To this2 P2.The lipid density in the vanilloid pocket of the TRPV1 structure was modeled as PtdIns, but it was argued that the site can accommodate other phosphoinositides, including PtdInsP2 . Because2 bound at the putative activating site . Analysis of RMSD values (C and D) revealed average fluctuations higher than 4 Å for both phosphoinositide lipids, suggesting an overall larger mobility of both structure ensembles compared with their relative behavior at the inhibiting site (A and B). This is not surprising, given the shallow nature of the activating site. The latter is located close to the pore, at the boundaries of the TM domain (C and D) also revealed significantly larger fluctuations for PtdIns  compared with PtdInsP2 .Next, we performed a set of similar MD simulations with PtdIns or PtdInsPD values , C and Ding site , A and BM domain , C and D2 over PtdIns.Overall our computational and experimental findings suggest that two phosphoinositide binding sites exist in the TRPV1 channel; one, the inhibiting site, is overlapping with the vanilloid pocket and binds primarily PtdIns. The other, the activating site, may favor binding of PtdInsP2 and PtdIns(4)P. We found that PtdIns, but not PtdIns(4)P, partially inhibited TRPV1 in the presence of PtdInsP2 in excised inside out patches. The inhibition by PtdIns showed an inverse correlation with the concentration of capsaicin, which is expected from a competitive inhibitor acting through a shared binding site.The main goal of this work was to experimentally and computationally test the model in which phosphoinositides act as competitive antagonists binding to the vanilloid binding site , and to We computationally identified residues in the vanilloid binding site that are predicted to interact with PtdIns, but not with capsaicin. We found that the D509A and I703A mutants shifted the capsaicin dose response to the left, as expected if PtdIns is a competitive antagonist of capsaicin binding. The mutant channels also became more sensitive to low pH, which is compatible with PtdIns not only acting as a competitive vanilloid antagonist but also as a negative allosteric regulator. This is in line with phosphoinositides also reducing heat sensitivity in lipid vesicles  as well A). This residue is also in contact with PtdInsP2 in the activating binding site (B), which may induce complex effects when this residue is mutated. Consistent with this, the L574A mutation showed slightly right-shifted concentration dependence of capsaicin activation (A), which could be the consequence of allosteric effects by reduced PtdInsP2 binding on capsaicin activation. Nevertheless, this mutant showed reduced inhibition by PtdIns , further supporting our finding that PtdIns competes with capsaicin for an overlapping, yet not identical, lipid binding site . Interestingly, two of these residues are negatively charged (D509 and E570) and exert important destabilization effects on the negatively charged head-group of PtdInsP2 (A). Moreover, the additional steric bulk offered by the phosphate groups of PtdInsP2 hardly fits within the inhibiting site, inducing the lipid head to interact with a cluster of peripheral residues  located between the VSD and the TRP domain (B). While another residue, L574, was shown to clearly favor PtdIns over both PtdInsP2 and capsaicin (A), the nearby R575 residue, which is located at the interface with the activating site, clearly prefers PtdInsP2 (B). Although further work will be required to fully characterize the extent of PtdInsP2 binding at the inhibiting site, our current data strongly support the hypothesis that at this site, PtdIns is more stable compared to PtdInsP2.Further analysis of contact residues at the inhibiting site also revealed that 14 residues, namely R409, H410, D509, S510, Y511, S512, Y554, R557, E570, K571, I696, L699, Q700, and I703, established contacts with both PtdIns and PtdInsP2 . Interes2 A, furthesP2 A. MoreovP domain B. While apsaicin A, the nesP2 B. Althou2 was ever reported in excised patches, the purified TRPV1 incorporated into lipid vesicles showed reduced sensitivity to capsaicin when various phosphoinositides were incorporated at 4% P3 had no effect P3 had similar EC50 values Ped at 4% . These eed at 4% , 17. Ptdo effect . This or0 values , PtdInsP2 or PtdIns(4)P to the extracellular membrane surface in outside out patches inhibited TRPV1 currents Pcurrents . This efcurrents  but inhicurrents .2, this lipid may exert indirect inhibitory effects on the channel via intermediary proteins Pproteins  or modifproteins , but det2 is a well-established positive cofactor of TRPV1, and it was shown by several laboratories that it potentiates TRPV1 activity in excised patches P patches , 13, 16.tches P2 binding site in TRPV5 was recently determined experimentally P2 showed a conformational change indicating clear channel opening, when compared with the structure of the channel without this lipid.The location of the PtdInsPmentally . TRPV5 i2 binding site responsible for opening TRPV5 is very similar to that computationally predicted to be responsible for activation in TRPV1 Pin TRPV1 . Furtherchannels , and thechannels . By a sivailable .2, our two binding site model is compatible with many seemingly controversial data in the literature.While the final proof for the location of the activating phosphoinositide binding site(s) in TRPV1 will likely come from future cryo-EM studies with added PtdInsP2 in a cellular environment. Our data not only resolve a substantial controversy but also identify a novel, specific role for PtdIns in regulating ion channel activity.Overall, we identify PtdIns as a negative regulator of TRPV1 activity by binding to a site that overlaps with the binding site for vanilloids. PtdIns bound to this site may serve to set the basal sensitivity of the channel. We propose a two phosphoinositide binding site model, where the distinct nonoverlapping activating binding site is mainly occupied by PtdInsPXenopus laevis frogs, as described earlier  overnight for ∼16 h at 18 °C in a temperature-controlled incubator. Defolliculated oocytes were selected and maintained in OR2 solution supplemented with 1.8 mM CaCl2 and 1% penicillin/streptomycin (Mediatech) at 18 °C.Animal procedures were approved by the Institutional Animal Care and Use Committee at the New Jersey Medical School, and all animal procedures were performed in accordance with the approved guidelines. Oocytes were prepared from female  earlier . BrieflyX. laevis oocytes, and measurements were performed 5 to 7 days after injection, using borosilicate glass pipettes (World Precision Instruments) of 0.4 to 0.7 MΩ resistance, filled with a solution containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM Hepes, (pH=7.4), supplemented with 0.5 μM or 4 μM capsaicin (as indicated in the figure legends). After establishing GΩ-resistance seals on devitellinized surfaces of oocytes, the inside-out configuration was established, and currents were measured using a ramp protocol from −100 to +100 mV applied every second. The main perfusion solution contained 96 mM KCl, 5 mM EGTA, and 10 mM Hepes, pH adjusted to 7.4. Currents were recorded with an Axopatch 200B unit and analyzed with the pClamp 9.2 software (Molecular Devices). Measurements were performed at 18 to 20 °C. Various stimulating solutions were applied to the internal side of the inside-out membrane patch using a custom-made gravity-driven perfusion system. DiC8 phosphoinositides were purchased from Cayman Chemical. Capsaicin was purchased from Sigma. Experiments were performed in a random order.Excised inside-out patch clamp measurements were performed as described earlier . Briefly2, and 5 mM Hepes, pH 7.4, and currents were recorded with thin-wall inner filament-containing glass pipettes (World Precision Instruments) filled with 3 M KCl in 1% agarose. Currents were measured with an GeneClamp 500B amplifier and analyzed with the pClamp 9.2 software (Molecular Devices) using the same ramp protocol as described earlier for excised patch measurements. Various stimulating solutions were applied using a gravity-driven perfusion system. Experiments were performed in a randomized order.Measurements were performed as described earlier . Briefly2. The cells were transiently transfected at 80% confluency with cDNA encoding the rat TRPV1 tagged with GFP on its N terminus . TIRF images were obtained at room temperature using a Nikon Eclipse Ti2 microscope. Fluorescence excitation was performed using a 15-mW solid state 488-nm laser at 90% of the maximal power through a CFI Apochromat TIRF 60× oil objective (NA of 1.49), and the images were captured using an ORCA-Fusion Digital CMOS camera. The images were analyzed using Nikon NIS-Elements AR Analysis software and Image J.Human embryonic kidney HEK-293) cells  were maintained in MEM (Life Technologies) with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO3 cells (t test (two tailed) or analysis of variance, with Bonferroni post hoc test for normally distributed data (Shapiro–Wilk test). For nonnormally distributed data, Wilcoxon or Mann–Whitney tests were used as appropriate. The specific tests for each experiment are described in the figure legends.Data are represented as mean ± standard deviation plus scatter plots. Statistical significance was calculated either with We used the structure of TRPV1 in lipid nanodiscs solved in the presence of a phosphoinositide at the vanilloid binding site   as the shttp://www.gromacs.org). The top four binding modes (one each TRPV1 subunit) were selected to constitute our final structural complex of TRPV1 with PtdIns bound at the inhibiting site. In addition, we modified the structures of PtdIns lipids in this final complex to match the geometry of PtdInsP2 and by applying topology and atom types in agreement with the force field available through the CHARMM-GUI interface (SAPI25) (2 to the activating site.We used the docking tool Glide  Schrödi. The doc(SAPI25) . An anal3, with a total number of 330,000 atoms. We used all-atom MD simulation to equilibrate the systems P2 at the inhibiting or the activating sites plus a TRPV1 in complex with capsaicin) through a multistep protocol. First, we performed energy minimization of the systems (1000 steps). Second, we applied position restraints on protein (backbone and sidechain atoms) and lipids (head groups) that were gradually released during the first 50-ns simulation time . Last, upon releasing the restraints, we performed additional ∼425-ns production runs. During all simulations, we used the velocity Verlet integration method to solve the equations of motion, with a time step of 2 fs using the Particle mesh Ewald method for calculating the electrostatic potential. We applied the Langevin temperature and Langevin piston coupling schemes. We set the temperature to 300 K and the pressure to 1 atm. We generated four independent MD trajectories, with PtdIns or PtdInsP2 bound at the inhibiting or the activating sites. In all cases, we used the CHARMM36 force field  to isolate densities of phosphoinositide lipids occupying the inhibiting site in the TRPV1 structure solved by cryo-EM (PDB-ID: 5irz). Specifically, the cryo-EM map was uploaded on the experimental structure file and the “Volume Viewer” and the “Volume Eraser” tools were used to extract the density blobs of bound lipids, which were saved as BRIX files. The experimental densities of lipids were then compared by superimposition with atomic densities derived from MD simulations.We used the software Chimera  occupying the inhibiting or the activating sites using the VolMap Plugin available in VMD(9) (version 1.9). For all calculations, we used a resolution of 0.5 Å, with an atom size of 1.0 Å and weights corresponding to the atomic mass. Maps were computed for all frames of each trajectory and subunit; frames were then combined by the averaging method. The computed atomic densities of lipids were then compared by superimposition with the experimental densities obtained by cryo-EM. Similarly, we generated the occupancy map of water molecules located within 6 Å of residue Q700 and capsaicin. The generated map was rendered as wireframe representation, colored in cyan P in cyan .E (bold licorice). Residue contact lists, with relative frequency of occurrence within the distance threshold, were then generated individually per subunit and finally combined over each simulation trajectory (TRPV1 with PtdIns or capsaicin at the inhibiting site). Last, relative frequencies were plotted as stacked bar charts, color-coded by ligand type (PtdIns is blue and capsaicin is gray), using Microsoft Excel. By comparing the two distributions, we identified eight protein sidechains that were selectively in contact with PtdIns (but not with capsaicin) in at least 50% of the analyzed simulation frames (dotted lines). These residues, namely D509, S510, K571, L574, I696, L699, Q700, and I703, are located in the inhibiting site of TRPV1.We first used custom script (Tcl) to select protein sidechain atoms within a distance threshold (5 Å) of headgroups in either the phosphoinositide lipid PtdIns (PI) or capsaicin. Headgroups were defined as all heavy atoms in the substructures depicted in Figure 52 in the inhibiting site. We first calculated all contacts within a 5 Å cutoff established by PtdIns or PtdInsP2. We then extracted all contact maintained for at least 50% of the frames in each simulation, by averaging over the four TRPV1 subunits. Results are reported in 2, respectively.In addition to computationally identify amino acid residues that are in contact with PtdIns, we identified residues interacting with PtdInsPE, bold licorice) of the phosphoinositide lipid PtdIns or capsaicin occupying the inhibiting site and the contact residues within 5 Å of PtdIns. Distances were calculated using the “g_mindist” function available with the program GROMACS (http://www.gromacs.org). Distance plots were generated in XMGRACE . Substructures were sketched using Sketch Tool available in Maestro .We calculated the minimum distance between the headgroups . Systems were prepared for the calculations using the Protein Preparation Wizard (We calculated the change in binding affinity of TRPV1 for PtdIns upon mutation of each PtdIns-contacting residue into alanine. To do so, we used the “Residue-Scanning and Mutation” protocol available in BioLuminate  Schrödi. All resn Wizard  (Schrödin Wizard . AffinitAll data are contained in the manuscript and in the This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Classic galactosemiais caused by loss-of-function mutations ingalactose-1-phosphate uridylyltransferase (GALT) that lead to toxicaccumulation of its substrate, galactose-1-phosphate. One proposedtherapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzedby galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1)are primarily ATP-competitive with limited clinical utility to date.Here, we determined crystal structures of hGALK1 bound with reportedATP-competitive inhibitors of the spiro-benzoxazole series, to revealtheir binding mode in the active site. Spurred by the need for additionalchemotypes of hGALK1 inhibitors, desirably targeting a nonorthostericsite, we also performed crystallography-based screening by soakinghundreds of hGALK1 crystals, already containing active site ligands,with fragments from a custom library. Two fragments were found tobind close to the ATP binding site, and a further eight were foundin a hotspot distal from the active site, highlighting the strengthof this method in identifying previously uncharacterized allostericsites. To generate inhibitors of improved potency and selectivitytargeting the newly identified binding hotspot, new compounds weredesigned by merging overlapping fragments. This yielded two micromolarinhibitors of hGALK1 that were not competitive with respect to eithersubstrate  and demonstrated good selectivity overhGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findingsare therefore the first to demonstrate inhibition of hGALK1 from anallosteric site, with potential for further development of potentand selective inhibitors to provide novel therapeutics for classicgalactosemia. At the hub of the Leloirpathway is galactose-1-phosphate uridylyltransferase ,which converts galactose-1-phosphate  and UDP-glucose (UDP-Glc)into glucose-1-phosphate and UDP-galactose.2 Inherited mutations of the GALT gene lead to theautosomal recessive disorder classic galactosemia (OMIM 230400),3 affecting 1:16 000–60 000live births. Classic galactosemia patients generally sicken in theneonatal period, with liver, kidney, intestinal, and central nervoussystem toxicity exacerbated by the high galactose content in humanand formula milk.5 If lactose, the primary exogenoussource of galactose, is not removed from the patient’s diet,progressive liver and brain damage lead to death or severe disability.The current mainstay treatment is life-long dietary galactose restriction,which resolves acute life-threatening symptoms but fails to preventthe long-term, late-onset complications that include learning andspeech difficulties, neurological impairments manifesting as ataxia,and premature ovarian insufficiency.4The Leloir pathwayis essential for the metabolism of dietary galactose,6 Gal-1-P has been shown to inhibit various metabolic enzymeswith key cellular functions, including inositol phosphatase,7 UDP-Glc pyrophosphorylase and galactosyltransferases,11 glycogen phosphorylase, and phosphoglucomutase.13 Inhibition of human GALK1 (hGALK1) to prevent Gal-1-P productiontherefore could provide a therapeutic benefit for GALT deficiency.14 This is supported by the evidence that galk1 knockout in Drosophila rescued the galactosemic neurologicalphenotype,15galk1 knockdownin GALT-deficient yeast abolished sensitivity to galactose levels,17 and patients with inherited GALK1 deficiency (OMIM 230200) presentmilder phenotypes  and do not accumulateGal-1-P.18 This “substrate reduction”approach, to mitigate the toxic accumulation of a metabolite thatarises from a metabolic block by inhibiting an enzyme upstream ofit, is gaining therapeutic potential for other inborn errors of metabolism.20In classic galactosemia, GALT enzymatic deficiency causes the accumulationof Gal-1-P, the product of the enzyme galactokinase 1 upstream of GALT in the Leloir pathway. Gal-1-P is proposed to bethe major, if not sole, pathogenic driver of disease.21 which catalyzes the MgATP-dependent phosphorylationof the C-1 hydroxyl of α-d-galactose to yield Gal-1-Pin the Leloir pathway. The crystal structure of hGALK1 in complexwith galactose and the nonhydrolyzable ATP analogue AMPPNP (PDB 1WUU) supported an orderedkinetic mechanism, whereby ATP stabilizes the active site loops  campaign using a luminescence-basedactivity assay yielded a series of spiro-benzoxazole derivatives,such as T1 and T2 .A series of spiro-benzoxazoleinhibitors of low-micromolar potencywas previously reported for hGALK1, and T1 and T2 share the chemical scaffold of a benzoxazole ring connectedto a tetrahydroquinazolinone ring by an amide linker, and both compoundsare found to occupy the ATP binding site in a similar manner , previously applied in HTS campaignsfor hGALK1.30 In this method, the level of ATP remainingafter the hGALK1 reaction is measured as a function of luciferin turnoverand subsequent light generation caused by the ATP-dependent activityof luciferase enzyme.31 We observed dose-dependentinhibition of hGALK1, with IC50’s  similar to publishedvalues24 . This assay setup is not suited for measuring hGALK1 activity athigher ATP concentrations, however, as the luciferase enzyme is subjectedto feedback inhibition by its product oxyluciferin.32We reproduced the inhibition of hGALK1 by the spiro-benzoxazoleinhibitors using the Kinase-Glo luminescence-based assay  to measurethe level of galactose remaining after being depleted as a substratefor hGALK1. In this method, hydrogen peroxide generated by the oxidationof galactose by GAO is used by horseradish peroxidase to convert thenonfluorescent substrate Amplex Red into its highly fluorescent productresorufin; therefore, the resultant fluorescent signal is inverselyproportional to hGALK1 enzyme activity. We optimized several parametersof this assay to allow measurement of hGALK1 activity for the firsttime .ATP-titration experiments with T2 showed no significantchange in Vmaxapp (P = 0.1801) and a significantincrease in Kmapp (P <0.0001), collectively indicating competitive inhibition with ATP by T2, which is supported by fitting in GraphPad Prism  and diffractionquality (consistently better than 2.5 Å) for the screening campaign.Additionally, this crystal system is biased for the detection of fragmentsbinding to nonorthosteric sites of hGALK1 outside of the ATP and galactosepockets. hGALK1 crystals were each soaked with an individual fragmentfrom the DSi-Poised library26 at a millimolarconcentration.AshGALK1 belongs to the kinase superfamily, we recognize thatdeveloping inhibitors that are not ATP-competitive could be beneficialfor downstream optimization of pharmacokinetics/dynamics. To thisend, we pursued the fragment screening approach, motivated by itsemerging promise in revealing novel binding pockets and providinggood chemistry starting points.Table S2).38 We observed fragments 1 and 2 bound atthe entrance to the active site ATP binding pocket (Table S2), positioned within 5 Å of the T2 carbonylgroup  and lower  clusters,according to whether they extend above or below the β7−β8loop.Severalfragments occupy a hotspot distal from the active site ATP/galactosepockets, on the opposite face of the protein 2C. This Fragments from the upper cluster consist of an aromaticring separated by an amide, ester, or urea linker from another ring,which is usually aromatic 2, inset.3 from the upper hotspot cluster, reducing hGALK1 activityby 24 ± 1.6% at 5 mM fragment, an effect that is statisticallysignificant  ortheir inhibitory effects were not statistically significant . The lackof inhibition by fragments 1 and 2 at theactive site entrance suggest that these fragments are only bound inthe structure due to the proximity of T2 present in thecrystal system.For other fragments tested, either there is noobserved inhibition or a nonorthosteric site . Galactose-titration experiments withfragment 3 showed a concentration-dependent, 5-fold decreasein Vmaxapp (P < 0.0001) and a 6-foldincrease in Kmapp (P <0.0001), collectively indicating mixed model inhibition with galactoseby fragment 3, which is supported by fitting in GraphPadPrism (Table S5).We next investigated whether the inhibitoryeffect of fragment 3 showed a concentration-dependent,4-fold decrease in Vmaxapp (P < 0.0001)without change in the Kmapp (P = 0.4932), collectively indicatingnoncompetitive inhibition with ATP by fragment 3, whichis supported by fitting in GraphPad Prism (Table S5). Collectively, these results suggest that fragment 3 does not bind to the active site of hGALK1, consistent with itscostructure, and that the hotspot is a true allosteric binding site.Additionally, ATP-titrationexperiments with fragment 39 We overlaid fragment 3 from theupper cluster and fragments from the lower cluster in the costructuresand searched commercial catalogues for compounds that incorporatechemical groups from both fragment clusters (Supporting Information for more details), with the expectation that such compounds couldengage with both upper and lower components of the hotspot. We procuredthree compounds 11–13  and Amplex Red assays . Dose-dependent sigmoidal curves and 25–198 μMfor 12  (Table S6). Therefore, 11 and 12, bearing a 6,7-dichloro-quinoxaline scaffold, share similarIC50 values, with 12 achieving a slightlyhigher ligand efficiency. Determination of IC50 valueswas not possible for 13 due to its lower potency, with50% inhibition achieved by approximately 1 mM of 13 inboth assays.Compounds nase-Glo S4A and A11–13 with respect to galactose and ATP were then determinedby comparisonof nonlinear least-squares fitting to inhibition equations as describedfor fragment 3. Compounds 11 and 12 were noncompetitive with respect to both ATP and galactose, whereas 13 demonstrated mixed inhibition with respect to galactoseand noncompetitive inhibition with respect to ATP . Altogether, our kinetics dataindicate that inhibition of hGALK1 activity by fragment 3 and follow-up compounds 11–13 isconsistent with their binding away from the active site, in agreementwith the fragment-bound crystal structures.The modes of inhibition for 3 and follow-upcompounds 11–13 to hGALK1 in solution,we performed surface plasmon resonance (SPR) measuring dissociationconstants (KD) of each compound upon bindingto hGALK1 covalently immobilized on a sensor chip. Weak binding wasobserved for fragment 3, with KD in the high-micromolar to low-micromolar range . For example, predicted docking poses of 11  and 12  show the 6,7-dichloro-quinoxalinegroup occupying the lower binding pocket while the flexible groupinteracts with surface loops around the upper binding pocket. Predicteddocking poses of 13  indicate this compound interacts more with the upper binding pocketbut forms fewer interactions with the lower binding pocket.3 or compounds 11–13, to determine the relative selectivityof these inhibitors. While fragment 3 demonstrates asimilar degree of inhibition against hGALK1, hGALK2, and hMVK, compounds 11–13 by contrast were selective for hGALK1,displaying less than 10% inhibition of hGALK2 and hMVK at 1 mM . At the sequence level, these two regions are also poorly conserved. Finally, the β7−β8loop and the α8−α9 turn differ in conformationamong all hGALK1 protomers within the asymmetric unit, indicatingconformational plasticity in this region that can be modulated byligand binding .GALK1 is one of several small-molecule kinases from the GHMP class.We measured the activity of two other human GHMP kinases, N-acetylgalactosamine kinase  and mevalonate kinase , in the presence of fragment  at 1 mM 4E. This 40 Future MD and structural studies are therefore merited to investigatewhether these regions, or others, are involved in a long-range allostericcommunication for hGALK1, to guide further development of small moleculesthat can target such mechanism(s) for higher potency.This work reveals chemical startingpoints bound to a non-orthosteric hotspot region, which can be exploitedfor future development of hGALK1-specific allosteric inhibitors. Allostericinhibitors often promote structural rearrangements to an enzyme’sactive site that can decrease the enzyme’s affinity for itssubstrates, affect the formation of the enzyme–substrate complex,or change the stability of the transition state. In hGALK1, moleculardynamics studies have pinpointed Arg105 in the active site and tworegions (residues 174–179 and 231–240), where conformationalflexibility is important for catalysis.T1 and T2 were purchased from Interbioscreen.All fragments were purchasedfrom Enamine.Compounds 41 mutations K252A:E253A, with an engineered N-terminalHis6-tag subcloned into the pET21d vector, was transformed into E. coli BL21(DE3) cells. hGALK1 was cultured in TerrificBroth with 0.1 mM IPTG induction at 18 °C. Cell pellets wereharvested, homogenized in lysis buffer , andcentrifuged to remove insoluble material. The supernatant was purifiedby nickel affinity (Thermo Fisher Scientific) followed by size exclusion chromatography into crystallizationbuffer . Purified protein was concentrated to24 mg mL–1. Constructs encoding full-length hGALK2(Met1-Ala458) and hMVK (Met1-Leu396) were subcloned into the pNIC-NHStIITvector encoding an engineered N-terminal His6–Strep–Strep-tagfollowed by a TEV protease cleavage site. Each construct was expressedand purified as described for hGALK1.A hGALK1 construct,encoding full-length protein harboring the surface entropyd]oxazol-2-ylamino)-7′,8′-dihydro-1′H-spiro-5′(6′H)-one , 24 mg mL–1 of purified hGALK1was preincubated with 5 mM T1 dissolved in N-methylpyrrolidone , and crystals weregrown by sitting-drop vapor diffusion at 20 °C, equilibratedagainst a well solution of 0.1 M Morpheus Amino Acids Mix, 0.1 M MorpheusBuffer system 1 pH 6.5, and 50% v/v Morpheus Precipitant Mix 1. Toco-crystallize hGALK1 with galactose and 2′-(benzo[d]oxazol-2-ylamino)-7′,8′-dihydro-1′H-spiro-5′(6′H)-one , 24 mg mL–1 of purified GALK1was preincubated with 2.25 mM T2 dissolved in NMP  and crystals grown by sitting-drop vapor diffusionat 20 °C, equilibrated against a well solution of 0.1 M MorpheusCarboxylic Acids Mix, 0.1 M Morpheus Buffer system 2 pH 7.5, and 50%v/v Morpheus Precipitant Mix 4. Crystals were cryo-protected with50% NMP and flash cooled in liquid nitrogen. Diffraction data werecollected at Diamond Light Source beamline I04 and indexed, integrated,and scaled via the automated Xia2 pipeline.42 Molecular replacement, using the hGALK1–galactose–AMPPNPstructure (PDB 1WUU) as a search template, and subsequent model building and refinementwere performed in the CCP4 program suite.43To co-crystallize hGALK1 withgalactose and 2′-. Hundreds ofcrystals were mass-produced by sitting-drop vapor diffusion at 20°C, equilibrated against well solutions of 0.1 M MOPS/sodiumHEPES pH 7.0–8.0 (based on Morpheus Buffer system 2), 40–50%Morpheus Precipitant Mix 4, and 0.1 M Morpheus Carboxylic acids mix.For soaking, 50 nL of each fragment  was added to a crystallizationdrop using an ECHO acoustic liquid handler dispenser at the DiamondLight Source beamline I04-1. Crystals were soaked for 2 h with fragments before being harvested usingthe SHIFTER technology,44 cryo-cooled inliquid nitrogen, and measured using the “automated unattended”mode of the i04-1 beamline. The XChemExplorer pipeline45 was used for structure solution with parallelmolecular replacement using DIMPLE,46 followedby map averaging and statistical modeling to identify weak electrondensities generated from low occupancy fragments using PanDDA.47 Model building and refinement were performedusing the WinCoot and REFMAC software integrated into the XChemExplorerpipeline.45 Figures were prepared usingICM-Pro software (Molsoft LLC).Purified hGALK1at 24 mg mLinvitro was determined using the Kinase-Glo luminescent kinaseassay (Promega), according to the manufacturer’s protocol.Determination of suitable assay parameters is described in the Supplementary Methods and shown in Figure S1. To measure activity and inhibitionof hGALK1, 10 μL/well of reaction containing 10 nM hGALK1, 100μM galactose, and 35 μM ATP in an assay buffer  was incubated with 5 mM of fragments; varyingconcentrations of spiro-benzoxazole compounds T1 and T2  or compounds 11–13  were dispensedinto 384-well assay plates (Greiner). Similarly, hGALK2 activity andinhibition was measured with reactions containing 50 nM hGALK2, 100μM N-acetylgalactosamine, and 35 μM ATP in the above-describedbuffer and hMVK activity and inhibition was measured with reactionscontaining 250 nM hMVK, 100 μM mevalonate, and 35 μM ATPin the above-described buffer. Following a 1-h RT incubation, 10 μLof Kinase-Glo Plus detection reagent was added , and after a further 20 minutes RT incubation, luminescencewas detected using a Pherastar FSX plate reader (BMG Labtech) containinga luminescence optics module. To determine the inhibition mode withrespect to galactose, 10 nM hGALK1 and 35 μM ATP in an assaybuffer was added to an array of six fragment (0–15 mM) or 12compound (0–2 mM) concentrations against six galactose concentrations(0–250 μM), with a final reaction volume of 10 μL/welldispensed into 384-well assay plates (Greiner), and measured as describedabove. All reactions were performed in technical triplicates for twodifferent preparations of hGALK1. Reaction rate, defined as totalATP consumed over the 1-h hGALK1 reaction, was determined using thefollowing equation, where Lumreaction = mean value of measuredluminescence of each reaction and Lum35 = measured luminescencewith no galactose :hGALK1 activity F test P values calculated by the software.Data were plottedusing GraphPad Prismsoftware; curve fitting was performed with a nonlinear least-squaresregression fit to the mixed, competitive, noncompetitive, and uncompetitiveinhibition models from the GraphPad Prism enzyme kinetics–inhibitionequations and the best fit selected by comparison of Akaike’sInformation Criterion (AICc) probability scores and extra sum-of-squares Supplementary Methods and shown in Figure S3. The reactionconditions were 250 nM hGALK1, 50 μM galactose, and 100 μMATP for IC50 calculation and 250 nM hGALK1, 50 μMgalactose, and 0–250 μM ATP for determination of inhibitionmode. Amplex Red reagent was added at a 1:1 volume to the hGALK1 reactionafter 1-h incubation at RT. Fluorescence emission was measured at585 nm, with excitation at 570 nm, after a further incubation periodof 40 minutes, using a Pherastar FSX plate reader with a FI 540 590optics module. Reactions were performed in technical triplicates fortwo biological replicates, and data were plotted and fitted as describedfor the Kinase-Glo assay except that the reaction rate, defined astotal galactose consumed over the 1-h hGALK1 reaction, was determinedusing the following equation, where Fluorreaction = meanvalue of measured fluorescence of each reaction and Fluor50 = measured fluorescence with no galactose :The Amplex Red assay reagentcontained horseradish peroxidase , its substrateAmplex Red , andgalactose oxidase . The assay buffer used wasthe same as in the Kinase-Glo assay. Determination of suitable assayparameters is described in the 3, 11–13) by 1:1 dilution from 200 μMto 0.195 μM, and the subsequent solutions were passed over thechip at a flow rate of 30 μL/min. Data are from an n = 1 experiment.PurifiedhGALK1 (30 μg/mL), diluted in acetate buffer at pH 5, was attachedvia covalent coupling to a density of 13000 RU on a CM5 chip. Theassay buffer was 50 mM sodium phosphate, pH 7.5, 200 mM NaCl, 0.5mM TCEP, 5% DMSO, and 0.05% TWEEN20. A serial dilution (11 concentrations)was prepared in the above buffer for each analyte (


SI-1), fosfomycin trometamol(SI-2), and hydrolysis of fosfomycin called 1,2-dihydroxypropylphosphonic acid (SI-3). The inhibition efficiency ofall these chemicals was evaluated against calcite and gypsum scalescompared to commercial oilfield scale inhibitor hydroxyphosphonoaceticacid (HPAA) according to the NACE Standard TM0374-2007.In addition, the calcite scale inhibition efficiency of all aminomethylene-freephosphonate SIs (SI-1 to SI-3 and HPAA) was investigated based on the Heidrun oilfield, Norway.Moreover, we have reported the calcium compatibility of these chemicalsat various concentrations of SIs and calcium ions at 80 °C over24 h. All new aminomethylene-free phosphonate SIs showed good gypsumand calcite inhibition performance. It was also found that all testedchemicals derived from fosfomycin demonstrated excellent compatibilitywith calcium ions of up to 1000 ppm throughout the 24 h experimentperiod compared to HPAA.Aminomethylenephosphonate-basedscale inhibitors (SIs) have beenwidely studied and recognized for several decades to mitigate variousoilfield scales. However, most of these compounds afforded severaldrawbacks, such as poor biodegradability and intolerance with theproduction system. As environmental regulations become more rigid,new production chemicals must adhere to certain criteria to qualifyfor use in the oil and gas industry, particularly in areas with strictregulations, such as the Norwegian Sea. The low toxicity of fosfomycinencouraged us to test fosfomycin and related molecules as new aminomethylene-freephosphonate SIs for calcite and gypsum scales. The tested chemicalsare fosfomycin disodium salt ( The life cycle of the petroleumreservoir plays a vital role in the scale formation process.2 Scale formation is more common in mature oilfields,where seawater is reinjected into the reservoir to enhance oil recovery(EOR). Scale can be precipitated over time, eventually hindering theproduction process, triggering a chain of problems, thereby decreasingoilfield productivity, causing economic and potential loss of thewell. So, there is a clear need in preventing the formation of scaleprecipitates.4Scale formation leadsto critical flow assurance problems in oiland gas production installations.3), barite (BaSO4), gypsum (CaSO4·2H2O), and celestite (SrSO4) areamong the most common divalent metal ion precipitates encounteredin the oilfield industry. Calcite is the most commonly encounteredscale deposit and the most thermodynamically stable polymorph of calciumcarbonate.5 Water found within carbonateand calcite-cemented sandstone petroleum reservoirs usually containshigh concentrations of Ca2+ and Mg2+ ions.7 Furthermore, seawater carries high concentrations of sulfate (SO42–) ions. The difference in the ionic natureof seawater and formation water when mixed during EOR leads to scaleformation. Sulfate scales are more commonly formed when formationwater and injected seawater are mixed or when two different watersmix in topside flowlines. For example, the gypsum scale is commonlyfound in heat exchangers and oilfield applications.8Calcite .ATMP), diethylenetriaminepentamethylene phosphonic acid (DTPMP), and ethylenediamine tetramethylene phosphonic acid (EDTMP) demonstrate low biodegradability, implying that they cannot beused in offshore regions with strict environmental regulations, suchas the North Sea  showed outstanding inhibition performance for calcite scale anda good efficiency against oilfield barite scaling using a high-pressuredynamic tube-blocking rig at 100 °C and 1200 psi  based on theHeidrun oilfield, Norwegian Sea, Norway.1200 psi 1. Howeve16 Furthermore, the drawbacksof commercial aminomethylenephosphonate SIs motivated us to synthesizeand evaluate new SIs based on the aminomethylene-free phosphonategroup.11 This study aims to synthesizeaminomethylene-free phosphonates derived from fosfomycin as the startingcompound and evaluate their performance as oilfield SIs. Fosfomycinbelongs to the class of phosphonic antibiotics used for the treatmentof urinary tract infections (UTIs).17 Asfar as our research is concerned, and to the best of our knowledge,this chemical has not been reported in the open literature for oilfieldscale inhibition applications. The initiative of using fosfomycinas the starting compound rises from its low toxicity, good bioavailability,and the presence of functional groups in its structure backbone thatare known to be favorable features in scale inhibitors. This set ofcharacteristics could provide good inhibition efficiency and improvedbiodegradation performance in contrast to commercial SIs. Similarly,our research group has developed a series of low toxicity bisphosphonates,commonly used as bone-targeting drugs, as new SIs against carbonateand sulfate oilfield scales.18As environmental regulations become more rigid, new productionchemicals must adhere to certain criteria to qualify for use in thepetroleum industry. Therefore, there is a great need to develop suitablechemicals that allow a high scale inhibition efficiency and an improvedenvironmental footprint to fulfill the requirements set by variousstakeholders.SI-1) wasevaluated as a scale inhibitor against gypsum and calcite scales.In addition, the starting material (SI-1) will be usedto synthesize the bioavailable version of fosfomycin, commerciallyknown as fosfomycin trometamol (SI-2)  2.19 Thising SI-3 2. The inHPAA) was supplied from ShanDong XinTai Water TreatmentTechnology Co., Ltd., China. The structures of the synthesized productswere characterized using nuclear magnetic resonance (NMR) spectroscopyand Fourier-transform infrared (FTIR) spectroscopy. 1Hand 31P NMR chemical shifts were obtained in deuteriumoxide (D2O) using a 400 MHz Bruker NMR spectrometer. Thedata was processed using TopSpinTM 3.2 software. Additionally, anAgilent Cary 630 FTIR spectrometer equipped with a diamond compositeATR  crystal was used. FTIR data wasprocessed using MicroLab PC software.Fosfomycin was purchasedfrom Tokyo Chemical Industry Co., Ltd. In addition, all other chemicalsused in this project were purchased from Tokyo Chemical Industry Co.,Ltd., Sigma-Aldrich (Merck), VWR chemicals, and ACROS Organics. Allsolvents were used as purchased without further purification. Hydroxyphosphonoaceticacid : IR νmax (cm–1): 1089 (PO3). 1H NMR  δ ppm: 3.20–3.13 , 2.75–2.69 , 1.38–1.37 . 31P NMR  δ ppm: 10.00.(3-Methyloxiran-2-yl)phosphonic acid  in 16 mL of methanol. Another 100 mL one-neckflask with the same arrangement was set up at 50 °C and loadedwith oxalic acid dihydrate , tromethamine , and 9 mL of methanol. After complete dissolution,the temperature of the second reaction was raised to 65 °C. Afterthat, this solution was gradually added to the first reaction at thesame temperature using a dropping funnel. After completion, the finalsolution was left to cool down to room temperature for 3 h and furthercooled in a fridge at 4 °C overnight. The next day, the milkysuspension was filtered under vacuum using a Büchner funnel.The filtrate was concentrated using a rotary evaporator. The productwas further washed with 15 mL of a 1:1 mixture of acetone-ethanolunder vigorous stirring at room temperature for 3 h. A suspensionof white crystals was obtained and filtered under vacuum using a Büchnerfunnel. The obtained crystals were washed with absolute ethanol toafford a white powder (SI-2). The synthesis route ofthis reaction is shown in SI-2): Yield: 86%. IR νmax (cm–1): 3048 (NH2), 2945, 2822 (OH), 1138 (CO),1035 (PO3). 1H NMR δ ppm: 3.60 )2–C(NH2)–CH2−), 3.27–3.20, 2.87–2.80 (PO3H2)), 1.37–1.35. 31P NMR  δppm: 12.29.(2-(2-Amino-3-hydroxy-2-(hydroxymethyl)propoxy)-1-hydroxypropyl)phosphonicacid (SI-3). Thesynthesis route of SI-3 is presented in A 50 mL one-neck flask equipped with a refluxcondenser at 100 °Cand magnetic stirring was loaded with fosfomycin disodium salt  in 6 mL of deionized water. The pH of the solutionwas adjusted to 2.89 using 0.1 M HCl and stirred overnight at 100°C. The temperature was then decreased to room temperature, andwater was removed using a rotary evaporator to afford a hygroscopiccompound. Next, the product was washed with absolute ethanol undervigorous stirring for 4 h at room temperature. The solvent was thenremoved under vacuo to leave a white solid (SI-3): Yield: 76%. IR νmax (cm–1): 3249 (OH), 1041 (PO3). 1H NMR  δ ppm: 3.99–3.92–CH3), 3.46–3.42 –PO3H2), 1.22–1.20. 31P NMR  δppm: 17.33.1,2-Dihydroxypropylphosphonic acid  and variousCa2+ ions concentrations  inthe presence of 30,000 ppm NaCl (3 wt %) were dissolved in 20 mL ofdistilled water. The pH of the mixture solutions was adjusted in therange of 4–6. Then, all prepared vessels were located in anoven at 80 °C over 24 h. The haziness and/or precipitation oftested SIs with Ca2+ ions were checked at mixing, after30 min and 1, 4, and 24 h via visual observation.Organophosphorous compoundsare widely used as SIs for squeeze treatment applications.SI-1) was tested as the SI under oilfield conditions. Second,its orally bioavailable derivative, fosfomycin trometamol (SI-2), was synthesized by the reaction of fosfomycin disodium salt (SI-1) and a tromethamine acid salt with oxalic acid in thepresence of alcoholic media.22 Finally,due to the instability of SI-1 in acidic media, it wasdesired to hydrolyze the epoxide ring to afford an aliphatic compoundand compare its performances as new aminomethylene-free phosphonateSI (SI-3). This reaction consisted of bringing the pHof a fosfomycin solution down to 2.89 under reflux conditions at hightemperature.26Three aminomethylene-free phosphonate-basedSIs were studied as antiscaling agents for calcite and gypsum scales.First, the commercial and natural product antibiotic fosfomycin  and a C=O band (1650–1800 cm–1). The FTIR spectra of SI-2 showed a remarkable broadpeak at 3048 cm–1, representing the NH stretchingvibration, and further at 2945 and 2822 cm–1 asa result of the OH stretch. A characteristic peak for the phosphonategroup was presented at 1035 cm–1. In the case of SI-3, a broad peak for the OH stretch was obtained at 3249cm–1, and a sharp absorption peak at 1041 cm–1 was attributed to the phosphonate group.The structures ofthese compounds were characterized using NMR and FTIR spectroscopictechniques. In the FTIR spectra of 1H NMR for SI-1 in D2O displayedmultiple peaks in the range of δ 3.20–3.13 ppm, representingthe −CH–CH3 proton. A doublet–doublet peak was observed at δ 2.75–2.69ppm, attributed to the −CH–PO3H2 proton. Finally, a very sharpdoublet peak was displayed at δ 1.38 and δ 1.37 ppm representingthe methyl group (−CH–CH3). For SI-2, a sharp singlet peak was shownat δ 3.60 ppm representing the methylene groups (CH2(OH))2–C(NH2)–CH2−). Furthermore, the determining factor in differentiating SI-3 from SI-1 was the chemical shift obtainedin the 31P NMR spectra. While SI-1 showeda singlet signal at δ 10.00 ppm, SI-2 and SI-3 shifted the signal to δ 12.29 and δ 17.33ppm, respectively.The SI-1, SI-2, and SI-3) was screened againstgypsum and calcite scales using the NACE Standard TM0374-2007 protocol.25 A series of numerous concentrations of SIs of 100,50, 20, 10, 5, 2, and 1 ppm were evaluated using static bottle testsat 80 °C for 5 h. In addition, a commercial SI HPAA was also tested under the same conditions to compare the performanceof our proposed SIs with a commercially available product that possessesa similar chemical structure.The scale inhibitionperformance of aminomethylene-free phosphonates  against thegypsum scale using static bottle tests. In general, all tested aminomethylene-freephosphonate scale inhibitors showed good inhibition performance forthe gypsum scale. The commercial scale inhibitor HPAA gave a very good inhibition efficiency at high concentration SIdoses (100–20 ppm) and a moderate inhibition activity at lowSI concentrations (10–1 ppm). For example, the inhibition efficiencyof HPAA was 97% at 100 ppm, as shown in SI-1 showed only about 1% better performance than HPAA at100 and 50 ppm. In addition, SI-1 afforded a reasonableinhibition efficiency of 85% at 20 ppm. However, the inhibition efficiencywas dropped down to 31% at 10 ppm. It was also investigated that SI-1 showed a weak inhibition performance at low SI concentrationscompared to HPAA. The modified fosfomycin (SI-2) containing one phosphonate group and three hydroxyl moieties inits structure backbone afforded a moderate gypsum inhibition performancecompared to HPAA. For example, the calcium inhibitionactivity was 91% at 100 ppm SI-2. It was also found thatthe inhibition activity of SI-2 was slightly droppedthroughout the whole test until reaching <10% inhibition at 2 and1 ppm  and the commercial SI (HPAA) are presented in HPAA remained above 90% until 5 ppm. In addition, it was found that theinhibition performances of HPAA were dropped to 36 and13% at 2 and 1 ppm, respectively.In this study, the scale inhibitionperformance of all tested aminomethylene-free phosphonates was screenedagainst the Heidrun calcite scale using the NACE Standard TM0374-2007protocol. The Heidrun calcite inhibition efficiencies of aminomethylene-freephosphonates (SI-1 (based epoxide ring) showed 92% inhibition at 100 ppm.However, this inhibition decreased to 71% at 50 ppm. Moreover, SI-1 afforded a weak inhibition at low inhibitor doses againstthe Heidrun calcite scale. For the modified fosfomycin, SI-2 showed moderate inhibition performances of 68 and 55% at 100 and50 ppm, respectively. It was also found that the inhibition performancewas dropped continuously at lower concentrations, affording poor efficiency.Furthermore, the new linear aminomethylene-free phosphonate SI-3 showed very good inhibition efficiency against the calcitescale at high SI concentrations (100–10 ppm). For example,the inhibition efficiency of SI-3 reached 97% at 100ppm. Moreover, SI-3 showed poor inhibition performancesat lower inhibitor concentrations compared to other new aminomethylene-freephosphonates SIs (SI-1 and SI-2) and commercialSI HPAA, as shown in For the new aminomethylene-free phosphonate SIs, fosfomycin 3H2, COOH, and SO3H) in the inhibitor structural backbone led to weak inhibitionperformance, particularly at lower SI doses. In addition, the testingconditions of the Heidrun calcite water system most likely influencedthe scale inhibitor performance compared to standard calcite. Forstandard calcite, the Ca2+ ions concentration in the mixedbrine is 1657 ppm, and the Mg2+ concentration is 220 ppm.For Heidrun calcite, the Ca2+ concentration is 1020 ppmand the Mg2+ concentration is 265 ppm. Although the Mg2+ concentration for Heidrun conditions is slightly higherthan that proposed by NACE Standard TM0374-2007, it did not seem tonegatively affect the tested SIs’ inhibition performance. Thismay be due to the lower Ca2+ concentration for Heidrunwater chemistry as the harmful effect of Mg2+ ions on theinhibition efficiency is stronger at higher Ca2+ concentrations.6Clearly, for both calcite and gypsumoilfield scales, the new aminomethylene-freephosphonate SIs are moderate inhibitors. The limited number of functionalinhibition groups  at 80 °C. The compatibility results are presented in Table S2). The concentrations of tested SIs werevaried from 100 to 50,000 ppm, while the doses of Ca2+ ionswere changed from 100 to 1000 ppm in the presence of 30,000 ppm NaClto match the salinity of the petroleum reservoir.As mentionedearlier, mostorganophosphorus-based SIs showed poor compatibility activity withcalcium ions, giving a CaHPAA exhibited reasonablecalcium tolerance at allSI doses  and 100 ppm Ca2+ ions displayed outstanding calcium compatibility.No precipitates were determined at all inhibitor and calcium ion concentrations.For example, SI-1 and SI-2 gave superiorcalcium compatibility performance at all SI concentrations and 10,000ppm calcium ions (Table S2). The presence ofether linkages in their structure backbones may enhance the abilityto overcome complexing with Ca2+ ions, as investigatedpreviously in our research.20 It was alsofound that SI-3  displayedcloudiness at 10,000, and 50,000 ppm inhibitors and 10,000 ppm Ca2+ ions, as shown in Table S2.The newly tested aminomethylene-free phosphonates  have been comparedand screened with commercial SI hydroxyphosphonoacetic acid (HPAA) through a static bottle test and calcium compatibility.For the gypsum scale, SI-1 and SI-2 showedgood performance only at the highest concentrations of the SI. However, SI-3 showed good performance throughout all concentrationsof SIs tested. In addition, HPAA exhibited very goodscale inhibition efficiency. The slightly different performance between HPAA and SI-3 is attributed to the presence ofthe carboxyl group in the structure of HPAA. For thecalcite scale, the inhibition efficiency of all tested SIs was relativelymoderate for simplified calcium carbonate, according to the NACE StandardTM0374-2007 protocol. However, these chemicals gave good inhibitionactivity against the Heidrun calcite scale, Norway. We speculate thatthe primary reason for the weak inhibition of these SI classes maybe due to the limited number of functional inhibition groups  in the inhibitorstructural backbone.Three new aminomethylene-free phosphonate-basednontoxic fosfomycindrugs have been developed as scale inhibitors in the petroleum industry.The new aminomethylene-free phosphonates  gave superiorcompatibility activity at all inhibitor connections  and high calcium ion doses up to 10,000 ppm. We believe thatthe ether linkage in their structure backbones improved the calciumtolerance properties. Moreover, SI-3  displayed outstanding calcium tolerance up to 1000 ppmcalcium ions. However, SI-3 afforded haziness at 10,000and 50,000 ppm inhibitors and 10,000 ppm Ca2+ ions. Weplan to study the thermal stability and adsorption/desorption activitiesof these nontoxic phosphonate scale inhibitors for squeeze treatmentapplications.Commerical inhibitor


Traumatic nerve injury activates cell stress pathways, resulting in neuronal death and loss of vital neural functions. To date, there are no available neuroprotectants for the treatment of traumatic neural injuries. Here, we studied three important flavanones of citrus components, in vitro and in vivo, to reveal their roles in inhibiting the JNK -JUN pathway and their neuroprotective effects in the optic nerve crush injury model, a kind of traumatic nerve injury in the central nervous system. Results showed that both neural injury in vivo and cell stress in vitro activated the JNK-JUN pathway and increased JUN phosphorylation. We also demonstrated that naringenin treatment completely inhibited stress-induced JUN phosphorylation in cultured cells, whereas nobiletin and hesperidin only partially inhibited JUN phosphorylation. Neuroprotection studies in optic nerve crush injury mouse models revealed that naringenin treatment increased the survival of retinal ganglion cells after traumatic optic nerve injury, while the other two components had no neuroprotective effect. The neuroprotection effect of naringenin was due to the inhibition of JUN phosphorylation in crush-injured retinal ganglion cells. Therefore, the citrus component naringenin provides neuroprotection through the inhibition of the JNK-JUN pathway by inhibiting JUN phosphorylation, indicating the potential application of citrus chemical components in the clinical therapy of traumatic optic nerve injuries. Citrus aurantium L., named Zhishi, is used in traditional Chinese medicine [Traumatic neural injuries always lead to immediate neural degeneration and subsequent neuronal death. To date, there are no available neuroprotectants for the treatment of traumatic neural injuries. The immature fruit of medicine  for its medicine ,3. Narin15H12O5 and a molecular weight of 272.25 g/mol) is a plant bioflavonoid that is naturally found in citrus fruits (up to 10% of its dry weight) [28H34O15 and a molecular weight of 610.56 g/mol [21H22O8 and a molecular weight of 402.39 g/mol) is a poly-methoxylated flavone that originates from citrus peel, with a content of about 0.08% [Naringenin  . Hesperiut 0.08% ,12. Receut 0.08% , and an ut 0.08% , resultiut 0.08% ,15.Jun knockout significantly increased RGC survival [Chop (the key pro-apoptotic transcription factor of the ER stress pathway) knockout also increased RGC survival [Jun knockout showed better RGC survival (93%) than Chop knockout (51%) [JNK, also known as stress-activated protein kinase (SAPK), is preferentially activated by a variety of environmental stresses. JNK activity is higher in the brain than in any other tissues ,17. Thersurvival ,21. Our survival ,23. Howeut (51%) . TherefoAll these neuroprotective effects in traumatic optic nerve injury models are based on transgenic knockout mice; however, this transgenic method is not practical in the clinic. Therefore, it is urgent and necessary to look for safe chemical compounds to provide neuroprotection after traumatic optic nerve injury in the clinic. Natural chemical compounds from citrus food are the best candidates for fulfilling the requirements of both safe and potential neuron neuroprotection. Citrus components are reported to have neuroprotective effects, however, whether these components can regulate the JNK-JUN pathway and increase RGC survival after traumatic nerve injury remains elusive. In this study, we investigated the roles of three important components of Citrus flavanones—naringenin, hesperidin, and nobiletin, in the neuroprotection of RGCs in traumatic ONC injury mouse models, and revealed the underlying molecular mechanisms of their neuroprotective effects in regulating the JNK-JUN pathway.To identify whether the JNK-JUN pathway is activated after traumatic ONC injury, we generated traumatic ONC injury mouse models and verified the phosphorylation level of JUN. Three days after ONC, we co-stained the whole retina with an antibody against phosphorylated JUN and a neuron marker-βIII tubulin (Tuj1). The results showed that the phosphorylation of JUN in RGCs was significantly increased after ONC injury compared with no JUN phosphorylation in wild type (WT) retinas A. We the50) of naringenin and nobiletin in HEK 293T cells was 409.6 μM and 142.4 μM (50 of hesperidin (data not shown). Therefore, all three citrus components were able to inhibit stress-induced JUN phosphorylation, with naringenin exhibiting the best inhibitory effect.Since optic nerve injury activated the pro-apoptotic JNK-JUN pathway and resulted in neuronal death, to assess whether citrus components could inhibit the JNK-JUN pathway in vitro, we induced the pro-apoptotic JNK-JUN pathway in HEK293T cells and performed a series of concentration tests of the citrus components. HEK293T cells were incubated with 500 mM sorbitol for 30 min to activate the JNK-JUN pathway and induce the phosphorylation of JUN . Western142.4 μM G,H, resp142.4 μM A,C. Due Since all three citrus components were able to inhibit JUN phosphorylation in vitro, we examined whether they had neuroprotective effects in traumatic ONC injury mouse models. We labeled the retinal RGCs with an antibody against the RGC-specific marker RBPMS. Results showed that at 14 dpc, the control injured retina showed a survival of 29% RGCs compared to naive retinas. However, after injection of 2 μL of 10 μM naringenin into the vitreous chamber of the eyeball after ONC injury, the RGC survival rate was significantly increased to 40% A,B, whicSince naringenin was able to promote RGC survival after ONC, we then examined whether the neuroprotective effect of naringenin was fulfilled through inhibiting the JNK-JUN pathway in vivo. We injected 2 of 10 μM naringenin into the vitreous chamber of the mouse eyeball after ONC, and stained the whole retina with an antibody against phosphorylated JUN. The results showed that the p-JUN positive RGC number was significantly decreased in ONC retina with naringenin treatment compared with ONC retina with control PBS treatment A, and thDdit3  and Jun [Ddit3 deficient mice showed 51% RGC survival, whereas Jun deficient mice showed 93% RGC survival, compared with control 29% RGC survival in ONC alone mice. Jun deficiency showed an overwhelming neuroprotective effect compared to that of Ddit3 deficiency [In this study, we first showed that all three citrus components inhibited stress-induced JUN phosphorylation, and among the three components, naringenin had the best JUN phosphorylation inhibition effect. All three compounds inhibited JUN phosphorylation in HEK 293T cells, however, only naringenin showed neuroprotective effects in vivo, increasing the RGC survival in traumatic ONC injury mouse models. Further JUN phosphorylation study in ONC retina with naringenin treatment confirmed the inhibition of JNK-JUN pathway by naringenin in RGCs. Previous studies have also reported that all three citrus components play protection roles in cultured cell lines . These s and Jun . Other r and Jun ,28, consficiency .Jun knockout are still unknown. Therefore, in this study, we evaluated the neuroprotective effect of citrus components via inhibition of JUN phosphorylation. Due to the superior neuroprotective effect of Jun deficiency, we focused on studying the effects of the three citrus components on the inhibition of JUN phosphorylation. Although the in vitro study demonstrated that naringenin, nobiletin, and hesperidin were all able to inhibit JUN phosphorylation, only naringenin showed neuroprotection in in vivo experiments. Further study revealed that only naringenin inhibited JUN phosphorylation in ONC RGCs, while the other two compounds had no inhibition effects in vivo (data not shown). Considering the complete inhibition of JUN phosphorylation by naringenin and partial inhibition by the other two compounds in HEK 293T cells, naringenin still had inhibition effects on JUN phosphorylation in vivo. The weakened effects of p-JUN inhibition by these compounds may be due to the complicated environment in vivo where the JNK-JUN pathway may be affected by many other factors. Previous studies have shown a substantial increase in RGC survival after crush injury; however, all these neuroprotection studies were based on transgenic mice with gene knockout or overexpression [Whether Citrus compounds can inhibit the JNK-JUN pathway and whether the inhibition of JUN phosphorylation still have the same neuroprotective effect as pression ,24,29,30In conclusion, our study demonstrated that the three flavanone components of citrus extract inhibited the stress-induced phosphorylation of JUN, with complete inhibition by naringenin and partial inhibition by nobiletin and hesperidin. Experiments using traumatic ONC injury mouse models demonstrated that naringenin showed a significant neuroprotective effect by increasing RGC survival after neural injury, and the neuroprotection effect of naringenin in vivo was fulfilled by inhibiting the JNK-JUN pathway in injured RGCs.We performed the experiments in 6-weeks-old C57BL/6J mice. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Beijing Institute of Technology and Peking University.Naringenin , nobiletin , hesperidin , mouse neuronal class β-III tubulin , rabbit anti-RBPMS , rabbit anti-p-JUN , mouse anti-GAPDH , Cy2/Cy3-conjugated antibodies , Fluoromount-G , phosphate buffered saline (PBS) , Tris HCl , NaCl , sodium dodecyl sulfate (SDS) , NP-40 , sodium deoxycholate , sodium pyrophosphate , sodium fluoride , sodium orthovanadate , protease inhibitor cocktail , paraformaldehyde , goat serum , Triton X-100 .Mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine/g + 0.08 mg ketamine/g). Optic nerves on both sides were sequentially exposed intraorbitally and crushed with a jeweler’s forceps  for 2 s, approximately 1 mm behind the eyeball. Care was taken to avoid damaging the underlying ophthalmic arteries. Erythromycin eye ointment was applied to protect the cornea after surgery.Mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine/g + 0.08 mg ketamine/g). For intravitreal injection of chemical compounds or PBS control, a micropipette was inserted into the eyeballs of 6-weeks-old mice just behind the ora serrata and advanced into the vitreous chamber to avoid damage to the lens. After ONC injury, approximately 2 μL of the vitreous was removed before injection of 2 μL of chemical compounds or PBS control into the vitreous chamber. The chemical compounds for injection were as follows: 10 μM naringenin, 10 μM nobiletin, and 5 μM hesperidin. The drug injections were performed right after crush injury.2 incubator. Twenty-four hours after plating, the cells were first treated with 500 mM of sorbitol for 30 min and then with sequential concentrations of each compound for 1 h. Finally, the cells were suspended, washed with ice-cold PBS, and centrifuged at 3000 rpm for 10 min.HEK293T cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  at 37 °C in a 5% CO2 incubator. Twenty-four hours after plating, the cells were treated with a series of concentrations of each compound for 24 h and then treated with MTS-based CellTiter 96® AQueous assay kits  and finally measured by the absorbance at 490 nm in 96-well plate reader which is directly proportional to the number of living cells in culture. Each compound concentration was assessed with 6 replicates.HEK293T cells were cultured in DMEM supplemented with 10% FBS in 96-well plates at 37 °C in a 5% COg for 10 min, and supernatants were subjected to 10% SDS–polyacrylamide gel electrophoresis. After transfer onto a polyvinylidene difluoride (PVDF) membrane , the membranes were blocked with 5% milk, probed with primary and secondary antibodies, and exposed using an Amersham Imager 600 chemiluminescence imager . The band intensity was analyzed using ImageJ software . The primary antibodies used were rabbit anti-phospho-JUN and mouse anti-GAPDH.The cell pellets were suspended and lysed in radio-immunoprecipitation assay buffer  on ice for 30 min. The lysates were centrifuged at 12,000× Retinas were dissected out from 4% paraformaldehyde-fixed eyes and washed extensively in PBS before blocking in staining buffer  for 30 min. Antibodies were diluted in the same staining buffer. Floating retinas were incubated with primary antibodies overnight at 4 °C and washed three times for 30 min each with PBS. Secondary antibodies were then applied and incubated for 1 h at room temperature. Retinas were again washed three times for 30 min each with PBS before a cover slip was attached with Fluoromount-G.For RGC counting, three fields were randomly collected from peripheral regions of each retina to estimate RGC survival. The investigator who counted the cells was blinded to the preparing and imaging of the retina samples. The percentage of RGC survival was calculated as the ratio of surviving RGC numbers of injured eyes compared to that of uninjured naive eyes.t-test was used for two-group comparisons, and one-way ANOVA with Bonferroni’s multiple comparisons test was used for multiple comparisons. p ≤ 0.05 was considered statistically significant. Animal experiments were repeated two times and HEK293T cell experiments were performed with 4–6 replicates.Data are presented as mean ± SEM, and Student’s