Xenopus laevis oocytes. We found that coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by approximately threefold, likely because of an increase in average channel open probability. Furthermore, TMPRSS2-dependent ENaC stimulation was not observed using a catalytically inactive TMPRSS2 mutant and was associated with fully cleaved γ-ENaC in the intracellular and cell surface protein fractions. This stimulatory effect of TMPRSS2 on ENaC was partially preserved when inhibiting its proteolytic activity at the cell surface using aprotinin but was abolished when the γ-inhibitory tract remained attached to its binding site following introduction of two cysteine residues (S155C–Q426C) to form a disulfide bridge. In addition, computer simulations and site-directed mutagenesis experiments indicated that TMPRSS2 can cleave γ-ENaC at sites both proximal and distal to the γ-inhibitory tract. This suggests a dual role of TMPRSS2 in the proteolytic release of the γ-inhibitory tract. Finally, we demonstrated that TMPRSS2 knockdown in cultured human airway epithelial cells (H441) reduced baseline proteolytic activation of endogenously expressed ENaC. Thus, we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo.The epithelial sodium channel (ENaC) is a heterotrimer consisting of α-, β-, and γ-subunits. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α-ENaC and γ-ENaC; however, the proteases involved in the removal of the γ-inhibitory tract remain unclear. In several epithelial tissues, ENaC is coexpressed with the transmembrane serine protease 2 (TMPRSS2). Here, we explored the effect of human TMPRSS2 on human αβγ-ENaC heterologously expressed in  The epithelial sodium channel (ENaC) belongs to the ENaC/degenerin family of ion channels . As ratein vivo remain elusive  was the first membrane-anchored serine protease demonstrated to activate ENaC in coexpression experiments and was therefore named channel-activating protease 1 (CAP1)  , 51, 52.1 (CAP1) , 54. How1 (CAP1) . Interes1 (CAP1) , 56. In 1 (CAP1) , 45, 46.Xenopus laevis oocytes dramatically reduced ENaC-mediated currents and ENaC protein expression  on ENaC function. An initial study found that coexpression of human TMPRSS2 with rat ENaC in pression . This fipression . In contpression . Thus, tpression , 60, 61.pression . Thus, Tpression , 64, 65,X. laevis oocyte expression system to provide evidence for proteolytic ENaC activation by TMPRSS2. Moreover, we identified putative TMPRSS2 cleavage sites in the channel’s γ-subunit. To investigate a possible regulatory effect of TMPRSS2 on ENaC function in polarized epithelial cells, we also studied the effect of TMPRSS2 knockdown on ENaC-mediated transepithelial sodium transport in H441 human distal airway epithelial cells.In this study, we used the X. laevis oocytes with or without human TMPRSS2. ENaC function was assessed by measuring amiloride-sensitive inward currents (ΔIami) using the two-electrode voltage clamp technique. Figure 1A shows representative continuous whole-cell current traces recorded at a holding potential of −60 mV in individual oocytes expressing ENaC alone (left panel), coexpressing ENaC and TMPRSS2 (middle panel), or coexpressing ENaC and a catalytically inactive mutant TMPRSS2 . Results from similar experiments are summarized in Figure 1B. Recordings were started in the presence of 2 μM amiloride, which in this concentration is known to reversibly inhibit human ENaC heterologously expressed in oocytes by >90%  application of chymotrypsin (2 μg/ml) further increased the inward current by ∼2.5-fold, is in good agreement with this concept (B and C). Reapplication of amiloride at the end of the measurement returned the current to its initial level. This confirms that the inward current stimulated by chymotrypsin is mediated by ENaC. Importantly, in oocytes coexpressing ENaC and TMPRSS2 , baseline ΔIami was ∼2.5-fold larger than that in oocytes expressing ENaC alone . Moreover, in oocytes coexpressing ENaC and TMPRSS2, application of chymotrypsin had no additional stimulatory effect (A–C). This indicates that in these oocytes, the channels present at the cell surface were fully cleaved. Thus, coexpression of ENaC and TMPRSS2 strongly activates ΔIami and mimics proteolytic ENaC activation by chymotrypsin in oocytes expressing ENaC alone.It is well established that in the oocyte expression system a sizeable fraction of ENaC is not fully cleaved when it reaches the cell surface. These latter channels can be activated by extracellular application of prototypical serine proteases like trypsin or chymotrypsin , 14. OurFig. 1S441A, in which the critical serine residue belonging to the catalytic triad was substituted by an alanine. Importantly, TMPRSS2S441A failed to stimulate ENaC . Moreover, the relative stimulatory effect of chymotrypsin in oocytes coexpressing ENaC and TMPRSS2S441A was not significantly different from that observed in control oocytes expressing ENaC alone . Thus, the stimulatory effect of TMPRSS2 coexpression on ENaC-mediated currents depends on the proteolytic activity of TMPRSS2.It has been shown that mutating the catalytic triad of TMPRSS4 (CAP2) or matriptase (CAP3) abolished the stimulatory effect of these membrane-anchored proteases on ENaC , 45. Thet-butyloxycarbonyl–Gln-Ala-Arg–7-amino-4-methylcoumarin; R&D Systems)  , which wSystems) , can alservation  that endS441A, we analyzed intracellular and cell surface expression of wildtype TMPRSS2 and TMPRSS2S441A using western blot analysis and a biotinylation approach (E). In the intracellular protein fraction , wildtype TMPRSS2 was detected predominantly as ∼27 kDa band most likely representing the catalytic chain of mature TMPRSS2  form of TMPRSS2S441A could hardly be detected. This latter finding is in good agreement with previous reports suggesting autocatalytic processing of TMPRSS2, which can be blocked by the S441A mutation  ∼27 kDa form  consistent with our protease activity measurements with Boc–QAR–AMC. Interestingly, in oocytes coexpressing ENaC and TMPRSS2S441A, the mutant protein was detectable at the cell surface in both mature (∼27 kDa) and zymogen (∼65 kDa) forms. Moreover, cell surface expression of the catalytically inactive TMPRSS2S441A was overall stronger than that of wildtype TMPRSS2. We have no explanation for the increased cell surface expression of the mutant protein. Nevertheless, our findings demonstrate that the lack of ENaC stimulation by coexpression of TMPRSS2S441A is not because of impaired protein expression or plasma membrane trafficking of the mutant protease. Thus, the failure of TMPRSS2S441A to stimulate ENaC currents is most likely because of the absence of its proteolytic activity.To exclude that the lack of proteolytic activity and ENaC activation was due to impaired protein expression or trafficking of TMPRSS2approach E. In the TMPRSS2 . In compmutation . As expeE). Therefore, we performed control experiments coexpressing ENaC with untagged TMPRSS2. These experiments confirmed the stimulatory effect of coexpressed TMPRSS2 on ENaC and also the appearance of trypsin-like proteolytic activity at the cell surface of TMPRSS2-coexpressing oocytes . To investigate this, we used an established approach to assess average ENaC PO in the oocyte expression system (S520C). This cysteine residue can be covalently modified by the sulfhydryl reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). This has been shown to increase the PO of mutant αβS520Cγ-ENaC close to 1  and summarized in B and C, application of MTSET for 5 min strongly stimulated ΔIami in oocytes expressing αβS520Cγ-ENaC by approximately threefold, which is consistent with our recently published data obtained using a similar experimental protocol . This indicates, that in oocytes coexpressing TMPRSS2, average ENaC PO is nearly maximal and cannot be further increased by the application of MTSET. Moreover, the increased PO of ENaC because of TMPRSS2 coexpression can fully explain the stimulatory effect of TMPRSS2 on ΔIami. In contrast, in oocytes coexpressing αβS520Cγ-ENaC and TMPRSS2S441A, the stimulatory effect of MTSET was similar to that observed in control oocytes expressing αβS520Cγ-ENaC alone . Thus, catalytically inactive TMPRSS2S441A does not increase ENaC PO.Proteolytic channel activation by TMPRSS2 is likely to cause an increase of ENaC open probability . Indeed, in the presence of aprotinin, ΔIami averaged 2.0 ± 1.3 μA (n = 40) in TMPRSS2- and ENaC-coexpressing oocytes compared with 0.8 ± 0.5 μA  in oocytes expressing ENaC alone. In control experiments performed in parallel in the absence of aprotinin, the stimulatory effect of TMPRSS2 was even more pronounced with ΔIami averaging 3.7 ± 2.3 μA (n = 40) in oocytes coexpressing TMPRSS2 and 0.8 ± 0.4 μA  in oocytes expressing ENaC alone (A and B). This suggests that in the presence of aprotinin, proteolytic ENaC activation by coexpressed TMPRSS2 is incomplete and that cell surface protease activity contributes, at least in part, to channel activation by TMPRSS2 in control cells maintained without aprotinin.Interestingly, a substantial stimulatory effect of TMPRSS2 coexpression on ΔIprotinin , A and BaC alone , A and Bami (A and B). This is consistent with the finding that no measurable aprotinin-sensitive trypsin-like proteolytic activity was observed at the cell surface of these oocytes (C). In contrast, the substantial trypsin-like proteolytic activity at the cell surface of oocytes coexpressing TMPRSS2 and ENaC was completely abolished by aprotinin (C). Thus, the partially preserved stimulatory effect of TMPRSS2 on ENaC in oocytes treated with aprotinin is unlikely to be due to residual proteolytic activity at the cell surface. Instead, it is probably because of intracellular proteolytic activity of TMPRSS2, which activates ENaC and is not affected by extracellular aprotinin.In oocytes expressing ENaC alone, incubation with aprotinin did not significantly alter baseline ΔIami , A and B oocytes C. In conprotinin C. Thus, A) in the intracellular protein fraction and at the cell surface of oocytes with and without coexpression of TMPRSS2, we used western blot analysis in combination with a biotinylation approach. In the nonbiotinylated intracellular protein fractions, a ∼87 kDa γ-ENaC band corresponding to uncleaved γ-ENaC could be detected in oocytes with and without TMPRSS2 coexpression . As expected from previous studies, an additional prominent ∼76 kDa band was present in the intracellular protein fraction from oocytes expressing ENaC alone. This cleavage product is thought to result from partial γ-ENaC cleavage by endogenous furin or furin-like convertases at the so-called furin cleavage site (R138) and can be detected using an antibody recognizing a C-terminal γ-ENaC epitope  . This latter cleavage product has been shown to correspond to fully cleaved γ-ENaC associated with proteolytic channel activation  . This is in good agreement with the robust stimulatory effect of TMPRSS2 coexpression on ΔIami observed in parallel electrophysiological experiments using oocytes from the same batches . TMPRSS2 coexpression resulted in the appearance of an additional small intracellular γ-ENaC band of ∼18 kDa , which was not detected at the cell surface , and may correspond to a degradation product. A possible functional role of this additional γ-ENaC cleavage product remains to be elucidated.To detect γ-ENaC and its typical cleavage products A in the Fig. 4A) , 23, 76.Fig. 4A) , 28. At Taken together, the functional experiments with aprotinin and the western blot results support the hypothesis that TMPRSS2 cleaves γ-ENaC not only at the cell surface but at least in part also intracellularly before the channel reaches the plasma membrane.A). The molecular structure of the binding site occupied by γ-11 suggests that the serine residue (S155) of γ-11 and the glutamine residue (Q426) of the binding site lie in close proximity to each other . We reasoned that substitution of these amino acid residues by cysteines  would lead to the formation of an additional disulfide bond between the γ-inhibitory tract and its binding site. Moreover, we hypothesized that stabilizing the interaction of the γ-inhibitory tract to its binding site by this additional disulfide bond would prevent channel activation despite proteolytic cleavage of γ-ENaC by TMPRSS2.The final cleavage of γ-ENaC in the region distal to the furin site causes the release of a γ-inhibitory tract consisting of ∼40 amino acid residues. The release of this γ-inhibitory tract is critical for channel activation . ExtraceFig. 5S155C;Q426C-ENaC was similar to that of wildtype γ-ENaC with or without TMPRSS2 coexpression (B). Moreover, cleavage of the mutant γ-subunit by TMPRSS2 coexpression was preserved intracellularly and at the cell surface (B). However, TMPRSS2 coexpression did not increase baseline ΔIami in oocytes with αβγS155C;Q426C-ENaC (C and D) unlike in matched oocytes with wildtype αβγ-ENaC, where TMPRSS2 coexpression had its usual stimulatory effect (E and F). This suggests that despite γ-ENaC cleavage, the γ-inhibitory tract remains attached to its binding site because of the introduced disulfide bond, thereby preventing activation of the mutant channel. Disulfide bonds can be reduced by incubating oocytes with agents like DTT  increased ΔIami by approximately threefold (C and D). This stimulatory effect was similar to that of TMPRSS2 on baseline ΔIami in wildtype ENaC (E and F). DTT had no stimulatory effect on ΔIami in oocytes expressing αβγS155C;Q426C-ENaC alone or in oocytes expressing wildtype αβγ-ENaC with or without TMPRSS2 coexpression (C–F). This indicates that the observed DTT-mediated stimulation of ΔIami in oocytes coexpressing αβγS155C;Q426C-ENaC and TMPRSS2 was due to the specific reduction of the disulfide bond between S155C and Q426C in γ-ENaC and the release of the γ-inhibitory tract from γ-ENaC, which had been cleaved by TMPRSS2 as evidenced by our western blot data (B).Western blot experiments performed under reducing conditions see the  section pression B. Moreov surface B. Howeve26C-ENaC , C and Dy effect , E and Flike DTT . Indeed,hreefold , C and Dype ENaC , E and Fpression , C–F. Thlot data B.To conclude, these data clearly demonstrate that the stimulatory effect of TMPRSS2 on ENaC is due to proteolytic cleavage of γ-ENaC and subsequent release of the γ-inhibitory tract.A), which may potentially play a role as TMPRSS2 cleavage sites. To assess whether TMPRSS2 has a preference for one of these putative cleavage sites, we used a molecular docking approach with three different 6-mer peptides corresponding to three segments of the γ-ENaC sequence: 169-GKARDF-174, 177-GRKRKV-182, and 187-IHKASN-192 (A). We simulated the binding of these 6-mer peptides to a homology model of the catalytic domain of human TMPRSS2 (B and C). Importantly, the homology model of TMPRSS2 revealed a well-defined S1 pocket in the vicinity of the catalytic triad, which is highly negatively charged because of the presence of an aspartate residue (D435) at its bottom (B and C). This corresponds well to the expected selectivity of TMPRSS2 for substrates with a positively charged arginine or lysine residue in the P1 position (A). First, the side chain of an arginine residue or a lysine residue in the P1 position of a peptide should occupy the S1 pocket and have a distance to the side chain of the aspartate (D435) at the bottom of the S1 pocket of less than 4 Å, compatible with the formation of a salt bridge between these two residues. Second, the distance between the carbonyl carbon atom of the scissile peptide bond and the hydroxyl group of the catalytic serine residue (S441) should be less than 4 Å, which is required for initiation of proteolysis . In contrast to discarded binding modes, the selected binding modes were structurally similar and formed a prominent cluster (A). This was confirmed by calculation of root-mean-square deviation (RMSD) values between corresponding binding modes, which were on average significantly lower for binding modes that fulfilled the selection criteria compared with those calculated for binding modes that did not meet the selection criteria (E). This indicates that the strong P1–S1 interaction stabilizes the backbone conformation of the whole GKARDF-peptide within the catalytic domain of TMPRSS2. Interestingly, we observed that only the side chain of the arginine but not that of the lysine occupied the S1 pocket of TMPRSS2, which is consistent with the preference of TMPRSS2 for arginine over lysine observed experimentally (E). Overall, more than 30% of the generated binding modes fulfilled the selection criteria, suggesting that TMPRSS2 may effectively cleave γ-ENaC in the 177-GRKRKV-182 region, most likely after the arginine residue R178, but possibly also after K179 or R180. Finally, we simulated the binding of the IHKASN-peptide to the catalytic domain of TMPRSS2. As demonstrated in Figure 6F, the lysine side chain occupied the S1 pocket of TMPRSS2, but this was observed only in less than 10% of all generated binding modes. This finding suggests that TMPRSS2 may cleave γ-ENaC also after the lysine residue (K189), but this cleavage site seems to be less preferable than R172 or R178.Analysis of the γ-ENaC sequence revealed the presence of several positively charged residues, arginines and lysines, distal to the γ-inhibitory tract A, which KASN-192 A. We sim TMPRSS2 , B and Cs bottom , B and Cposition . To evalposition A. First,teolysis . One repcriteria D and S2A cluster A. This wcriteria E. This imentally . To summmentally E. OveralB, the substitution of the lysine residue by an alanine did not substantially affect the binding of the simulated peptide to the catalytic domain of TMPRSS2. Similar to the GKARDF-peptide, the mutant GAARDF-peptide also formed the close arginine–aspartate interaction within the S1 pocket in more than 40% of the generated binding modes (A and B). In contrast, when the critical arginine residue was replaced by an alanine, as in the GKAADF-peptide, the lysine–aspartate interaction within the S1 pocket was observed only in less than 10% of all generated binding modes . Finally, the binding modes of the GAAADF-peptide lacking both positively charged residues demonstrated highly variable conformations  and high RMSD values  and generally resembled the discarded binding modes of the GKARDF-peptide , which would not favor proteolysis. In summary, these computer simulations predict that in the absence of the critical arginine (R172), TMPRSS2 may cleave γ-ENaC after an otherwise less preferable lysine residue (K170). Thus, TMPRSS2 may cleave γ-ENaC at multiple sites distal to the γ-inhibitory tract. Therefore, it may be necessary to replace several arginine and lysine residues in this region with alanines to fully abolish the stimulatory effect of TMPRSS2 on ENaC.In addition, we simulated how a substitution of a positively charged arginine or lysine residue within a putative cleavage site by an alanine may affect the interaction of γ-ENaC with TMPRSS2. This analysis was performed using the GKARDF-peptide. As shown in Fig. S2RKRK178AAAA). Previously, it has been shown that mutating this site fully abolished the stimulatory effect of PRSS8 (prostasin) on ENaC . Thus, an intact prostasin site is not required for TMPRSS2-dependent ENaC activation. To test a possible involvement of alternative cleavage sites in this region, we generated another mutant  in which the prostasin site remained intact but three positively charged residues proximal to the prostasin site  and one lysine residue distal to the prostasin site (K189) were replaced with alanines. The stimulatory effect of TMPRSS2 on this mutant ENaC was only slightly reduced compared with that on wildtype ENaC . This indicates that TMPRSS2 can cleave γ-ENaC at the prostasin site when other positively charged residues distal to the γ-inhibitory tract are not available because of their replacement by alanines. Interestingly, when all arginine and lysine residues in the prostasin site and its vicinity were replaced with alanine residues , the stimulatory effect of TMPRSS2 on ENaC was largely abolished, albeit not completely .To confirm our computer simulation results experimentally, we generated several mutant γ-ENaC subunits in which positively charged residues distal to the γ-inhibitory tract were substituted by alanines . First,  on ENaC . InteresC and similar western blots shown in RKRK178AAAA or γK168A; K170A; R172A; K189A but not in oocytes expressing γRKRK178AAAA; K168A; K170A; R172A; K189A. This indicates that TMPRSS2 can efficiently cleave γ-ENaC distal to the γ-inhibitory tract, unless all positively charged residues are mutated in this region. The failure of TMPRSS2 to produce a detectable increase of the ∼67 kDa cleavage product in oocytes expressing γRKRK178AAAA; K168A; K170A; R172A; K189A-ENaC is in good agreement with the largely reduced stimulatory effect of TMPRSS2 on ΔIami in this group of oocytes.In parallel experiments, we analyzed the cleavage fragments of γ-ENaC in whole-cell lysates of oocytes expressing wildtype γ-ENaC or different γ-ENaC mutants. As illustrated by representative western blots shown in Figure 8RKRK178AAAA; K168A; K170A; R172A; K189A-ENaC suggests that additional TMPRSS2 cleavage sites exist in the γ-subunit, which may contribute to proteolytic channel activation .Collectively, our functional data confirm the computer modeling results and indicate that TMPRSS2 can cleave γ-ENaC at multiple sites distal to the γ-inhibitory tract without a clear preference for a particular position. Moreover, the preserved minor stimulatory effect of TMPRSS2 on αβγR138A) , thereby releasing the γ-inhibitory tract resulting in complete proteolytic ENaC activation. Indeed, in αβγR138A-ENaC–expressing oocytes, the stimulatory effect of TMPRSS2 on ΔIami was similar to that in matched oocytes expressing wildtype αβγ-ENaC (A and B). This indicates that TMPRSS2 can cleave mutant γR138A-ENaC both proximally and distally to the γ-inhibitory tract. This conclusion was further supported by western blot analysis in which we used an antibody against a C-terminal γ-ENaC epitope  and in addition another antibody against a V5 tag attached to the N terminus of γ-ENaC . As expected, cleavage of γ-ENaC by endogenous proteases was effectively prevented by the R138A mutation. Indeed, in oocytes expressing αβγR138A-ENaC, we detected neither a ∼76 kDa C-terminal fragment  nor a corresponding ∼15 kDa N-terminal fragment . In contrast, these fragments were present in matched αβγ-ENaC–expressing oocytes. Importantly, coexpression of TMPRSS2 and αβγR138A-ENaC resulted in the reappearance of these fragments, clearly demonstrating cleavage of γ-ENaC in the region proximal to the γ-inhibitory tract by TMPRSS2. As expected, the C-terminal ∼67 kDa fragment reflecting distal γ-ENaC cleavage was detectable in both wildtype and αβγR138A-ENaC–expressing oocytes when TMPRSS2 was coexpressed. In these oocytes, we also detected a ∼21 kDa N-terminal fragment , which corresponds to γ-ENaC cleaved only distally but not proximally to the γ-inhibitory tract . This suggests that distal γ-ENaC cleavage by TMPRSS2 may occur independently of the proximal cleavage event. In addition, we detected a ∼12 kDa N-terminal γ-ENaC fragment in both groups of oocytes coexpressing TMPRSS2 . This fragment probably represents a degradation product of γ-ENaC, but its identity and possible function remain to be determined.The lack of a strong substrate specificity of TMPRSS2 (except that a positively charged arginine or lysine residue must be present at the P1 position) suggests that TMPRSS2 may cleave γ-ENaC also proximal to the γ-inhibitory tract in a polybasic region  , 23, 76.αβγ-ENaC , A and BIn summary, our data demonstrate that TMPRSS2 can cleave γ-ENaC at the polybasic site proximal to the γ-inhibitory tract. Thus, TMPRSS2 may play a dual role in proteolytic ENaC activation because of its ability to cleave γ-ENaC both proximally and distally to the γ-inhibitory tract.RKRR138AAAA . The failure of the γRKRR138AAAA mutant to prevent the stimulatory effect of TMPRSS2 suggests that an additional cleavage site exists in the region proximal to the γ-inhibitory tract. Interestingly, the first amino acid residue of the γ-11 sequence (R153) has previously been described as a plasmin cleavage site . This finding suggests that R153 can serve as an additional TMPRSS2 cleavage site in γ-ENaC. This additional cleavage site may also explain our previous observation . Indeed, introducing an additional arginine-to-alanine substitution (R153A) to this mutant γ-ENaC . This finding further supports the conclusion that γ-ENaC cleavage at R153 may be accomplished by TMPRSS2 and may partially activate ENaC.In the previous section, we demonstrated that R138 in the 135-RKRR-138 polybasic tract is not essential for TMPRSS2-dependent γ-ENaC cleavage proximal to the critical γ-11 inhibitory sequence. To investigate a possible involvement of the three remaining positively charged amino acid residues , we generated a mutant subunit γR138AAAA . Interesage site . We hypoervation , that a Collectively, our data obtained in the oocyte expression system demonstrate that TMPRSS2 is an ENaC-activating membrane-anchored protease, which can cleave γ-ENaC at the cell surface and intracellularly at multiple cleavage sites localized distally and proximally to the γ-inhibitory tract leading to complete proteolytic ENaC activation.A). The commercial TMPRSS2-specific antibody used in these experiments was validated using whole-cell lysates from oocytes expressing HA-labeled TMPRSS2  and tight junction formation . PRSS8  is critical for epithelial monolayer formation  measurements in modified Ussing chambers, which allowed stable ISC recordings for more than 30 min as described previously (ISC recordings and summary data obtained from TMPRSS2-knockdown cells (model 1) and matched wildtype H441 cells are shown in ISC values in TMPRSS2-knockdown H441 cells were similar to those in wildtype cells averaging 3.9 ± 4.4 μA/cm2 (n = 35) and 4.6 ± 4.0 μA/cm2 (n = 32), respectively (A). To test whether TMPRSS2 knockdown reduces proteolytic ENaC activation in H441 cells, we applied a prototypical protease trypsin to the apical bath solution. Trypsin was chosen in these experiments because its substrate specificity is similar to that of TMPRSS2  with an average effect of 0.3 ± 0.9 μA/cm2 . This confirms previous reports that under baseline conditions, ENaC localized in the apical membrane of H441 is largely cleaved by endogenous proteases and, therefore, cannot be further activated by apical application of exogenous proteases  with an average ISC increase of 3.0 ± 2.0 μA/cm2 . This indicates that in TMPRSS2-knockdown cells, proteolytic ENaC activation is incomplete and that channels present at the apical cell surface can be further stimulated by application of trypsin. In the presence of trypsin, subsequent application of amiloride (10 μM) caused a robust ISC decrease, which was significantly larger in TMPRSS2-knockdown cells than in wildtype cells averaging −5.8 ± 2.8 μA/cm2 (n = 20) and −3.3 ± 1.9 μA/cm2 (n = 18), respectively (A and B and S6B). Importantly, when trypsin was applied in the presence of amiloride, it stimulated ISC neither in wildtype cells  nor in TMPRSS2-knockdown cells . This confirms that the prominent stimulatory effect of trypsin on ISC observed in TMPRSS2-knockdown cells under baseline conditions is due to increased ENaC-mediated transepithelial sodium transport. Interestingly, in TMPRSS2-knockdown H441 cells, the inhibitory effect of amiloride application on baseline ISC was similar to that in wildtype cells, averaging −3.1 ± 1.9 μA/cm2 (n = 15) and −2.6 ± 2.0 μA/cm2 (n = 14), respectively (D and S6C). This is consistent with the finding that baseline ISC values in TMPRSS2-knockdown H441 cells were similar to those of wildtype cells and confirms that a large portion of baseline ISC in these cells is due to ENaC-mediated electrogenic Na+ transport. Using the same experimental strategy, we also investigated TMPRSS2-knockdown H441 cells from model 2 (A and B). Importantly, like in wildtype cells, application of trypsin did not significantly alter ISC (C–F). This indicates that incomplete proteolytic ENaC activation observed in TMPRSS2-knockdown cells is not caused by an unspecific effect of exposing cells to the CRISPR/Cas9 procedure.To assess ENaC-mediated electrogenic transepithelial sodium transport in H441 cells, we performed equivalent short-circuit current  coexpression of TMPRSS2 and ENaC stimulated ENaC-mediated whole-cell currents by approximately threefold, mainly because of a large increase of ENaC average open probability; (2) a catalytically inactive TMPRSS2 mutant (S441A) failed to activate ENaC, whereas inhibition of the catalytic activity of TMPRSS2 at the cell surface by aprotinin only partially reduced the stimulatory effect of TMPRSS2 on ENaC; (3) in oocytes coexpressing ENaC and TMPRSS2, fully cleaved γ-ENaC was not only present at the cell surface, but a substantial fraction of intracellular γ-ENaC was also fully cleaved; (4) the stimulatory effect of TMPRSS2 on ENaC was completely abolished by introducing a disulfide bond, to prevent the release of the γ-inhibitory tract from its binding site, and was rescued by reducing this bond with DTT; (5) TMPRSS2 cleaved γ-ENaC at multiple sites not only in the region distal but also proximal to the γ-inhibitory tract; and (6) TMPRSS2 knockdown in H441 cells led to incomplete proteolytic activation of endogenously expressed ENaC. Taken together, these findings indicate that TMPRSS2 can proteolytically activate ENaC by cleaving its γ-subunit possibly at more than one cleavage site. Importantly, TMPRSS2 accomplishes γ-ENaC cleavage not only at the cell surface but also intracellularly, including the critical final cleavage step at the distal cleavage site. Thus, in oocytes coexpressing TMPRSS2 and ENaC, a substantial portion of channels reaching the plasma membrane are fully cleaved and active. Moreover, our findings in H441 airway epithelial cells provide proof of principle that TMPRSS2 can play a role in proteolytic ENaC activation not only in the oocyte expression system but also in polarized epithelial cells. Thus, TMPRSS2 is a likely candidate protease involved in proteolytic ENaC regulation in vivo.In this study, we investigated the functional interaction of human αβγ-ENaC with human TMPRSS2 in the S520Cγ-ENaC revealed that TMPRSS2 coexpression increased average PO of ENaC to an extent that can fully explain the observed stimulatory effect of TMPRSS2 on ENaC whole-cell currents. Interestingly, in the first study, in which the effect of TMPRSS2 on ENaC was investigated, ENaC-mediated currents and ENaC protein levels were found to be dramatically reduced in oocytes coexpressing rat ENaC and human TMPRSS2  has been reported to produce a similar C-terminal γ-ENaC fragment probably by cleaving at a highly conserved arginine residue (R515) in the region preceding the second transmembrane domain of γ-ENaC. Whether this C-terminal γ-ENaC fragment is a degradation product or has a functional role is unclear  based on the analysis of the recently published cryo-EM structures of ENaC  are preferential cleavage sites of TMPRSS2 in γ-ENaC. This prediction corresponds well with the reported optimal TMPRSS2 cleavage sequence identified using short peptide screening analysis . Accordiet al. , TMPRSS2et al. . Thus, bet al. , which set al. . HoweverR138A) indicated that TMPRSS2 may cleave γ-ENaC in the preceding 135-RKR-137 sequence. According to the reported TMPRSS2 substrate specificity , Middle East respiratory syndrome–associated coronavirus (MERS-CoV) , and thenditions . It remaISC in TMPRSS2-knockdown cells was similar to that in H441 control cells but could be further stimulated by application of trypsin. This stimulatory effect of trypsin was not observed in H441 control cells and indicates that in TMPRSS2-knockdown cells, a channel population exists in the apical membrane, which is only partially cleaved. This supports the conclusion that under normal conditions, TMPRSS2 contributes to proteolytic ENaC activation in H441 cells. Despite incomplete proteolytic ENaC activation, TMPRSS2-knockdown cells attained a baseline ISC similar to that of control cells possibly because of compensatory upregulation of ENaC-mediated transepithelial transport by other mechanisms. These were not addressed in the present study but may be of interest to be explored in future experiments. Importantly, the ability of the TMPRSS2-knockdown cells to generate a normal baseline ISC suggests that TMPRSS2 knockdown does not severely compromise overall transepithelial ion transport function and epithelial integrity in H441 cells.Nevertheless, our finding that proteolytic ENaC activation is significantly reduced in TMPRSS2-knockdown H441 cells makes TMPRSS2 a likely candidate to be involved in proteolytic ENaC activation in human distal airway epithelial cells under physiological conditions. Baseline ENaC-mediated ISC recordings in H441 cells were performed under conditions of apical surface layer (ASL) expansion because of the presence of apical bath solution in the Ussing chambers. It has been reported that ASL expansion stimulates proteolytic ENaC processing in human airway epithelial cells probably by diluting protease inhibitors  encoding human α-ENaC, β-ENaC, and γ-ENaC were kindly provided by H. Cuppens. Full-length cDNA encoding human TMPRSS2 was obtained from Source BioScience UK Limited (I.M.A.G.E. Clone ID: 6199625) . cDNAs wX. laevis in accordance with the principles of German legislation, with approval by the animal welfare officer for the University of Erlangen-Nürnberg, and under the governance of the state veterinary health inspectorate . Animals were anesthetized in 0.2% MS222 (Sigma–Aldrich), and ovarian lobes were obtained by a small abdominal incision. Oocytes were isolated from ovarian lobes using a type-2 collagenase from Clostridium histolyticum (Sigma–Aldrich). Oocytes were injected with the same amount  of cRNA per ENaC subunit  per oocyte. Unless stated otherwise, 2 ng of human TMPRSS2 cRNA were injected. After cRNA injection, oocytes were kept in a low sodium ND9 solution  supplemented with 100 units/ml sodium penicillin and 100 μg/ml streptomycin sulphate. Oocytes were studied 48 h after cRNA injection. Bath solution exchanges with a gravity-fed system were controlled by a magnetic valve system  in combination with a TIB14 interface (HEKA). An individual oocyte was placed in an experimental chamber with a narrow flow channel  with a U-shaped cross section of ∼8 mm2. The oocyte was positioned in the experimental chamber close to the site of solution inflow and was held in place by the impaling microelectrodes. To achieve rapid and reproducible solution exchanges at the oocyte, the perfusion rate was carefully adjusted for each experimental solution to ∼10 ml/min, resulting in a flow velocity of ∼20 mm/s. The flow channel drained into a reservoir (2 cm × 1 cm) from which the solution was continuously removed via a suction tube. The suction tube was adjusted to maintain the fluid level in the flow channel at ∼2 mm. Oocytes were clamped at a holding potential of –60 mV using an OC-725C amplifier (Warner Instruments) connected by an LIH-1600 (HEKA) to a personal computer. Pulse 8.78 software (HEKA) was used for data acquisition. Whole-cell current recordings started typically within 10 s after impalement of an oocyte with microelectrodes. ENaC-mediated whole-cell currents (ΔIami) were determined by washing out amiloride (2 μM) with amiloride-free bath solution and subtracting the whole-cell currents measured in the presence of amiloride from the corresponding whole-cell currents recorded in its absence. ND96 solution was used as standard bath solution .Isolation of oocytes and two-electrode voltage-clamp experiments were essentially performed as described previously , 102. DeTo separate cell surface proteins from intracellular proteins, a biotinylation approach was used essentially as described previously , 70, 103In some experiments, cleavage fragments of γ-ENaC were analyzed in whole-cell lysates prepared essentially as described previously . The antTMPRSS2-dependent proteolytic activity at the cell surface was quantified using the fluorogenic substrate Boc–QAR–AMC and the experimental protocol previously established in our laboratory for the detection of trypsin-like proteolytic activity at the surface of oocytes expressing mouse PRSS8 . Each in1Z8G) as a template (http://www.tcoffee.org)  was built using the default modeling in the automodel-class suite of MODELLER 9.12 package  , 105 andtemplate , 107. Thfee.org) , 109. TMfee.org) , 114. Thfee.org) . The lenfee.org) . Putativfee.org) . Peptidefee.org) , 118. Thfee.org) , 121.https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) and cloned into the pLenti-CRISPR-V2 vector as described previously . To generate CRISPR/Cas9-V2-based lentiviral particles targeting TMPRSS2 or luc, human embryonic kidney 293T cells were transfected with vesicular stomatitis virus glycoprotein expression plasmid (pVSV-G), the HIV gag/pol packaging plasmid pCMVΔR8.9, and lentiviral vector pLenti-CRISPR-V2 encoding sgRNAs targeting TMPRSS2 or luc at a mass ratio of 1:2:2 using calcium phosphate. Cell-culture supernatants were collected 48 hpi, passed through 0.45 μm pore size filters, and concentrated by centrifugation through size-exclusion filters  and stored at −80 °C. Three days postinfection, transduced H441 cells were selected using 2.5 μg/ml puromycin containing medium. Wildtype (passages 64–72), luc-knockdown cells (passages 66–69), and TMPRSS2-knockdown H441 cells (passages 66–74) were cultured in parallel as described previously . This medium was in addition supplemented with 0.5 μg/ml puromycin for TMPRSS2- and luc-knockdown H441 cells.The National Cancer Institute-H441 (H441) (American Type Culture Collection HTB-174) human lung epithelial cell line was obtained from American Type Culture Collection and used as wildtype control. Two TMPRSS2-knockdown H441 cell lines and a luc-knockdown H441 cell line (nontargeting control) were generated from wildtype cells by lentiviral transduction using pLenti-CRISPR-V2–based lentiviral particles targeting TMPRSS2 or firefly luciferase (luc). The latter is not expressed in human cells. Two different sgRNA sequences  targeting exon 6 or exon 9 of human TMPRSS2, respectively, were designed using GPP sgRNA Designer  was added to the apical compartment. After 5 days at the air–liquid interface, the monolayers were transferred into Ussing chambers to measure the equivalent short circuit current (ISC) essentially as described previously  in H441 growth medium. At day 4 after seeding, this medium was replaced in the basolateral compartment by a differentiation H441 medium . The apical side of the epithelial monolayer was kept at an air–liquid interface. Epithelial monolayer formation was controlled by measuring the transepithelial electrical resistance as previously reported . For theeviously , 124. Tog to remove cell debris. Samples were boiled for 5 min at 95 °C and subjected to 10% SDS-PAGE using 25 μg of total protein per lane. Western blot analysis was performed under reducing conditions. After separation, proteins were transferred to polyvinylidene difluoride membranes by semidry electroblotting and probed with mouse monoclonal antibody against serine protease domain of human TMPRSS2  at a dilution of 1:2500 or mouse antihuman PRSS8 antibody  at a dilution of 1:250. Goat anti-mouse horseradish peroxidase–labeled antibody  at a dilution of 1:50,000 was used as a secondary antibody. For immunofluorescence staining experiments, H441 cells were fixed on permeable supports with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with Roti Immunoblock (Carl Roth). These cell preparations were stained by overnight incubation with mouse monoclonal anti-ZO-1 (zonula occludens-1) antibody , at a dilution of 1:250 dissolved in PBS, supplemented with 0.5% bovine serum albumin, and 0.04% sodium azide. This was followed by an incubation for 1 h with a goat antimouse DyLight488 antibody (Thermo Fisher Scientific) at a dilution of 1:400. After staining, the cell preparations were mounted on slides using Dako Glycergel Mounting Medium (Agilent), and images were acquired using a ZEISS microscope (Axiovert 200M) and apotome technology.Whole-cell lysates of H441 cells were obtained by scraping cells from their permeable supports and transferring them into a lysis buffer  supplemented with protease inhibitor mixture . The lysates were sonicated and centrifuged for 10 min at 1000t test or by a nonparametric test: Kruskal–Wallis (with Dunn’s post hoc test) or Mann–Whitney test as indicated. N indicates the number of different batches of oocytes, and n indicates the number of individual oocytes studied per experimental group. Statistical analysis was performed using GraphPad Prism, version 5.04 .Data are presented as mean ± SD. Normal distribution of data was assessed using D’Agostino–Pearson omnibus test. Statistical significance was assessed by an appropriate parametric test: ANOVA (with Bonferroni post hoc test) or Student’s All data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Carallia brachiata—a medicinal terrestrial plant from Rhizophoraceae. Furofuran lignans [(−)--buddlenol D (1) and (−)--buddlenol D (2)] isolated from the stem and bark inhibited nitric oxide : 9.25 ± 2.69 and 8.43 ± 1.20 micromolar for 1 and 2, respectively) and prostaglandin E2  productions in lipopolysaccharide-induced RAW264.7 cells. From western blotting, 1 and 2 suppressed LPS-induced inducible nitric oxide synthase and cyclooxygenase-2 expression in a dose-dependent manner (0.3–30 micromolar). Moreover, analysis of the mitogen-activated protein kinase (MAPK) signaling pathway showed decreased p38 phosphorylation levels in 1- and 2-treated cells, while phosphorylated ERK1/2 and JNK levels were unaffected. This discovery agreed with in silico studies which suggested 1 and 2 bound to the ATP-binding site in p38-alpha MAPK based on predicted binding affinity and intermolecular interaction docking. In summary, 7′′,8′′-buddlenol D epimers demonstrated anti-inflammatory activities via p38 MAPK inhibition and may be used as viable anti-inflammatory therapies.Excessive macrophage activation induces the release of high levels of inflammatory mediators which not only amplify chronic inflammation and degenerative diseases but also exacerbate fever and retard wound healing. To identify anti-inflammatory molecules, we examined  Additionally, non-infectious stimuli such as cell damage and tissue injury also trigger these responses and lead to local inflammation. Uncontrolled acute inflammation gradually destroys tissues and organs, diminishes their functions, and evolves into chronic inflammation and degenerative diseases such as osteoarthritis, rheumatoid arthritis, and even cancer2. When exposed to chemoattractants, tissue monocytes evolve to macrophages which engulf and destroy microbes in phagolysosomes using various hydrolytic enzymes and substances, including nitric oxide (NO). Moreover, NO is released into the extracellular fluid and functions as an inflammatory mediator which enhances phagocyte migration to injured or infected sites to amplify immune responses. However, excessive NO production limits recovery processes and destroys surrounding tissues4. Toll-like receptors (TLRs), especially TLR-4 which is a pathogen recognition receptor, and mitogen-activated protein kinase (MAPK) signaling pathways have important roles during inflammatory responses. Their deactivation mitigates acute sepsis, chronic inflammation, and degenerative diseases6. Eritoran, a structural lipopolysaccharide (LPS) lipid A mimic, and SB203580, a pyridinylimidazole derivative, both target TLR-4 and p38 MAPK, respectively. Additionally, curcumin and its analog L48H37 are effective in inhibiting TLR-48. Furthermore, p38 MAPK inhibition is beneficial for Alzheimer’s disease treatment9. Natural compounds such as icariin, apigenin, and astaxanthin also decrease amyloid-β-induced neurotoxicity via p38 MAPK inhibition9.Inflammation is a cascade event generated by innate immunity responses against microbial infectionCarallia brachiata (Lour.) Merr. (Rhizophoraceae) is one such natural source whose stem and bark are used for antipyretic remedies. Similarly, in Ayurveda  the stem bark is also used to treat oral ulcers and stomatitis10. In a previous study, ethyl acetate (EtOAc) and methanol (MeOH) bark extracts showed significant wound healing properties in incision and excision wound rat models11. Furthermore, MeOH and hydro-ethanolic leaf extracts exhibited analgesic, anti-inflammatory, and anti-diabetic activities in in vivo models13. Based on these traditional uses and pharmacological activities, the anti-inflammatory activity of this plant appears to underlie its mechanism of action.Natural products are vital sources of medicines and have been used extensively by different populations; in Thailand, traditional medicines have been used for centuries. Based on Thai traditional medicine, O-β-apiofuranosyl-(1 → 6)-β-glucopyranoside], and flavonoid glycosides such as apigenin-7-O-α-rhamnoside-(1 → 2)-β-glucopyranoside were isolated from leaves of C. brachiata16. Also, p-hydroxy benzoic acid and two proanthocyanidins [carallidin and mahuanin A], which possessed anti-radical scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals and inhibited xanthine oxidase, were isolated from bark17. To the best of our knowledge, no evidence exists in the literature to support the compounds contributing to the activities addressed in traditional uses of this plants, especially for anti-inflammatory activity which is linked antipyretic and wound healing properties. In this work, we identified compounds which exerted anti-inflammatory activities, examined their effects on p38 MAPK signaling pathway, and developed in silico models to identify ligand binding targets.In terms of chemical investigations, two pyrrolidine alkaloids [hygroline and (+)-pseudohygroline], a megastigmane diglycoside , together with (+)-7″R,8′′S:7″′R,8′′′S-hedyotisol A (3), (−)-syringaresinol (4), two modified stilbene dimers [(+)-diptoindonesin D (5) and (+)-parviflorol (6)], a proanthrocyanidin [(−)-mahuanin A (7)], 4-hydroxy-2-methoxyphenyl-6-O-syringoyl-β-D-glucopyranoside (8), and three benzoic acid derivatives  were identified by spectroscopic analysis and compared with the literature  values of 0.44 and 0.48, respectively, when triply developed in 20% acetone/dichloromethane (Me2CO/CH2Cl2) -C. brachiata stem and bark, the crude MeOH and its partitioned extracts were preliminary tested for NO inhibition in murine macrophages (RAW264.7). The EtOAc extract was selected for further isolation. All isolates were screened for NO inhibition at a final concentration of 30 μM. Only buddlenols D 1 and 2, which exhibited > 50% inhibition, underwent half maximal inhibitory concentrations (IC50) analysis. In pre-treated RAW264.7 cells with 1 and 2 prior to stimulation with 500 ng/mL LPS, 1 and 2 inhibited NO production in a concentration-dependent manner (0.3–30 µM) and exhibited no cytotoxicity  was used as a positive control showed percentage of NO inhibition at 78.69 ± 1.48%  production via the arachidonic acid pathway. Buddlenols D 1 and 2 were evaluated for PGE2 inhibition as a putative fever suppressant. Cell pretreatment with 1 and 2 inhibited LPS-induced PGE2 production in a concentration-dependent manner; IC50 values for 1 and 2 were 6.15 ± 0.39 and 5.70 ± 0.97 µM, respectively, whereas dexamethasone (10 µM) exhibited an inhibition percentage of 91.21 ± 1.17%  almost completely ablated iNOS and COX2 expression , and c-Jun N-terminal kinase (JNK) phosphorylation. Treatment with 500 ng/mL LPS significantly increased p-p38, p-ERK1/2, and p-JNK levels detected at 24-h incubation when compared with non-stimulated cells. Dexamethasone (10 µM) also significantly suppressed these levels when induced by LPS and suggested its anti-inflammatory activity may be related to the inhibition of p38, ERK1/2, and JNK phosphorylation. In cells pre-treated with buddlenols 1 and 2, p38 phosphorylation was significantly reduced when compared with LPS-treated cells. However, ERK1/2 and JNK phosphorylation levels were not significantly changed. Therefore, the effects of 1 and 2 on NO and PGE2 suppression may be due to inhibited p38 phosphorylation  site as their fitness scores for the allosteric pocket were much lower than BIRB-796, a p38-α non-ATP site inhibitor. For TLR-4, 1 and 2 exhibited approximately a 0.8-fold decrease in fitness score when compared with ZINC25778142, a TLR-4 inhibitor. Additionally, we investgatged protein–ligand intermolecular interactions in the ATP-binding site of p38-α MAPK and TLR4-MD2 (myeloid differentiation protien 2) interface using the most likely occurring conformation (highest fitness score) .The ADMET data Table  showed t1 and 2, adduct ions [M+Cl]− at m/z of 679.2166 and 679.2156 were observed by HR-ESI–MS, and calculated for their molecular formula, C33H40O13 of 1 and 2, respectively. From Heteronuclear Multiple Bond Correlation (HMBC) spectra, hydroxy protons at δH 7.05 and 7.15 of 1 were placed on C-4″  and C-4′ , respectively. Additionally, a C8″-O-C4 ether linkage of both substructures was suggested by carbon chemical shifts at C-8″ (δC 88.0) and C-4 (δC 135.9)18. From NMR analysis, HMBC correlations , and 13C-NMR signals of C-8″ (δC 89.4) and C-4 (δC 136.2) were also observed in 2. Relative configurations on C-8″ and C-7″ of 1 and 2 were guided by NOESY correlations. The Nuclear Overhauser Effect Spectroscopy (NOESY) spectrum of 1 showed a prominent correlation of δH 4.18 (H-8″) and δH 4.98 (H-7″) and a relatively weak correlation of δH 4.18 (H-8″)/δH 4.38 (OH-7″), while a strong correlation of δH 4.00 (H-8″) and δH 4.34 (OH-7″) and a relatively pale correlation of δH 4.00 (H-8″)/δH 4.96 (H-7″) were observed in NOESY spectrum of 2. Moreover, J7′′,8′′ of 1 and 2 were observed at 4 and 6.8 Hz, respectively, and corresponded with erythro- and threo- forms of an acyl glycerol moiety19. Thus, 7′′,8′′-erythro and 7′′,8′′-threo buddlenols D were deduced for 1 and 2, respectively, and supported chemical shift differences for 7″ and 8′′ positions (ΔδC8′′-C7′′) as suggested by Xiong et al.19; . From circular dichroism (CD) spectra of 1 and 2, positive Cotton effects were observed at 239 and 233 nm, respectively, indicating the 8″S configuration of both compounds19. Additionally, negative benzene 1Lb-band Cotton effects observed at 283 and 282 nm for 1 and 2, respectively, were consistent with 4  and revealed 7R,7′R,8S,8′S configurations in these molecules20. The absolute configuration of benzylic carbon (C-7″) bearing a hydroxy group in 1 and 2 was suggested by Cotton effects in Rh2(OCOCF3)4-induced CD experiments, based on the secondary alcohol bulkiness rule. E band signs (≈ 350 nm) were observed as negative and positive Δε for 1 and 2, respectively, suggesting the 7″R configuration for 1 and 7″S configuration for 221. Moreover, the assigned configurations of 1  and 2  were consistent with the electronic circular dichroism (ECD) spectrum calculated based on Density Functional Theory (DFT) 22. Later, the absolute configuration of two buddlenol D isomers  were thoroughly investigated in work by Xiong et al.19. Both 7′′R,8′′S- and 7′′S,8′′S-buddlenols D were isolated from Sinocalamus affinis (Poaceae) and Melodinus cochinchinesis (Apocynaceae) using reverse phase chromatography23. In our study these 7″,8″-buddlenol D epimers were separated by silica gel column chromatography eluted in 20% Me2CO/CH2Cl2. Buddlenol D and syringaresinol are furofuran lignans comprised of synapyl alcohol units, while hedyotisol A comes from coniferyl alcohol monomers. In our study, phenolic compounds  which possessed substructural guaiacyl and syringyl moieties, oligostilbenes , and a proanthocyanidin (7) demonstrated the influence of shikimate pathway on the biosynthesis of compounds stored in C. brachiata stem and bark. Although 7″,8″-buddlenol D epimers were isolated from the aforementioned plants, their biological activity and medicinal use were not previously addressed.Buddlenol D was first isolated from 2, a pyrogenic mediator, via suppressed COX2 expression by 1 and 2 supported the antipyretic properties of C. brachiata stem and bark for traditional use, and was putatively related to the finding that a higher core temperature correlated with higher PGE2 production24. Additionally, 1 and 2 significantly inhibited iNOS expression induced by LPS treatment. As LPS-induced iNOS and COX2 expression, these results suggested 1 and 2 exerted anti-inflammatory activities by suppressing these proinflammatory enzymes and reducing NO and PGE2 levels. MAPK signal transduction, including ERK1/2, JNK, and p38 MAPK, modulates inflammatory processes in response to stimuli via different receptors25. ERK1/2, JNK, and p38 MAPK phosphorylation activities are the major downstream signaling phenomena after TLR4 activation. Therefore, molecules regulating TLR4-mediated ERK1/2, JNK, and p38 MAPK phosphorylation may exhibit anti-inflammatory potential. From our data, we hypothesized the underlying mechanisms of 1 and 2 may occurred via modulated p38 MAPK phosphorylation. In contrast, dexamethasone, which is an anti-inflammatory drug and used as a positive control inhibited all ERK1/2, JNK, and p38MAPK phosphorylation26 and exerted potent anti-inflammatory activity by suppressing NO and PGE2 production and down-regulating iNOS and COX2 expression27.We also reported the anti-inflammatory activity of 7″,8″-buddlenol D epimers. The inhibition of PGE1 and 2 inhibited p38 MAPK phosphorylation, a specific p38 inhibitor SB203580 was used to identify anti-inflammatory activity. In RAW264.7 cells pre-treated with SB203580 prior to LPS stimulation, p38 MAPK phosphorylation suppression was clearly observed and resulted in NO inhibition  activities of 1 and 2 support the use of C. brachiata extracts for oral stomatitis and wound healing32 . Hence, molecular docking studies suggested that 1 and 2 could selectively impede p38-α MAPK at the ATP-binding site.Concerning the sharing features of ATP-binding pocket among kinases, we thus observed the binding capability toward other kinases involved in the focused signaling cascade. As listed in Table 1 and 2 might possess low GI absorption profile, based on in silico calculation 1 and 2 exhibited good metabolic profiles. Compounds 1 and 2 could be potential compounds for further structural development for better pharmacokinetic profiles and suitable drug delivery system. Moreover, after oral administration of 1 and 2, they might lose the syringyl unit during the digestion process and could be transformed to enterodiol derivatives by gut microbiome37. There was evidence that enterodiol exhibited p38 down regulation as one of the mechanisms inhibiting the growth of colorectal cancer cells38.Although, S-deoxydihydroglyparvin which is a sulfur-containing propanamide derivative isolated from Glycosmis parva (Rutaceae) leaves39. As furofuran lignans such as epimagnolin B from Magnolia fargesii (Magnoliaceae), (−)-sesamin-2,2′-diol from Isodon japonicus (Lamiaceae), and zanthpodocarpins A and B from Zanthoxylum podocarpum (Rutaceae) displayed anti-inflammatory activity via NO inhibition in cultured cells42, we suggest this compound class may exhibit anti-inflammatory activity. From our study, we propose 7″,8″-buddlenol D epimers function as p38 MAPK inhibitors.In our previous work, we reported a natural p38 MAPK inhibitor (+)-C. brachiata were collected in January 2018 from the botanical gardens of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok. The samples were identified by a study author (C. Chansriniyom). The herbarium specimen (CC-CB-010118) was deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University. In addition, the handling and notification of stems and barks of C. brachiata were carried out in accordance with the Plant Variety Protection Act B.E. 2542 (1999): Section 53, the Kingdom of Thailand.Stems and barks from C. brachiata stems and barks (4.0 kg) were successively macerated in MeOH (3 × 15 L) to yield a 203.80 g MeOH extract which was partitioned with hexane to generate a hexane extract (7.34 g). The remaining organic layer was added to water and further partitioned with EtOAc and n-butanol (n-BuOH) to obtain EtOAc (27.39 g), n-BuOH (50.39 g), and aqueous (113.89 g) extracts.Dried and coarsely powdered ® XS 420, Advion Inc., NY, USA) and was eluted in a gradient solvent of MeOH, EtOAc and hexane at an initial ratio of 0:40:60 (respectively), then progressed to 30:70:0 (respectively), and terminated in 100% MeOH. Based on chromatographic patterns fractions were combined to generate seven fractions (CBE1–CBE7).The EtOAc extract (27.39 g) underwent silica gel column chromatography (Flash column chromatography (Flash CC), PuriFlash2Cl2 system ]. CBE7-3 (874 mg) was separated into 48 fractions using Medium Pressure Liquid Chromatography  . CBE7-3-12 (178.2 mg) was purified using MPLC  to obtain 1 (6 mg) and 2 (2.4 mg). CBE7-3-20 (88.9 mg) was separated by MPLC  to afford 3 (6 mg). CBE7-4 (466 mg) was purified using two MPLC steps  to achieve 8 (6.3 mg).CBE7 (6.58 g) was separated into seven sub-fractions using Flash CC +. HR-ESI–MS m/z: 679.2166 [M+Cl]−, calcd. for C33H40O13Cl 679.2163. [α]20D: − 12.96° . CD nm (MeCN): 204.8 (Δε − 13.88), 208.7 (Δε − 1.50), 213.1 (Δε − 5.35), 217.9 (Δε + 3.23), 225.7 (Δε − 4.14), 234.3 (Δε + 2.06), 239.0 (Δε + 0.50), 244.6 (Δε − 0.73), 283.3 (Δε − 0.09). Rh2(OCOCF3)4-induced CD nm (CH2Cl2): 355.4 (Δε − 0.83) -4′,4′′-Dihydroxy-3,3′,3′′,5,5′,5′′-hexamethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol : white solid. 1H-NMR  δ: 7.15 , 7.06 , 6.76 , 6.74 , 6.68 , 4.96 , 4.73 , 4.68 , 4.34 , 4.25 , 4.00 , 3.90 , 3.87 , 3.82 , 3.79 , 3.67 , 3.36 , 3.11 ; 13C-NMR  δ: 153.9 , 148.8 , 148.4 , 139.3 (C-1), 136.3 (C-4′), 136.2 (C-4), 136.1 (C-4″), 133.2 (C-1′), 132.8 (C-1″), 105.4 , 104.5 , 104.0 , 89.4 (C-8″), 86.8 (C-7′), 86.6 (C-7), 74.1 (C-7″), 72.6 , 61.6 (C-9″), 56.7 , 55.4 ; IR (ATR) cm−1: 3466 (broad), 2939, 2844, 1610, 1594, 1520, 1459, 1216, 1116; UV λmax (MeOH) nm (log ε): 206 (4.64), 236 sh (3.93), 272 (3.24). ESI–MS m/z: 667 [M+Na]+. HR-ESI–MS m/z: 679.2156 [M+Cl]−, calcd. for C33H40O13Cl 679.2163. [α]20D: − 9.78° . CD nm (MeCN): 203.9 (Δε − 12.58), 207.7 (Δε + 3.04), 210.1 (Δε + 1.19), 214.8 (Δε + 6.30), 219.0 (Δε − 0.24), 221.4 (Δε + 0.42), 225.5 (Δε − 0.98), 233.1 (Δε + 0.61), 237.8 (Δε − 0.62), 282.2 (Δε − 0.04). Rh2(OCOCF3)4-induced CD nm (CH2Cl2): 347.2 (Δε + 0.81) -.Mouse macrophage cells RAW264.7  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum in 5% CO5 cells/mL) in 100 µL were seeded in a 96‑well plate and incubated for 24 h at 37 °C. After this, cells were treated with different compound concentrations and incubated for 24 h. Then, medium containing compound was replaced by phenol red-free medium containing LPS   and the cells were further incubated for 24 h. NO levels were determined by collecting 100 µL supernatants and mixing with 100 µL modified Griess reagent (Sigma-Aldrich). Reaction mixtures were incubated for 15 min at room temperature and absorbance measured at 560 nm using a microplate reader. NO levels were quantified by interpolation using a nitrite standard curve. Cells treated with dexamethasone (10 µM) were used as a positive control group.Cells (5 × 105 cells/mL) in 100 µL were seeded in a 96‑well plate and incubated for 24 h at 37 °C. Then, cells were treated with different compound concentrations and incubated for 24 h. After that, medium containing compound was replaced by medium containing LPS  and the cells were incubated for another 24 h. Supernatants were collected and subjected to enzyme-linked immunosorbent assay for PGE2  following manufacturer’s instructions.Cells , anti-COX2 (CST#2282), anti-phospho-ERK (CST#4376), anti-ERK (CST#4695), anti-phospho-JNK (CST#4668), anti-JNK (CST#9252), anti-phospho-p38 (CST#4511), anti-p38 (CST#9212), and followed by incubation with a horse radish peroxidase-conjugated secondary antibody. Protein bands were detected using a chemiluminescent substrate and images acquired using ImageQuant LAS 4000 . Relative protein band intensity was normalized to β-actin or GAPDH expression using ImageJ. SB203580  is a specific p38-α inhibitor and was used as a positive control for p38 phosphorylation inhibition.44, 3ZSH33, 1KV245, 4QTB46, 1PME47, 2NO348, 3NPC49, 4W4Y50, 4KIK51, respectively. In addition to protein preparation, the 3-dimensional structures of 1 and 2 were manually constructed using the Gaussian 09 program and optimized using the HF/6-31d basis set implemented in the program52. For docking studies, the Gold docking program was used, and protocols were as follows: (1) the ligand binding site was defined as 6 Å for sphere docking and (2) ChemScore was used for the scoring function. Binding between proteins and compounds was visualized in Accelrys Discovery Studio 2.5 (Accelrys Inc.).Protein–ligand docking was conducted for eight targeted proteins under nine systems-TLR4, p38-α MAPK (ATP-binding site), p38-α MAPK (non-ATP-binding site), ERK1, ERK2, JNK1, JNK2, JNK3 and IκB kinase β using crystallized structures (retrieved from RCSB Protein Data Bank), listed as follows: 2Z6534 and the docked pose was then observed its orientation and intermolecular interactions with the key reported residues including D209, S211, and D234. Our docking protocols were validated for all systems 53 while their toxicity profiles were predicted using Protox-II platform (https://tox-new.charite.de/protox_II/)54 and the DataWarrior software package55.Pharmacokinetic properties  of compounds p < 0.05.All data were expressed as the mean ± standard error of the mean from at least three independent experiments. Differences among groups were evaluated using one-way analysis of variance, followed by post-hoc tests with Bonferroni correction. Statistical significance was accepted at C. brachiata stem and bark. Their action mechanisms were mediated by inhibited p38 MAPK phosphorylation which attenuated iNOS and COX2 expression and inhibited NO and PGE2 production in LPS-induced RAW264.7 macrophages. These findings support the use of C. brachiata for antipyretic, oral stomatitis, and wound healing purposes. Therefore, 7″,8″-buddlenol D epimers can be used as biological markers for C. brachiata extract.For the first time, we characterized the anti-inflammatory activities of 7″,8″-buddlenol D epimers from Supplementary Information.


Given its efficient suppression of IgE levels, good safety and pharmacokinetic/pharmacodynamic profile, ligelizumab clearly warrants further studies for the potential management of FA.Food allergy (FA) is a growing healthcare problem worldwide and the rising prevalence in many countries can be attributed to lifestyle, environmental, and nutritional changes. Immunoglobulin E (IgE)-mediated FA is the most common form of FA affecting approximately 3%–10% of adults and 8% of children across the globe. Food allergen–induced immediate hypersensitivity reactions mediated by IgE and high-affinity IgE receptor (FcεRI) complexes on mast cells and basophils are a major hallmark of the disease. FA can affect several aspects of health-related quality of life and impose a substantial financial burden on patients and healthcare systems. Although currently there is one United States Food and Drug Administration (FDA) and European Medicines Agency (EMA)–approved treatment for peanut allergy , the main treatment approaches are based on allergen avoidance and symptom management. Thus, there is an urgent need for more effective and ideally disease-modifying strategies. Given the crucial role of IgE in FA, anti-IgE monoclonal antibodies are considered promising therapeutic agents. Talizumab was the first humanized anti-IgE antibody to demonstrate substantial protection against allergic reactions from accidental peanut exposure by substantially increasing the peanut reactivity threshold on oral food challenge. However, development of talizumab was discontinued and further trials were performed using omalizumab. In double-blind, Phase 2, placebo-controlled trials in patients with multi-FAs, sustained dosing with omalizumab, or omalizumab in combination with oral immunotherapy, enabled rapid desensitization to multiple trigger foods. In this review, we describe the development of ligelizumab , a next generation, humanized monoclonal anti-IgE antibody, its existing clinical evidence, and its potential in the management of FA. When compared with omalizumab, ligelizumab binds with ∼88-fold higher affinity for human IgE and recognizes a different epitope that substantially overlaps with the binding site of FcεRI. These properties translate into a high potency to block IgE/FcεRI signaling in both  OIT is based on the administration of increasing doses of the allergenic food until a maintenance dose is reached, at which point many patients continue regular ingestion to maintain desensitization to prevent reactions from accidental ingestions. However, OIT carries significant risks; allergic reactions to accidental food allergen exposure may not be completely eliminated and allergic reactions to maintenance dosing may occur, and long-term need for continued maintenance dosing in most is required to sustain desensitization.There is an urgent requirement to address the unmet needs of patients with FA, particularly of those with multiple FAs as they are more likely to experience accidental exposure and often need to avoid several food groups, which can lead to harmful effects on nutrition, growth, and quality of life (QoL).,,The development of FA begins with a period of sensitization that may occur through multiple pathways, often including early cutaneous exposure to food protein through a compromised skin barrier. Langerhans cells in the epidermis may take up these food proteins and induce a type 2 helper T cell (Th2) response and IgE production by B cells.,IgE binds via its fragment crystallizable (Fc) domain to its two major receptors, the high-affinity IgE receptor (FcεRI) and the low-affinity IgE receptor (FcεRII) or CD23 .,de novo synthesis of leukotrienes, prostaglandins, and platelet-activating factor that contribute to the characteristic symptoms of an allergic reaction and anaphylaxis.Re-exposure to the allergen, most often through oral ingestion, leads to the crosslinking of FcεRI-bound IgE on mast cells and basophils resulting in cell degranulation and release of vasoactive and pro-inflammatory mediators.p < 0.001), with subjects showing an average ∼16-fold increase in their peanut reaction threshold.As IgE plays a significant role in the pathology of FA, the investigation of an anti-IgE therapy for FA is well reasoned.,,Omalizumab is the only monoclonal anti-IgE antibody approved for clinical use, currently for the treatment of severe allergic asthma, CSU, and severe nasal polyposis. It has also been investigated for the treatment of FA, both as monotherapy and in combination with OIT. Studies involving omalizumab have evaluated treatment for peanut, milk, and multi-food allergies, and all have produced encouraging results.54p = 0.18). Sustained unresponsiveness to oral food challenge by month 32 demonstrated that ∼48.0% of patients maintained desensitization in the omalizumab add-on group vs 35.7% in the placebo group (p = 0.42).p = 0.004).p = 0.03).In an unblinded study, marked changes in peanut challenge thresholds were observed after only 1–3 doses of omalizumab.in vitro laboratory investigation. Ligelizumab recognizes an epitope that spans across the Fc domain Cε3 regions of human IgE and thereby blocks IgE binding to FcεRI and CD23.,,Ligelizumab, a derivative of talizumab, is a humanized immunoglobulin-G1 (IgG1/κ) monoclonal antibody that binds to human IgE with ∼88-fold higher affinity than omalizumab, as observed through Details of completed and ongoing ligelizumab clinical trials in asthma and CSU are listed in p < 0.001). A dose-dependent reduction in circulating free IgE levels and basophil cell surface–bound IgE levels was also observed with ligelizumab. In addition, ligelizumab demonstrated a dose- and time-dependent reduction in basophil FcεRI and IgE levels.p = 0.002 and p < 0.0001 for 72 mg and 240 mg, respectively) and a ∼3-fold greater reduction in reactivity  to the allergen compared with omalizumab (dosed as per the dosing table posologyp = 0.10).Two Phase 2 clinical trials have investigated ligelizumab in allergic asthma following one Phase 1 trial in atopic subjects. In a Phase 1 trial, ligelizumab showed greater reduction in skin prick wheal allergic responses in atopic subjects compared with omalizumab  met their primary endpoint of superiority vs placebo (change from baseline in weekly Urticaria Activity Score) at Week 12 in the treatment of CSU; however, superiority vs omalizumab was not demonstrated.With regard to CSU, a Phase 2b study in adult patients demonstrated a dose-dependent response to ligelizumab with more patients achieving complete control of disease activity compared with omalizumab and placebo at Week 12 .,,,,,,,In a Phase 1 study in patients with atopy, the most commonly reported adverse events (AEs) were headache and upper respiratory tract infection. Mild-to-moderate urticaria was reported as the most significant AE in this study, with an incidence of ∼17% in any ligelizumab dose group; the event resolved spontaneously or with the use of antihistamines. Only 2 (5%) patients on ligelizumab discontinued due to AEs of asthma exacerbations and flu-like illness, but neither were considered to be related to the study drug.,In early Phase 1 trials, ligelizumab showed dose- and time-dependent suppression of allergen-elicited wheals with a standardized skin prick test.in vitro and in vivo studies. Consequently, considering its potent suppression of both serum IgE and skin prick test, and known safety profile, ligelizumab is a promising candidate to be investigated in the management of patients with IgE-mediated FA.Although the financial and psychosocial burden of FA is high, and the prevalence of FA in many countries is on the rise, existing treatment options for FA are extremely limited. Due to the important role played by IgE/FcεRI pathway in FA, IgE suppression with monoclonal antibodies, such as talizumab and omalizumab, has been shown to provide clinical benefits in patients with FA. Allergen avoidance and patient nutrition care continues to be a key approach for food allergy management. In addition, anti-IgE therapy has the potential to complement the standard of care by diminishing the risk to the patient. Ligelizumab , is a next generation humanized monoclonal antibody that shows greater overlap with the binding site of FcεRI and binds to human IgE with ⁓88-fold higher affinity compared with omalizumab. Accordingly, ligelizumab has displayed high potency to block IgE/FcεRI signaling in both AE, adverse event; CSU, chronic spontaneous urticaria; EAACI, European Academy of Allergy and Clinical Immunology; FA, food allergy; Fc, fragment crystallizable; FcεRI, high-affinity IgE receptor; FcεRII, low-affinity IgE receptor; GI, gastrointestinal; HRQoL, health-related quality of life; IgE, immunoglobulin E; IL, interleukin; OIT, oral immunotherapy; PD, pharmacodynamic; pDC, plasmacytoid dendritic cells; PK, pharmacokinetic; Q2W, once every 2 weeks; Q4W, once every 4 weeks; QoL, quality of life; Th1, type 1 helper T cell; Th2, type 2 helper T cell.10.13039/100004336Novartis Healthcare, Hyderabad, India, for providing medical writing support which was funded by Novartis Pharma AG, Switzerland in accordance with Good Publication Practice (GPP3) guidelines (http://www.ismpp.org/gpp3).The authors thank Mohammad Fahad Haroon (PhD), Sahaja Banda, and Anupama Boddupalli of Not applicable.All authors made substantial contributions to the conception and design of this article; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval for the version to be published; and agree to be accountable for all aspects of the work.Not applicable.World Allergy Organization Journal and has not been previously submitted to another journal, nor is it currently under consideration by another journal.This manuscript is being submitted only to The authors' consented to the publication of this review.Robert A Wood receives research support from NIAID, Aimmune, Astellas, DBV, FARE, Genentech, HAL-Allergy, Novartis, and Regeneron.R. Sharon Chinthrajah reports grants from NIAID, CoFAR, Aimmune, DBV Technologies, Astellas, Regeneron, FARE, and MCHRI; is an advisory member for Alladapt Therapeutics, Genentech, Allergenis, Novartis, Sanofi, and Nutricia; and received personal fees from Before Brands, all outside the submitted work.Alexander Eggel reports consulting activities for Novartis and received research and travel grant support from Novartis.Ivan Bottoli, Paolo Tassinari, Aurelie Gautier, and Maximilian Woisetschlaeger are employees of Novartis Pharma AG, Basel, Switzerland.Pablo Altman is an employee of Novartis Pharmaceuticals Corporation, East Hanover, New Jersey, United States.


Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs  were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening. A G protein-coupled receptors (GPCRs) phosphorylation assay for cell culture plates can be used for ligand profiling and inhibitor screening, as evidenced by the identification of two GRK inhibitor compounds. The unique ability of GRKs to recognize activated receptors results in agonist-dependent phosphorylation at intracellular serine and threonine residues3. In addition, second messenger-regulated kinases can also participate in GPCR phosphorylation, including protein kinases A and C and other serine/threonine kinases6. Phosphorylation of GPCRs is a biologically and pharmacologically important process that primarily initiates desensitization and internalization of a receptor8. Phosphorylation also increases the interaction of GPCRs with intracellular adapter proteins such as β-arrestins, which can trigger a second wave of signaling12.G protein-coupled receptors (GPCRs) are vital signal transducers that mediate the effects of a variety of chemical and physical stimuli, and represent major drug targets for many human diseases. GPCRs have a uniform structure with seven-transmembrane domains and are therefore also referred to as seven-transmembrane receptors (7TMRs). Activation by their endogenous ligands or exogenous agonizts leads to GPCR conformational changes that are recognized by a family of kinases termed G protein-coupled receptor kinases (GRKs)13. Currently, the prevailing approach is based on antibodies that specifically recognize the phosphorylated state of a GPCR18. When available, phosphosite-specific anti-GPCR antibodies are excellent tools to resolve the temporal and spatial dynamics of receptor phosphorylation, identify relevant kinases and phosphatases, detect receptor activation and profile the pharmacological properties of new ligands22. However, phosphosite-specific antibodies are predominantly used in immunoblotting approaches that are technically challenging, limited to small sample sizes and difficult to quantify.Initial GPCR studies were based on radioactive whole-cell phosphorylation assays, which require high levels of radioactivity and cannot be used to identify individual phosphorylated serine and threonine residues. Later, studies were directed to phosphoproteomics analysis, through which limited quantitative information has been obtainedIn pharmaceutical and academic research, high-throughput assays are often required to facilitate the screening of large chemical libraries. Although receptor binding, G-protein signaling and arrestin recruitment can be assessed with available methodologies, adequate technologies for the determination of GPCR phosphorylation are currently not available. We were therefore motivated to take advantage of phosphosite-specific anti-GPCR antibodies in the development of a quantitative phosphorylation immunoassay that can be performed entirely in multiwell cell culture plates and is conducive to medium to high-throughput applications Fig. . Because2,N-MePhe4,Gly-ol]-enkephalin (DAMGO) and then lysed in detergent buffer containing protease and protein phosphatase (PPase) inhibitors. After plate centrifugation, lysates were transferred to U-bottom 96-well plates. MOPs were then immunoprecipitated using mouse anti-HA antibody-coated magnetic beads. After washing the beads under magnetic force, either phosphosite-specific rabbit antibodies that specifically recognized the S375-phosphorylated form of MOP (pS375-MOP) or antibodies that detected MOP in a phosphorylation-independent manner (np-MOP) were added to the cells -tagged mouse µ-opioid receptors (MOPs) were seeded into poly-L-lysine-coated F-bottom 96-well plates and grown to >95% confluency. The cells were cultured in the presence or absence of the agonist DAMGO (51.7 Ci/mmol) (Perkin Elmer NET902250UC) as described previously52. Briefly, cells grown to confluency were harvested in PBS and stored at −80 °C. Saturation binding experiments were performed with 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 ml containing 30–40 µg of membrane protein. Membranes were incubated with different concentrations of [3H]DAMGO (0.05–9 nM) at 25 °C for 60 min. Nonspecific binding was determined in the presence of 10 µM unlabeled DAMGO. Reactions were terminated by rapid filtration through Whatman glass GF/C fiber filters. The filters were washed three times with 5 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) with a Brandel M24R cell harvester. Bound radioactivity retained on the filters was measured by liquid scintillation counting using a Beckman-Coulter LS6500. All experiments were repeated three times with independently prepared samples. Nonlinear regression analysis of the saturation binding curves was performed with GraphPad Prism.Binding experiments were performed on membranes prepared from wild-type and HA-MOP-transfected HEK293 cells using [m of ATP for GRK2 and GRK5  and 4 µl of substrate working solution in assay buffer were transferred into assay plates (Corning #4513). Compound was added via an Echo acoustic dispenser (BeckmanCoulter) in a concentration range from 10 to 0.0025 µM. Reaction was started by addition of 2 µl ATP. After 1 h incubation at room temperature, the reaction was stopped with 10 µl detection mix containing the anti-phospho peptide antibody and 10 mM EDTA. After a second incubation period of 1 h at room temperature the FRET signal was measured at 340 nm excitation, 665 nm and 615 nm emission with an Envision spectrophotometer (Perkin Elmer) with 50 µs delay and 300 µs integration time. IC50 values were calculated with concentration–response-curves with Quattro Workflow.The kinase inhibitory activity of LDC8988 and LDC9728 against GRK2 and GRK5 was assessed using the Lance kinase activity assay, which is based on the detection of a phosphorylated Ulight-peptide substrate by a specific europium-labeled anti-phospho peptide antibody. Binding of a kinase inhibitor prevents phosphorylation of the Ulight-substrate resulting in loss of the FRET signal. The assay was performed at an ATP concentration representing the KAll assays were performed in duplicates and repeated in least five independent experiments. Concentration–response-curves were generated with GraphPad Prism 9.3.1 software. Western blot assays were repeated at least three times. Western blot signals were imaged and quantified using a Fusion FX7 imaging system (Peqlab) and quantified using ImageJ.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


A key component among the regulatory mechanisms is the binding of sugar phosphate derivatives that inhibit activity. Removal of inhibitors via the action of Rubisco activase is required to restore catalytic competency. In addition, specific phosphatases dephosphorylate newly released inhibitors, rendering them incapable of binding to Rubisco catalytic sites. The best studied inhibitor is 2-carboxy-d-arabinitol 1-phosphate (CA1P), a naturally occurring nocturnal inhibitor that accumulates in most species during darkness and low light, progressively binding to Rubisco. As light increases, Rubisco activase removes CA1P from Rubisco, and the specific phosphatase CA1Pase dephosphorylates CA1P to CA, which cannot bind Rubisco. Misfire products of Rubisco’s complex reaction chemistry can also act as inhibitors. One example is xylulose-1,5-bisphosphate (XuBP), which is dephosphorylated by XuBPase. Here we revisit key findings related to sugar phosphate derivatives and their specific phosphatases, highlighting outstanding questions and how further consideration of these inhibitors and their role is important for better understanding the regulation of carbon assimilation.Regulating the central CO We review the complex regulation of Rubisco by sugar phosphate derivatives and their phosphatases, and highlight unresolved questions for a better understanding of the regulation of carbon assimilation. This regulation is associated with longer periods of shade (>30 min), whereas short shade periods are more likely associated with rapid carbamylation/decarbamylation of Rubisco  removes the phosphate group of CA1P, resulting in non-inhibitory 2-carboxy-d-arabinitol (CA)  .14C labelling of the potential for FBP to be converted to HBP, and that dephosphorylation of HBP could then produce hamamelose  plants have been shown to contain high levels of the CA1P precursor CA , with Rubisco-catalysed XuBP carboxylation believed to occur at rates fractions of a percent of that of RuBP carboxylation  and carboxy-tetriol-1,5-bisphosphate  for these compared with for CA1P itself  than CA1Pase from wheat (10 µM), a species with little CA1P  motif identical to the catalytic site of a phosphoglycerate mutase (PGM). This feature is frequently observed for enzymes whose catalytic reaction involves phosphate transfer, including Calvin–Benson–Basham cycle enzymes such as FBPase  domain sugar phosphatase, XuBPase (uite low . While sin vivo substrate(s) of the SOQ1 HAD domain and its potential impact on Rubisco regulation are currently unknown  have been shown experimentally to be regulated in multiple ways . HoweverIn vitro analyses of CA1Pase activity have shown that several chloroplast metabolites can stimulate activity and increase Vmax, including RuBP, FBP, and 3-phosphoglycerate (3-PGA), with as much as a 9-fold increase in the case of FBP (in vivo role in regulating CA1Pase activity. Consistent with these observations is the decreased in vitro activity of CA1Pase both produced recombinantly and purified from leaves with the addition of inorganic phosphate (Pi); however there is evidence to suggest species differences in this sensitivity (in vivo consequences of this are difficult to estimate due to the known variability in leaf Pi content with factors such as leaf age and species (e of FBP . That thin vivo heat stress experiments with wheat, a species which does not possess large amounts of CA1P (in vitro (in vitro experiments, redox status greatly enhanced protein activity, but this was dependent upon glutathione state, pre-incubation either with other reducing agents such as DTT or air oxidation, and in some cases the assay pH (CA1Pase from tobacco has been shown to be resilient to incubation at moderately high temperatures, with activity remaining unaffected after an hour at temperatures up to 30 °C ( of CA1P , showed  of CA1P . DTT hasThe inhibition of Rubisco by the nocturnal inhibitor CA1P, and subsequent dephosphorylation of CA1P by CA1Pase, has a number of links to the light-dependent side of photosynthesis. Synthesis of CA1P, by an as yet unknown enzyme, occurs in darkness and it progressively inhibits Rubisco in prolonged dark periods when its removal by Rca is limited by stromal ADP/ATP ratios. Increasing light then provides the energy requirements for removal of CA1P by Rca and coincides with promotion of CA1Pase activity to degrade CA1P and render it non-inhibitory. In contrast, treatment with methyl viologen, a PSI electron acceptor, decreased CA1P degradation in the light . In addi3 plants, with plants investing considerable resources to produce Rubisco and the ancillary proteins required for its synthesis and maintaining its activity (reviewed in 3 plants and the central role it plays in carbon metabolism. Rubisco degradation and replacement is an area less understood and might be linked to a protective role for sugar phosphate inhibitors (reviewed in Rubisco protein is very abundant in chloroplasts, particularly within Cin vitro experimentation, the closure of Loop 6 has been proposed to limit the accessibility of the large subunit for proteolysis, which would conserve Rubisco protein (2+ and CO2, to form a carbamate within the catalytic site prior to CA1P binding. Stromal protease extracts were also unable to degrade Rubisco that had been activated and incubated with CA1P (One theory posed for the role of CA1P as a nocturnal inhibitor is to prevent attack of Rubisco by proteases through the conformational changes that occur when the catalytic site changes to bind a sugar phosphate such as CA1P . Based oith CA1P . Tobaccoin vitro are representative of the interaction between stromal proteases and inhibited Rubisco.As in planta for improved photosynthesis. Regulation of the phosphatases CA1Pase and XuBPase responsible for inhibitor degradation also warrants deeper investigation of these highly conserved components of Rubisco regulation. This conservation, the rapid development of CRISPR/Cas9 technologies in plants, and the large variation evident inter- and intraspecies provide encouragement for better understanding this regulation as well as its potential role in improving photosynthetic efficiency and crop productivity.The inhibition of Rubisco by tightly binding sugar phosphates, either actively synthesized or derived from misfire of its complex reaction mechanism, can have large impacts on Rubisco activity by limiting carboxylation capacity. Key to this regulation of Rubisco activity is the action of Rca in removing these inhibitors from the Rubisco catalytic site, followed by their dephosphorylation by sugar phosphatases. Despite extensive study of dark inhibition of Rubisco by CA1P, many questions remain about the role of this seemingly ubiquitous, yet highly variable, process. The potential for a role in modulating Rubisco abundance as well as activity may make this a necessary consideration for manipulating Rubisco