To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.Dynein is the primary minus-end-directed microtubule motor protein. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the “activated dynein complex.” The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and the adaptor. Ndel1 and its paralog Nde1 are dynein- and Lis1-binding proteins that help control dynein localization within the cell. Cell-based assays suggest that Ndel1–Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear. Using purified proteins and quantitative binding assays, here we found that the C-terminal region of Ndel1 contributes to dynein binding and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in the C-terminal domain of Ndel1 increase its ability to inhibit dynein–dynactin–adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein-binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Together, our work suggests that  Cytoplasmic dynein-1 (dynein) is a microtubule-associated molecular motor that is responsible for nearly all minus-end-directed force generation in most eukaryotes , 3, 4, 5A and S1A). The largest subunit, called the heavy chain, contains a motor domain that is a ring of six AAA (ATPase Associated with various cellular Activities) domains (A). AAA1, AAA3, and AAA4 are active ATPase modules (A and S1A) (A) (A) (A) (A)  , 8. In tand S1A) A 9). Dy. DyA andS1A) (A) A 10, 11A and S1A (A) (A) A 12, 13A and S1A (A) (A) , 15. In  (A) (A) A 16, 17A and S1A (A) (A) , 23, 24.B and C)  Ndel1 or Nde1, structures of the N-terminal half of Ndel1 reveal that it is a long coiled coil . The C-tB and C) . Many prB and C) , 28, 29.B and C) .In addition to localizing dynein within the cell, there is evidence that Ndel1 and Nde1 directly support dynein activation. In human cells, concurrent depletion of Ndel1 and Nde1 causes Golgi dispersal, whereas inhibition of Ndel1 and Nde1 by a function-blocking antibody causes acidic cargo dispersal , 31. BotIn vitro experiments conducted with purified mammalian and yeast proteins show that Ndel1–Nde1 promote Lis1–dynein association  (A). The first ∼50 amino acids of Ndel1–Nde1 interacts with the disordered N-terminal tail of the intermediate chain (A)  (A) (A)  , 22, 24.A) A. The fite chain A 37, 38, 38A (A)hain (A) , 45, 46.led CC1) A  (A) , 48. TheCC1) (A) A 49, 50, 50A (A)via competition with p150 for intermediate chain binding. Despite the C terminus of Ndel1 being dispensable for inhibiting dynein activation, it increases the potency of inhibition. Furthermore, phosphomimetic mutations in the C terminus of Ndel1 increase its ability to inhibit complex formation. Our work also revealed that Ndel1 and Lis1 binding is negatively regulated by the C-terminal half of Ndel1, suggesting that Ndel1 is autoinhibited with respect to Lis1 association. We found that although Ndel1 can bind both Lis1 and dynein simultaneously, Lis1 cannot bind Ndel1 and dynein at the same time. This competition significantly attenuates the ability of Lis1 to promote activated dynein complex assembly in the presence of Ndel1. Our work suggests that Ndel1 has two distinct modes of preventing dynein activation: disfavoring dynein complex assembly by binding the intermediate chain and preventing Lis1-mediated activation.We set out to investigate how Ndel1 affects dynein activity and test the model that Ndel1 tethers Lis1 to dynein to promote activation. Using single-molecule imaging and protein biochemistry, we found that Ndel1 disfavors formation of the activated dynein complex, most likely B and Fig. S1D). These constructs include a truncation of Ndel1 containing only the N-terminal coiled coil (NT-Ndel1), a well-characterized Ndel1 mutant that disrupts binding to the intermediate chain in both the FL-Ndel1 and NT-Ndel1 backgrounds (FL-Ndel1E48A and NT-Ndel1E48A), and a construct with the coiled coil containing the purported heavy chain–binding site deleted from the C-terminal region of Ndel1 (Ndel1ΔCTCC) (B) , FL-Ndel1, and several Ndel1 mutant or deletion constructs  B  (B) , 50. AllTCC) (B) .via the HaloTag and quantified dynein depletion via SDS-PAGE. We found that FL-Ndel1 bound dynein with a Kd of ∼33 nM (C and S1E). Both NT-Ndel1 and Ndel1ΔCTCC displayed a reduced affinity for dynein  . Together, these results suggest that regions in the N-terminal and C-terminal halves of Ndel1 both contribute to interaction with dynein. FL-Ndel1E48A did not appreciably bind dynein (H), which indicates that the interaction between the C-terminal half of Ndel1 and dynein is not sufficiently strong to promote binding in the absence of a productive interaction between Ndel1 and the intermediate chain.First, we determined the binding affinities between each Ndel1 construct and dynein to determine the relative contribution of each of the dynein-binding sites of Ndel1. To do this, we conjugated increasing amounts of Ndel1 to magnetic beads f ∼33 nM C and S1Ed dynein H, which I). This finding is consistent with what has been observed with yeast proteins . Because many FL adaptors display autoinhibition, we used a well-characterized truncation of BicD2 containing amino acids 25 to 398  . We did not observe FL-Ndel1 comigrating with processive events, suggesting that Ndel1 does not alter motility by remaining bound to the moving complex (B).Next, we investigated if Ndel1 affects the motility of activated dynein complexes. To do this, we assembled TMR- or Alexa647-labeled dynein, purified dynactin, and the purified recombinant adaptor, BicD2, into complexes (DDB) D. Becausd BicD2) , 55, 56. D–F and A. We did complex B.E48A (C). As expected, NT-Ndel1E48A did not affect any motility parameters (D and G). NT-Ndel1 also reduced the landing rate of processive events, but to a lesser extent than FL-Ndel1 (compare an approximately fivefold reduction in landing rate with FL-Ndel1 to an approximately twofold reduction with NT-Ndel1) (H).To determine the relative contribution of the C-terminal binding site of Ndel1 to its effect on dynein motility, we repeated the smTIRF motility experiments with NT-Ndel1 and NT-Ndel1E48A C. As exprameters . Interesrameters , D and GT-Ndel1) . NT-NdelT-Ndel1) H.Kd measurements that show that the C-terminal half of Ndel1 increases its association with dynein (C). Thus, our data suggest that the C-terminal region of Ndel1 increases the potency of inhibition, but that the N-terminal coiled coil of Ndel1 is necessary and sufficient.Together, these data suggest that Ndel1 reduces the number of activated dynein complexes as well as the velocity and run length of processive DDB. Because both NT-Ndel1 and FL-Ndel1 reduced velocity and total processive events, we reason that the inhibitory effect of Ndel1 is mediated by its N-terminal intermediate chain–binding site. The increased inhibition observed with FL-Ndel1 compared with NT-Ndel1 is consistent with the h dynein C. Thus, A–C, S2D, and S1D). As with BicD2, we used a truncation of NINL (amino acids 1–702) to avoid autoinhibition (C). Unlike with DDB, however, FL-Ndel1 had little effect on the velocity or run length of processive DDN complexes (A and B).We next asked if Ndel1 had an inhibitory effect on dynein activated with different adaptors. Here, we reconstituted dynein motility with the adaptor ninein-like (NINL) and dynactin (together refered to as DDN) and determined if FL-Ndel1 affected DDN motility . To test this hypothesis, we conjugated FL-Ndel1 via the C-terminal 6X-His tag to magnetic beads and monitored its ability to bind and deplete dynein from solution. Here, we incubated FL-Ndel1-beads with dynein alone; dynein, dynactin, and BicD2; or dynein, dynactin, and NINL. FL-Ndel1 depleted over 80% of the dynein in solution in the absence of dynactin and adaptor (E). Inclusion of dynactin and BicD2 resulted in less binding between dynein and Ndel1, with a depletion of ∼65% of the dynein (E). Remarkably, only ∼11% of the total dynein was depleted by FL-Ndel1 in the presence of dynactin and NINL (E). Because dynactin and either adaptor reduced dynein’s binding affinity for FL-Ndel1, these results suggest that Ndel1 competes with dynactin and adaptors for dynein binding. This result supports the model that Ndel1 disfavors complex formation. These results also highlight that DDN is more refractory to Ndel1 inhibition than DDB, which may explain why DDN velocity and run length are less affected by Ndel1 than DDB.We reasoned that the reduction in processive events caused by Ndel1 with both adaptors could be explained if Ndel1 acted upstream of activation and disfavored the formation of the activated dynein complex D. To tes adaptor E. Incluse dynein E. Remarkand NINL E. BecausA and S3A)  , 64. Phoand S3A) , 61. In ervation , 64, 65.PM) (A and S1D). First, we determined that FL-Ndel1PM binds dynein with a twofold higher affinity than FL-Ndel1 (B and S3B) using the quantitative binding assay outlined previously. Next, we determined if FL-Ndel1PM functioned like FL-Ndel1 in DDB motility assays (C–F). At the concentration of Ndel1 used in previous smTIRF assays (300 nM), FL-Ndel1PM caused a decrease in the run length of processive events that was comparable to what we observed with FL-Ndel1 . FL-Ndel1PM reduced the velocity and landing rate to a slightly greater extent than FL-Ndel1 . To probe how much more effective the FL-Ndel1PM construct was, we reduced the concentration of FL-Ndel1PM and FL-Ndel1 to 50 nM and repeated the smTIRF motility experiments with DDB. At the reduced concentration, neither FL-Ndel1PM nor FL-Ndel1 had a statistically significant reduction in velocity or run length . Remarkably, inclusion of 50 nM FL-Ndel1PM reduced the processive landing rate of DDB by 10-fold, whereas 50 nM FL-Ndel1 reduced the landing rate by only 1.7-fold (C and F). We conclude that the phosphomimetic mutations in Ndel1 promote its binding to dynein and thereby increase its ability to inhibit the formation of the activated dynein complex.To explore the effect of phosphorylation on the ability of Ndel1 to modulate dynein motility, we purified a FL-Ndel1 construct with the four sites phosphorylated by CDK1  mutated to aspartic acid to mimic phosphorylation (FL-Ndel1PM) A and S1DFL-Ndel1 B and S3By assays , C–F. AtC and E, , and S2A1.7-fold , C and FPM. We conjugated each construct of Ndel1 to magnetic resin via the N-terminal HaloTag and monitored depletion of Lis1 from solution. We performed the initial binding experiments at 36 mM KCl, which matches the experimental conditions for all previous binding experiments and the smTIRF motility assays. At 36 mM KCl, the Kd between Lis1 and Ndel1 was far too low to accurately measure in the bead-based assay (data not shown). Therefore, we increased the ionic strength of the final assay buffer to 150 mM KCl and repeated the binding experiments. We found that all constructs of Ndel1 bound Lis1 with high affinity. Both FL-Ndel1 and FL-Ndel1PM had a Kd of ∼4 nM for Lis1 . These data suggest that phosphomimetic mutations of Ndel1 at the CDK1 target sites do not affect binding to Lis1. Interestingly, NT-Ndel1 exhibited an increased affinity for Lis1, with a Kd of ∼1.2 nM , which shows that removing the C terminus of Ndel1 increases its association with Lis1. This result is consistent with crosslinking-mass spectrometry data that suggest that the C-terminus of Ndel1 can fold back toward the N-terminal coiled coil and partially occlude the Lis1-binding site . AlphaFold predictions of just the C-terminal half of Ndel1  indicate that the putative heavy chain–binding site assumes a coiled-coil fold (B and C). Given the two conformations of the C-terminal region of Ndel1 predicted by AlphaFold, it is possible that Ndel1 exhibits a conformational equilibrium that regulates binding to Lis1 (E).The ability of Ndel1 to bind to Lis1 is well documented; however, the structural basis for their interaction is poorly defined , 66, 67.ing site . To furting site , 70, 71.oil fold , B and C to Lis1 E.Figure Saccharomyces cerevisiae Ndel1 and Lis1  have a synergistic effect on Pac1-mediated alteration of dynein velocity . As previously observed, Lis1 alone resulted in DDB complexes that moved with a faster velocity and exhibited a higher landing rate (C and D) (E). Next, we performed the DDB smTIRF motility experiments with equimolar FL-Ndel1PM and Lis1. If Ndel1 tethers Lis1 to dynein as has been proposed, we would anticipate that Lis1 and FL-Ndel1PM should activate dynein motility  to a greater extent than what was observed with Lis1 alone. Instead, we observed that the negative FL-Ndel1PM effect was attenuated by the positive Lis1 effect, with resulting events moving at velocities that were not significantly different than DDB alone (C). Remarkably, we observed that the inhibitory effect of FL-Ndel1PM outweighed the activating effect of Lis1 with respect to landing rate. Compared with DDB alone, Lis1 resulted in an approximately 4-fold increase in landing rate, whereas FL-Ndel1PM and Lis1 together exhibited approximately 2-fold reduction in landing rate (D). These results suggest that Ndel1 and Lis1 do not interact synergistically to increase dynein activation and that the Ndel1-mediated inhibition of DDB assembly prevents Lis1 from promoting complex formation.velocity . Becausevelocity , 72. Incvelocity , 36, 37. C–E and F. As preing rate , C and DC and D) , 21, 22.C and D) E. Next, DB alone C. Remarking rate D. These C). It is also well established that Lis1 binds dynein’s motor domain at the interface of AAA3 and AAA4 (AAA3/4) and along the stalk that emanates from AAA4 and connects dynein’s ATPase ring to the microtubule-binding domain (A) (F and G). We conjugated NT-Ndel1, FL-Ndel1, or FL-Ndel1PM to magnetic beads and monitored the amount of dynein depleted by each Ndel1 construct in the absence and presence of Lis1. Each Ndel1 construct depleted different amounts of dynein from solution, which is consistent with the measured Kd values (C and G). This suggests that Lis1 does not regulate the Ndel1–dynein interaction.We have observed that Lis1 binds Ndel1 directly, and that the N and C termini of Ndel1 both contribute to dynein binding C. It is g domain A 16, 18, 18C. Itmain (A) . We set main (A) , F and Gd values C and 3B.es C and G. This svia an N-terminal HaloTag. We incubated dynein alone or with tagless FL-Ndel1  and asked if Ndel1 affects the amount of dynein depleted by Lis1. In these experiments, in the absence of FL-Ndel1, we observed ∼40% of the dynein was depleted by Lis1, which is consistent with the Lis1–dynein Kd measured (I and S4G). Inclusion of FL-Ndel1 increased the amount of dynein depleted by Lis1 to ∼60% (I). This suggests that Ndel1 increases the association of dynein and Lis1, which is consistent with previous reports . Second, it is possible that Lis1 cannot bind dynein and Ndel1 at the same time and, instead, Ndel1 “daisy chains” Lis1 to dynein . This would lead to an increased apparent affinity between Lis1–dynein because Ndel1 binds dynein with a higher affinity than Lis1 does (compare a Kd of 33 nM for dynein–Ndel1 to 101 nM for dynein–Lis1), and Ndel1 and Lis1 are always bound in these experiments given their high-affinity interaction .There are two possible explanations for the apparent increased affinity between Lis1 and dynein in the presence of FL-Ndel1. First, Ndel1, dynein, and Lis1 could assemble in a tripartite structure, with Lis1 bound to dynein at the AAA3/4 and stalk sites, whereas Ndel1 bridges the dynein motor to interact with the intermediate chain and Lis1 simultaneously J, top. So dynein J, bottomE48A, which disrupts binding to dynein intermediate chain. If the dynein–Lis1–Ndel1 complex is tripartite, FL-Ndel1E48A would reduce the Lis1–dynein binding to the levels observed when no Ndel1 was included (∼40%). However, if Lis1 cannot bind dynein and Ndel1 at the same time and the increased affinity is due to an Ndel1 daisy chain, then FL-Ndel1E48A would inhibit Lis1–dynein binding. When we performed the experiment with FL-Ndel1E48A, we observed almost no dynein depletion by Lis1 (I). This result supports the daisy-chain model  and suggests that Lis1 cannot bind Ndel1 and dynein at the same time. This finding is consistent with a recent study that also showed that Lis1 does not bind Ndel1 and dynein concurrently   , suggestC) and found that phosphomimetic mutations in the C-terminus that mimic CDK1 phosphorylation positively regulate binding to dynein (B). Because many kinases target similar residues as CDK1, we propose that phosphorylation by multiple kinases may promote increased dynein–Ndel1 interaction (A). Further work is needed to understand how the C-terminal coiled-coil of Ndel1 promotes dynein binding and to determine the structural effect of phosphorylation. Our finding that the C-terminal half of Ndel1 negatively regulates association with Lis1 (A) suggests that FL-Ndel1 is autoinhibited with respect to Lis1 binding. Structural predictions and previous studies suggest that the C-terminus of Ndel1 contains a stretch of amino acids that can exist as a coiled coil or fold back and interact with the interface that Ndel1 uses to bind Lis1  (E). More studies will be required to test this hypothesis.We set out to explore how the core dynein regulator, Ndel1, affects activation of dynein motility. We found that the C-terminal half of Ndel1 functions in a regulatory capacity to modulate binding to both dynein and Lis1. First, we confirmed that the C-terminal coiled coil of Ndel1 participates in dynein binding C and fouo dynein B. Becauseraction A. Furtheith Lis1 A suggestand S4D) , 68. We and S4D) E. More sC–F and B–F). Therefore, we suggest that the C-terminal half of Ndel1 acts to stabilize the interaction with dynein and provide a mechanism for the magnitude of inhibition to be modulated by phosphorylation. Exploring the role of the p150–intermediate chain interaction in activated complex formation will be critical to understand how Ndel1 fits into the current model of dynein activity. We also observe that Ndel1 inhibits the formation of activated dynein complexes to different extents, depending on the identity of the adaptor . We observe that though Ndel1 can bind dynein and Lis1 at the same time, Lis1 does not bind both Ndel1 and dynein concurrently. This competition is likely because dynein and Ndel1 have close or overlapping binding sites on Lis1. Structural studies will be required to fully test this theory.In addition, our work shows that Ndel1 also inhibits dynein activation by preventing the direct association of dynein and Lis1 I. We obsPM was a more potent inhibitor of activated dynein complex assembly, we propose that phosphorylation of the C-terminal domain of Ndel1 also regulates its ability to recruit and inhibit dynein. In this model, Ndel1 must unbind from dynein and Lis1 for dynein activation to occur  at a ratio of 3:1 (Fugene reagent:DNA) according to the manufacturer’s directions. Cells were incubated at 27 °C for 3 days without agitation in a humid incubator. Next, the supernatant containing the virus (V0) was harvested by centrifugation . About 1 ml of the V0 virus was used to transfect 50 ml of Sf9 cells at 1 × 106 cells/ml to generate the next passage of virus (V1). Cells were incubated at 27 °C for 3 days with shaking at 105 rpm. Supernatant containing V1 virus was collected by centrifugation . All V1 were protected from light and stored at 4 °C until further use. To express protein, 4 ml of V1 virus were used to transfect 400 ml of Sf9 cells at a density of 1 × 106 cells/ml. Cells were incubated at 27 °C for 3 days with shaking at 105 rpm and collected by centrifugation . The pellet was washed with 10 ml of ice-cold PBS and collected again via centrifugation before being flash frozen in liquid nitrogen and stored at −80 °C until needed for protein purification.Dynein, Lis1, and Ndel1 constructs were expressed in Sf9 cells as described , 77. Brig, 88 min, 4 °C) in a Type 70Ti rotor (Beckman). The supernatant was mixed with 2 ml of IgG Sepharose 6 Fast Flow beads (Cytiva) equilibrated in Dynein-lysis buffer and incubated for 3 to 4 h with rotation along the long axis of the tube. The beads were transferred to a glass gravity column, washed with at least 200 ml of dynein-lysis buffer and 300 ml of tobacco etch virus (TEV) buffer . For fluorescent labeling of SNAP tag, dynein-bound beads were mixed with 5 μM SNAP-Cell-TMR or SNAP-AlexaFluor-647 (New England Biolabs) for 10 min at room temperature. Unconjugated dye was removed with a 300 ml wash with TEV buffer at 4 °C. The beads were resuspended in 15 ml of TEV buffer supplemented with 0.5 mM Pefabloc and 0.2 mg/ml TEV protease and incubated overnight with rotation along the long axis of the tube. Cleaved proteins in the supernatant were concentrated with a 100 K molecular weight cutoff (MWCO) concentrator (EMD Millipore) to 500 μl and purified via size-exclusion chromatography on a TSKgel G4000SWXL column (TOSOH Bioscience) with GF150 buffer  supplemented with 0.1 mM Mg–ATP as the mobile phase at 0.75 ml/min. Peak fractions were collected, buffer exchanged into a GF150 buffer supplemented with 0.1 mM Mg–ATP and 10% glycerol, and concentrated to 0.1 to 0.5 mg/ml using a 100 K MWCO concentrator. Small-volume aliquots were flash frozen in liquid nitrogen and stored at −80 °C.All steps for protein purification were performed at 4 °C unless indicated otherwise. For dynein preparation, Sf9 cell pellets were thawed on ice and resuspended in 40 ml of dynein-lysis buffer  supplemented with one cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per 50 ml. Cells were lysed with a Dounce homogenizer (ten strokes with a loose plunger followed by 15 strokes with a tight plunger). The lysate was clarified by centrifugation  with GF150 buffer as the mobile phase at 0.75 ml/min. For Lis1, the GF150 mobile phase was supplemented with 10% glycerol. SNAP-Lis1 was labeled with SNAP-AlexaFluor 647 (Promega) by incubating with 5 μM dye for 10 min at room temperature prior to size-exclusion chromatography. Peak fractions were collected, concentrated to 0.2 to 1 mg/ml with a 30 K MWCO concentrator, frozen in liquid nitrogen, and stored at −80 °C. Ndel1 purifications were supplemented with 10% glycerol before freezing.Lysis and clarification steps for the Ndel1 and Lis1 purifications were similar to the dynein purification except Lis1-lysis buffer  was used in place of dynein-lysis buffer. The clarified supernatant was mixed with 2 ml of IgG Sepharose 6 Fast Flow beads  and incubated for 2 to 3 h with rotation along the long axis of the tube. The beads were transferred to a gravity column, washed with at least 20 ml of Lis1-lysis buffer, 200 ml of Lis1-TEV buffer  supplemented with 100 mM potassium acetate and 0.5 mM Pefabloc, and 100 ml of Lis1-TEV buffer. For fluorescent labeling of Ndel1, the Ndel1-bound beads were mixed with 5 μM Halo-JFX646 (Lavis Lab) for 10 min at room temperature after the lysis buffer wash. TEV protease was added to the beads at a final concentration of 0.2 mg/ml, and the beads were incubated overnight with rotation along the long axis of the tube. Cleaved Lis1 or Ndel1 in the supernatant was collected and concentrated to 500 μl with a 30 K MWCO concentrator. The concentrated protein was then purified via centrifugation  in a Type 70 Ti rotor (Beckman). The supernatant was mixed with 1.5 ml of anti-FLAG M2 affinity gel (Sigma–Aldrich) and incubated overnight with rotation along the long axis of the tube. The beads were transferred to a glass gravity column, washed with at least 50 ml of wash buffer , 100 ml of wash buffer supplemented with 250 mM potassium acetate, and then washed again with 100 ml of wash buffer. About 1 ml of elution buffer (wash buffer with 2 mg/ml of 3xFLAG peptide) was used to elute dynactin, which was then filtered via centrifuging through an Ultrafree-MC VV filter (EMD Millipore) in a tabletop centrifuge according to the manufacturer’s instructions. The filtered dynactin was then diluted to 2 ml in buffer A  and loaded onto a MonoQ 5/50 GL column (Cytiva) at 1 ml/min. The column was prewashed with 10 column volumes (CVs) of buffer A, 10 CVs of buffer B  and then equilibrated with 10 CVs of buffer A. A linear gradient was run over 26 CVs from 35 to 100% buffer B. Pure dynactin eluted between 75 and 80% buffer B. Peak fractions were collected, pooled, buffer exchanged into a GF150 buffer supplemented with 10% glycerol, concentrated to 0.02 to 0.1 mg/ml using a 100 K MWCO concentrator, aliquoted into small volumes, and then flash frozen in liquid nitrogen.Dynactin was purified from human embryonic kidney 293T cell lines stably expressing p62-Halo-3xFLAG as described . Brieflyg for 30 min in Type 70 Ti rotor. The clarified supernatant was incubated with 2 ml of IgG Sepharose 6 Fast Flow beads (Cytiva) for 2 h on a roller. The beads were transferred into a gravity-flow column, washed with 100 ml of activating-adaptor-lysis buffer supplemented with 150 mM potassium acetate and 50 ml of cleavage buffer . The beads were then resuspended and incubated in 15 ml of cleavage buffer supplemented with 0.2 mg/ml TEV protease overnight with rotation along the long axis of the tube. The supernatant containing cleaved proteins was concentrated using a 30 kDa MWCO concentrator to 1 ml, filtered by centrifuging with Ultrafree-MC VV filter (EMD Millipore) in a tabletop centrifuge, diluted to 2 ml in buffer A  and injected into a MonoQ 5/50 GL column at 1 m/min. The column was prewashed with 10 CVs of buffer A, 10 CVs of buffer B  and again with 10 CVs of buffer A. To elute, a linear gradient was run over 26 CVs from 0 to 100% buffer B. The peak fractions containing Halo-tagged activating adaptors were collected and concentrated to using a 30 kDa MWCO concentrator to 0.2 ml, diluted to 0.5 ml in GF150 buffer, and further purified using size-exclusion chromatography on a Superose 6 Increase 10/300 GL column (Cytiva) with GF150 buffer at 0.5 ml/min. The peak fractions were collected, buffer-exchanged into a GF150 buffer supplemented with 10% glycerol, concentrated to 0.2 to 1 mg/ml using a 30 kDa MWCO concentrator, and flash frozen in liquid nitrogen.BicD2 and NINL containing amino-terminal HaloTags were expressed in BL-21[DE3] cells (New England Biolabs) at an absorbance at 600 nm of 0.4 to 0.6 with 0.1 mM IPTG for 16 h at 18  °C. Frozen cell pellets from a 1.5 l culture were resuspended in 40 ml of activating-adaptor-lysis buffer  supplemented with 1× cOmplete EDTA-free protease inhibitor cocktail tablets and 1 mg/ml lysozyme. The resuspension was incubated on ice for 30 min and lysed by sonication. The lysate was clarified by centrifuging at 66,000To predict the structure of FL-Ndel1 and the C-terminal half of Ndel1, we used the COSMIC2 web platform to run the AlphaFold2 Colab Notebook on FL-Ndel1 or FL-Ndel1 (amino acids 194–345) , 70, 71.via SDS-PAGE to confirm complete depletion of Ndel1. The Ndel1-conjugated beads were washed once with 1 ml GF150 with 10% glycerol and 0.1% NP-40 and once with 1 ml of binding buffer  supplemented with 36 mM KCl . About 5 nM dynein or Lis1 was diluted in binding buffer, which resulted in binding buffer with 36 mM KCl . About 25 μl of the dynein or Lis1 mixture was added to the beads prebound with Ndel1 and gently agitated for 45 min. After incubation, 20 μl of the supernatant was removed, and 6.67 μl of NuPAGE LDS Sample Buffer (4×) and 1.33 μl of beta-mercaptoethanol was added to each. The samples were boiled for 5 min before running on a 4 to 12% NuPAGE Bis–Tris gel. Depletion was determined using densitometry in ImageJ. For dynein–Ndel1 and dynein–Lis1 binding data, curves were fit in Prism 9  with a nonlinear regression for one site binding with Bmax set to 1. Because the Lis1–Ndel1 interaction is high affinity and the concentration of Lis1 used in the Lis1–Ndel1 binding assays is comparable to the observed Kd, we fit curves for Lis1–Ndel1 binding data with a quadratic binding equation, as described  in 2 ml Protein Lo Bind Tubes (Eppendorf) using the following protocol. Beads were washed twice with 1 ml of GF150 without ATP supplemented with 10% glycerol and 0.1% NP-40. Ndel1 was diluted in this buffer to 0, 30, 60, 90, 120, 300, and 600 nM. About 25 μl of diluted Ndel1 was added to the beads and gently shaken for 1 h. About 20 μl of supernatant were then analyzed escribed .Experiments measuring binding between dynein, Lis1, and Ndel1 were performed with the same method and buffers as aforementioned. For the experiment with Lis1 on the beads, 50 nM of Halo-Lis1 was conjugated to the beads and incubated with 5 nM dynein with and without 100 nM of WT and E48A Ndel1. For the experiment with Ndel1 on the beads, 30 nM of Halo-Ndel1 was conjugated to beads and incubated with 5 nM dynein in the presence and absence of 30 nM Lis1.For experiments investigating the competition between Ndel1 and dynactin–activating adaptor, the methods and buffers were the same as aforementioned. About 90 nM Ndel1 was conjugated to 30 μl of NEBExpress Ni–NTA Magnetic Beads (NEB) and incubated with 5 nM dynein. The appropriate samples were further supplemented with 10 nM dynactin and 150 nM of either BicD2 or NINL. Three or more replicates were performed for all binding assays.Single-molecule imaging was performed with an inverted microscope  with a 100× 1.49 numerical aperture oil immersion objective . The microscope was equipped with a LUNF-XL laser launch (Nikon), with 405, 488, 561, and 640 nm laser lines. The excitation path was filtered using an appropriate quad bandpass filter cube (Chroma). The emission path was filtered through appropriate emission filters (Chroma) located in a high-speed filter wheel (Finger Lakes Instrumentation). Emitted signals were detected on an electron-multiplying CCD camera . Image acquisition was controlled by NIS Elements Advanced Research software (Nikon).Single-molecule motility and microtubule-binding assays were performed in flow chambers assembled as described previously , 79. EitTo assemble dynein–dynactin–activating adaptor complexes, purified dynein (10–20 nM concentration), dynactin, and the activating adaptor were mixed at 1:2:10 M ratio and incubated on ice for 10 min. These dynein–dynactin–activating adaptor complexes were then incubated with Ndel1 and/or Lis1 or modified TEV buffer (to buffer match for experiments without Ndel1 or Lis1) for 10 min on ice. Dynactin and the activating adaptors were omitted for the experiments with dynein alone. The mixtures of dynein, dynactin, activating adaptor or dynein alone, and Nde1l/Lis1 were then flowed into the flow chamber assembled with taxol-stabilized microtubules. The final imaging buffer contained the assay buffer supplemented with 20 μM taxol, 1 mg/ml casein, 71.5 mM β-mercaptoethanol, 0.05 mg/ml glucose catalase, 1.2 mg/ml glucose oxidase, 0.4% glucose, and 2.5 mM Mg–ATP. The final concentration of dynein in the flow chamber was 0.5 to 1 nM for experiments with dynein–dynactin–activating adaptor complexes and 0.05 nM for dynein-alone experiments. For standard motility experiments, our final imaging buffer contained 37.5 mM KCl.For single-molecule motility assays, microtubules were imaged first by taking a single-frame snapshot. Dynein labeled with fluorophores (TMR or Alexa647) was imaged every 300 ms for 3 min. At the end, microtubules were imaged again by taking a snapshot to assess stage drift. Movies showing significant drift were not analyzed. Each sample was imaged no longer than 10 min. For single-molecule microtubule binding assays, the final imaging mixture containing dynein was incubated for an additional 5 min in the flow chamber at room temperature before imaging. After 5 min incubation, microtubules were imaged first by taking a single-frame snapshot. Dynein labeled with fluorophores (TMR or Alexa64) were imaged by taking a single-frame snapshot. Each sample was imaged at two different fields of view, and there were between 15 and 25 microtubules in each field of view.x-direction and 300 ms in the y-direction. Bright protein aggregates, which were defined as molecules 4× brighter than the background, were excluded. Velocity and run length were reported for individual dynein complexes. For the analysis of landing rate, the number of processive events measured on individual microtubules was divided by the length of the microtubule, the concentration of dynein, and the total imaging time. Landing rate was calculated for five microtubules per field of view for each sample. Data were then normalized to the landing rate of the positive control (dynein–dynactin–adaptor complex without Ndel1 and/or Lis1) collected on the same slide. Data plotting and statistical analyses were performed in Prism 9. Three technical replicates, including at least two individual dynein preparations , were performed for each smTIRF motility experiment. Data from individual replicates are shown in different colors  in A–C, D–F, and C–E.Kymographs were generated from motility movies, and dynein velocity and run length were calculated from kymographs using custom ImageJ macros as described . Only ruAll data from this publication can be found in the This article contains The authors declare that they have no conflicts of interest with the contents of this article.


In plastic surgery, tissue expansion is widely used for repairing skin defects. However, low expansion efficiency and skin rupture caused by thin, expanded skin remain significant challenges in promoting skin regeneration during expansion. S100 calcium-binding protein A9 (S100A9) is essential in promoting wound healing; however, its effects on skin regeneration during tissue expansion remain unclear. The aim of the present study was to explore the role of S100A9 in skin regeneration, particularly collagen production to investigate its importance in skin regeneration during tissue expansion.in vitro. The effects of S100A9 on the proliferation and migration of skin fibroblasts were also observed. TAK-242 was used to inhibit the binding of S100A9 to TLR-4; the levels of collagen I (COL I), transforming growth factor beta (TGF-β), TLR-4 and phospho-extracellular signal-related kinase 1/2 (p-ERK1/2) in fibroblasts were determined. Furthermore, fibroblasts were co-cultured with stretched S100A9-knockout keratinocytes by siRNA transfection and the levels of COL I, TGF-β, TLR-4 and p-ERK1/2 in fibroblasts were investigated. Additionally, the area of expanded skin, thickness of the dermis, and synthesis of COL I, TGF-β, TLR-4 and p-ERK1/2 were analysed to determine the effects of S100A9 on expanded skin.The expression and distribution of S100A9 and its receptors—toll-like receptor 4 (TLR-4) and receptor for advanced glycation end products were studied in expanded skin. These characteristics were investigated in skin samples of rats and patients. Moreover, the expression of S100A9 was investigated in stretched keratinocytes in vivo as well as increasing ECM deposition during expansion.Increased expression of S100A9 and TLR-4 was associated with decreased extracellular matrix (ECM) in the expanded dermis. Furthermore, S100A9 facilitated the proliferation and migration of human skin fibroblasts as well as the expression of COL I and TGF-β in fibroblasts via the TLR-4/ERK1/2 pathway. We found that mechanical stretch-induced S100A9 expression and secretion of keratinocytes stimulated COL I, TGF-β, TLR-4 and p-ERK1/2 expression in skin fibroblasts. Recombined S100A9 protein aided expanded skin regeneration and rescued dermal thinning in rats These findings demonstrate that mechanical stretch promoted expanded skin regeneration by upregulating S100A9 expression. Our study laid the foundation for clinically improving tissue expansion using S100A9.  HighlightsDermal thinning could cause skin rupture which inhibited skin regeneration during tissue expansion.S100A9, which was up-regulated and secreted in the expanded epidermis, elevated collagen synthesis and enhanced ECM deposition in expanded skin via TLR-4 and activation of ERK1/2 protein, causing an increase in dermal thickness.We showed that S100A9 might be a possible therapeutic target for dermal thinning during skin expansion.Skin tissue expansion has been widely used for repairing skin defects and congenital malformations . NeumannTissue expansion frequently results in dermal thinning, which may further lead to skin rupture , 8. TherIn the present study, to investigate the role of S100A9 in skin regeneration during tissue expansion, we examined S100A9 distribution in expanded human and rat skin tissues. We further explored its effects on skin regeneration, particularly collagen production, which is a novel candidate option for accelerating skin regeneration during tissue expansion in plastic surgery.ad libitum access to water. All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals by the National Research Council (US) Committee . All experimental procedures were approved by the ethics committee of the Fourth Military Medical University (IACUC-20191207); every effort was made to reduce the number and suffering of animals. All animal experiments were conducted in the Laboratory of the Department of Plastic Surgery at Xijing Hospital, Fourth Military Medical University.Adult male Sprague–Dawley rats weighing 200–250 g were purchased from the Fourth Military Medical University’s Animal Center. Rats were housed in a clean, temperature-controlled environment with a 12-h light/dark cycle, fed a standard laboratory diet and provided 2 square area in the middle of the expanded flap was tattooed for tracing the expanding areas. The expander was implanted beneath the scalp, following which 1 ml of saline was injected into it. The control group was a sham expansion group implanted with a silicone sheet. Expanders in the expanded group were routinely inflated , whereas the control group received no injections. The first saline injection was designated as day 0. Tissues were harvested on day 28 and used for mRNA and protein analysis. Animals that completed the study were intraperitoneally euthanised using large amounts of pentobarbital.The rats were randomly divided into two groups: control (n = 8) and expanded (n = 8). The rat scalp expansion model was created using our previously described procedures . BrieflyHuman tissue samples including 11 expanded skin and 4 normal skin biopsies were collected from various volunteers. Expanded skin samples had been expanded for ~3 months before collection. The control skin comprised normal skin samples. The harvested skin samples were fixed in 4% paraformaldehyde before being embedded in paraffin. Sections of 4-μm thick were stained with haematoxylin and eosin (H&E). Masson’s trichrome and Sirius red staining were used to assess the dermal thickness, collagen content and collagen types.http://www.annoroad.com/) using Illumina HiSeq 4000 [Our previous study described the procedures for RNA-seq of expanded rat skin . BrieflySeq 4000 . The seqAfter being deparaffinized and rehydrated in varying alcohol concentrations, the tissue sections were placed in a sodium citrate solution at 96°C for 20 min before being blocked for 1.5 h in phosphate-buffered solution (PBS) containing 5% bovine serum albumin and incubated with rabbit anti-rat S100A9 , rabbit anti-rat TLR-4 , rabbit anti-rat RAGE  and rabbit anti-human S100A9  overnight at 4°C in humid chambers. The sections were incubated in the same solution for 2 h at room temperature (22–24°C) on the following day before incubation with a goat anti-rabbit Alexa Fluor 488 secondary antibody  or goat anti-rabbit Alexa Fluor 594 secondary antibody  at room temperature for 1 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole at 1 : 1000 dilution .2 humidified atmosphere at 37°C.HaCaT cells and human skin fibroblasts were cultured in Dulbecco’s modified Eagle’s medium . All culture media were supplemented with 10% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin and cultured in a 5% CO5 cells/cm2. A uniaxial sinusoidal stretch of 12% at a frequency of 15 cycles/min with sine wave was applied using a stretching apparatus  driven by a computer-controlled stepping motor, as previously demonstrated [HaCaT cells were seeded on a silicone chamber coated with fibronectin (100 μg/ml) at a density of 10nstrated . The relAfter stretching, the S100A9 concentrations in the supernatants of cultured HaCaT cells were collected at 0, 3, 6, 12 and 24 h and measured using an S100A9 enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.The effects of S100A9 on human skin fibroblast proliferation were evaluated using the cell counting kit-8 (CCK-8) assay . Briefly, fibroblasts were seeded in 96-well plates and starved for 12 h. Then, the medium was supplemented with culture medium with or without S100A9 ; the cells were tested with CCK-8 reagent at 0, 24, 48 and 72 h.5 cells/well. After 24 h, S100A9 solutions of varying concentrations  were added. Each group had three parallel wells. At 0 and 24 h, the cells were cultured with 200 μL of 1× EdU medium  for 4 h and fixed with 200 μL of cell-fixation solution  for 15 min at room temperature. Subsequently, the cells were washed and rinsed with 200 μL of PBS containing 0.5% TritonX-100  for 10 min before staining with 1× Apollo staining reaction solution  for 30 min in the dark. The cells were treated with 100 μL of 1× Hoechst 33342 reaction solution  for 30 min before being sealed with 200 μL of the anti-fluorescence quenching agent. Approximately 6–10 fields of view were randomly selected for each well and photographed using a fluorescence microscope .The 5-ethynyl-2′-deoxyuridine (EdU) assay was used to assess fibroblast proliferation. Human skin fibroblasts were cultured in the 24-well plates at a density of 1 × 10Human skin fibroblasts were seeded in six-well plates. After cell density reached 90% confluency, the cells were starved for 12 h. The cell monolayer was scratched and lined with a pipette tip before being treated with S100A9 (500 ng/ml) cultured in 2% Dulbecco’s modified Eagle’s medium for 24 h. The cells were washed with PBS and cultured in serum-free medium at 0 and 24 h. The distance between the cells on both sides of the scratch was calculated using ImageJ software (1.6.0 20). The tests were performed at least three times.5 cells/well) were seeded into the upper chamber in 200 μl serum-free medium; 500 μl complete medium containing 10% fetal bovine serum was placed in the lower chamber as a chemoattractant. Following incubation for 24 h, cells were treated with or without S100A9 (500 ng/ml) for 0 and 24 h. The membranes were fixed in 4% paraformaldehyde for 15 min and stained with crystal violet for 20 min. The cells on the upper side of the membranes were gently wiped away with a cotton swab; cells on the lower side of the membranes were observed and photographed using a microscope .Moreover, the transwell assay was used to evaluate the migration ability of fibroblasts. The transwell cell culture chamber contains a polycarbonate membrane with a pore size of 4 μm . The cells (2 × 10−ΔΔCt (threshold cycle) method [Total RNA was extracted from the rat scalp samples and cells using TRIzol  and reverse transcribed into cDNA with the PrimeScript™ RT reagent kit  and TB Green™ Premix Ex Taq™ . A quantitative polymerase chain reaction (qPCR) analysis was performed using Bio-Rad CFX Manager 3.0. The mRNA expression levels were measured using the 2) method . The priThe proteins of tissue samples and cells were lysed in radioimmunoprecipitation assay buffer . A bicinchoninic acid assay (BCA) kit  was used to quantify protein concentrations. Proteins were separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis  and transferred to polyvinylidene difluoride membranes . After blocking for 1 h in PBS containing 5% bovine serum albumin, the membranes were incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat S100A9 , rabbit anti-rat TLR-4 , rabbit anti-rat RAGE , rabbit anti-rat COL I , rabbit anti-rat TGF-β , rabbit anti-rat extracellular signal-related kinase 1/2 (ERK1/2) , rabbit anti-rat phospho-ERK 1/2 (p-ERK1/2)  and rabbit anti-rat GAPDH . After washing three times in Tris-buffered saline with 0.1% Tween-20, the membranes were incubated with either goat anti-rabbit secondary antibody  or goat anti-mouse secondary antibody  for 1 h. The membrane was then washed three times before visualization using a biological image shooting software .The human skin fibroblasts were seeded in 6-well plates. After the cell density reached 90% confluency, the cells were treated with PBS, S100A9 (500 ng/ml), TAK-242  or S100A9 + TAK-242 (500 ng/ml S100A9 + 1 μM TAK-242). After 24 h, the cells were collected and prepared for qPCR and western blot assay.The HaCaT cells were plated at 40–50% density and grown to ~75% confluency. On the following day, the cells were treated with 20 nM negative control small interfering RNA (siRNA) (FAM-siRNA) or S100A9 siRNAs dissolved in a non-serum medium according to the manufacturer’s protocol . The S100A9 siRNA sequences were as follows, sense strand 5’-CAUCAACACCUUCCACCAAUATT-3′ and anti-sense strand 5’-UAUUGGUGGAAGGUGUUGAUGTT-3′. The cells and their proteins were collected 24 h after transfection. Subsequently, qPCR and western blot were applied to investigate the effects of S100A9 siRNA.The human skin fibroblasts were co-cultured with normal HaCaT cell culture supernatants, stretched HaCaT cell culture supernatants, negative control siRNA + stretched HaCaT cells, and S100A9 siRNA + stretched HaCaT cells, respectively. These fibroblasts’ proteins were collected 24 h after co-culture and used to test the expression of COL I, TGF-β, TLR-4, ERK1/2, and p-ERK1/2.For animal experiments, rats were randomly divided into four groups (n = 6): the PBS-injected expanded group (injected with 100 μl of PBS into expanded skin); S100A9-injected expanded group (injected with 100 μl of 5 μg/ml recombinant S100A9 protein into expanded skin according to a previous study ); TAK-24post hoc test (Tukey’s test). A p value of < 0.05 (*), < 0.01 (**), or < 0.001 (***) was used to determine statistical significance.All experiments were performed at least three times. All data are presented as the mean ± SD values. Statistical analyses of differences between groups were performed using a Student’s unpaired t-test, one-way analysis of variance (ANOVA), or two-way ANOVA (GraphPad Prism 6.0). Following one-way or two-way ANOVA, we performed the vs 533.90 ± 11.40 μm, p < 0.001, vs 46.52 ± 3.94%, p < 0.01, Mechanical stretch caused significant alterations in dermal thickness and collagen content. The macroscopic appearance of rats from the control and expanded groups are shown in p < 0.001, To determine the characteristics of S100A9 in expanded rat skin, we used RNA-seq analysis to detect its expression. The transcriptomic data revealed that S100A9 was overexpressed in expanded rat skin . The qPCp < 0.01, p < 0.01, Exploring their protein expression and cellular locations will aid future research. Using immunofluorescence staining, we investigated the cellular localization of S100A9 and discovered that it was chiefly distributed in the cytoplasm of keratinocytes . In addip < 0.01, p < 0.05, p < 0.001, Furthermore, the expression and cellular location of S100A9 and TLR-4 were explored in expanded human skin. The qPCR results showed upregulated mRNA levels of S100A9 and TLR-4  compared with the PBS-treated group (4.36 ± 0.24 cm2), TAK-242–treated skin (4.38 ± 0.44 cm2) and S100A9 + TAK-242–treated skin  and nuclear factor-κB (NF-κB) pathways [To investigate the effects of recombined S100A9 protein on ECM deposition, we measured the mRNA and protein levels of COL I and TGF-β in S100A9-treated expanded rat skin. Compared with the S100A9-treated skin, the expression of these genes was reduced in all the remaining three groups . Considepathways . The proin vivo by binding to the TLR-4 receptor and activating the ERK1/2 signalling pathway. In vitro, S100A9 was increased in stretched HaCaT cells. S100A9 accelerated human skin fibroblast proliferation, migration and collagen synthesis. Furthermore, the co-culture of fibroblasts with the supernatants of stretched HaCaT cells induced a higher expression of collagen in skin fibroblasts, indicating that S100A9 synthesised and secreted from expanded epidermal keratinocytes may function in promoting expanded dermal regeneration via a paracrine mode by binding to fibroblast TLR-4.Tissue expansion provides patients with additional skin for defect repair. However, issues such as low expansion efficiency and skin rupture challenge the application of this technique . To addrS100A9 is a low-molecular-weight protein that participates in multiple cellular processes , 32. TypIn vitro studies have revealed that S100A9 promoted fibroblast proliferation and migration. All these factors contribute to dermal thickening because the dermis primarily comprises ECM and fibroblasts. These findings suggested that the in vivo application of S100A9 promoted dermal thickening and skin regeneration during tissue expansion.Several studies have shown that cytokines play a role in cellular processes contributing to skin regeneration during tissue expansion. S100A9 is essential for ECM deposition , 37. In However, dermal thinning was accompanied by an increase in endogenous S100A9 expression during growth; this was attributed to two factors. On the one hand, tissue expansion resulted in dermal thinning and repeated skin injuries. Endogenous S100A9 protein was gradually secreted under this microenvironment and acted as a ‘fireman’ to rescue dermal thinning. However, dermal thinning could not be reversed at the time. On the other hand, endogenous S100A9 was insufficient to completely reverse dermal thinning. To test our hypothesis, we injected the expanded skin with recombined S100A9 protein. The expanded dermis thickened as expected, possibly owing to the increased S100A9 protein level in the expanded skin. These findings suggested that measures to facilitate endogenous S100A9 secretion in expanded skin should be implemented at the earliest opportunity to promote skin regeneration. Moreover, using exogenous S100A9 protein during skin expansion would be beneficial.in vitro findings. S100A9 promoted ERK1/2 phosphorylation in human lung fibroblasts [Secreted S100A9 binds to the receptors TLR-4 , 39 and roblasts . Furtherroblasts . Previouroblasts , therebyroblasts  and Fos roblasts . BecauseThe findings of the present study demonstrated that S100A9 could be activated by mechanical stretch during tissue expansion; its activation alleviated dermal thinning, increased skin area, and improved tissue ECM production by binding to the TLR-4 receptor and activating ERK1/2. Therefore, S100A9 could be a promising intervention target or medication for reversing dermal thinning and promoting skin regeneration during tissue expansion.Figure_S1_tkad030Click here for additional data file.Figure_S2_tkad030Click here for additional data file.Table_S1_tkad030Click here for additional data file.Table_S2_tkad030Click here for additional data file.


ATP-binding cassette subfamily C member 6 (ABCC6). On a molecular level, PXE shares similarities with Hutchinson–Gilford progeria syndrome, such as increased activity of senescence-associated- beta-galactosidase or high expression of inflammatory factors. Thus, this study’s aim was the evaluation of activated STAT3 and the influence of JAK1/2-inhibitor baricitinib (BA) on inflammatory processes such as the complement system in PXE. Analysis of activation of STAT3 was performed by immunofluorescence and Western blot, while inflammatory processes and complement system factors were determined based on mRNA expression and protein level. Our results assume overactivation of JAK/STAT3 signaling, increased expression levels of several complement factors and high C3 protein concentration in the sera of PXE patients. Supplementation with BA reduces JAK/STAT3 activation and partly reduces inflammation as well as the gene expression of complement factors belonging to the C1 complex and C3 convertase in PXE fibroblasts. Our results indicate a link between JAK/STAT3 signaling and complement activation contributing to the proinflammatory phenotype in PXE fibroblasts.Previous studies revealed a link between inflammation and overactivation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling in syndromes associated with aging. Pseudoxanthoma elasticum (PXE), a rare autosomal-recessive disorder, arises from mutations in  The main characteristic of PXE is the ectopic calcification of connective tissue, particularly elastic fibers. Clinical manifestations of PXE are seen in the skin, eyes or cardiovascular system. Symptoms of patients with PXE in the early stage of the disease are yellow papules at flexural body sites, which coalesce, leading to sagging and wrinkled skin . In addil vision .ATP-binding cassette subfamily C member 6 (ABCC6), resulting in a loss of function of the encoded ABC-transporter protein [ABCC6 gene have been identified in PXE patients to date, but the physiological substrate of ABCC6 remains unknown, which makes the development of an effective therapy difficult [The origin of PXE are mutations in the gene of  protein . ABCC6 i protein . Over 50ifficult .Our previous studies showed an association between PXE and premature aging . FurtherMCP-1 and IL-6, as well as the regulatory protein suppressor of cytokine signaling (SOCS3). SOCS3 is highly expressed in inflamed tissue and reduces activation of JAK1/2 [As shown before, IL-6 is considered one of the most prominent proinflammatory cytokines and plays an important role in chronic inflammatory processes ,20. On tf JAK1/2 ,24. An af JAK1/2 ,26. As sf JAK1/2 ,28.SERPING1, which inhibits the classical pathway by blocking the enzymatic activity of the C1-complex. The alternative pathway is regulated by complement factor H (CFH), which inhibits the cleavage of C3, a necessary step for complement activation [It is known that, in addition to IL-6 signaling, STAT3 activation can also be induced by complement factor 3 (C3) . C3 belotivation ,34. Deretivation .Because of the association between PXE, premature aging and inflammatory processes, we hypothesized that JAK/STAT3 signaling and the complement system might play a role in PXE pathogenesis. For a better understanding of inflammatory processes in PXE pathogenesis, we investigate the activation of STAT3 and the expression of the complement system in dermal fibroblasts of PXE patients (PXEF). Furthermore, we analyze the PXE phenotype after blocking JAK/STAT3 signaling by the specific inhibitor baricitinib (BA) to determine the role of JAK/STAT3 signaling and the complement cascade in inflammatory processes in PXE.STAT3 and suppressor of cytokine signaling 3. Furthermore, the effect of BA was determined by measuring the mRNA expression of factors that are involved in the generation of SASP, such as IL-6, MCP-1 and CXCL1, as well as the protein concentration of IL-6 and MCP-1. Furthermore, the mRNA expression of complement system factors complement factor 1r (C1r), 1s (C1s), 2 (C2), C3, 4A (C4A), FB (CFB), CFH and SERPING1 and protein concentration in the supernatant of C3, C3a and C1r under standard conditions and treatment with BA were determined. Additionally, C3 protein concentrations in sera from PXE patients and sex- and age-matched healthy controls were examined.This study was designed to evaluate the activation status of JAK/STAT3 signaling and the effect of blocking JAK/STAT3 signaling by specific JAK1/2 inhibitor BA on primary human dermal fibroblasts of PXE patients. Accordingly, PXEF and fibroblasts from sex- and age-matched healthy controls (NHDF) were cultivated with 10% lipoprotein-deficient fetal calf serum (LPDS). Additional treatment with BA was performed to evaluate the effects on PXEF. The activation status of JAK/STAT3 signaling and the effect of BA on this activation status were evaluated in PXEF by immunofluorescence, Western blot and mRNA expression of The diagnosis of PXE in all patients was consistent with the consensus criteria, as reported previously . Sera frPrimary PXEF from patients were isolated from skin biopsies as described previously . All cha2 in 60 mm culture dishes  or on coverslips (Ø 18 mm) coated with 5 µg/cm2 rat collagen  for immunofluorescence experiments and were cultivated in medium supplemented with 10% FCS. After 24 h, the medium was changed to Dulbecco’s modified essential medium supplemented with 10% LPDS and 0.01% (v/v) dimethyl sulfoxide , with BA in a final concentration of 1 µM or for immunofluorescence with IL-6 (50 ng/mL) or BA (1 µM) and IL-6 (50 ng/mL). Fibroblasts were cultivated for an additional 72 h, prepared for further experiments and analyzed.The PXEF and NHDF were cultivated in Dulbecco’s modified essential medium  containing 10% fetal calf serum , 2% L-glutamine (200 mM)  and 1% antibiotic/antimycotic solution . After reaching 85–95% confluence, fibroblasts were sub-cultivated. Fibroblasts between passages 8 and 12 were used in this study. Biological samples were prepared in triplicate. In an experimental context, fibroblasts were seeded in a final density of 177 cells/mmg for 1 h at 4 °C. The supernatant was transferred into a new tube and used directly or stored at −20 °C until further use. The LPDS was sterile filtered  before preparation of medium. After delipidation, the concentration of free cholesterol was lowered by about 78%, low-density lipoprotein by about 95% and high-density lipoprotein by about 57%, but the triglyceride concentrations remained unchanged, as described previously [The preparation of LPDS was carried out according to . In brieeviously .A commercially available NucleoSpin RNA Kit  was used for the isolation of RNA. DNA isolation was carried out by using a NucleoSpin Blood Extraction Kit . Isolation procedure was performed as described by the manufacturer. For both, the procedures were determined on a NanoDrop2000 spectrophotometer .ß-actin (ACTB), glyceraldehyde-3-phosphatase-dehydrogenase (GAPDH) and ß2-microglobulin (ß2M) were used for normalization. Finally, a melting curve was measured after amplification for the analysis of purity. Three technical replicates were performed for each biological sample. All measurements were carried out by using a LightCycler480 . The ΔΔCt method, considering PCR efficiency, was used for the calculation of relative mRNA expression. All sequences of the primer used for qPCR are listed in A total of 1 µg RNA was transcribed into cDNA by using the SuperScript II Reverse Transcriptase , as described by the manufacturer. Regarding all gene expression measurements, 2.5 µL cDNA (1:10 diluted in water), 0.25 µL forward and reverse primer  with a final concentration of 25 µM, 2 µL water and 5 µL LightCycler 480 SYBR Green I Master reaction mixture  were used. The protocol for quantitative real-time PCR (qPCR) is shown in Fibroblasts were analyzed via immunofluorescence for the evaluation of active pSTAT3. After treatment with BA, cells were washed once with 1× PBS and fixed with 4% formaldehyde  for 10 min. Afterwards, fibroblasts were washed three times for 5 min each and permeabilized in cold methanol . After three washing steps, each for 5 min, unspecific binding sites were blocked for 1 h at room temperature in 1× PBS supplemented with 5% goat serum  and 0.3% Triton X-100 . After the blocking step, fibroblasts were incubated with primary anti-pSTAT3 (pTyr705) antibody  overnight at 4 °C. The next day, the fibroblasts were washed three times for 5 min each with 1× PBS and incubated with secondary goat anti-rabbit antibody  for 1 h at room temperature. After an additional three washing steps (each 5 min), fibroblasts were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, the coverslips were washed three times in 1× PBS and mounted with ProLong Diamond Antifade Mountant . Six pictures per cell culture condition were analyzed for the assessment of immunofluorescence by using the software ImageJ 1.53. Briefly, the area of the nuclei was bordered, and the fluorescence intensity was measured and relativized to the area.g for 5 min and stored at −80 °C for further experiments. An amount of 20 µg of protein and 5 µL multicolor broad range protein ladder  were separated by using 8–16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The protein blots were transferred on polyvinylidene fluoride membrane  in a Mini Gel Tank . After transfer, membranes were blocked in 5% skimmed milk in Tris-buffered saline supplemented with 0.1% (v/v) Tween 20 for 1 h. Thereafter, membranes were incubated with primary antibodies against pSTAT3 in 5% skimmed milk  or GAPDH  overnight at 4 °C. The next day, membranes were washed three times in Tris-buffered saline with Tween 20 and incubated for 1 h at room temperature with secondary antibodies conjugated with horseradish peroxidase against rabbit  or mouse . After three additional washing steps, the protein band detection was performed using an enhanced chemiluminescence detection system . Band densitometry-based quantification of three biological replicates was carried out by using ImageJ 1.53.In addition to immunofluorescence analysis, active pSTAT3 was analyzed by Western blot. Fibroblasts were cultivated on 60 mm culture dishes as described above. After cultivation, fibroblasts were washed once in 1× PBS and lysed in radioimmunoprecipitation assay buffer  supplemented with 1× protease inhibitor cocktail  and 1× phosphatase inhibitor cocktail . Lysates were centrifuged at 2000× Analysis of the protein concentration of IL-6 in cell culture supernatants was carried out by using the Immunoanalyzer Cobas e411 . Determination of MCP-1, C3, C3a and C1r in cell culture supernatants and in sera of PXE patients were conducted by using a commercially available enzyme-linked immunosorbent assay . The protein concentrations measured were normalized to DNA content.p-values of 0.05 or less were considered statistically significant.All data are shown as mean ± standard error for the qPCR, protein concentrations in cell culture supernatants and quantification of immunofluorescence and Western blot. GraphPad Prism 9.0 was used, and the non-parametric two-tailed Mann–Whitney U test was performed for statistical analysis. The level of active pSTAT3 was evaluated by immunofluorescence and Western blot to determine the basal activation of STAT3 in PXEF and NHDF and the effect of BA on pSTAT3 expression. Accordingly, fibroblasts were treated with DMSO as a vehicle (untreated), with BA for blocking JAK/STAT3 signaling, with IL-6 to induce JAK/STAT3 signaling (positive control), or in combination. Analysis of the signal (intensity/area) for active pSTAT3 showed a significant 28-fold higher signal of active pSTAT3 in untreated PXEF (intensity/area: 4.5 ± 1.81) compared to untreated NHDF (intensity/area: 0.2 ± 0.05). Treatment with BA reduced the level of active pSTAT3 in PXEF by 98% (intensity/area 0.1 ± 0.02) and for NHDF by 50% (intensity/area: 0.1 ± 0.02) compared to untreated fibroblasts. There was no significant difference in the level of active pSTAT3 in IL-6-treated PXEF (absolute intensity/area: 6.3 ± 1.16) compared to untreated PXEF, but a significant 84-fold higher signal could be seen for IL-6-treated NHDF (intensity/area: 13.4 ± 1.96) in contrast to untreated NHDF. Furthermore, a significantly reduced signal for active pSTAT3 was shown in BA + IL-6-treated PXEF  and NHDF  in comparison to the corresponding untreated fibroblasts A,B.Analysis via Western blot also showed similar effects. A significant 14-fold higher signal for pSTAT3  could be detected in untreated PXEF (1.5 ± 0.26 AU) compared to untreated NHDF (0.1 ± 0.03 AU). Additionally, a significant reduction of the pSTAT3 signal in BA-treated (0.08 ± 0.02 AU) compared to untreated PXEF by 94% was detected. No difference in the pSTAT3 signal could be seen in untreated NHDF compared to BA-treated NHDF (0.09 ± 0.01 AU) and to BA-treated PXEF .STAT3 and SOCS3 were analyzed in addition to immunofluorescence and Western blot. As described in STAT3 and SOCS3 in untreated PXEF  compared to untreated NHDF. Gene expression of both targets was significantly decreased in PXEF  after treatment with BA, while only SOCS3 expression was significantly decreased by 42% in NHDF after BA treatment compared to corresponding untreated fibroblasts ) and protein level (190.0 ± 71.7 pg/µL/(µg DNA)) in PXEF in comparison to untreated PXEF. Only a significant reduction of IL-6 gene expression by 16% was for BA-treated NHDF, but there was no significant difference in the IL-6 protein level ) compared to untreated NHDF  and the protein level in supernatants (PXE: 227.0 ± 86.7 pg/µL/(µg DNA), 19-fold; NHDF: 12.1 ± 3.04 pg/µL/(µg DNA)) showed a significant increase in untreated PXEF compared to untreated NHDF. A treatment with BA revealed no significant differences in ted NHDF A,B.MCP-1 expression could be detected in untreated PXEF compared to untreated NHDF . MCP-1 protein concentration was also increased in PXEF supernatants )). The BA-treated PXEF showed a significant reduction of MCP-1 gene expression by 68% (2.6 ± 0.36) and a tendential reduction of the MCP-1 protein level by 64% (401.4 ± 98.5 pg/µL/(µg DNA)) in comparison untreated PXEF. For BA-treated NHDF, there was a significant reduction in the MCP-1 gene expression by 52% (0.49 ± 0.03) and on protein level by 51% (87.9 ± 19.5 pg/µL/(µg DNA)) compared to untreated NHDF  in contrast to NHDF (0.98 ± 0.05) (CXCL1 expression in NHDF by 13% (0.85 ± 0.12) in contrast to untreated NHDF. The CXCL1 mRNA expression was only tendentially reduced by 33% (3.5 ± 0.82) in BA-treated PXEF in comparison to untreated PXEF (In addition to the gene expression of  ± 0.05) E. A treated PXEF E.To determine the role of the complement system and its association with JAK/STAT3 signaling in PXE pathogenesis, factors of the complement system were analyzed in PXEF.C1r mRNA expression significantly by 58% in contrast to untreated NHDF (0.5 ± 0.15)  and protein concentration in supernatants (PXE: 1.1 ± 0.30 (ng/mL)/µg DNA, 4-fold; con: 0.27 ± 0.07 (ng/mL)/µg DNA) of C1r were significantly increased in untreated PXEF in comparison to untreated NHDF A,B. BA-t ± 0.15) A,B.C1s , C2  and CFB  were significantly increased; additionally, C4A  expression was tendentially increased in untreated PXEF compared to untreated NHDF (C1s (1.7 ± 0.25) and C4A (0.81 ± 0.05) gene expression significantly by 57% and 51% in contrast to untreated PXEF. The mRNA expression of C2 (3.9 ± 0.72) and CFB (1.8 ± 0.17) were only tendentially reduced by 68% and 45% in BA-treated PXEF compared to untreated PXEF. After BA supplementation, the gene expression of C1s (0.63 ± 0.06) and C2 (0.84 ± 0.1) was significantly decreased by 40% and 19.7%, respectively, in NHDF in comparison to the untreated NHDF , while SERPING1 gene expression was significantly enhanced  in untreated PXEF compared to untreated NHDF  and reduced SERPING1 gene expression tendentially by 30% (2.3 ± 0.21) in comparison to untreated PXEF. No significant difference for both gene expressions could be detected for BA-treated in contrast to untreated NHDF.In addition, the gene expression of complement inhibitors analyzed G,H. The ted NHDF G,H. A suNext, we analyzed downstream factors of the complement system, such as C3 and C3a, the proteolytic product of C3 convertase, to determine the complement system activation level and its association with JAK/STAT3 signaling in PXEF.The gene expression  and protein concentration (PXE: 5.3 ± 2.3 (ng/mL)/µg DNA, 35-fold; con: 0.15 ± 0.05 (ng/mL)/µg DNA) in the supernatant were significantly increased for C3 in untreated PXEF compared to untreated NHDF A,B. The To further analyze the systemic role of the complement system, C3 protein concentrations were measured in sera from PXE patients and healthy controls. We detected a significantly increased protein concentration of C3 in sera of PXE patients compared to healthy controls (PXE: 3.5 ± 0.19 mg/mL, 1.4-fold; con: 2.55 ± 0.18 mg/mL; Our previous studies revealed a link between premature cellular senescence and the increased production of inflammatory factors of the SASP in PXEF ,15. One STAT3 mRNA level. We found a significant increase of active pSTAT3 in nuclei and STAT3 gene expression in PXEF. Only active pSTAT3 is able to translocate into the nucleus [STAT3 expression in PXEF but not in NHDF. As shown in different studies, BA is a specific JAK inhibitor, preventing phosphorylation and activation of STAT3 [STAT3 in BA-treated PXEF. Interestingly, there was no difference in the level of active pSTAT3 in untreated and IL-6-treated PXEF. This assumes an activation of JAK/STAT3 signaling in untreated PXEF, which could be reached by high IL-6 levels.For this, we evaluated the activation status of JAK/STAT3 signaling by analyzing active pSTAT3 on protein level and  nucleus , indicatof STAT3 ,41, whicSOCS3, a negative regulator of JAK/STAT3 signaling in PXEF [SOCS3 expression, STAT3 activation and inflammation [SOCS3 gene expression in PXEF could be a sign of local inflammatory processes caused by activation of JAK/STAT3 signaling. Blocking JAK/STAT3 signaling correlated with a decrease of SOCS3 gene expression in PXEF, supporting this hypothesis.Furthermore, we showed an increased gene expression of  in PXEF , which i in PXEF . Previouammation . Thus, iTo evaluate the role of JAK/STAT3 signaling in chronic inflammatory processes in PXE, we next determine the effect of blocking JAK/STAT3 signaling on gene expression and protein level of SASP factors, which were shown to be dysregulated in PXE .IL-6 gene expression and the IL-6 protein level in supernatant in PXEF. IL-6 induces its own expression by JAK/STAT3 signaling [Examination of IL-6 revealed an increased gene expression and protein level in the supernatant of untreated PXEF in comparison to NHDF, which confirms our previous study . Blockinignaling , so blocignaling ,44. Our 1 expression but not CXCL1 gene expression in PXEF. Several studies have already shown that IL-6 stimulates MCP-1 expression via JAK/STAT3 signaling [In the case of MCP-1 and CXCL1, we showed increased gene expression and protein levels in untreated PXEF. As both chemokines play a pivotal role in chronic inflammatory processes ,46, thisignaling ,50,51,52Besides investigating the effect of blocking JAK/STAT3 signaling on SASP factors, we focused on the complement system and its role in PXE pathogenesis. Therefore, we analyze the influence of JAK/STAT3 inhibition on the expression of different complement factors.C1r C1s and CFB, as well as an increased C1r protein concentration in supernatants in untreated PXEF compared to NHDF. C1s and C1r are components of the C1 complex, part of the classical complement cascade, while CFB plays an important role in the activation of the alternative complement cascade [CFB might indicate the activation of a classic complement cascade as well as an alternative complement cascade, which is initiated by the formation of the C1 complex or spontaneous hydrolyzation of C3 [C1r and C1s in keratinocyte-derived cutaneous squamous carcinoma cells, which is associated with chronic inflammation, supporting the hypothesis of possible chronic inflammatory processes in PXEF [C1s in PXEF, indicating a regulation of classic complement cascade but not alternative cascade via JAK/STAT3 signaling in PXEF.First, we analyzed complement factors initiating the complement cascade. We found an increased gene expression of  cascade , but boton of C3 . A previon of C3 . Riihilä in PXEF . BlockinSERPING1, an inhibitor of the classic complement cascade, by blocking the C1 complex, and CFH, an inhibitor of the alternative complement cascade. Regarding SERPING, we discovered a significantly high mRNA expression in PXEF, which is not affected by BA supplementation. As shown in the literature, SERPING1 belongs to the acute phase proteins, which are highly expressed in inflamed tissues and correlate positively with the gene expression of C1 factors [CFH gene expression, we identified a reduction in PXEF, while the CFH expression was upregulated after supplementation with BA. CFH is one of the key regulators of the complement system. Previous studies revealed a connection between a potential risk of AMD and polymorphisms in CFH, resulting in a downregulation of CFH expression and overactivation of the complement system [CFH and increased gene expression and protein level secretion of complement factors could indicate the missing regulation of the complement system, resulting in complement activation in PXE, which could partially be regulated by inhibiting JAK/STAT3 signaling.Next, we analyzed the gene expression of  factors . Therefot system ,56. Const system . Thus, aC3 expression by STAT3 in PXEF. Yuan et al. described that JAK/STAT3 signaling is induced by C3 overexpression [C3 expression could induce JAK/STAT3 signaling in addition to IL-6-mediated signaling in PXEF. We also analyzed the C3 concentration in sera from PXE patients to determine a possible link between local C3 production and systemic C3 circulation. We found significantly increased C3 protein levels in sera from PXE patients. High C3 levels could also be seen in patients with inflammatory bowel disease, which is associated with chronic inflammation [We then analyzed the expression of proinflammatory C3, which is a central protein in the complement cascade. We discovered a strongly increased expression of C3 and C3a in untreated PXEF. The increased level of C3a, a product of C3 convertase, additionally indicates the high activity of the complement system in PXEF. Blocking JAK/STAT3 signaling did not affect the C3 gene expression or protein level of C3 and indicated no regulation of pression , so incrammation . TherefoC2 and C4A, which form the C3 convertase together with C3b after cleavage of C3 by C1 complex [To investigate the role of JAK/STAT3 signaling on downstream processes of the complement cascade in PXEF, we analyzed gene expression of  complex . We founABCC6 deficiency in PXEF to a permanent activation of JAK/STAT3 signaling and activation of the complement cascade. Our results indicate that JAK/STAT3 activation partly modulates chronic inflammatory processes, but this seems to be limited in PXEF. Thus, further studies are necessary to evaluate the association of IL-6 to the permanent activation of JAK/STAT3 signaling. Other JAK/STAT signaling pathways and downstream factors of the complement system should be analyzed in PXEF to uncover the origin of chronic inflammation observed in PXE.In conclusion, this is the first study linking


The application of halogen bonding in pharmaceuticalchemistryremains a challenge. In this work, novel halogen-bonded cocrystalsbased on azole antifungal active pharmaceutical ingredients (APIs)and the ditopic molecule 1,4-diiodotetrafluorobenzene are reported.Their crystal structural features, spectroscopic properties, and thermalstability were studied. The components are bound through I···Nfrom the triazole moieties present in all of the compounds. The molecularelectrostatic potential (MEP) surfaces and quantum theory of atomsin molecules (QTAIM) calculations are used to rationalize the presenceof hydrogen and halogen bonds in the resulting structures and theirenergetic analysis. The relative halogen bond ability of the differentgroups of voriconazole, fluconazole, and itraconazole was analyzedusing MEP surfaces, demonstrating this approach to be an interestingtool to predict halogen-bonding preferences. New halogen-bonded cocrystals of azoleantifungal APIs withthe ditopic 1,4-diiodotetrafluorobenzene were synthesized. Their crystalstructures, intermolecular interactions, and thermal stability werestudied with combined DFT calculations. Halogen atoms can be present in the APIbackbone because of the need to tune their lipophilic behavior whencrossing lipid membranes and to increase their activity before drugdecomposition in the body. Thus, halogen···halogencontacts and halogen bonds can also be present and responsible forthe supramolecular network in molecular solids. All of these interactionscan be exploited in the search for new functional materials and applications.4Noncovalent interactions,mostly hydrogen bonding, are responsiblefor many tridimensional networks in the solid state. Other supramolecularinteractions include halogen bonds, being also a highly directionaland linear interaction.5 A few halogen-bondedpharmaceutical cocrystals have been described in the literature.12 We have recently reported several halogen-bonded cocrystals of nucleobasecompounds as adenine and uracil derivatives, including an antineoplasticsubstance.14 Now, in this work, we expandthis strategy to a different class of molecules, antifungal APIs,selectively used to eliminate infections caused by fungal pathogens.We have studied the feasibility of azole antifungals for halogen bonding.With this purpose in mind, first, different azole antifungals werecarefully considered taking into account the presence of multiplefunctional groups  in those molecules. Finally, the following three antifungalmolecules voriconazole (VZ), fluconazole (FLZ), and itraconazole (ITZ) were selected as model compounds.Cocrystals are molecular solids containing at least two neutralsolid compounds.32All three APIs contain halogen atoms (F or Cl) in their skeletonas well as one or two triazole rings see 1. Except21 Herein, inspired by that work, the ditopic coformer 1,4-diiodotetrafluorobenzene(DITFB), in which iodine atoms are activated by the presenceof fluorines,11 has been used to explorethe preparation of multicomponent solids and understand the possiblecompetition between hydrogen and halogen bonds in these pharmaceuticalactive molecules.In 2013, a cocrystal of itraconazole with succinicacid was revisitedand a weak halogen bond was identified between C–Cl···Ofrom the triazolone moiety and a chlorine atom.33The new multicomponent solids were preparedby solvent-assistedgrinding and by slow evaporation crystallization. Later, characterizationby X-ray diffraction , infrared spectroscopy,and thermal methods was performed. The use of DFT and QTAIM has allowedus to measure the strength of the halogen bonds besides other interestingintramolecular H-bonds. Moreover, the MEP surface analysis allowsus to identify those groups that are better halogen bond acceptorsand rationalize the cocrystal formation.22.1VZ), fluconazole , and itraconazole were kindly donated by local pharmaceuticalcompanies and used without further purification. 1,4-Diiodotetrafluorobenzene(DIFTB) was purchased from Cymit Química S.L.. All of the solvents used in the present work wereanalytical grade and were obtained from local suppliers.Voriconazole and 1,4-diiodotetrafluorobenzene were ground together with three drops of methanolin a Retsch MM400 mixer mill using a 10 mL agate jar with two 5 mmagate balls at 30 Hz for 30 min. The resulting solid was recoveredand analyzed by PXRD. Slow evaporation of this solid in methanol affordedsingle crystals.2.2.2Fluconazole  and 1,4-diiodotetrafluorobenzene were ground together with three drops of methanolin a Retsch MM400 mixer mill using a 10 mL agate jar with two 5 mmagate balls at 30 Hz for 30 min. The resulting solid was recoveredand analyzed by PXRD. Suitable single crystals were obtained by slowevaporation of this solid in a methanol solution.2.2.3ITZ-DITFB system.Suitable single crystals of this phase wereobtained by slow evaporation of a mixture of precursors (1:1 molarratio) in methanol. The crystal resolution afforded a new compoundwith a molar stoichiometry 2:1 for the 2.2.4Itraconazole  and 1,4-diiodotetrafluorobenzene were ground together with three drops of methanolin a Retsch MM400 mixer mill using a 10 mL agate jar with two 5 mmagate balls at 25 Hz for 30 min. The resulting solid was recoveredand analyzed by PXRD.2.3VZ·DITFB was selected forsingle-crystal X-ray diffraction experiments and mounted at the tipof a nylon CryoLoop on a Bruker D8 QUEST ECO (Photon II detector)diffractometer using graphite-monochromated Mo Kα radiation(λ = 0.71073 Å). Single crystals of compounds 2(FLZ)·DITFB and 2(ITZ)·DITFB were selected for single-crystalX-ray diffraction experiments and mounted at the tip of a nylon CryoLoopon a Bruker Apex-II CCD diffractometer using graphite-monochromatedMo Kα radiation (λ = 0.71073 Å). Crystallographicdata for 2(FLZ)·DITFB and 2(ITZ)·DITFB were collected at294(2) and 298(2) K, respectively. Data reduction was performed usingSAINT v6.45A and SORTAV in the diffractometer package.34 Data were corrected for Lorentz and polarizationeffects and for absorption by SADABS.35 The structural resolution procedure was made using SHELXT.36 Non-hydrogen atoms were refined anisotropically.Hydrogen atoms were introduced in calculated positions and refinedriding on their parent atoms. Selected crystal and data collectionparameters are reported in the corresponding A SiemensD5000 powder diffractometer was used for all X-ray powder (XRPD) measurementswith experimental parameters as follows: Cu Kα radiation (λ= 1.5418 Å), scanning interval 2–50° 2θ, stepsize 0.02°, and exposure time 1 s per step. A single crystalof compound 37Calculated X-ray powder patterns were obtained fromsingle-crystalstructure data using Mercury 4.3.1 software.2.4–1.IR spectrawere collected using a Jasco 4700LE spectrophotometer with attenuatedtotal reflectance accessory at a resolution of 4.0 cm2.52 atmosphere as protective and purge gas (their respectiveflow velocities were 20 and 40 mL/min).A simultaneous thermogravimetricanalysis (TGA)–differential scanning calorimetry/differentialthermal analysis (heat flow DSC/DTA) system (NETZSCH-STA 449 F1 Jupiter)was used to perform thermal analysis on the solids. Samples (3–8mg) were placed in open alumina pan and measured at a scan speed of10 °C/min from ambient temperature to 300 °C under a N2.638 and the PBE0-D3/def2-TZVP level of theory.40 The crystallographic coordinates were used to estimate the interactionsin the solid state. The interaction energies of the halogen-bondingcontacts were computed by calculating the difference between the energiesof isolated monomers and their assembly. The binding energies werecorrected for the basis set superposition error (BSSE) by using theBoys–Bernardi method.41 Bader’s“atoms in molecules” theory (QTAIM)42 was used to study and estimate the association energiesof the intramolecular H-bonding interactions by using the AIMAll program.43 The molecular electrostatic potential (MEP)surfaces (isosurface 0.001 a.u.) were computed using Gaussian-16 software.38The energeticanalysis of the intermolecular halogen-bonding interactions was performedusing the Gaussian-16 suite of programs44 derived from the electronicdensity (ρ).45 The sign of the secondHessian eigenvalue (λ2) multiplied by the electrondensity was employed for the identification of attractive/stabilizing (blue-greencolored isosurfaces) or repulsive (yellow-red colored isosurfaces)interactions using 3D plots. The NCIplot index parameters used inthis article are RGD = 0.45; ρ cutoff = 0.04 a.u.; color range:−0.04 a.u. ≤ sign(λ2) ρ ≤0.04 a.u.To analyze the nature of interactions(attractive or repulsive) and uncover them in real space, the NCIPLOTvisualization index was used. It plots the reduced density gradient(RDG) regions33.1FLZ, several polymorphs, solvates, and hydrogen-bonded cocrystalsare described in the literature.32 Structurally, this is the simplest conazole of the three molecules,as two triazole rings besides a hydroxyl group are the only availablefunctional groups in this drug. The solvent-drop grinding of FLZ form II and DITFB in 2:1 molar ratio affordeda diffractogram exhibiting a new phase (2(FLZ)·DITFB, Powder XRD was used to identify the formation of new crystallinephases with respect to other forms available for precursors. For VZ only one polymorphicform has been previously described in the literature, although severalH-bonded cocrystals and salts have been reported.19 Moreover, with respect to FLZ, one triazolering has been substituted by a fluoropyrimidine ring and a methylgroup. The powder pattern of a 1:1 mixture of precursors ground witha few drops of methanol resulted in a new phase (VZ·DITFB). Finally, the third antifungal tested, ITZ, shows several polymorphic forms and cocrystals containingdifferent carboxylic acids or amino acids as coformers.25 This is a longer and flexible molecule with many more functionalgroups than FLZ or VZ. Liquid-assisted grinding(LAG) of mixtures of ITZ form I and DITFB in different molar ratios  afforded powder diffractogramswith low crystallinity in which new peaks were observed at 5.0, 6.7,7.6, 10.0, 20.9, or 23.0°, although a few peaks of ITZ or DITFB could be detected. Furthermore, slow evaporationof the 1:1 solid in methanol (or in a toluene–nitromethanemixture) rendered a few days later a concomitant mixture of plateletand needle single crystals. The calculated powder pattern from theseplatelet crystals gave a different phase, 2(ITZ)·DITFB, from the precursors or the one observedby grinding, with a 2:1 molar ratio composition (ITZ ·DITFB (2:1) LAG in ITZ·2(DITFB); see Unlike the previous conazole, for Figure S1, we show the comparisonfor FLZ, VZ, or ITZ, the halogenatedcoformer, and the new cocrystals. The presence of the OH group wasidentified for the two small conazoles, FLZ and VZ, and their new solid phases. Moreover, in the case of theircocrystals, the typical vibrations for C=C or C=N frompyrimidyl and triazole rings were also followed. Thereby, a band splittingwas observed in the 2(FLZ)·DITFB cocrystal for the C=C bond with respect to FLZ. Moreover, in both cocrystals, shifts in the C=N (triazole)bands were observed. In the case of ITZ or its solidforms, the shift of the C=O vibrational mode resulted in characteristicpeaks. While for the 2(ITZ)·DITFB crystal, it appeared at 1701 cm–1, for the ITZ·DITFB (1:2) LAG composition, it shiftedto 1672 cm–1. The decreasing wavenumber value withrespect to pure ITZ (at 1693 cm–1)indicates a different interaction of the triazolone ring in the newform. Taking into consideration the molar ratio and the appearanceof this new band, the interaction mode shown in Figure S2 is proposed. Finally, the presence of the typicalbands approximately at 1463, 940, and 760 cm–1 forthe Ha-bond donor was confirmed in all of the multicomponent solids.The FTIR spectra of the three conazoles and theirrespective cocrystalswere measured and compared to study the effect of the halogen bondformation. In 3.23.2.1VZ·DITFB crystallizesin the monoclinic space group P21 containingone molecule of voriconazole and a molecule of the halogenated coformerin the asymmetric unit. In former voriconazole, an intramolecularhydrogen bond between the alcohol and one nitrogen from the pirimidinylring is found besides other H-bonds, all together responsible forthe molecular arrangement observed in the crystal packing.15 In cocrystal VZ·DITFB, the alcohol forms the intramolecular H-bond between O(1)-H···N(4)(distance: 2.784(8) Å), although it is longer/weaker than thatobserved in VZ  Å).An intermolecular H-bond with another voriconazole molecule throughO(1)-H(1)···N(2) is also observed. Moreover, the triazolering establishes a new HaB with the coformer by N(3)···I(2)interactions (Table S1) or F···Fcontacts among the fluorine atoms from voriconazole and the coformermolecules ···H-O(1) interactions,distance: 2.773(2) Å), which are shorter than the equivalentH-bond observed in FLZ form II  Å, This cocrystal crystallizes in thetriclinic space group a-axis 2·DITFB is the first cocrystalof ITZ containing strong and symmetric HaB interactions.This new cocrystal crystallizes in the triclinic space group P1̅ containing a molecule of ITZ anda half molecule of DITFB in the asymmetric unit, whichresults in a 2:1 stoichiometry. In this structure, symmetrical Ha-bondsamong the triazole moiety and DITFB were establishedthrough the N(2)···I(1) interactions, but unlike in ITZ cocrystals with succinic or terephthalic acid, hereinthe coformer is not found between two ITZ molecules (DITFB and the methylenegroups by F(2)···H-C(8) interactions. All of them arecollected in Table S3. As a result, pillarsof ITZ molecules, in which the conazole rings are connectedthrough the H-bonds, are related through the HaB established withthe halogenated coformer besides additional F···H contacts. Most of the new cocrystals showed melting points lowerthat their former precursors. Only the (FLZ)2·DITFB resulted to have a meltingpoint higher than fluconazole or the coformer  donor molecule. Moreover,we have also evaluated the intramolecular H-bonds and π-stackinginteractions in FLZ), voriconazole(VZ), and itraconazole (ITZ) skeletons.The MEP surfaces are represented in DITFB, the MEP maxima are located at the iodine σ-holes ,thus confirming the strong electrophilicity of this molecule. In compound FLZ, the MEP values at the N-atoms of the five-membered triazolering are more negative than those at the pyrimidine ring, thus revealingthat the triazole ring is a better halogen bond acceptor than thepyrimidine ring. Moreover, one of the N-atoms of the latter is notavailable to form HaBs because it is forming an intramolecular OH···NH-bond . TheMEP value at the sp3 N-atom piperazine ring is significantlyless nucleophilic .Primarily, the MEP surfaces of all coformers havebeen analyzed to explore the relative HaB acceptor ability of thedifferent N-atoms present in the fluconazole (bond see 1b. The MVZ·DITFB and (FLZ)2·DITFB cocrystals 2·DITFB, thus confirming the moderately strong nature of theHaB contacts, in line with the blue color of the NCIplot isosurface.Each HaB in (FLZ)2·DITFB is weaker than in VZ·DITFB likely due to anti-cooperativity effects. That is, the halogen-bondingability of the I-atom in DITFB weakens upon the formationof a HaB in the opposite side of the molecule. Such effects have beenrecently demonstrated by Bedeković et al.48 in cocrystals of perfluorinated iodobenzenes with pyridines.In addition, Cinĉić et al.49 have also analyzed anti-cooperativity effects in three-centeredhalogen bonds with bifurcated acceptors present in molecular crystals,cocrystals and salts. An additional effect that explains the differentstrengths of the HaBs in (FLZ)2·DITFB and VZ·DITFB cocrystals is that the MEP value at the interacting N-atom of VZ is more negative than that of FLZ (see 50 (using the Vr energypredictor). The QTAIM/NCIplot confirms the existence of the OH···Nintramolecular H-bond in VZ·DITFB cocrystalscharacterized by a bond CP, bond path, and blue RDG isosurface. Thestrength of this intramolecular H-bond is −5.2 kcal/mol. Interestingly,the combined QTAIM/NCIplot analysis also reveals the existence oftwo additional CH···F bonds that further stabilizethe conformation observed in the solid state . Finally, an extended green RDG isosurface is observedbetween both aromatic rings, evidencing the formation of a π–πstacking interaction. In the (FLZ)2·DITFB cocrystal the CH···Fcontacts are also observed with similar energies .In order to characterize the halogen-bonding assembliesof thecocrystals, we have used a combination of QTAIM and NCIplot methods.The formation energies of the assemblies have been computed usingthe supramolecular approach. The combined QTAIM/NCIPlot analysis ofthe crystals 5 shows t FLZ see 4. We hav(ITZ)2·DITFB cocrystal is depictedin (FLZ)2·DITFB and weaker than that of VZ·DITFB due to utilization of both σ-holes of DITFB.Three intramolecular H-bonds are observed in ITZ influencingits conformation, which are CH···N(triazole), CH···Cl(dichlorophenyl),and CH···O(triazolone). The CH···O H-bondis the strongest one  in line with the largeand negative MEP value at the O-atom of the triazolone ring 2·DITFB to −8.1 kcal/mol in VZ·DITFB, thus confirming the strong nature of thesecontacts and their importance in the formation of the cocrystals.We have also evaluated the strength of the intramolecular H-bondsthat influence the conformation of the antifungal drugs in the cocrystals.These interactions are weaker than the HaBs. We expect that the resultsreported herein may inspire not only researchers working in the cocrystallizationof active pharmaceutical ingredients, but also theoreticians and thoseworking in crystal engineering and supramolecular chemistry.New cocrystals of threeantifungal drugs have been synthesizedand characterized. For the three APIs, these are the first halogen-bondedcocrystals described. In all of them, the structures consist of I···N(triazole)halogen-bonded molecular complexes, which confirms that the triazoleis a reliable Ha-bond acceptor. For