Bruton’s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI–Syk–Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis. The tyrosine kinases Src, Syk and Btk have similar domain structures, although they represent distinct types of molecular switches [Protein tyrosine kinases, in particular Src family kinases (SFKs), spleen tyrosine kinase (Syk) and Bruton’s tyrosine kinase (Btk), play central roles in the physiology and pathophysiology of immune and hematopoietic cells, including platelets ,2. In suswitches .Under basal conditions, Src, Syk and Btk are thought to be in a folded and auto-inhibited state, while their activation leads to an unfolding structure linked to tyrosine phosphorylation within the activation loops . SFKs ha3) with the recruitment of the PH-domain proteins PLCγ2, Btk, Akt and PDK1 [In unstimulated cells, Syk and Btk are also present in an autoinhibited state and require recruitment from the cytosol to the membrane for activation. With GPVI-mediated platelet activation, SFKs dually phosphorylate a receptor-associated ITAM (immunoreceptor tyrosine-based activation motif), which then recruits target proteins containing two SH2 domains, such as Syk. This unfolds the Syk kinase with simultaneous phosphorylation of its crucial tyrosine residues (Y348/352 and Y525/526), resulting in the activation of the kinase [and PDK1 . Btk, meand PDK1 .The kinase Btk was identified as the gene responsible for the human immunodeficiency disorder X-linked agammaglobinemia (XLA) ,12. The 2+ mobilization from intracellular stores, dense granule secretion and aggregation of platelets, which were impaired in Btk-deficient platelets from XLA patients [xid animals), platelet functions in response to GPIb-V-IX (GPIb) were reduced [The kinases SFKs, Syk and Btk are central for human platelet activation, in particular in response to collagen, convulxin (Cvx) and other GPVI agonists. In early studies, Btk was shown to mediate collagen-induced PLCγ2 phosphorylation, Capatients . In a Bt reduced . Recent  reduced ,21,22,23 reduced . While t reduced , there a reduced .2 (PGI2) [Previously, we demonstrated using a phosphoproteomic approach that the phosphorylation of Syk and Btk at S/T residues is regulated by both the platelet agonist adenosine diphosphate (ADP) and the platelet inhibitor prostaglandin I2 (PGI2) . Subsequ2 (PGI2) ,29. Spec2 (PGI2) . On the 2 (PGI2) .So far, similar studies concerning the tyrosine kinase Btk in platelets do not exist. With B cell lines, it was shown that the PKC-mediated phosphorylation of Btk S180  negatively affects the Btk membrane localization and activity . Here, wWe previously described and characterized the fact that GPVI induces phosphorylation of Syk serine 297 (Syk pS297), tyrosine 352 (pY352) and tyrosine 525/526 (pY525/526) in studies with Cvx-stimulated human platelets . In thisPreviously, enhanced Syk pS297 in platelets was also observed with the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu) . To testThere is evidence of Src-PKC interactions and that PKC may induce Src activation/phosphorylation . To furtIt was interesting to examine the interaction of PDBu- and Cvx-regulated Btk pS180. We analyzed the effects of PDBu on Cvx-regulated Btk and Syk phosphorylation. Platelets pre-incubated with the vehicle control (0.1% DMSO) or with 0.2 PDBu for 1 min prior to Cvx (50 ng/mL) stimulation induced a similar aggregation response compared with aggregation using Cvx alone (50 ng/mL) . HoweverPreviously, we observed that GPVI-evoked Syk pS297 was blocked by the established PKC inhibitor GF109203X (GFX) . TherefoWe showed earlier that activation of PKA by the prostacyclin analog iloprost abolished the ADP- and Cvx-induced platelet aggregation responses, as well as PKC activation, whereas iloprost did not inhibit the Cvx-induced activatory Syk tyrosine phosphorylation ,28. HerePreviously, we reported that inhibition of the serine/threonine protein phosphatase 2A (PP2A) using okadaic acid (OA) induced a stoichiometric Syk pS297. In addition, OA was found to inhibit the Cvx-induced tyrosine phosphorylation of Syk, LAT and PLCγ2 [Considering that PLCγ2 is a regulated substrate of Btk, we analyzed the effects of the Btk inhibitor acalabrutinib and PKC inhibitor GF109203X (GFX) on the Cvx-induced phosphorylation of PLCγ2. Out of five donors, acalabrutinib completely abolished the Cvx-induced platelet aggregation response for three donors, and partially inhibited the response for two donors A. As alrThis paper reports several novel aspects of platelet collagen receptor GPVI-stimulated Btk activation, including tyrosine Y223, Y551) and serine (S180) phosphorylation, which are rapid, reversible and regulated by PKC, PKA and PP2A. The SH3-domain-located Btk Y223, which was not strongly regulated here, is an established autophosphorylation site, which may recruit proteins containing the SH2 domain or regulate the ligand-binding property of the Btk  and seri,32 and, Here, we showed that GPVI stimulation transiently increased phosphorylation of both Btk S180 and Y551, which was strongly stimulated within 1 min and then rapidly reversed within 5 min, whereas Cvx-stimulated platelet aggregation was not reversed. Consistent with previous studies , the phoAnother novel result of this study was that Btk S180, which is located within the N-terminal regulatory TH domain see , is a suA further novel finding was that PDBu alone significantly and also transiently increased the Btk pY551 without affecting any other Syk or Btk tyrosine phospho-sites. We then showed that PDBu-induced Btk pY551 was strongly inhibited by GFX and, unexpectedly, also by the Src/SFK inhibitor PP2. In contrast, Syk inhibitor PRT-060318 and Btk inhibitor acalabrutinib had only a little, if any, effect. PDBu-induced Syk pS297 and Btk pS180 were abolished only by GFX but not by PP2 or other tyrosine kinase inhibitors. Moreover, PDBu-stimulated aggregation of washed human platelets was abolished by GFX but not affected by Src/SFK, Syk and Btk inhibitors, indicating that Syk and Btk were not required for this PDBu/PKC aggregation response. PDBu-induced Syk pS297/Btk pS180 was directly PKC-mediated, whereas the PDBu-induced Btk pY551 was mediated by a PKC-stimulated Src/SFK without the participation of Syk. Interestingly, bidirectional phosphorylation/activation of PKCs and SFKs was well reported and described for platelets and other cell types .Btk can also be activated by chemokine receptor-coupled G-proteins and their dissociated Gα and Gβγ subunits, which may bind directly to PI3K or even Btk, resulting in the activation of Btk and Akt . We receCvx-stimulated phosphorylation of Syk S297 and Btk S180 was also transient and almost completely reversed after 5 min of stimulation, indicating the activity of potent serine/threonine protein phosphatases. Earlier, we established PP2A as a major Syk S297 protein phosphatase in platelets . We conc3/Ca2+ elevation and PKC activation [2+ mobilization in human platelets, indicating increased PLCγ2 activity [2+/calmodulin and/or Ca2+-dependent pathways.The functional effects of Btk are defined primarily by its substrates, although Btk and other tyrosine kinases have also a role as adapter proteins . A crucitivation . Here, ttivation . We alsoactivity . We concBtk is an important component of platelet activation mediated by GPVI, GPIb-IX-V, CLEC-2 and certain GPCRs, which often contribute to “hyperactive platelet disorders”, atherothrombosis and thrombo-inflammation . HoweverFinally, multiple genetic receptor variants, which signal via platelet SFKs, are known . RecentlPlatelet activators/inhibitors: Convulxin (Cvx), GF 109203X (GFX) and okadaic acid (OA) were obtained from Enzo Life Sciences . Phorbol ester phorbol 12, 13-dibutyrate (PDBu) was supplied by Sigma-Aldrich . Iloprost was obtained from Ratiopharm . Acalabrutinib and PP2 were purchased from Abcam . PRT-060318 was from Sellckem .Sources of antibodies: General Syk antibody was from Sigma-Aldrich . General Btk, Btk pS180, Btk pY223, Btk pY551, Syk pS297, Syk pY352, Syk pY525/526, general PLCγ2, PLCγ2 pY759 and PLCγ2 pY1217 antibodies were provided by Cell Signaling Technologies . Secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibodies were obtained from BioRad Laboratories .®-20 and Precision Plus Protein Dual Color Standard was Bio-Rad Laboratories . Bovine serum albumin (BSA) was from Capricorn Scientific . ClarityTM Western ECL Substrate and β- mercaptoethanol were from BioRad Laboratories . D(+)-Glucose, ethanol (EtOH), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), glycerol, hepes and sodium chloride (NaCl) were from Carl Roth . Dimethyl sulfoxide (DMSO) was from Merck . Reagents: TweenEquipment: Apact4S Plus aggregometer was from DiaSys Greiner . Fusion FX7 apparatus was from Vilber Loumat . Automatic Haematology analyser Sysmex® KX-21N was from Sysmex Corporation Japan/Sysmex Germany. Centrifuge Allegra X-30R  was from Beckmann Coulter . Blood was collected from healthy volunteers without taking platelet-affecting drugs and without presenting inflammatory, infectious or allergic symptoms for at least 14 days, including normal blood counts. Informed consent was obtained from all healthy donors prior to participation in the study. This study was approved in accordance with the Declaration of Helsinki by the local Ethics Committee of the University Medical Center Mainz, as detailed below.g for 10 min at room temperature (RT). The supernatant of PRP was diluted with CGS buffer  and then centrifuged at 400× g for 10 min at RT. The pellet was resuspended in 3 mL CGS buffer and then centrifuged at 400× g for 10 min at RT. Finally, the platelet pellet was resuspended in HEPES buffer . The platelet concentration was adjusted to 3 × 108/mL, and the platelets were rested for 15 min at 37 °C.Human platelets were washed and isolated, as previously described ,29. Brie8/mL) were prepared in cuvettes and treated with 50 ng/mL Cvx or 0.9% NaCl (vehicle control), 0.2 µM PDBu or 0.1% DMSO (vehicle control), or 1 µM OA or 0.1% EtOH (vehicle control) under stirring. Washed human platelets were preincubated with 0.2 µM PDBu or 0.1% DMSO (vehicle control) for 1 min, with 5 µM GFX or 0.1% DMSO (vehicle control) for 5 min, with 5 nM iloprost or 0.9% NaCl (vehicle control) for 5 min, with 1 µM PRT-060318 or 0.1% DMSO for 5 min, or with 10 µM PP2 or 0.1% DMSO for 5 min before 50 ng/mL Cvx stimulation. For Western blotting, aggregation was stopped by adding 100 µL of 3× Lämmli buffer  glycerol, 6% (w/v) SDS, 0.06% (w/v) bromophenol blue, 1:10 β-mercaptoethanol) at 0 min, 1 min, 2 min and 5 min. Samples were boiled at 95 °C for 10 min with gentle shaking.Using an Apact4S Plus aggregometer, washed human platelets  Tween-20) for 1 h at RT, and then incubated overnight with desired antibodies in 1× TBS-T/5% BSA at 4 °C. The incubated membranes were washed three times with 1× TBS-T and treated for 2 h at RT with relevant secondary horseradish peroxidase (HRP)-conjugated antibodies in 1× TBS-T/5% BSA. After three washes with 1× TBS-T, the membranes were developed via electrochemiluminescence (ECL) detection. The phosphoantibodies used were against Btk pS180, pY223, pY551, pSyk pS297, pY352 and pY525/526, PLCγ2 pY759 and pY1217, anti-Syk, anti-Btk and anti-PLCγ2.Samples for Western blotting analysis were prepared by adding 3× Lämmli buffer to washed platelet suspensions. The lysed platelet proteins were separated via electrophoresis using 8% gels, as previously described. Proteins separated via SDS-polyacrylamide electrophoresis were transferred to polyvinylidene difluoride membranes (PDVF). The membranes were blocked with 5% bovine serum albumin (BSA) in 1× TBS-T , from n ≥ 3 independent experiments with platelets from at least three healthy donors. Statistical analysis was performed using GraphPad Prism 9.0.1 . One-way or two-way ANOVA, followed by Sidak’s multiple comparison test, was used for the comparison of two groups. A


Rapid Arctic warming may increase erosion and stream channel formation, which alters the flux of sediments, carbon, and nutrients in these sensitive ecosystems. Yet, understanding landscape change is hampered by a lack of predictive tools applicable to permafrost settings. However8. These collapse features remove protective vegetation and expose easily erodible soils to concentrated surface runoff leading to new channels with flow paths that closely follow the degrading ice wedge network8.In non-permafrost watersheds, erosional gullies commonly develop in response to increases in erosive capability of surface water, decreases in the resisting forces of the land to erosion, or a combination of both. In these systems, gullying may be triggered by land use, climate change, or by natural cycles of erosion and deposition. In permafrost settings, gully formation by surface water flow commonly occurs by melting of ice wedges rather than erosion of the ground surface. Melting of ice wedges by flowing water creates tunnels that in turn collapse7 show that the development of channels within such settings is spatially discontinuous and independent of both topographic steepness of the valley and grain size of the channelized material, two common predictors for channel development in non-permafrost settings. Chartrand et al.7 further note the sources of water driving the melting of ice wedges are strongly influenced by thaw of permafrost and changing hydrology, such as snow melt magnitude and timing. All these factors make this type of channelization difficult to predict and require new models developed for these systems.Documented in high-resolution topographic data, Chartrand et al.9, where ground ice may be more distributed and less predictable in location than the ice wedge complexes of Muskox Valley7. In these settings, subsurface flow also accelerates the melting of ground ice and physically erode soils from beneath the overlying, and often intact surface vegetation. Where pipes reconnect to the ground surface, large volumes of sediment and eroded organic matter may be deposited on top of undisturbed ground downslope6. Once sufficient volumes of ice melt and soil are excavated, the ground surface may collapse and drive channel development through coalescing disturbances7. Such channelization appears most commonly along valley axes where converging topography concentrates surface runoff.These challenges are not limited to settings with ice wedges. Subsurface erosion of soils due to water flowing through tunnels or pipes has also been observed in more southerly and discontinuous permafrost settings7, but they also document gullies, landslides, and linear surface water flow features called water tracks that all may play a role in expanding channel networks in permafrost watersheds. Along hillslopes across the Arctic, shallow landslides and deeper failures accelerate erosion and increase sediment and carbon delivery to streams and rivers11. Similar to ice wedge melt, these landslides tend to be isolated and generate local channel development rather than an integrated watershed-scale expansion of drainage networks. Attempts to predict the location and timing of these landslides with tools developed for temperate watersheds have met with limited success6. Results to date suggest that failures appear to be strongly connected to local ground ice conditions and seasonal warming, rather than topographic slope and hydrology5.Rapid channelization along ice wedges is the most dramatic change highlighted in the Chartrand et al. study7 shows numerous linear water track features. Water tracks are ubiquitous features in the Arctic that preferentially route water down hillslopes. The origin of water tracks is not well understood12 but it has been hypothesized that they may represent incipient drainage networks that will facilitate channel expansion in response to warming-induced erosion13. Erosion within water tracks have recently been observed and may be increasing14. However, a widespread integration of erosion along water tracks that would cause widespread channel expansion has not yet been documented. At present, the occurrence of water track erosion appears to be largely related to local ground collapse due to melting of ice12 rather than a systematic or predictable shift in processes governing channel development.Figure 2 of the Chartrand et al. study7 provide a watershed-scale picture of diverse processes that influence the flow of water and erosion. These processes range from failures on hillslopes to rapid erosion of ice wedge complexes along the valley axis. This study also highlights the complex interactions of ground ice, permafrost thaw, rapid warming, sediment grain sizes, and even stochastic flood events on channel formation. At present, we have very few tools for predicting where and how individual erosional processes occur in permafrost settings, let alone the ability to integrate these processes to predict channel network evolution across entire watersheds. The presence of ground ice strongly influences the vulnerability of permafrost landscapes to erosion and channel formation. Despite recent advances in the automated detection and mapping of ice wedge polygons15, we still lack the ability to map more distributed ground ice or predict which polygons may be vulnerable to surface water infiltration and melting.To address these research gaps, in their study of Muskox Valley, Canada, Chartrand et al.7, that identify patterns and timing of channel expansion and link these changes to climatic and hydrological drivers, will be needed to develop mechanistic models of landscape evolution in permafrost settings. These models will need to incorporate thermal controls on landscape responses to warming and couple hillslopes to channels. Integrated models that capture hillslope and channel coupling will allow for predictions of how increased disturbance and erosion of permafrost soils will alter the storage, transport rates, and release pathways of sediment, carbon, and nutrients across watersheds in a rapidly warming Arctic.More detailed watershed studies, such as the Muskox Valley study by Chartrand et al.


It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure and function of small-molecule TIL-type protease inhibitors. In this study, site-directed saturation mutagenesis at the P1 position was conducted to investigate the effect of P1 sites on the inhibitory activity and specificity of BmSPI38 and BmSPI39. In-gel activity staining and protease inhibition experiments confirmed that BmSPI38 and BmSPI39 could strongly inhibit elastase activity. Almost all mutant proteins of BmSPI38 and BmSPI39 retained the inhibitory activities against subtilisin and elastase, but the replacement of P1 residues greatly affected their intrinsic inhibitory activities. Overall, the substitution of Gly54 in BmSPI38 and Ala56 in BmSPI39 with Gln, Ser, or Thr was able to significantly enhance their inhibitory activities against subtilisin and elastase. However, replacing P1 residues in BmSPI38 and BmSPI39 with Ile, Trp, Pro, or Val could seriously weaken their inhibitory activity against subtilisin and elastase. The replacement of P1 residues with Arg or Lys not only reduced the intrinsic activities of BmSPI38 and BmSPI39, but also resulted in the acquisition of stronger trypsin inhibitory activities and weaker chymotrypsin inhibitory activities. The activity staining results showed that BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) had extremely high acid–base and thermal stability. In conclusion, this study not only confirmed that BmSPI38 and BmSPI39 had strong elastase inhibitory activity, but also confirmed that P1 residue replacement could change their activity and inhibitory specificity. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors. Proteases exist widely in organisms, and participate in the decomposition of proteins to maintain the normal life activities of organisms. Protease inhibitors are the main regulators of protease catalytic activity in vivo, which can bind protease molecules and inhibit their physiological activity, thereby terminating the unwanted proteolytic process . The intBombyx mori, is a silk-spinning insect with important economic value, which has accumulated a large amount of basic research, and has become one of the ideal models of Lepidoptera in the fields of the biochemistry, genetics, and genomics [Beauveria bassiana, inhibit the germination of B. bassiana conidia, and thus enhance the antifungal capacity of silkworm [The silkworm, genomics ,4,5,6. Ugenomics ,8,9. In genomics . Furthersilkworm ,12. Protsilkworm ,14.Aspergillus melleus protease, but have no inhibitory effect on trypsin and chymotrypsin [2nd and Cys6th may be the important reason for them to obtain inhibitory activity against microbial proteases. Cys2nd and Cys6th were introduced into the corresponding sites of BmSPI38 and BmSPI39 by site-directed mutagenesis. The results showed that the introduction of cysteines not only affected the polymerization state of BmSPI38 and BmSPI39, but also significantly reduced their inhibitory activity against microbial proteases [2nd and Cys6th did not change the inhibitory specificity of BmSPI38 and BmSPI39. By systematically summarizing the sequence characteristics and activities of reported TIL family protease inhibitors, it is found that the inhibitory specificity of these inhibitors may have a certain rule. The inhibitory activity and specificity of TIL family protease inhibitors may be determined jointly by the two missing cysteines (Cys2nd and Cys6th), and the physicochemical properties of P1 and P1′ residues in the reactive center [The TIL domain of most TIL family protease inhibitors in nature usually contains 10 conserved cysteine residues, forming 5 intramolecular disulfide bonds. The disulfide bridge positions are 1–7, 2–6, 3–5, 4–10, and 8–9 ,16,17. Sotrypsin ,12. It wroteases . Unfortue center . HoweverIt is well known that small-molecule serine protease inhibitors bind to proteases as substrate analogues, and at the same time the peptide bond in their reactive center is cleaved. Previous studies have shown that P1 residues in the reactive center of serine protease inhibitors largely determine their inhibitory specificity, and any change in P1 residues may affect the specificity and inhibitory ability of the inhibitor ,24,25,26In this study, mutant proteins were obtained by site-directed mutagenesis and prokaryotic expression techniques, and the effects of amino acid substitutions at the P1 position on the inhibitory activity and specificity of protease inhibitors BmSPI38 and BmSPI39 were investigated in vitro. This was carried out in order to provide a basis or reference for the inhibitory activity and specificity modification of TIL family protease inhibitors, and to promote the production and application of such inhibitors.B. mori, have elastase inhibitory activity, an in-gel activity staining technique was used to examine the inhibitory activity of recombinant BmSPI38 and BmSPI39 proteins towards porcine pancreatic elastase. Three strong inhibitory-active bands were detected for both recombinant BmSPI38 and BmSPI39, indicating that they could strongly inhibit elastase activity  and BL21(DE3) cells for induced expression, respectively. The P1 mutant proteins recombinantly expressed in E. coli cells were separated and detected by 16.5% reducing SDS-PAGE. The results showed that the P1 mutant proteins of BmSPI39 were mainly expressed in soluble form in BL21(DE3) cells, and obvious protein expression bands were detected at the size of its monomer (about 9.3 kDa) and dimer (about 18.6 kDa)  C. All P18.6 kDa) D. These E. coli cells , BmSPI38(G54K), BmSPI39, BmSPI39(A56R), and BmSPI39(A56K) were purified by immobilized-nickel affinity chromatography. In addition to the monomer forms of wild-type and mutant proteins, the dimers, trimers, and a small amount of higher-order multimeric forms were also detected in SDS-PAGE A. ProteaTo further compare the intrinsic inhibitory activity of P1 mutants of BmSPI38, different molar concentrations of protease inhibitors were incubated with subtilisin, proteinase K, and elastase, respectively, and the residual enzyme activities were measured. With the increase in BmSPI38, BmSPI38(G54R), and BmSPI38(G54K) concentrations, the residual enzyme activities of subtilisin, proteinase K, and elastase gradually decreased, indicating that all of them could strongly inhibit the activities of the above three proteases A,C,E. OvTo further investigate the effect of replacing Ala at the P1 position with a strong basic amino acid on the intrinsic activity of BmSPI39, the protease inhibitors with different molar concentrations were incubated with subtilisin, proteinase K, or elastase, respectively, and the residual enzyme activities were determined. With the increase in the molar ratio of inhibitor to protease, the residual enzyme activities of subtilisin A, proteiPrevious studies have confirmed that wild-type BmSPI38 and wild-type BmSPI39 have extremely high acid–base stability and thermal stability ,12. To eElastase is a serine protease with broad specificity that hydrolyzes connective tissue components such as elastin, proteoglycan, fibronectin, and collagen I, II, III, and IV, etc. Elastin is the major component of elastic fibers. Elastic fibers mainly exist in ligaments and vascular walls, endowing tissues with elasticity and tensile capacity, and are important components of tissues and organs such as skin, lungs, large arteries, and ligaments. The excessive production of elastase can lead to different degrees of tissue damage, and can cause or aggravate emphysema ,32, acutAntheraea assamensis, B. mori, and Philosamia ricini have a certain inhibitory effect on porcine pancreatic elastase [B. mori silk, and TIL protease inhibitors represented by BmSPI39 and BmSPI38 enter the cocoon layer during the process of silk secretion, which endows the cocoon with strong anti-microbial protease hydrolysis properties [Silk protein has broad application prospects in many fields due to its unique characteristics of biocompatibility, biodegradability, self-assembly, mechanical stability, controllable structure, and morphology ,52. The elastase . The treelastase . Studiesoperties ,14,53,542nd and Cys6th resulted in a dramatic decrease in the inhibitory activities of BmSPI38 and BmSPI39 against subtilisin and proteinase K, but failed to alter their inhibitory specificity [It is found that BmSPI38 and BmSPI39 are very different from typical TIL protease inhibitors in structure and activity ,12. The cificity . Obviouscificity ,24,25,26B. mori fungal protease inhibitor-F (BmFPI-F) has similar sequence characteristics and inhibitory activities with BmSPI38 and BmSPI39, and only has one TIL domain containing eight cysteine residues, which can inhibit the activities of microbial proteases such as subtilisin, proteinase K, and A. melleus protease [Streptomyces subtilisin inhibitor (SSI) was carried out, and all mutants also showed different degrees of subtilisin inhibitory activity [Studies have shown that different P1 residues may affect the conformation of protease inhibitors ,56,57. Hprotease . When thprotease . The satactivity . These factivity . The repactivity , which wCenchritis muricatus, which can inhibit not only trypsin activity, but also subtilisin and elastase activity [Microplitis demolitor bracovirus, has only eight conserved cysteines in the TIL domain. The mutation of the P1 residue of Egf1.0 from Arg to Ala resulted in the loss of its inhibitory activity against prophenoloxidase activating proteinase-3  [Cotesia vestalis teratocytes can secrete a TIL-type protease inhibitor CvT-TIL, which can strongly inhibit the activation of prophenoloxidase in the hemolymph of the host insect by interacting with PAP3 of the phenoloxidase (PO) cascade pathway [Trypsin, chymotrypsin, and elastase are serine proteases with high sequence and structural similarity, but with different substrate specificity . Trypsinactivity . The subactivity . SSI mutactivity . The winactivity . This rutrypsin) . Cotesia pathway . CvT-TIL pathway . It shouIt should be noted that the mutant proteins of BmSPI38 and BmSPI39 cannot be excluded from acquiring other new activities and functions due to the sensitivity of in-gel activity staining of protease inhibitor, and the limited types of proteases tested. For example, no inhibitory bands of BmSPI38(G54R), BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) against chymotrypsin were detected by in-gel activity staining, while the protease inhibition assays confirmed that the above four mutants had weak inhibitory activities of chymotrypsin. Because BmSPI38(G54R) has relatively weak inhibitory activity against trypsin, only the physicochemical properties of BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) with strong activity were investigated in this study. Similar to the wild-type BmSPI38 and BmSPI39 ,12, the The results presented above indicate that the amino acid properties of P1 residues play an important role in the inhibitory activity and specificity of BmSPl38 and BmSPl39, which may be related to the conformational changes around the reactive center. Future studies should be carried out to obtain more mutants, reveal the structure–function relationship between BmSPl38, BmSPl39, and their target proteases, and obtain more potent and specific inhibitors using BmSPl38 and BmSPl39 as scaffolds for biomedicine and agroforestry pest control.E. coli Trans-T1, BL21(DE3), and Origami 2(DE3) were purchased from TransGen Biotech , Sangon Biotech  and Invitrogen , respectively. ® FastPfuTransStart DNA polymerase (FastPfu), ® FastPfuTransStart Fly DNA polymerase (FastPfu Fly) and EasyPfu DNA Polymerase (EasyPfu) were purchased from TransGen Biotech. Proteinase K from Tritirachium album limber and elastase from the porcine pancreas were purchased from Roche  and Sangon Biotech, respectively. Subtilisin A from Bacillus licheniformis, trypsin and α-chymotrypsin from the bovine pancreas, N-acetyl-D,L-phenylalanine-β-naphthylester (APNE) and Fast Blue B Salt were purchased from Sigma-Aldrich . Fluorescein isothiocyanate (FITC)-labeled casein was purchased from Thermo Fisher Scientific . Recombinant expression plasmids BmSPI38-p28 and BmSPI39-p28 were preserved by the College of Biological Science and Engineering, Shaanxi University of Technology.BmSPI38-p28 and BmSPI39-p28 recombinant expression plasmids were transferred into dam+E. coli strain Trans1-T1. After overnight culture at 37 °C 220 r/min, methylated BmSPI38-p28 and BmSPI39-p28 plasmids were extracted again. Then, the BmSPI38-p28 or BmSPI39-p28 plasmid was used as a template for PCR amplification with FastPfu DNA polymerase to introduce target mutations. The PCR products were purified using ®EasyPure PCR Purification Kit (TransGen Biotech) or ®EasyPure Quick Gel Extraction Kit (TransGen Biotech), and then digested with the Dpn I restriction endonuclease (Thermo Fisher Scientific) at 37 °C for 30 min to completely remove the methylated template DNA. The Dpn I digested products were transformed into Trans1-T1 competent cells, and the monoclones were selected for sequencing verification. As shown in FastPfu Fly or EasyPfu) or templates (the mutant expression plasmid that has been successfully constructed) in the PCR. Finally, all the mutant expression vectors were constructed.According to the manual of the QuikChange II XL Site-Directed Mutagenesis Kit , 19 pairs of mutagenic primers were designed for site-directed saturation mutagenesis at the P1 position of BmSPI38 and BmSPI39, respectively. The templates, desired mutations, DNA polymerases, and primer sequences involved in P1-site mutations of BmSPI38 and BmSPI39 are shown in E. coli Origami 2(DE 3) and BL21(DE3) competent cells for induced expression, respectively. When the OD600 of the culture reached 0.6–1.0, protein expression was induced using IPTG at a final concentration of 0.2 mmol/L at 37 °C for 5 h or at 16 °C for 20 h. The E. coli cells were harvested by centrifugation at 6000× g for 30 min, washed, and suspended with binding buffer . The E. coli cells were lysed by sonication, and bacterial precipitates and supernatants were collected by centrifugation at 16,000× g for 30 min. The expressions of mutant proteins were analyzed by 16.5% SDS-PAGE. The mutated proteins were purified with Ni2+-NTA (nitrilotriacetic acid) affinity chromatography  as described for the wild-type proteins [2+-NTA affinity chromatography column. It was then washed and eluted sequentially by binding buffer supplemented with 20, 50, 100, and 400 mmol/L imidazole. The eluted fraction enriched with the target protein was pooled, and the imidazole was removed by over-night dialysis at 4 °C in binding buffer. The second round of immobilized-nickel affinity chromatography was performed. The purified mutant proteins were finally collected and dialyzed to 10 mmol/L PBS buffer (pH 7.4) for activity detection.The expression plasmids of the P1 mutants of BmSPI38 and the expression plasmids of the P1 mutants of BmSPI39 were transferred into proteins ,12. The 2O, and then were allowed to stand in the dark at 37 °C for 30 min. The gels were stained for 15 min at 37 °C in the dark with a mixture of substrate  and a staining solution  at a ratio of 1:10. The gels were stained fuchsia due to the diazotization-coupling reaction of β-naphthol that was produced by protease hydrolyzed substrates (APNE) on the gel. Due to protease inhibition, the positions of protease inhibitors will not be stained and will be shown as white bands.In-gel activity staining of protease inhibitor was performed as previously described, with a slight modification ,66. AfteDepending on the hydrolytic capacity of different serine proteases to FITC-casein substrate, 3 pmol of subtilisin, 3 pmol of proteinase K, 750 pmol of elastase, 15 pmol of trypsin, or 15 pmol of chymotrypsin were used per reaction (per well) in the protease inhibition assay. To determine whether P1 residue replacement could alter the inhibitory specificity of BmSPI38 and BmSPI39, a relatively high dose of the protease inhibitors was incubated with the specified proteases. The molar ratios of protease inhibitor to subtilisin, proteinase K, elastase, trypsin, and chymotrypsin were set as follows: 15:1, 15:1, 0.2:1, 150:1, 150:1. In order to further compare the inhibitory capacity of the mutant proteins on different proteases, different molar concentrations of protease inhibitors were incubated with the specified proteases. The molar ratios of protease inhibitors to subtilisin or proteinase K were set as 0.2, 0.5, 1, 2, 5, 15, and 25. The molar ratios with elastase were set as 0.004, 0.02, 0.04, 0.08, 0.2, 0.4, and 0.6. The molar ratios with chymotrypsin were set as 1, 10, 25, 50, 100, and 150. The molar ratio of BmSPI38 mutants to trypsin were set as 1, 5, 25, 55, 100, and 150; the molar ratio of BmSPI39 mutants to trypsin were set as 0.2, 0.5, 1, 2, 5, 15, and 25. The protease inhibitor and protease were mixed and supplemented with 0.1 mol/L Tris-HCl (pH 7.5) to 100 μL, and then incubated at 37 °C for 30 min. Then, 100 μL FITC-casein was added and incubated at 37 °C for 60 min in darkness. The fluorescence intensity was measured at the excitation and emission wavelengths of 485/535 nm, and the residual enzyme activity was calculated. The inhibitory activity of the protease inhibitor against protease was assessed by the following formula: residual enzyme activity% = enzyme activity of experimental group/enzyme activity of control group × 100%. In the control group, the protease inhibitor was replaced by an equal volume of 10 mmol/L PBS buffer (pH 7.4).Equal masses of purified mutant proteins were mixed with Britton–Robinson buffer of different pH values (pH 3–11) and incubated at room temperature for 24 h. The purified mutant proteins were treated at different temperatures  for 10 min. Then, the treated protein samples were separated using 10% alkaline Native PAGE. Finally, the activities of mutant proteins were analyzed by in-gel activity staining.n = 3). Marks with different lowercase letters “a to d” indicate significant differences between treatment groups at p < 0.05, while marks with one identical letter indicate no significant differences between treatment groups at p < 0.05.All statistical analyses of the data were performed using the Data Processing System (DPS) software version 9.01. Statistically significant differences were assessed by one-way analysis of variance (ANOVA). The error bar represents the standard error of the mean (In conclusion, this study demonstrated, for the first time, that silkworm protease inhibitors BmSPI38 and BmSPI39 can strongly inhibit the activity of elastase, and confirmed that the replacements of P1 residues not only greatly affect the intrinsic activity of BmSPI38 and BmSPI39, but also alter their inhibitory specificity when P1 residues are replaced with strong basic amino acids. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors.


To predict the factors of residual stones after percutaneous nephrolithotomy (PCNL) by analyzing the characteristics of the renal anatomical structure in intravenous urography, so as to make a reasonable operation plan, reduce the risk of residual stones in PCNL, and improve the stone-free rate (SFR).t-test was used to analyze the age, the length and width of channel calices, the angle between the channel calices and the involved calices, and the length and width of the involved calices. The gender, the channel types, the number of channels, the degree of hydronephrosis, and the number of involved calices were analyzed by using the chi-square test. A score of p < 0.05 was considered statistically significant. At the same time, logistic regression analysis was carried out to explore the independent influencing factors of the SFR after PCNL.A retrospective study was performed between January 2019 and September 2020 for patients treated with PCNL. According to the results of a kidney ureter bladder review after PCNL, 245 patients were divided into a residual stone group  and a stone-free group . An independent sample p = 0.003), the angle between the channel calices and the involved calices (p = 0.007), the width of the involved calices (p < 0.001), the channel types (p = 0.008), and the number of involved calices (p < 0.001) were all significantly correlated with residual stones after PCNL. Logistic regression analysis showed that the width of the channel calices (p = 0.003), the angle between the channel calices and the involved calices (p = 0.012), the width of the involved calices (p < 0.001), the channel types (p = 0.008), and the number of involved calyces (p < 0.001) were all independent influencing factors of the SFR after PCNL.A total of 71 patients developed residual stones after surgery. The overall residual rate was 29.0%. The width of the channel calices (A larger caliceal neck width and angle can reduce the risk of residual stones. The more calyces that are involved, the higher the risk of residual stones. There was no difference between F16 and F18, but F16 had a higher SFR than F24. Percutaneous nephrolithotomy (PCNL) is considered the first choice of treatment for large ≥2 cm) and complex renal calculi , 2. Howe cm and cGSS classified the complexity of PCNL into four grades  according to the imaging characteristics of patients , 7. The However, the renal anatomy was not involved in the four scoring systems. In this study, we compared several renal anatomic factors associated with PCNL outcomes and studied the possible predictors of the stone-free rate (SFR) after PCNL.A total of 245 renal calculi patients  were retrospectively reviewed from January 2019 to September 2020 in our hospital. Demographic, perioperative, and anatomical data for all patients were retrospectively collected, and the data included age, gender, the length and width of channel calices, the angle between channel calices and involved calices, the length and width of the involved calices, the channel types, the number of channels, the degree of hydronephrosis, and the number of involved calices . All patWith the patient under general anesthesia, all operations were performed on the fluoroscopy table . The patient was initially placed in the lithotomy position, with a 5-Fr ureteral catheter retrograde attached to the renal pelvis under cystoscope. Then, the patient’s position was changed to prone. Under the guidance of ultrasound  was used for statistical analysis. A value of p < 0.05 was considered statistically significant.The two groups were compared in terms of demographic, perioperative, and anatomical data using the Student’s p = 0.356). The length of the channel calices was longer in the residual stone group (18.83 ± 7.76 vs. 17.89 ± 6.74 mm) than in the other group, but it was not statistically significant (p = 0.343). There was no significant difference in the number of tracts (p = 0.280) and the degree of hydronephrosis (p = 0.706) between the two groups (p = 0.003), involved calices , and angle . There were significant differences in channel types (p = 0.008) and the number of involved calices (p < 0.001) between the two groups , the angle between the channel calices and the involved calices (p = 0.012), the width of the involved calices (p < 0.001), the channel types (p = 0.008), and the number of involved calyces (p < 0.001) were the independent influencing factors of the SFR after PCNL  between the two groups. The number of calyces involved basically represents the complexity of the renal stone. The complexity of the stones increased with the number of calyces involved. Chen et al. reported that the residual rate of renal complex stones after PCNL is approximately 29.6%  had residual stones after PCNL. The gender and age differences were small and were not predicted to affect the outcomes. The length of the channel calyces and involved calyces had no effect on the PCNL results. Because the length of the working channel in PCNL can reach up to 20 cm as long as the angle and calyceal neck width are appropriate, the operation channel can reach any position of the calyces. As the study was a retrospective one, the selection of the number of channels was influenced by the surgeon's preference. In our series, surgeons prefer single-channel lithotripsy, which, however, will give rise to the possibility of producing biased results. As the surgical assistant injected 0.9% of sodium chloride into the ureteral catheter during puncture positioning to create an artificial hydronephrosis, the question whether the patients had hydronephrosis during the course of their disease has little bearing on the results of PCNL. There were significant differences in the number of involved calices (ly 29.6% , which ily 29.6% . In the However, there are several limitations in this study that should be noted. First, we used the IVU method to study renal anatomical factors instead of the more accurate 3D-CT image reconstruction, which may impact the analysis of the measured data. Second, we used a retrospective research method rather than a prospective study, and therefore, a possible bias in the research results cannot be ruled out. Third, this finding may have some bias due to the fact that the F16/18 is mainly used for stones of smaller size. So, the chances of a higher SFR will be greater. With the same stone volume, the use of F24 may Úlso result in better clearance.A larger caliceal neck width and angle may reduce the risk of residual stones. The more the involved calyces, the higher the risk of residual stones. There was no difference between F16 and F18, but F16 had a higher SFR than F24.


A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe+2) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). We evaluate each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms  Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, here called an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. We provide evidence that CcmF/H receives Fe+3 heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD/P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe+2) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site.We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out  Following release, the cyt c protein folds into its native state.Cytochromes are heme proteins involved in electron transport in the cell. Maturation of cytochromes often requires accessory factors for heme insertion and/or protein complex formation. The assembly of c-type cytochromes (cyt c) is complicated, in part because reduced heme Fe+ is posttiewed in , 4, 5). It has been known for over 2 decades that three pathways in nature exist for the biogenesis of c-type cytochromes, called systems I, II, and III . System A and B), which are more complicated than system III. System I and II are present in bacteria, archaea, and some mitochondria (C). Since all c-type cytochromes are in the periplasmic space, these pathways transport heme to outside the inner membrane and covalently attach it to the CXXCH motif (A and B). All cyt c apoproteins possess a SEC-dependent signal sequence for secretion to the outside. Roles in heme transport and attachment for the eight proteins (CcmA-H) in System I, and two (CcsB and CcsA) in system II, all membrane proteins, have been suggested . Here, we explore the structures to examine the structural basis for mechanisms and universal features of key domains. We focus on the CcsBA and CcmF/H complexes, which have been termed multifunctional molecular machines .This manuscript concerns the much older (evolutionarily) systems I and II , A and Bchondria C. Since CH motif , A and Bd , and works in two general steps (A): the first is to transport heme via CcmABCD, an ABC transporter complex, and attach the heme to CcmE, then release holoCcmE. Recent cryo-EM structures of CcmABCD showed where exogenously added heme bound to the CcmC WWD/P-His domain (C and D), whether via a channel through the CcmC protein or from the outer leaflet. Here, it is important to note that heme transport only requires CcmCD, not the entire CcmABCD complex (+3)   , 29. Ccmlex (+3) , and itslex (+3) , 34, 35.ynthesis A 36)..EschericB). This analyses and other evidence are collectively used to describe mechanisms of trafficking holoCcmE (heme) to the CcmF/H cyt c synthase active site (the WWD/P-His domain). Our analyses here indicates that heme (via holoCcmE) enters CcmF from above, via the soluble periplasmic milieu, directly into the WWD/P-His domain. We call this the periplasmic entry model, with heme delivery by holoCcmE directly to the active site. In contrast to the heme transport function of CcsBA , we have proposed a reduction function for CcmF whereby the heme in holoCcmE is reduced (to Fe+2) prior to attachment to CXXCH(29). CcmF thus is a cytochrome itself, using its stable transmembrane (TM)-bound heme to reduce the incoming heme of holoCcmE. We discuss key residues involved in the multifunctional features of CcmF/H, which can be considered a heme oxidoreductase and cyt c synthase machine.The structures of both CcsBA  and CcmFFig. 1+2) from inside to outside, and attaches it to CXXCH (B). We explore CcsBA from a wide distribution of organisms (C), fitting key conserved regions into cryo-EM densities to scrutinize the structural basis for universal mechanisms of these functions. This includes periplasmic chamber domains for access to the CXXCH motif, the binding of CXXCH, and conserved heme binding and export features. We structurally define two pockets for the two thiols of the CXXCH substrate, which also facilitate histidine (of CXXCH) liganding to the substrate heme at the active site. Above the active site we observe a beta stranded domain in all CcsB/A proteins. We suggest this “beta cap” functions to occlude the active site when the substrate heme is not present.CcsBA has been referred to as a bifunctional heme transporter and cyt c synthase machine , 38. Ccsto CXXCH B. We exprganisms C, fittinHelicobacter hepaticus is displayed in Fig. S1A, with conserved residues in the subfamily colored red. The topology is used as a framework for discussing three dimensional elements critical for function, some of which have never been addressed.We begin with CcsBA because its structures, with endogenous heme bound at the active site, offers insights that are later applied to CcmF/H. The protein topology and primary sequence of CcsBA from ccsB and ccsA involvement in cyt csynthesis in model organisms: ccsA and ccsB (ccs1) in Chlamydomonas reinhardtii chloroplasts (ccsA (resA) and ccsB (resB) for Bacillus subtilis . This natural fusion has facilitated overexpression, GST-tagging and hexahistidine tagging, and purification of CcsBA in recombinant E. coli  and CcsB-CcsA complexes (unfused). When referring to both, we use CcsB/A. Helicobacter, Wolinella, Bacteroides in this study).As background, early genetic studies defined roplasts , 42, 43,subtilis , 45, ccsertussis , 47, 48. E. coli , 49. Theochromes . These red CcsBA . We focuB). This conformation has a closed periplasmic chamber above the active site, thus is called the “closed” conformation or state. The other structure has heme present in both the TM-heme site and in the external WWD/P-His site. The periplasmic chamber in this conformation is “open” with access to CXXCH, which will bind adjacent to the heme in the WWD/P-His site.From cryo-EM studies, one of the two CcsBA structures has heme only in the TM-heme site B. This cHelicobacter) to beta proteobacterium (B. pertussis) to Gram positive  to cyanobacteria and chloroplasts. We address whether all elements are present in both the fused CcsBA and two-subunit unfused CcsB-CcsA complexes. As a convenient flow of the structural analyses, we begin with internal (cytoplasmic) heme as it is transported through CcsBA to the external active site, where heme is attached to CXXCH and the holocyt c released for proper folding.Major questions on CcsBA concern what features are completely conserved in the subfamily, from Gram negative epsilon proteobacterium . In H. hepaticus CcsBA these are TM10-13.The general architecture of CcsBA proteins appears to have particularly conserved features, although some of the CcsB/A proteins have a different number of predicted TMs . RoseTTAe.g., (H. hepaticus densities (A).A key question for any transporter is the initial entry point of the substrate. For CcsB/A, heme enters from the cytoplasmic side (or inner leaflet), moving to the TM heme site that is defined by two TM histidine ligands . Because no CcsBA conformation was uncovered that has heme only in the P-heme site, we have proposed that heme in the TM heme site moves through a channel to the P-heme site by a mechanism analogous to the well-known potassium channel e.g., , 53, 54), 54e.g.,ensities A.C, red highlighted), fused CcsBA proteins possess a vestibule or possible entry site but no other distinguishing domains on the cytoplasmic side. Interestingly, the unfused CcsA-CcsB complexes possess a large cytoplasmic domain that is immediately adjacent to the proposed heme entry site (to the TM-heme site) (A). The general architecture of this cytoplasmic domain is similar among the unfused CcsB-CcsA complexes (B). The cytoplasmic domain is in large part composed of an additional C-terminal extension of the ccsB sequences. We suggest that this additional domain could facilitate heme delivery to the TM-heme site. It is entirely feasible that heme could be inserted directly into the vestibule or directly into the TM-heme site from the ferrochelatase, the final enzyme in heme biosynthesis that inserts the reduced iron. Such an interaction could be heme-driven or by CcsB/A:ferrochelatase protein:protein contacts. Indeed, using the web-based STRING database on protein:protein interactions, we discovered that in H. hepaticus, ferrochelatase is predicted to interact with CcsBA (C).We have analyzed a diverse set of CcsBA and CcsB/CcsA proteins for the presence of the putative vestibule and cytoplasmic domains that could assist delivery of heme. Our analyses of other ccsBA gene(s) involved the use of RoseTTAFold to predict structures, then to fit them into our cryo-EM densities and analyze. Of the eight CcsBA or CcsB-CcsA proteins chosen  A. The geomplexes B. The cyth CcsBA C.in vivo function when these recombinant CcsBA variants in E. coli are grown in the presence of exogenous imidazole. This chemical complementation suggests two stable ligand sites that can bind the free imidazole and thus correct heme binding. Importantly, less than 10% of the heme remains with the TM-His variants upon purification. This suggests that a vestibule would have a weak affinity for heme and that heme must go through the TM-site to the WWD/P-His site. All CcsBA proteins possess TM-His1 and TM-His2, with TM-His2 present in CcsB and TMHis1 in CcsA.The first heme liganded binding site in CcsBA has been demonstrated to require two histidines that are present in TM12 (TM-His1) and TM3 (TM-His2) . MutatioThe eight CcsBA predicted (RoseTTAFold) structures fit the densities surrounding the TM-heme binding site, including the TM-His1 and TM-His2 residues . In eachH. hepaticus CcsBA cryo-EM structures, indicate that heme moves from the TM-heme site to the external WWD/P-His site. Consistent with this conclusion is the presence of a channel in both the open and closed conformations in the cryo-EM densities. Examination of the eight CcsBA proteins fit to these densities all show this channel  to the CcsBA protein. These cys/heme crosslinks were detected and mapped in both CcsBA and CcmC, with results suggesting a conserved common structural motif near the heme vinyl groups.Various experiments have shown that the WWD/P-His domain constitutes a heme binding domain that faces the periplasmic space. Both histidines are required for und heme . That isi.e., with no P-heme).Analyses of the CcsBA open conformation with endogenous heme in the external binding site indeed confirms the above predictions from biochemical results. The WWD/P-His domain binds heme and represents the active site; this heme will be attached to the CXXCH acceptor. Both periplasmic histidines act as ligands to the heme iron in this state. Thus, the structure of CcsBA (open state) is the only family member so far with a resolvable P-His2 loop and a P-His2 ligand, no doubt stabilized by the heme liganding. We have proposed that the P-His2 loop is flexible, able to move in and out of the active site . This isH. hepaticus open conformation density for fitting the other CcsB/A proteins in this active site (A and B). It is clear that P-His1 of each CcsB/A fit into the densities . Although the P-His2 imidazole can be envisioned as a ligand , the P-His2 loop itself is less poorly predicted by RoseTTAFold, supporting the suggestion of a flexible loop. The P-His1 residue is at the outer leaflet of TM 10 helix, with the P-His1 loop between TM9 and 10. The overall structures in this active site, including core TMs are strikingly similar in all CcsB/A proteins.We used the ive site , A and BB shows the same site after computationally removing the P-His2 loop so that the WWD domain can be visualized (green). In fact, the cryo-EM structures show the WWD interaction with heme is nearly identical in CcsBA (open) and the CcmC with added heme. Even more remarkable is that the aforementioned residues in the WWD that cross-linked heme (after cysteine substitution) are adjacent to the 2 or 4 vinyl groups of heme  illustrates the conservation of the WWD domain. We have argued previously that the WWD domain is for binding heme and likely for releasing it once the covalent thioether bonds are formed. Additionally, the WWD side chains may be conserved to aid the chemistry of covalent bond formation (thioethers for CXXCH substrate and the CcmE His130 adduct for CcmC). Even though these are spontaneous reactions, the WWD environment may optimize them.Fig. S5 of heme , 26. FitA and B displays the human cyt c sequence CSQCH docked into the active site above the external heme in the CcsBA open state. Two pockets are present in which the first cysteine (C1) and second cysteine (C2) are docked. These are adjacent to vinyl 2 and 4, respectively, thus a mechanism for the stereochemical attachment is revealed by this binding and the histidine liganding (of his in CXXCH). For reference in the topological sequence of CcsBA and numbering, refer to Fig. 3A) is formed by residues (colored purple) that include the outer TM13 (conserved V889 and N890) as well as residues at the C terminus of the WWD sequence . The C2 pocket  is formed by residues (colored green) that includes a periplasmic loop between TM9 and 10  as well as conserved residues at the N terminus of the WWD sequence . Conservation of nearly all these residues suggests a highly conserved binding site for the CXXCH substrate  machines are present, in addition to the canonical CcsBAs that recognize CXXCH. Wolinella succinogenes is one example of these bacteria, and its three substrates are recognized by three different fused CcsBA proteins . Structures of these unusual CcsBA proteins could address such questions on alternative substrate recognition.In addition to the canonical cyt c heme binding motif CXXCH, some bacteria possess very rare c-type cytochromes that have alternative substrates, CXXCK and CXproteins , 56. TheH. hepaticus revealed a large periplasmic chamber which opens for CXXCH substrate access only when heme is at the active site. We discussed the molecular basis for the opening upon heme binding to the WWD/P-His site and speculated that closing could protect the channel and/or TM-heme . As in H. hepaticus, fused CcsBAs have chambers . Part of this chamber is comprised of what we here designate the beta cap domain, a more conserved region that appears to occlude (cap) the active site upon closing . The unfused CcsB-CcsA complexes possess a smaller periplasmic region, all possessing the CcsB beta cap , the beta cap representing the conserved domain structurally and likely functionally. Fig. S6B shows the best-fit analyses of the beta cap sequences, with the loop region labeled. Within the loop is a conserved tyrosine (Tyr245 in H. hepaticus) and phenylalanine (Phe 240 in H. hepaticus) that begins the loop  that are separated by two TMs . PD1 andFig. S6the loop . These mructures .+3 to Fe+2). We previously have suggested the chemistry behind formation of the His130 adduct to heme vinyl 2 in CcmE, favoring oxidized heme (+3) is the substrate for CcmF/H, we proposed that the role for the stable TM-heme in CcmF is to reduce holoCcmE at the active site  the donor heme for the synthase reaction is not transported from the TM heme site but instead comes from periplasmic holoCcmE, (2) a function for a “stable” (accessory) heme in the TM site is to reduce the external “donor” heme at the CcmF/H active site  is in the same location whether closed (no P-heme) or open (P-heme present). Likewise, in CcmC, the P-His1 imidazole position is not appreciably changed in various CcmC states and to do so would require significant movement of the entire TM in which P-His1 is located . Favoring the periplasmic model, there is access for heme (holoCcmE) from above in CcmF at the homologous site in CcsBA , without envisioning a 17 Å conformational change, and such that P-His1 liganding would occur without major movements. For the periplasmic entry, the WWD residues also would be located similarly to those in CcsBA and CcmC . Furthermore, the leaflet buoy entry model proposes that heme moves the WWD and is in fact on the opposite side of the WWD domain shown for CcsBA.In the CcmF leaflet entry model, heme docked in the vestibule/cleft is located at least 17 Å from P-His1 . AlthougFig. 62.B). Part of the hydrophoblic tails of a surface phospholipid also enters the putative vestibule . Although Brausemann et al. do not describe the details of heme docking, it seems likely they have computationally removed the dodecyl maltoside and phospholipid before molecular docking. Thus, a question remains on whether there is a vestibule/cleft and opening under physiological conditions . It may be pertinent that theoretical CcmF structures using RosseTTAFold  in CcmE enters first into the CcmF active site from above. The vinyl half of heme (adduct to His130) is consequently located near the surface within the CcmF active site formed by WWD/P-His. This makes sense since the vinyls will be attached to the incoming CXXCH, as occurs in CcsBA (from above). This in fact is how the active site heme is oriented in CcsBA (A). Only the P-His2 loop would be needed to move into its position, thus replacing the Tyr134 of the holoCcmE. Since the P-His2 loop is not resolved in the CcmF structure, it is likely flexible. As further support for this orientation and location in the periplasmic model, the Sutherland lab has recently published WWD cysteine/heme crosslinking studies on CcmF (A).The periplasmic entry model hypothesizes that the polar part of heme  will take place. Structures of various conformations as the CcmF/H synthase complex, with and without the holoCcmE heme donor, will be needed.Taken together, we suggest that the “periplasmic entry” model for CcmE, from the periplasmic surface, is consistent with the available data. Based on Cryo-EM structures of CcsBA  and struet al. . These two tryptophans not only occlude the channel from TM-heme to P-heme, they are absent in CcsBA. Thus, the proposed order of reduction is as follows: unknown reductant→TM-heme→W214→W251→P-heme. e.g., .Figure The CcmH protein is known to act as a thiol reduction protein (as is CcmG or in its place DTT) for reduction of the cysteines in CXXCH , 66, 67.Subsequent to formation of both thioethers, the holoCXXCH product (cyt c) is released from the active site. The release mechanism is expected to be similar to CcsBA. We have shown that in human HCCS, the heme distortion produced by two thioether attachments induces release, likely weakening the binding of heme to the cyt c synthase . Both CcA shows the overall structures of each member, with hemes in each class. Four core TMs form a central channel; in the case of CcsBA and possibly CcmC, this channel allows heme movement from inside to the outside WWD/P-His site. In the case of CcmF, two tryptophans and other side chains occlude the channel. The four core TMs also link the WWD domain, a conserved periplasmic loop that defines the family. P-His1 is localized to the outer TM of one of these four core TMs in each member, thus defining a structural and functional (heme liganding) state for all members. P-His2 is flexible, mobile, and part of a periplasmic loop following the last core TM in all three family members.There are three structural features present in all three family members  that stand out: a nearly identical core of four TMs, the WWD domain, and two periplasmic histidines (P-His1 and P-His2). Figure 2et al. . Importantly, the beta cap domain is situated directly above the active site (P-heme) in this chamber. For unfused CcsB-CcsA complexes, the beta cap domain is often not part of a chamber, but could also occlude the active site when heme is not present. Interestingly, as pointed out by Brausemann et al. , CcmF apet al. . CcmC doet al. , 26, whiet al.  will resvia CcmE) will require more structures and other experiments.Based on our CcsBA structures  as well e.g., repulsion of the thioethers), and (iii) ligand switching (the CXXCH histidine replacing the P-His2 ligand). In vitro reconstitution (cyt c synthase and release) was recently achieved with purified CcsBA, opening up the possibility that the recognition (of CXXCH) and release features can be dissected.Release of the heme-attached substrates remains speculative, although the cryo-EM structures of CcmABCD certainly point the way for ATP hydrolysis-mediated movement of the P-His2 loop for release of holoCcmE from CcmABCD . In contIn vivo and in vitro studies on protein:protein interactions are needed. The studies on mitochondrial protein interactions, including those specifically with ferrochelatase, are a good example  .Theoretical structures of the CcmABCD, CcmFH/E, and CcsBA complexes have been predicted using increasingly improved structural prediction programs over the last 5 years  e.g., , 22, 60), 60e.g.,in vitro biosynthesis  (The generation of cryo-EM densities of purified k-24942) . ExperimH. hepaticus) were generated through RoseTTAFold , and Pymol (https://pymol.org/2/). For CcsB/A analyses, we first compared the theoretical structures generated to the known cryoEM structure of H. hepaticus by overlaying the theoretical structures with the structure of H. hepaticus through the PyMOL “align” feature. From here Chimera’s “fit in map” was used to dock the structures into the H. hepaticus open electron density. Structures of fused CcsBA  were modeled using their already naturally fused CcsBA sequence. See ccsB/A from each organism. The unfused CcsB/A structures  were modeled with CcsA and CcsB predicted separately. Proteins for these unfused structures were similarly overlayed by PyMOL's “align” feature followed by the Chimera “fit in map” feature placing them into the open map density. The fit to density was further investigated in Coot  with CcsB in the unfused structures being rigidly docked using WinCoot’s rigid body fit zone to better align CcsB to density and prevent obvious physically improbable overlap with CcsA. All fittings were done rigidly, and only whole peptides were moved to avoid rearrangement of the position of TMs and residues relative to one another. All calculations of surfaces were done in Pymol using the MSMS algorithm. The surfaces were then considered relative to the electron cloud densities of H. hepaticus for the figures.All theoretical structures for CcsB/A proteins . In the present study, individual residues in the CXXCH binding site are analyzed and discussed.As described previously, docking of the CXXCH (human cyt c CSQCH) into the CcsBA structure was performed by removing the P-His2 loop of CcsBA and using AutoDock Vina to dock the CXXCH. The model chosen was the energetically plausible model that best liganded heme  was measured using UCSF Chimera, measuring the largest vertical and horizontal distance of each CcmF pore.et al. (130DENY134) modeled with WinCoot, and heme positioned according to the periplasmic model of entry, based on CcsBA open state analogies. The flexible loop containing P-His2 was constructed in WinCoot using the sequence of CcmF and modeled based on homology to the flexible loop of the H. hepaticus CcsBA structure (open state). After holoCcmE binding, electron transfer/reduction and apoCcmE ejection, CXXCH binding, attachment, and release are modeled, similar to the methods and mechanisms described for the H. hepaticus CcsBA structural studies.For the video showing the activities of CcmF, the crystal structure of CcmF from Brausemann et al.  was usedAll data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Anthracycline antibiotics (ANT) are among the most widely used anticancer drugs. Unfortunately, their use is limited due to the development of drug resistance and cardiotoxicity. ANT metabolism, performed mainly by two enzymes—aldo-keto reductase 1C3 (AKR1C3) and carbonyl reductase 1 (CBR1)—is one of the proposed mechanisms generated by the described effects. In this study, we evaluated the CBR1 inhibitory properties of ASP9521, a compound already known as potent AKR1C3 inhibitor. First, we assessed the possibility of ASP9521 binding to the CBR1 catalytic site using molecular docking and molecular dynamics. The research revealed a potential binding mode of ASP9521. Moderate inhibitory activity against CBR1 was observed in studies with recombinant enzymes. Finally, we examined whether ASP9521 can improve the cytotoxic activity of daunorubicin against human lung carcinoma cell line A549 and assessed the cardioprotective properties of ASP9521 in a rat cardiomyocytes model (H9c2) against doxorubicin- and daunorubicin-induced toxicity. The addition of ASP9521 ameliorated the cytotoxic activity of daunorubicin and protected rat cardiomyocytes from the cytotoxic effect of both applied drugs. Considering the favorable bioavailability and safety profile of ASP9521, the obtained results encourage further research. Inhibition of both AKR1C3 and CBR1 may be a promising method of overcoming ANT resistance and cardiotoxicity. Streptomyces genus. Since then, several natural and synthetic ANT have been introduced to therapy as effective cell-cycle nonspecific compounds, which act mainly via the inhibition of topoisomerase II and intercalation into double-strand DNA [Anthracycline antibiotics (ANT) are a group of therapeutics widely used in the treatment of various types of cancers, both solid tumours and haematological malignancies . The hisrand DNA . Doxorubrand DNA . Researcrand DNA ,4. To darand DNA . HoweverOne of the main new hypotheses explaining the limited use of ANT points out a crucial role of the activity of ANT metabolic enzymes, leading to the formation of secondary alcohols by the reduction of carbonyl moiety in the ANT C13 position . In a seThere are two main groups of enzymes leading to the formation of C13-reduced ANT metabolites: aldo-keto reductases (AKR) and carbonyl reductases (CBR). Enzymes belonging to both groups are monomeric, cytosolic, NAD(P)H-dependent proteins responsible for the two-electron reduction of various endogenous substrates, including prostaglandins, quinones and steroids ,9. AmongASP9521 is a potent indole-based AKR1C3 inhibitor. Considering nanomolar activity of ASP9521 against AKR1C3 and thus the possible inhibition of testosterone synthesis, the compound was tested in the treatment of castrate-resistant prostate cancer. It successfully passed in vitro and preclinical development evaluation and entered into clinical development but was then withdrawn due to a lack of clinical efficacy. However, the clinical evaluation indicated its acceptable safety and tolerability profile ,19. StruTaking into account the above facts, in the present study, we focused on the assessment of the potential CBR1 inhibitory activity of ASP9521. We hypothesised that ASP9521 can be an inhibitor of both main ANT reductases, making it a molecule with an as-yet undescribed mechanism of action. First, we docked ASP9521 to CBR1 to assess whether the compound can form interactions that are important for ligand recognition. We compared the binding mode with the one observed for known CBR1 ligands. Then, we performed an MD simulation to check whether ASP9521 holds the interactions observed in docking through a 20 ns simulation period and to evaluate the stability of the ligand within the CBR1 catalytic site. After in silico evaluation, the inhibitory activity was assessed in studies with recombinant CBR1 proteins, and then, the cytotoxic effect of simultaneous administration of DNR and ASP9521 was determined on A549 cancer cells. Finally, ASP9521 was examined as a potential cardioprotective agent preventing rat cardiomyocyte H9c2 cells from damage caused by DNR or DOX.To evaluate whether ASP9521 has the potential to bind to CBR1, we initially performed molecular docking to the CBR1 model. The ligand was docked successfully and its binding mode within the CBR1 catalytic site was in line with the one described for known CBR1 ligands , includiMolecular docking revealed the potential of ASP9521 to be a CBR1 ligand, thus we decided to confirm ASP9521 binding mode durability in MD simulation. The best-scored CBR1-ASP9521 complex obtained in molecular docking  was used in a 20 ns MD simulation to assess ligand stability within the CBR1 catalytic site. For this purpose, we measured distances between amide oxygen and carbon atoms of ASP9521 and two elements of the CBR1 enzymatic complex responsible for proton transfer to the CBR1 substrate. The latter elements were the phenol group of the Tyr193 side chain and the C4 pyridine atom of NADPH . ThroughThe Simulation Interactions Diagram tool detected and recorded all protein–ligand interactions during the 20 ns of the simulation .The most important interactions for a CBR1 ligand recognition–H-bonds with Ser139 and Tyr193–have been observed for most of the simulations time. The interaction between the amide oxygen atom of ASP9521 and the side chain of Ser139 was observed for 92.9% (1858/2000 frames–fr.) of the simulation time; and H-bond with Tyr193 for 87.4% (1748 fr.). These observations support the hypothesis that the interactions with ASP9521 remain stable within the CBR1 catalytic site and that the molecule does not dissociate when the protein–ligand complex undergoes dynamic changes over time. Unlike molecular docking, MD almost did not evidence the presence of interaction with a Met234 backbone. This interaction was observed only 1% (20 fr.) of the time—at the very beginning of the simulation. However, the hydroxy group of ASP9521 was more likely to interact with Met234 through a water bridge. This type of interaction was observed for 12% of the simulation time (238 fr.). This observation suggests that we cannot completely exclude the potential importance of Met234 in the binding mode of ASP9521 within CBR1. Instead of an H-bond with Met234, MD revealed another amino acid residue forming an interaction with the hydroxy group of 2-hydroxy-2-methylpropyl moiety of the ligand. For 86.2% of the simulation time (1724 fr.), we observed an H-bond with a backbone of Phe94. This observation suggests a second possible binding mode of ASP9521 within the CBR1 catalytic site in which the ligand does not occupy a space in the vicinity of Met234 and Ala235 (as observed in the case of ANT), but instead, a piperidine ring with 2-hydroxy-2-methylpropyl moiety adopts a conformation, which forms an interaction with the Phe94 backbone a.We additionally measured and compared distances between the ASP9521 hydroxy group and the backbone oxygen atoms of Met234 and Phe94. At the beginning of the simulation, the distance to Met234 was 2.69 Å, while the average distance during the simulation was 5.29 Å. Oppositely, the initial distance to Phe94 was 4.22 Å, while the average distance during the simulation was 3.05 Å b, which The location of the ASP9521 piperidine ring with attached 2-hydroxy-2-methylpropyl moiety, forming H-bonds mainly with Phe94 instead of Met234, was the most substantial difference between the binding modes observed in docking and MD simulation. In the first nanosecond of the MD simulation, the whole fragment shifted towards the backbone of Phe94, and the conformation of ASP9521 was then similar to that observed in the AKR1C3 crystal structure 4WDX, with another indole-based derivative co-crystallised within the catalytic site of the enzyme. The H-bond formation with Phe94 backbone was intriguing, as this residue has been indicated as one of the interaction points between CBR1 and glutathione molecules in crystal structures 3BHJ and 4Z3D . GlutathMD simulation also revealed that four amino acid residues have been involved in forming hydrophobic interactions with the indole ring of ASP9521. The π-π stacking with Trp229, formed in the initial CBR1-ASP9521 complex resulting from docking, was observed for 11.6% of the simulation time . That interaction was supported by additional van der Waals contacts with hydrophobic side chains within 3.6 Å of ligand aromatic or aliphatic carbon atoms, present 77% of the time (1347 fr.). The same type of hydrophobic interaction has been detected with residues next to Ser 139: Ile140  and Met141 . Based on the MD results, we may assume that such locations of Ile140, Met141, and Trp229 and observed hydrophobic interactions stabilise the aromatic part of ASP9521 and facilitate proper positioning of the whole ligand within the CBR1 catalytic site. The π-cation interaction between the indole ring of ASP9521 and the side chain of Arg144 was detected for 17% of the simulation time (343 fr.). Additionally, Arg144 was involved in the formation of a water bridge with an oxygen atom of the methoxy group attached to the indole ring of ASP9521 .50 value of 44.00 μM confirmed its moderate inhibitory properties against the enzyme. The obtained value is higher than those for the most potent CBR1 inhibitors (IC50 of flavonoids and 8-hydroxy-2-iminochromene derivatives around 0.1–0.4 μM), nevertheless, the report on the dual AKR1C3 and CBR1 inhibitory activity of ASP9521 sheds new light on its mechanism of action as a representative of multi-functional compounds as well as on the issue of ANT metabolism and directly related risks [50 for flavone: over 10 μM vs. IC50 for 7-hydroxyflavone: 0.6 μM; in the case of the second compound, the 7-hydroxy group formed a H-bond with Met234), but we do not have such analysis for compounds targeting interaction with Phe94 [We verified the hypothesis and observations obtained through in silico simulations using an in vitro CBR1 inhibitory assay measuring the change in the rate of CBR1 enzymatic activity upon the addition of ASP9521. The determined ASP9521 ICed risks ,27. ASP9th Phe94 . This inTo verify the hypothesis that co-administration of ASP9521 can be beneficial for ANT treatment, we performed experiments on a human lung carcinoma cell line A549 and rat cardiomyocyte H9c2. Cell line selection was based on literature research confirming high expression levels of CBR1 and AKR1C3. Increased levels of either one or both of the mentioned enzymes have been shown to correlate with greater resistance of cells to DOX and DNR as well as with the increased risk of cardiotoxicity . 50 of DNR used separately or in combination with ASP9521 (25 μM). Obtained IC50 values were 0.442 μM (separately) and 0.379 μM (in combination with ASP9521). Therefore, ASP9521 led to a decrease in IC50 of 14.25%. These results confirm that the inhibition of ANT metabolism is a significant factor for effective anticancer properties. Previously, a number of studies have shown a relationship between increased expression of CBR1 and AKR1C3 in cancer cells and reduced anticancer activity of ANT. It is estimated that the concentration of DOXol, causing the death of 50% of cancer cells, is 50–150 times higher than for DOX [We first examined whether ASP9521 (25 μM) enhances the anticancer activity of DNR (0.05–1 μM) in an A549 cells model. ASP9521 used alone did not possess any cytotoxic activity. However, co-administration of ASP9521 with DNR, statistically improved the anticancer activity of the drug after 48 h of incubation . This en for DOX . SimilarThe second part of in vitro assessment was the evaluation of the cardioprotective properties of ASP9521 25 μM; . H9c2 ceBased on the results of the enzymatic CBR1 inhibitory assay, we may assume that AKR1C3 inhibition was mainly responsible for the observed improvement in DNR activity as well as the cardioprotective effect and that it outweighs the effect associated with CBR1 inhibition. However, future design and development of dual CBR1-AKR1C3 inhibitors should lead to a more precise assessment of dual inhibitory strategy in the context of potentiation of ANT activity.Small-molecule Drug Discovery Suite with Desmond GPU  was licensed for Jagiellonian University Medical College. ASP9521 was purchased from Tocris . NADPH and DNR were purchased from BIOKOM. The sulforhodamine B cytotoxicity assay kit was purchased from Merck .α=20 = 0.552 [Molecular docking was performed using Small-Molecule Drug Discovery Suite . The structure of ASP9521 was prepared using LigPrep tool . An energy-minimized 3D conformer in a proper protonation state predicted in the pH range 7.4 ± 0.2 was generated. The CBR1 model used for docking studies (based on the PDB structure encoded 1WMA) was prepared as described previously and characterized by the following enrichment parameters: AUC = 0.87, BEDROC = 0.552 . The mod = 0.552 . DockingThe best-scored CBR1-ASP9521 complex obtained via molecular docking  was chosen to undergo MD. The simulation was performed using Desmond GPU . A system for the simulation was prepared using the System Builder module. The CBR1-ASP9521 complex was placed in an orthorhombic box, the size of which was calculated using the buffer method, with the box boundaries positioned 10 Å from the complex in all dimensions. TIP4P solvent model was applied, and 0.15 M of sodium and chlorine ions was added to simulate a cellular environment within the box. After initial model relaxation, performed in a default mode, a 20 ns simulation was run utilizing the OPLS3e force field and NPT ensemble. The trajectory interval was set to 10 ps, meaning that every 10 ps, an intermediate state (frame) of CBR1-AKR1C3 complex was captured and saved. As an output, 2000 CBR1-ASP9521 frames were obtained and analysed using the Simulation Event Analysis tool to measure distances between key CBR1 elements—phenol group of Tyr193 side chain and C4 pyridine atom of NADPH—and selected ASP9521 atoms—amide oxygen atoms and amide carbon atoms, respectively. Those pairs of characteristics are crucial considering the CBR1 mechanism of action. Simulation Event Analysis was also used for measuring distances between the ASP9521 hydroxy group from 2-hydroxy-2-methylpropyl moiety and backbone oxygen atoms of two amino acid residues—Met234 and Phe94—to assess which residue is more likely to contribute to interactions with the ligand. The Simulation Interactions Diagram tool was used to assess the types and durations of molecular interactions between ASP9521 and CBR1 amino acid residues formed within the 20 ns simulation period: ionic bonds, H-bonds and water bridges, and π-aromatic interactions.Escherichia coli Rosetta (DE3) and described in detail previously by Koczurkiewicz-Adamczyk et al. [50 value, additional assessments were performed in the concentration range 0.1–100 μM . Incubation mixtures containing recombinant CBR1 , menadione (120 μM) and ASP9521 or vehicle in phosphate buffer (pH 7.4) were preincubated for 5 min in 37 °C on UV-transparent 96-well microplate. Next, NADPH solution was added to a final concentration of 200 μM to initiate the reaction. The reaction was monitored using absorbance measurement at λ = 340 nm for 10 min in a microplate reader . A decrease in absorbance, corresponding to NADPH oxidation, was used to determine the initial velocities of reactions via a linear regression method. The inhibitory properties of ASP9521 were determined by comparing the velocity of the reaction against vehicle control . DMSO concentration <1%, did not affect enzyme activity. Each experiment was performed in three repetitions.Expression and purification of human recombinant CBR1 were performed in k et al. . The ini2 concentration (5%).Human lung carcinoma cell line A549 (ATCC CCL-18) and rat cardiomyocyte H9c2 (ATCC CRL-1446) were cultured in appropriate culture media recommended by ATCC and supplemented with 10% foetal bovine serum  and 1% antibiotic mixture . The cells were cultured in a standard condition of temperature (37 °C) and COASP9521 was applied from DMSO stock solutions and diluted in the culture media to the working concentrations. DOX/DNR were applied to the culture media diluted to the working concentrations from a freshly made stock solution in DMSO (100 μM).4 per well. After 24 h, cells were incubated with DNR in the concentration range of 0.05–1 μM  and ASP9521 (25 μM) or a vehicle. After the next 48 h, an SRB assay was performed. Cells were fixed with 50% trichloroacetic acid at 4 °C for 1 h. Next, they were washed with water and stained with SRB solution. After 20 min, cells were washed 4 times with 1% acetic acid, and the remaining SRB was dissolved in a solubilization solution (10 mM Tris base solution). Absorbance was read at 565 nm , and viability percentage was calculated by dividing the absorbance of experimental wells by the absorbance of the control (×100%). Three separate experiments were performed.For evaluation of the chemosensitising effect of ASP9521, sulforhodamine B (SRB) assay was used. Cells were seeded in 96well plates at a density of 1 × 104 per well. After 24 h, cells were preincubated for 3 h with ASP9521 (25 μM). Then, the culture medium was changed, and cells were incubated with DOX/DNR (1 μM). Control cells were incubated in culture medium without analysed compounds. An MTT assay was performed as described previously [For evaluation of the cardioprotective effect of ASP952, 3--2,5-diphenyltetrazolium bromide (MTT) assay was used. Cells were seeded in 96-well plates at a density of 1 × 10eviously . The viaThe selection of concentrations of ASP9521, DOX, and DNR was based on the experimental data published previously ,29.t-test was used to check the statistical significance of the difference between the IC50 values for DNR and the combination of DNR with ASP9521 (A549 cells only). One-way ANOVA with Tukey’s post-hoc test was used to check the statistical significance of the difference in viability cells as a result of adding 25 μM ASP9521. The calculations were carried out in the GraphPad Prism program. Statistical significance was determined for the significance level of p < 0.05.The independent Student The observed structural features of ASP9521 predisposed the compound to be considered as a potential CBR1 inhibitor. Molecular docking and MD simulation revealed two possible binding modes of the compound within the CBR1 catalytic site, both implying its highly probable affinity. Further research is needed to assess whether interaction with either Met234 or Phe94 is preferable in terms of ligand recognition and ability to effectively inhibit the activity of the CBR1 enzyme. Moderate inhibitory activity of ASP9521 was confirmed in biological studies with recombinant CBR1. The obtained results, together with the previous evaluation of ASP9521, allowed us to define it as a dual AKR1C3-CBR1 inhibitor—a compound that inhibits both main reductases responsible for ANT metabolism. Such a cumulative and not-yet-described mechanism of action can be promising in terms of overcoming ANT drug resistance and reducing the cardiotoxicity of this group of drugs. However, further development of compounds with more balanced inhibitory properties between both AKR1C3 and CBR1 is necessary to confirm this hypothesis. ASP9521 improved the cytotoxic activity of DNR on human lung carcinoma cell line A549. The compound also prevented rat cardiomyocytes H9c2 from the toxic activity of DOX, DNR or their reduced metabolites. Taking into account that the compound was previously defined to be safe in the clinical trial and also bearing in mind its good bioavailability , ASP9521 remains a promising molecule for evaluation not only in monotherapy of cancer but also as adjuvant therapy with ANT.