To analyze Unkempt phosphorylation, we immunoprecipitated Unkempt from cells in the presence or the absence of the mTORC1 inhibitor rapamycin and used mass spectrometry to identify mTORC1-dependent phosphorylated residues. This analysis showed that mTORC1-dependent phosphorylation is concentrated in a serine-rich intrinsically disordered region in the C-terminal half of Unkempt. We also found that Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Furthermore, analysis in the developing brain of mice lacking TSC1 expression showed that phosphorylation of Unkempt is mTORC1 dependent in vivo. Finally, mutation analysis of key serine/threonine residues in the serine-rich region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Phosphorylation within this serine-rich region thus profoundly affects the ability of Unkempt to regulate cellular morphogenesis. Taken together, our findings reveal a novel molecular link between mTORC1 signaling and cellular morphogenesis.Mechanistic target of rapamycin (mTOR) is a protein kinase that integrates multiple inputs to regulate anabolic cellular processes. For example, mTOR complex 1 (mTORC1) has key functions in growth control, autophagy, and metabolism. However, much less is known about the signaling components that act downstream of mTORC1 to regulate cellular morphogenesis. Here, we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate of mTORC1. We show that Unkempt phosphorylation is regulated by nutrient levels and growth factors  Drosophila /RING domain protein Unkempt acts genetically downstream of mTOR in the developing retina and central nervous system in osophila , 20, 21.osophila , 23.Tsc1 to activate mTORC1 in the nervous system, we find that Unkempt phosphorylation is mTORC1 dependent in vivo. Finally, we show that phosphorylation in the serine-rich region suppresses the capacity of Unkempt to induce a bipolar morphology, a critical cellular activity of Unkempt.Here, we show that mammalian Unkempt is abundantly phosphorylated and that phosphorylation is acutely sensitive to mTORC1 activity. Unkempt physically interacts with mTORC1 by binding to regulatory-associated protein of mTOR (Raptor), which mediates direct phosphorylation by mTOR at multiple serine/threonine residues in the highly serine-rich intrinsically disordered region (IDR). Using mice with conditional knockout of Drosophila and cellular morphogenesis in mammals . A strict correlation between inhibition of mTORC1, as judged by phosphorylation of ribosomal protein S6 (rpS6), and decreased mobility of Unkempt was observed over a wide range of rapamycin concentrations (A). Moreover, a phospho-specific antibody against Unkempt  detected reduced levels of phosphorylated Unkempt in rapamycin-treated cells (A). Rapamycin treatment also caused increased phosphorylation of AKT at Ser473 as a result of the established feedback loop between S6K and the insulin receptor substrate (A).In serum-starved neuroblastoma SH-SY5Y cells, we found that stimulation of the mTORC1 pathway with insulin or serum readdition caused a mobility shift of Unkempt to a higher resolved band, whereas direct inhibition of mTORC1 kinase activity with rapamycin prevented this shift A. A stritrations A. Moreoved cells A. Rapamyubstrate , 26  and in mouse E14.5 primary cortical cells (C). After insulin stimulation, lambda protein phosphatase treatment of the SH-SY5Y cell lysate converted a higher resolving Unkempt band to a lower resolving species, below that in unstimulated cells (D). Together, these data suggest that Unkempt is phosphorylated in an mTORC1-dependent manner and exhibits a basal level of phosphorylation in unstimulated cells.Unkempt showed a similar mTORC1-dependent mobility shift in response to rapamycin in mouse Neuro-2a neuroblastoma cells B and in al cells C. After ed cells D. TogethE, compare lanes 1 and 4). However, the presence of amino acids amplified the effect of insulin stimulation, as seen both by increased mTORC1 activity and enhanced phosphorylation of Unkempt . Similarly, increased glucose concentration, while having little effect on its own , potentiated mTORC1 activity and the phosphorylation of Unkempt primed by insulin stimulation in the presence of amino acids . The effects of amino acids and glucose were mTORC1 dependent, as rapamycin prevented the phosphorylation of Unkempt in the presence of amino acids, glucose, and insulin together . Unkempt phosphorylation is therefore acutely sensitive to nutrients and growth factors that regulate mTORC1.mTORC1 can be regulated by multiple signaling inputs, including insulin, amino acids, and ATP levels . Moreover, treatment of SH-SY5Y cells with a dual mTORC1/mTORC2 inhibitor, KU0063794 , or rapamycin and KU0063794 together , produced a similar reduction in phosphorylation of Unkempt as treatment with rapamycin alone , further evidence that it is the activity of mTORC1 but not mTORC2 that regulates Unkempt phosphorylation. Furthermore, treatment of SH-SY5Y cells with a range of concentrations of the highly selective mTORC1 inhibitor DL001 . We also asked whether Unkempt phosphorylation might be regulated at the level of S6K; however, treatment of SH-SY5Y cells with the S6K inhibitor PF4708671 had no noticeable effect (B). Together, these data confirm that Unkempt phosphorylation is mTORC1 dependent.mTOR is a component of both mTORC1 and mTORC2. Prolonged rapamycin treatment can inhibit mTORC2 , but shoFig. S1U0063794 A, lane 3together A, lane 4in alone A, lane 5or DL001 , which de effect B. TogethC). Endogenous Raptor and mTOR coimmunoprecipitated with FLAG-HA-Unkempt . Unkempt therefore physically interacts with mTORC1.Unkempt has been previously shown to physically interact with Raptor , 28. To Fig. 2C). This is in line with a known property of the zwitterionic detergent CHAPS to preserve and the nonionic detergent Triton X-100 to disrupt the interaction between Raptor and mTOR . MS identified five serine residues in Unkempt  that were in vitro phosphorylated in the control condition but not in the rapamycin/FKBP12-treated condition  of FLAG-HA-Unkempt immunoprecipitated from cells treated or not with rapamycin, we identified 31 phosphorylated serines and five phosphorylated threonines grouped in three clusters within a predicted IDR of Unkempt A and B,  and S3. Rheb-GTP C and S3Bin vitro ; Fig. 3Ain vitro kinase assay (D and S1A). The P-Unk antibody did not recognize phosphorylated Unkempt in SH-SY5Y cells in which Unkempt has been knocked down using an shRNA  and in the se assay . S611 alse assay  . Unkempt deletion mutants lacking either the ZF domain (ΔZF) or the RING domain (ΔRING) retained mTORC1-dependent phosphorylation potential, whereas a mutant containing just the ZF domain alone (ZF) was not phosphorylated by mTORC1 hyperactivation (A and B). Notably, removal of the whole serine-rich region (ΔSerL) or of a shorter region between amino acids 539 and 640 (ΔSerS) almost completely prevented phosphorylation (A and B). This deletion analysis confirms that the serine-rich region in Unkempt contains the majority of the mTORC1-dependent phosphosites.To independently define the region of Unkempt that is targeted by mTORC1-dependent phosphorylation, we performed deletion analysis of Unkempt transiently expressed in human embryonic kidney 293 cells. Coexpression of constitutively active Rheb, which directly hyperactivates mTORC1 , caused tivation , A and Brylation , A and BA). To test these data further, we mutated pairs of mTORC1-dependent serine residues in the IDR  to alanine. However, mutation of these serine pairs did not prevent Unkempt phosphorylation (C). Therefore, we took a systematic approach and generated a series of mutants in which clusters of MS-identified phosphorylated serine and threonine residues in the IDR were mutated to alanine (C). Mutation of phosphosites from S467 to S546 to alanine had no obvious effect on mTORC1-dependent phosphorylation of Unkempt (D and E). However, additional mutation of phosphosites between S565–S578, S596–S611, and S631–S640 to alanine strongly reduced mTORC1-dependent phosphorylation of Unkempt (D and E). Based on this, we then tested mutants that only affect phosphosites located exclusively within the serine-rich region of the IDR (C). Interestingly, mutations of groups of these phosphosites , or their combinations (S596–S640 or S565–S640), reduced but did not completely prevent the overall mTORC1-dependent phosphorylation of Unkempt . These mutagenesis data validate the MS analyses and point to the serine-rich region of the IDR as the hot spot of mTORC1-dependent phosphorylation of Unkempt.MS analysis showed that specific serine and threonine residues in the IDR of Unkempt are targets of mTORC1-dependent phosphorylation A. To tesrylation C. Theref alanine C. Mutati Unkempt , D and E Unkempt , D and E the IDR C. Interein vivo, we utilized Nestin-Cre;Tsc1fl/fl mice, in which Nestin promoter–driven Cre recombinase is expressed in neural progenitors beginning at E10.5 causing loss of TSC1 expression throughout the developing brain . Although we did not detect a change in the electrophoretic mobility of Unkempt protein, using the phospho-specific antibody, we observed a dramatic increase in Unkempt phosphorylation in brain tissue from Nestin-Cre;Tsc1fl/fl embryos (A and C).In mice, Unkempt protein is expressed in the developing nervous system from at least as early as E10 . To analng brain . Consequothermia . The actothermia . We conf embryos , A and B embryos , A and CNestin-Cre;Tsc1fl/fl embryos were injected with rapamycin or vehicle at E15.5. Twenty-four hours after rapamycin injection, rpS6 phosphorylation was dramatically reduced in the embryonic brain (E and F). Rapamycin treatment also significantly decreased phosphorylation of Unkempt (E and G). To determine whether prolonged rapamycin treatment affected mTORC2 signaling  and did not change with 24-h rapamycin treatment (E and H), indicating that mTORC2 signaling was unaffected. These data show that Unkempt phosphorylation is mTORC1 dependent in the developing brain.To test whether inhibition of mTORC1 affects Unkempt phosphorylation during nervous system development, mice pregnant with ic brain , E and F Unkempt , E and Gignaling , we analignaling . PKCα phcontrols , A and Dreatment , E and HC–G and 6A). Strikingly, whereas alanine mutations largely preserved the morphogenetic activity of Unkempt, aspartate mutations severely compromised or abolished cell polarization by Unkempt (B–D and E and F). Curiously, the S467–S640>A mutant still responded to changes in mTORC1 activity, similarly to the wildtype Unkempt (E). This result suggests that none of the 23 serine/threonine residues, which we turned to alanines in this mutant (C), are strictly required to confer sensitivity of Unkempt to mTORC1. Instead, we suspect that regulation of morphology by mTORC1 could occur through phosphorylation of other residues in Unkempt  and/or through phosphorylation of residues in its interacting proteins. Together, these data point to a critical role of mTORC1-dependent phosphorylation in regulating the activity of Unkempt in cellular morphogenesis (G). These studies pave the way for investigation of the mechanistic principles by which Unkempt and its phosphorylation by mTORC1, and potentially other kinases, regulate cellular morphogenesis.In addition to being required for the early morphology of neurons, Unkempt also possesses a remarkable capacity to establish a similar and early neuronal-like bipolar morphology in cells of non-neuronal origin, indicating that Unkempt regulates a specific program of cell morphogenesis , 23. To  B–D and . All mut B–D and , suggest B–D and , 23. Mor B–D and , E and F Unkempt E. This rs mutant C, are stogenesis G. These in vitro and in the embryonic brain. Unkempt is a direct substrate of and physically interacts with mTORC1 via binding to Raptor. mTORC1-dependent phosphorylation of Unkempt was shown by MS to occur on multiple serine and threonine residues concentrated in the IDR. Using extensive mutagenesis, we showed that mTORC1-dependent phosphorylation of Unkempt requires residues in the serine-rich region of the IDR. Importantly, we showed that phosphomimetic mutations in this serine-rich region abrogate the ability of Unkempt to induce a bipolar morphology. Taken together, our findings show that Unkempt is a novel substrate of mTORC1 and that mTORC1-dependent phosphorylation acutely regulates Unkempt’s ability to remodel cellular morphogenesis (G).Here, we have shown that phosphorylation of the highly conserved ZF/RING domain protein Unkempt is regulated by mTORC1 ogenesis G.in vitro, although phosphorylation detected using a phospho-specific antibody is still highly sensitive to mTORC1 activity in vivo. During nutrient-deprived conditions, the nervous system is preferentially spared at the expense of other organs to maintain brain growth . This might not be limited to the control of the cellular morphogenesis but could extend to other known roles of Unkempt in neuronal differentiation and cell proliferation . However, despite this and our evidence that Unkempt is directly phosphorylated by mTORC1  and Tsc1fl/fl mice  were maintained in a C57BL/6 background. Mice were housed under a 12 h light/dark cycle with ad libitum access to food and water. Tsc1fl/fl mice were genotyped using primers 5′-GTC ACG ACC GTA GGA GAA GC-3′ and 5′-GAA TCA ACC CCA CAG AGC AT-3′. Nestin-Cre mice were genotyped using primers 5′-TTGCTAAAGCGCTACATAGGA-3′ and 5′-GCCTTATTGTGGAAGGACTG-3′.Mouse studies were carried out in accordance with UK Home Office regulations and the UK Animals (Scientific Procedures) Act of 1986 (ASPA) under a UK Home Office license (PPL 70/8719) and approved by the King’s College London Ethical Review Committee. Pregnant dams were injected intraperitoneally with 1 mg/kg rapamycin (Fluorochem Ltd) at E15.5, embryos removed at E16.5, and embryonic brains dissected and immediately frozen on dry ice.SH-SY5Y, Neuro-2a, and human embryonic kidney 293 cells were maintained in Dulbecco’s modified Eagle’s medium high glucose (Sigma–Aldrich) supplemented with 10% (v/v) fetal bovine serum (Sigma–Aldrich) and 1% (w/v) penicillin/streptomycin (Sigma–Aldrich). Details of culture conditions, drug treatments, Western blot, and antibodies are described in the Details of DNA constructs and transfections are described in the To induce overexpression of and immunoprecipitate FLAG-HA-Unkempt, previously described HeLa S3 cells stably expressing DOX-inducible FLAG-HA-Unkempt  were usein vitro mTORC1 kinase assays with Rheb-GTP were performed as described previously (In vitro mTORC1 kinase assays for LC–MS/MS are described in the Radiolabeled eviously . In vitrDetails of immunofluorescence and immunohistochemistry are described in the Three phosphopeptides were generated corresponding to residues 601 to 610 (containing phosphoserine 606), 607 to 615 (containing phosphoserine 611), and 601 to 615 (phosphoserines 606 and 611) in Unkempt. Rabbits were immunized with all three phosphopeptides, and phospho-specific antibodies were affinity purified using the phosphopeptide corresponding to residues 601 to 615 (containing phosphoserines 606 and 611). Antibody generation was performed by Covalab.y/x), where the length of the absolute longest cellular axis (y) was divided by the length of the longest axis perpendicular to the y-axis (x). Lengths of x- and y-axes of GFP-positive–inducible HeLa cells were measured at 48 h of treatment with DOX. At least 50 GFP-positive cells were quantified for each inducible cell line.Cellular morphologies were analyzed as described previously . BrieflyCLIP experiments were carried out essentially as described . Furthert test. p Values <0.05 were considered significant.Data were analyzed using GraphPad Prism, version 7 . Data distributions were assessed for normality and then analyzed using a Student’s All methods and data are described in the article or This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Computational studies showed reliable binding poses within 3CLpro for the three investigated small peptides, considering docking scores as well as the binding free energy values. Validation by in vitro experiments confirmed these results. In particular, IPAVF exhibited the highest inhibitory activity by returning an IC50 equal to 1.21 μM; it was followed by IAEK, which registered an IC50 of 154.40 μM, whereas MHI was less active with an IC50 equal to 2700.62 μM. On the other hand, none of the assayed peptides registered inhibitory activity against 3Cpro. Based on these results, the herein presented small peptides are introduced as promising molecules to be exploited in the development of “target-specific antiviral” agents against SARS-CoV-2. In the present work, and for the first time, three whey protein-derived peptides , endowed with ACE inhibitory activity, were examined for their antiviral activity against the SARS-CoV-2 3C-like protease (3CL On 11 March 2020, the World Health Organization (WHO) declared the novel coronavirus (COVID-19) outbreak a global pandemic for the rapid spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) worldwide [The advent of effective vaccines and the adoption of restrictive prophylaxis measures, as well as appropriate sanitization procedures, have improved the global fight against SARS-CoV-2 . Howeverpro), also named the main protease (Mpro), which plays an important role in the virus’s life cycle, is considered an attractive target for the discovery of promising antiviral agents [pro is involved in the replication process due to its two N-terminal domains, containing two β-barrel chymotrypsin-like folds ultimately responsible for the cleavage of the viral polyproteins to yield 16 mature      non-structural proteins [The SARS-CoV-2 3-chymotrypsin-like cysteine protease  involved in the viral replication process. Briefly, 3Cpro and 3CLpro are cysteine proteases and share a typical chymotrypsin-like folding, a nucleophilic cysteine residue in the active site, and a preference for a glutamine or glutamic acid residue in the primary binding residue (P1 site) of the substrate proteins. Picornaviral HRV 3C protease was studied in the recent past for its degree of homology with coronaviral 3C-like proteases (3CLpro) within the 3C coding region, including the strict conservation of the active-site residues, thus providing an additional rationale for targeting drug discovery efforts [pro and HRV 3Cpro [Human rhinoviruses , etiological agents of the common cold, also show a protease  has been authorized for emergency use for COVID-19 patients not requiring supplemental oxygen [Several studies have been carried out regarding the structural and functional characterization of these proteins, to be employed for high-throughput screening for the discovery of new effective inhibitors ,16,17. Tl agents ,19,20. Bl agents ,22,23. Rl oxygen ,25.pro inhibitor substrates have been mainly modified by introducing some reactive chemical groups, such as Michael acceptors, aldehydes, epoxy ketones, and so on [In order to increase the affinity and efficacy, 3CLnd so on . Howevernd so on ,16.Besides synthetic compounds, other studies on promising antiviral agents are focusing on the identification of bioactive molecules from natural sources ,27,28. A50 values equal to or lower than 25 μM [Recently, we investigated ACE inhibitory activity by peptide sequences, derived from the enzymatic hydrolysis of whey proteins and predicted by molecular docking and related prioritization studies. This approach allowed us to obtain three peptides—MHI, IAEK, and IPAVF—with ACE inhibitory ICan 25 μM . Importaan 25 μM ,34.pro functioning. With this in mind, we carried out an integrated theoretical and experimental investigation, involving first, molecular docking studies to rationally evaluate the putative chance of binding and then, in vitro testing for validation. Due to the occurrence of coinfection with a common virus  in SARS-CoV-2 positive specimens [Building on these observations, we explored the potential of our recently studied ACE inhibitory peptides  in interfering with SARS-CoV2 3CLpecimens ,37, peptSARS-CoV-2’s main protease crystal structure was fetched from the Protein Data Bank by retrieving the entry 7L0D . The proThe induced-fit docking protocol ,44 was eThe molecular mechanics/generalized born surface area  method https://www.rcsb.org, accessed on 10 January 2022) and are included in the For completeness, -additional molecular docking analyses were carried out against the HRV 3C protease by retrieving entry 2XYA  from theSynthetic peptides were purchased  and resuspended in MQ water, then assayed at the concentrations of 2, 1, 1, 0.6 0.2 and 0.02 mg/mL . In the case of peptides with a value of relative inhibition (RI) percentage that was higher than 40–50% at the lowest assayed concentration, further dilutions were made. Escherichia coli expression system, MW = 34 kDa) and a buffer composed of 20 mM Tris, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT, pH 7.3, was used for the inhibition assay. The protease inhibitor, GC376 MW 507.5 Da, with an IC50 value of 0.017 μM was used as the positive control. Initially, 30 μL of diluted SARS-CoV-2 3CL protease, at the final concentration of 15 ng, was pipetted into a 96-well plate containing pre-pipetted 10 μL quantities of each test compound . The mixture was incubated at room temperature for 30 min with slow shaking. Afterward, the reaction was started by adding the substrate (10 μL), dissolved in the reaction buffer to 50 μL final volume, at a concentration of 40 μM, then the plates were incubated for 4 h at room temperature with slow shaking. The plates were sealed. Fluorescence intensity (Fi) was measured with the Varioskan microtiter plate-reader , exciting at a wavelength of 360 nm and detecting at a wavelength of 460 nm. Each sample was assayed in triplicate.The in vitro screening of enzyme inhibition activities was evaluated by using 3CL Protease, untagged . According to the manufacturer’s protocol, a fluorescent substrate, SARS-CoV-2 3CL Protease , expressed in an RI %) was calculated as follows:C is the control and T is the test peptide or inhibitor.The percentage of relative inhibitions , a cysteine protease that recognizes the cleavage site of Leu-Glu-Val-Leu-Phe-Gln*Gly-Pro. Protease activity in the presence or absence of peptide inhibitors was determined colorimetrically, as reported, with an HRV 3C Protease Inhibitor Screening Kit  according to the manufacturer’s instructions. The kit also included a protease inhibitor as a positive control.The inhibition activity of synthetic peptides was assayed against human rhinovirus 3C protease  or 10 μL of inhibitor, or 10 μL of assay buffer in the case of enzyme control, and 50 μL of enzyme solution. After the incubation of the plate at room temperature for 15 min, 40 μL of the substrate solution was added to each reaction, and absorbance (OD = 405 nm) was measured in a kinetic mode for 1–2 h at 37 °C on the Varioskan Flash microplate reader. Each sample was assayed in triplicate. RI) was calculated as follows: C is the control and T is the test peptide or inhibitor.The percentage of relative inhibitions (p < 0.05) using the IBM SPSS Statistics release 20  to evaluate the main and interaction effects of concentration levels and the assayed peptide types on the inhibition percentage of viral protease. Whenever required, the simple main effects of peptides and the assay control inhibitor on viral protease inhibition percentage were also examined, applying a one-way ANOVA (p < 0.05). Multiple comparisons among individual means for each assayed peptide and concentration level were performed using honestly significant difference (HSD)Tukey’s test (p < 0.05). A multiple regression test was run to predict the inhibition percentage of 3CL protease activity from different peptide concentrations. Results related to the 3CL protease inhibition percentage were subjected to a square root arcsin transformation in order to meet the homogeneity-of-variance assumptions, following Levene’s test. The univariate general linear model (GLM) procedure, carrying out a two-way analysis of variance  of peptides on SARS-CoV-2 3CL protease was calculated by using the software package SigmaPlot12  and referred to the micromolar range.The half-maximal inhibitory concentration ; these latter were, in turn, purified and identified after the enzymatic hydrolysis of whey, for its transformation from a by-product of cheese manufacture to a high added-value compound . The ass2 pocket . In addi2 pocket ,34. Simi2 pocket .We preferred to use the induced fit docking protocol instead of the standard methods, the former being suited to exploring with higher accuracy and reliability the nature and the type of molecular interactions by considering both the ligand and binding site flexibility. In this respect, the effect of the induced fit on the key binding site residues  is assessed by computing the deviations from the crystallographic pose for each protein-oligopeptide complex; the following RMSD values, equal to 1.51 Å, 1.39 Å and 1.24 Å, are measured for IPAVF, IAEK, and MHI, respectively. Interestingly, we observed that Q189 and N142 are more sensitive to the induced fit, as these two residues experienced significant conformational changes, promoting a better fit and the easier accommodation of the three small peptides through the formation of cooperative hydrogen bonds. The N142 χ1 and Q189 χ1 and χ2 torsional angle shifts, due to the induced fit, were reported in From an energetic point of view, we employed the OPLS3e force field to quantify the docking scores, and the MM-GBSA method to account for the binding of free energies. Interestingly, the values of the docking scores as well as of the binding free energies calculated for the three peptides were much better than those calculated for the reference X-ray-solved cognate ligand, ML188. For the sake of comparison, all the values are reported in In addition, a more detailed analysis of the terms of the binding free energy function indicated that the Coulomb and van der Waal energy contributions were determinants for IPAVF, with values equal to −43.15 kcal/mol and −70.55 kcal/mol, respectively. On the other hand, the strongest hydrogen bonding contribution with a value of −6.13 kcal/mol was for IAEK, due to the presence of two charged side chains within its sequence.Taken together, all the above-described results concerning the in silico investigations made us confident of the potential antiviral action of these natural small peptides in contrasting SARS-CoV-2 main protease, and this encouraged us to run experimental validations.pro inhibitory activity using molecular docking. Twenty peptides  showed the best binding energy but this was less than those obtained in the present work. In particular, three among these peptides, originating from the in silico proteolysis of β-caseins, β-lactoglobulin, and αs2-casein, were able to form at least two hydrogen bonds and achieve π-alkyl hydrophobic interactions with the catalytic residues, C145 and H41, of SARS-CoV-2 Mpro, respectively. Other authors [pro inhibitory activities, showing several potential advantages in terms of high binding affinity, bioavailability, and cost-effective synthesis. Recently, Behzadipour et al.  screened authors ,34,55 hapro. However, peptides that were modified with a primary amide (Gln-γ-CONH2) were more active, revealing carbonyl oxygen–hydrogen bonds with the H161 and Oγ of T142 [pro and similar interactions were experienced, although these yielded more disappointing scoring values. Additional details are available in the In the past, a representative group of tripeptide aldehydes (CBZ-Leu-Phe-Gln-CHO) was prepared and analyzed to demonstrate poor inhibitory activity against 3C of T142 . Our thrpro and HRV 3Cpro, both employing the catalytic dyad (His-Cys) for their functionality.Based on the theoretical analyses, the inhibitory activity of the three investigated peptides was experimentally evaluated against SARS-CoV-2 3CLpro activity.Satisfactory experimental results were observed, following the in vitro inhibition screening of the assayed peptides regarding SARS-CoV-2 3CL50) of the three assayed peptides against SARS-CoV-2 3CLpro were measured and are shown in 50 value of IPAVF was the best and was comparable with those obtained for the ACE inhibitor peptides against SARS-CoV-2 3CLpro, after in silico analyses [50 are reported in The half-maximal inhibitory concentrations (ICanalyses . The exppro; some of these peptides were also predicted to play a role as dual binders toward SARS-CoV-2 3CLpro and monoamine oxidase A [Similar inhibitory activities toward SARS-CoV2 in the μM range have been identified for synthetic tetrapeptides, pentapeptides, and octapeptides, as recently reviewed by Heydari et al. . Moreovexidase A .It is noteworthy that no appreciable activity was observed when testing the three small peptides against HRV 3C protease, the RI percentage being lower than a value of ca. 5% at a concentration of 1 mg/mL. A synoptic view is shown in pro gene was recorded [pro inhibitor could also be effective against multiple variants of the same virus. During the COVID-19 pandemic, several variants of SARS-CoV-2 emerged that, nevertheless, showed mutations in the binding domain of the spike protein receptor ,60. In arecorded . This suAs already demonstrated for Nirmatrelvir , it is eIt is noteworthy that the three proposed peptides represent the minimum active sequences with the desired biological properties toward the SARS-CoV-2 3CL protease. This finding opens the way for the synthesis of peptides with improved activity. Their conversion into therapeutic peptides is, indeed, a further challenging task aimed firstly at tackling two intrinsic stumbling blocks: the low membrane permeability and the poor in vivo stability . InspireOn the other hand, IAEK, IPAVF, and MHI are naturally included within whey proteins ,35; althIn this work, the inhibitory activity of the whey-derived bioactive small peptides MHI, IAEK, and IPAVF against viral proteases was evaluated for the first time, indicating their possible role in blocking the replication processes of SARS-CoV-2. pro. These results were validated by in vitro experiments that confirmed the highest antiviral activity for IPAVF and IAEK against 3CLpro. These peptides of natural origin were previously obtained by the enzymatic hydrolysis of whey proteins and also displayed ACE inhibitory activity. It is noteworthy that their short sequences facilitate their synthesis as well as changes to their structure to improve stability and enhance activity. The results herein open the door to new opportunities for the development of dual-target small peptides that are endowed with antiviral 3CLpro and inhibitory ACE activities.Molecular docking studies predicted the relevant interactions between the peptides and key amino acid residues of the enzyme catalytic pocket of 3CL


Primary decreases in glutathione and its redox potential, methionine, and ethanolamine are alleviated with increasing PDHC inhibition, the alleviation accompanied by physiological changes. A comparison of 39 brain biochemical parameters after administration of four phosphinate and phosphonate analogs of pyruvate at a fixed dose of 0.1 mmol/kg reveals no primary, but secondary changes, such as activation of 2-oxoglutarate dehydrogenase complex (OGDHC) and decreased levels of glutamate, isoleucine and leucine. The accompanying decreases in freezing time are most pronounced after administration of methyl acetylphosphinate and dimethyl acetylphosphonate. The PDHC inhibitors do not significantly change the levels of PDHA1 expression and phosphorylation, sirtuin 3 and total protein acetylation, but increase total protein succinylation and glutarylation, affecting sirtuin 5 expression. Thus, decreased production of the tricarboxylic acid cycle substrate acetyl-CoA by inhibited PDHC is compensated by increased degradation of amino acids through the activated OGDHC, increasing total protein succinylation/glutarylation. Simultaneously, parasympathetic activity and anxiety indicators decrease.Mitochondrial pyruvate dehydrogenase complex (PDHC) is essential for brain glucose and neurotransmitter metabolism, which is dysregulated in many pathologies. Using specific inhibitors of PDHC in vivo, we determine biochemical and physiological responses to PDHC dysfunction. Dose dependence of the responses to membrane-permeable dimethyl acetylphosphonate (AcPMe The thiamine diphosphate (ThDP)-dependent pyruvate dehydrogenase PDH, 1.2.4.1) functions within the multienzyme pyruvate dehydrogenase complex (PDHC) that links cytosolic glycolysis with the mitochondrial tricarboxylic acid (TCA) cycle. When oxidizing the glycolytic product pyruvate, PDHC produces the TCA cycle substrate acetyl-CoA and NADH, which is oxidized in the mitochondrial electron transport chain. In addition, the PDHC reactive acetyl intermediates and the product acetyl-CoA donate acetyl moieties for post-translational modifications of proteins, which are of regulatory significance .2.4.1 fu. Althoug2), we characterize the dose dependence of the changes. Furthermore, we use a fixed dose of the different phosphonate analogs of pyruvate, shown in Different pharmacological tools are available for PDHC regulation. Thiamine (vitamin B1) and its pharmacological forms are precursors for a PDHC coenzyme, ThDP ,8. In ad2 , significant decreases in glutathione and its redox potential, ethanolamine, methionine and valine are observed (2 dosage (0.1 mmol/kg), decreases in these primary indicators, except for that of valine, are alleviated  of an electrocardiogram (ECG) decrease along with an increase in the locomotor activity, estimated by crossing the lines in the Open Field Test  analogs of pyruvate (2) esters to AcP. These considerations are in accord with the automatically obtained clusters of the inhibitors-induced changes in the brain biochemical parameters, shown in 2, with the latter three forming a common cluster.The biochemical and physiological effects of the phosphinate (AcMeP) and phosphonate  and duration of freezing (p = 0.012). A decrease after administration of PDHC inhibition, with the most significant decreases being vs. control animals, was observed after administration of the strongest in vitro inhibitor AcMeP and/or membrane-penetrating AcPMe2  [As discussed above, complementary regulation of activities of PDHC and OGDHC is found in the current study on the PDHC inhibition in the male brain cortex, where PDHC inhibition elevates OGDHC function. Similarly, in the previous study on the OGDHC inhibition, the changes in the PDHC activity mirror those in the OGDHC activity . The comn = 24) . The negn = 24) . Thus, cn = 24) ,31. AlonRemarkably, both OGDHC and PDHC inhibition induce increases in OGDHC activity and biphasic changes in glutathione metabolism. However, when OGDHC is inhibited, the OGDHC activity increase is a primary response, followed by the activity decrease. Accordingly, administration of both the OGDHC and PDHC inhibitors affects brain glutathione metabolism, yet the primary effect of the OGDHC inhibition is manifested in the glutathione disulfide levels , whereas+. As a result, under the studied conditions of the PDHC inhibition, the levels of the OGDHC activity, PDHA1 phosphorylation and NAD+ contribute the most to protein succinylation.A compensatory response to PDHC inhibition could include the dephosphorylation of the first component of the complex to increase PDHC activity. However, this does not occur, at least under the induced levels of the enzyme inhibition . The totReduced acetyl-CoA production by PDHC is known to decrease biosynthesis of acetylcholine in the brain or neurons ,33. ThisThus, the characterized metabolic action of specific inhibitors of PDHC unravels molecular mechanisms involved in the brain homeostatic response to perturbed PDHC function, the secondary metabolic perturbations and their physiological impact.1H (CDCl3), δ, ppm: 3.76 P(O)), 3.37 2), 1.46 , 1.42 . NMR 13C (CDCl3), δ, ppm: 100.7 , 51.7 P(O)), 49.8 2), 49.6 2), 19.0 , 10.7 . NMR 31P (CDCl3), δ, ppm: 49.5.Sodium salt of AcPH was synthesized as previously described . Sodium 1H (D2O), δ, ppm: 3.36 2), 1.43 , 1.27 . NMR 13C (D2O), δ, ppm: 101.4 , 49.8 2) 18.4 , 13.3 . NMR 31P (D2O), δ, ppm: 37.5.To obtain sodium methylphosphinate, a solution of methyl methylphosphinate  and NaI  in 11.4 mL of dry methylethylketone was refluxed for 2 h under argon. The resulting precipitate was filtered off, washed twice with 5 mL of dry acetone and dried in vacuo. Yield: 1.56 g (82.2%), m.p. 186–188 °C. NMR 1H (D2O), δ, ppm: 2.42 ), 1.36 . NMR 13C (D2O), δ, ppm: 224.5 P(O)), 27.9 ), 12.3 . NMR 31P (D2O), δ, ppm: 27.1.Sodium methylphosphinate  was further dissolved in a mixture of 5.9 mL of glacial acetic acid and 0.35 mL of water. The solution was stirred at ambient temperature for 24 h. The solvent was evaporated to dryness and residue was triturated with 30 mL of acetone and filtered off to give sodium AcMeP as a white solid. Yield: 0.8 g (94.3%), m.p. 195–197 °C. NMR 2 was synthesized from dimethyl phosphite and acetyl chloride as described previously [2 with NaI according to [3 resulting in disodium salt of AcP was done according to [2  and stirred at 0 °C under argon. The resultant solution was stirred at an ambient temperature overnight. The excess of bromotrimethylsilane was removed under reduced pressure. Aqueous NaHCO3  was added at 0 °C and the solution stirred at 0 °C for 1 h. Water was evaporated to dryness under reduced pressure and the residue was washed with 10 mL of absolute ethanol to yield a solid white product. Yield: 1.56 g (93%). NMR 1H (D2O), δ, ppm: 2.34 d , J = 3.6 Hz). NMR 13C (D2O), δ, ppm: 228.2 P(O)), 29.8 ). NMR 31P (D2O), δ, ppm: −0.1.AcPMeeviously . Partialrding to  resultedrding to . Bromotr+ was obtained from Gerbu , and oxidized glutathionefrom Calbiochem . Formate dehydrogenase for NAD+ assays was obtained from the Federal Research Center of Biotechnology/Innotech MSU . All other reagents were of the highest purity available and obtained from Sigma-Aldrich .NAD2, only) or 0.1 mmol/kg dosage , respectively. A physiological solution (0.9% NaCl) was administered to the control animal group. Then, 24 h after the administration, the rats were subjected to physiological tests and sacrificed by decapitation as described before [Animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the European Union Directives 86/609/EEC and 2010/63/EU and were approved by the Bioethics Committee of Lomonosov Moscow State University (protocols number 69-o from 09.06.2016 and 139-a from 11.11.2021). Wistar male rats were kept in standard conditions with 12 h light and 12 h dark day cycle, with free access to water and food. Phosphinate and phosphonate analogues of pyruvate were dissolved in deionized water to obtain 0.2 M or 1 M solutions and were administered intranasally at 0.02 mmol/kg  were identified according to the iterative Grubb’s test  in each experimental group and were excluded from statistical analysis. The number of animals in experimental groups (n) is indicated including outliers.Data are presented as mean ± standard error of the mean for each experimental group. Due to noticeable differences in the variances of the parameters in the experimental groups, the latter were compared using one-way Welch ANOVA with the recommended Games-Howell’s post hoc test, employed in GraphPad Prism 9.0  or userfriendlyscience package in R. The differences with p ≤ 0.05 were considered significant. Where present, the row and column clustering of heatmaps was done using Euclidean distance and the “ward.D” agglomeration method employed in the pheatmap package in R.According to the Shapiro–Wilk test, some of the biochemical parameters were not normally distributed. Thus, for analysis of the components of protein acylation system, Spearman’s correlation coefficients were calculated for each pair of the parameters. Heatmaps including correlation matrices were prepared using RStudio 1.4  and Adobe Illustrator 24.2 . The correlations with In the current work, metabolic and physiological consequences of a short-term inhibition of PDHC in the brain cortex of male rats are characterized. First of all, PDHC inhibition affects the glutathione redox state (decreased glutathione), phospholipid biosynthesis (decreased ethanolamine) and anabolic reactions dependent on adenosyl methionine (decreased methionine). These changes are attenuated with increasing PDHC inhibition, which induces upregulation of the activity of the TCA-cycle-limiting OGDHC and downregulation of the malate dehydrogenase activity. As a result, degradation of amino acids in the TCA cycle is promoted to support the energy demands. Furthermore, succinylation/glutarylation of the brain proteins increases. PDHA phosphorylation does not show a strong response to the observed perturbations. The metabolic rearrangement upon increasing the PDHC inhibition is associated with decreased anxiety and reduced parasympathetic activity.


New antithrombotic therapies with less effect on bleeding are needed for coronary artery disease. The Btk inhibitor ibrutinib blocks atherosclerotic plaque–mediated thrombus formation. However, it is associated with increased bleeding, possibly due to non–Btk‐mediated effects. Btk‐deficient patients do not have bleeding issues, suggesting selective Btk inhibition as a promising antithrombotic strategy.To compare the antithrombotic effects of the highly selective Btk inhibitor AB‐95‐LH34 (LH34) with ibrutinib.Glycoprotein VI and G‐protein coupled receptor‐mediated platelet function and signaling were analyzed in healthy human donor platelets by lumi‐aggregometry, flow adhesion, and western blot following 1 h treatment with inhibitors in vitro.LH34 showed similar inhibition of Btk‐Y223 phosphorylation as ibrutinib, but had no off‐target inhibition of Src‐Y418 phosphorylation. Similar dose‐dependent inhibition of aggregation to atherosclerotic plaque material was observed for both. However, in response to Horm collagen, which also binds integrin α2β1, LH34 exhibited less marked inhibition than ibrutinib. Both LH34 and ibrutinib inhibited platelet adhesion and aggregation to plaque material at arterial shear. Ibrutinib demonstrated the most potent effect, with complete blockade at high concentrations. Platelet activation (P‐selectin) and procoagulant activity (phosphatidylserine exposure) in thrombi were inhibited by LH34 and completely blocked by ibrutinib at high concentrations. Furthermore, plaque‐induced thrombin generation was reduced by higher concentrations of LH34 and ibrutinib.LH34 potently inhibits atherosclerotic plaque–induced thrombus formation and procoagulant platelet activity in vitro, with less off‐target inhibition of Src than ibrutinib, suggesting it is a promising antiplatelet therapy with the potential for reduced bleeding side effects. Ibrutinib blocks platelet activation by plaque, but contributes to increased bleeding risk.Selective Btk inhibitors may block platelet activation by plaque without contributing to bleeding.Selective Btk inhibitor AB‐95‐LH34 inhibits glycoprotein VI–mediated platelet activation by plaque.Btk inhibitors reduce platelet procoagulant activity in response to plaque.112 antagonist reduce recurrent atherothrombotic events at the expense of increased risk of major bleeding as they target pathways common to both thrombosis and hemostasis.Platelet inhibition is a crucial component of the treatment strategy for acute coronary syndromes (ACS). Dual antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitors, while providing equivalent or superior efficacy against atherothrombosis. A promising target, platelet glycoprotein VI (GPVI), has been shown to be critical in atherothrombosis, with blockade or depletion of GPVI exhibiting protection against thrombosis in several models.New antithrombotic strategies must cause fewer hemorrhagic complications than P2YInhibition of the Tec family kinase Btk has been shown to block GPVI signaling, preferentially inhibiting thrombus formation on human atherosclerotic plaque, but not to affect integrin α2β1‐ and von Willebrand factor‐mediated adhesion to collagen, which is important for hemostasis.Bleeding side effects have, however, frequently been reported in patients treated in oncologic indications with early generation Btk inhibitors .More selective Btk inhibitors that avoid these off‐target kinase effects therefore have the potential to inhibit platelet GPVI function, while avoiding increased bleeding risk. Partially enhanced selectivity for Btk was shown in kinase assays for the irreversible Btk inhibitor evobrutinib,In this study, we used the covalent Btk inhibitor AB‐95‐LH34  monoclonal antibody (mAb) was from Millipore. The horseradish peroxidase (HRP)‐conjugated goat α‐rat IgG (SC 2032) and α‐Syk pAb (SC‐1077) were from Santa Cruz Biotechnology. Phospho‐specific polyclonal antibody (pAb) against pY1217 was from Cell Signaling Technology, against LAT pY200 and Btk pY223 were from Abcam, against Btk pY551 was from BD Biosciences and against Src pY418 was from Life Technologies. AB‐95‐LH34, ibrutinib (PCI‐32765), and evobrutinib were from Novartis. Eptifibatide was from GSK. The fluoroscein isothiocyanate (FITC)‐conjugated rat α‐mouse CD41 mAb was from BD Biosciences. Alexa Fluor® 647 anti‐human CD62P (P‐Selectin) antibody was from BioLegend. Annexin V Alexa Fluor 568 conjugate was from Fisher Scientific. Alexa Fluor 488‐conjugated phalloidin was from Thermo Fisher Scientific. The FITC‐conjugated rat α‐mouse, HRP‐conjugated α‐mouse, and α‐rabbit secondary pAbs and Hyperfilm enhanced chemiluminescence (ECL) autoradiography film were from Amersham Biosciences. Glucose, Bolt running buffer, Bolt antioxidant, and ECL reagent were from Thermo Fisher Scientific. Collagen related peptide (CRP) was from CambCol Ltd. Collagen  was from Takeda. Thrombin receptor activating peptide (TRAP) was from Severn Biotech Ltd. ADP and arachidonic acid (ASPi) were from Roche Diagnostics Corporation. Non‐fatty acid free bovine serum albumin (BSA) was from First Link UK Ltd. ChronoLume® and ATP standard were from Chrono‐Log Corporation. Methanol and ethanol were from VWR Chemicals. Transblot Turbo western blotting buffer was from Bio‐Rad Laboratories. All other reagents were purchased from Sigma‐Aldrich.2.2Ethical approval for collecting blood from healthy volunteers was granted by Birmingham University Internal Ethical Review (ERN_11–0175) in accordance with the Declaration of Helsinki. Ethical approval for use of atherosclerotic plaque material from patients undergoing carotid endarterectomy was provided by the North‐West Haydock Research Ethics Committee (20/NW/0001).2.3Blood was taken by venipuncture from consenting patients or healthy, drug‐free volunteers, into 4% sodium citrate .2.4g, 20 min, room temperature) and platelet‐rich plasma (PRP) collected. PRP was then centrifuged  in the presence of 0.5 μmol/L prostacyclin (PGI2), and the supernatant discarded. The platelet pellet was resuspended in 24 ml modified‐Tyrode's‐HEPES buffer  and 3 ml ACD then centrifuged  in the presence of 0.2 μg/ml PGI2. Supernatant was discarded and platelet pellet resuspended in modified‐Tyrode's‐HEPES buffer to the required concentration. Platelets were rested for 30 min prior to use in any experimentsWarmed acid citrate dextrose (ACD) 1:10 (v/v) was added to citrated whole blood then centrifuged (200 × 2.58/ml) or PRP under stirring conditions (1200 rpm) at 37°C were measured in a lumi‐aggregometer  for 5 min. Aggregation was monitored by measuring changes in light transmission, with ChronoLume® (D‐luciferin‐luciferase mixture) added to enable simultaneous measurement of ATP release by luminescence. Inhibitors (50 nM ‐ 2 μM) or vehicle (dimethyl sulfoxide [DMSO]) were incubated with washed platelets or PRP for 1 h prior to stimulation.Aggregation and ATP secretion in washed platelets (2 × 102.6Aggregation was measured in whole blood diluted 1:1 (v:v) in warmed saline in a Multiplate multiple electrode aggregometry analyzer (Roche Diagnostics) for 5 min. Inhibitors (50 nM ‐ 2 μM) or vehicle (0.05% DMSO) were incubated with whole blood for 1 h prior to stimulation.2.77/ml) were plated on atherosclerotic plaque coated glass coverslips for 30 min at 37°C, before non‐adherent platelets were removed and adherent platelets fixed . Inhibitors (0.5 or 2 μM) or vehicle (0.02% DMSO) were incubated with washed platelets for 30 min prior to seeding. Platelets were then permeabilised, actin cytoskeleton stained with phalloidin Alexa Fluor 488, and imaged . A semi‐automated machine learning based analysis workflow using KNIME 3.4 (KNIME.com AG) and Ilastik 1.1.2 (University of Heidelberg) software was then used to assess area, perimeter, classification, and number of spread platelets.Washed platelets (2×102.88/ml) were pre‐treated with 9 μM eptifibatide to block integrin αIIbβ3 activation. Agonists were added while stirring at 1200 rpm in an aggregometer at 37°C for 180 s unless stated otherwise. LH34, ibrutinib, evobrutinib (50 nM ‐ 2 μM), or vehicle (0.05% DMSO) were incubated with washed platelets for 1 h prior to stimulation. Activation was terminated with 5X sodium dodecyl sulfate (SDS) reducing sample buffer. Lysates were separated by SDS‐polyacrylamide gel electrophoresis, electro‐transferred, and western blotted. Western blots were probed with the stated antibodies and imaged using ECL autoradiography film. For analysis of levels of phosphorylation, western blot films were scanned and band intensity measured using ImageJ 1.5  with values normalized to those seen in vehicle‐treated platelets.Washed platelets (4×102.9Atherosclerotic plaque was collected from carotid endarterectomies of 10 patients with symptomatic carotid artery stenosis (transient ischemic attack or stroke) and frozen (–80°C). Samples were then crushed into a fine powder and pooled. Phosphate buffered saline (PBS) was added to pooled powder and sonicated on ice. Plaque homogenate was then centrifuged and supernatant protein concentration determined.2.106, Alexa Fluor 647 anti‐human CD62P (P‐Selectin) antibody or annexin V Alexa Fluor 568 conjugate were preincubated with blood for 10 min at 37 °C. Blood was then perfused over the coated channels at shear rate 1000 s−1 for 10 min using a PHD 2000 Syringe Pump (Harvard Apparatus). Platelet adhesion and thrombus formation were captured every 30 s with an Evos FL Auto imaging system (Life Technologies) using a 20× objective for the entire flow period. Images were processed and analyzed to measure fluorescence intensity and surface coverage area (%) using semi‐automated scripts in ImageJ software.Flow studies were performed using citrated whole blood from healthy volunteers. Hirudin‐anticoagulated whole blood from healthy volunteers was used for visualization of annexin V and P‐selectin expression. Ibidi μ‐Slide VI 0.1 slide (ibidi) were coated with 1 mg/ml atherosclerotic plaque homogenate or 200 μg/ml Horm collagen overnight at 4°C. Excess substrate was removed and the channels were blocked with 4 mg/ml BSA in PBS at 4°C. Channels were flushed with PBS immediately prior to blood perfusion. Blood was incubated with indicated Btk inhibitor or vehicle (0.05% DMSO) for 1 h prior to perfusion. DiOC2.11Whole blood was diluted 1:9 (v/v) in staining solution (anti‐P‐selectin and activated‐integrin αIIβ3 antibodies in PBS) then stimulated by indicated agonists 1:10 (v/v) for 20 min in the dark at room temperature. Samples were then fixed with ice‐cold 1% paraformaldehyde (PFA) and analyzed on an Accuri C6 flow cytometer (BD Biosciences). Platelets were gated using forward and side scatter. Inhibitors (20 nM ‐ 5 μM) or vehicle (DMSO) were incubated in whole blood for 1 h prior to antibody addition and stimulation.2.12g, 10 min, room temperature). PRP was incubated with Btk inhibitors at 37°C for 1 h, then stimulated with recombinant tissue factor or plaque homogenate in an Immulon 2 HB 96‐well round‐bottom plate (Fisher Scientific). Measurement was carried out using the Fluoroskan Ascent plate reader (Thermo Fisher Scientific) equipped with Thrombinoscope software . Endogenous thrombin potential  and maximum thrombin generation  were then measured in duplicate samples at 37°C for at least 60 min .Thrombin generation was measured as previously described.2.13p < .05 unless otherwise stated. Graphs were plotted using GraphPad Prism 9 (GraphPad Software Inc.). Statistical analysis was performed using one or two‐way analysis of variance with corrections for multiple comparisons unless otherwise stated. All statistical analyses were performed using GraphPad Prism 9.All data are presented as mean ± standard error of the mean (SEM) with statistical significance taken as 33.1Previously we have shown inhibition of GPVI‐mediated aggregation by ibrutinib correlated with off‐target inhibition of Src Y418  phosphorylation.These reduced off‐target kinase effects of LH34 observed here compared to ibrutinib are in line with the selectivity of LH34 in kinase binding assays Figure .3.2To determine the effect of specific Btk inhibition on GPVI function, platelet aggregation and dense‐granule secretion were assessed by lumi‐aggregometry in healthy human donor washed platelets. LH34, ibrutinib, and evobrutinib showed dose‐dependent inhibition of aggregation and secretion to atherosclerotic plaque homogenate, with LH34 and ibrutinib showing greater inhibition at low concentrations than evobrutinib Figure . IsolatiPlatelet spreading on atherosclerotic plaque was also assessed following Btk inhibition Figure . Platele3.3Platelet aggregation to atherosclerotic plaque in whole blood was also inhibited by LH34, and showed similar dose‐dependent inhibition as ibrutinib, with evobrutinib again showing less inhibitory effect Figure . No inhiPlatelet activation in response to GPVI stimulation with CRP was measured following incubation of Btk inhibitors in whole blood for 1 h. LH34 and ibrutinib inhibited platelet α‐granule secretion and integrin αIIbβ3 activation at concentrations >2 μM. Evobrutinib however had no effect Figure . GPCR‐me3.4Ibrutinib has previously been shown to abolish platelet aggregate formation in whole blood perfused over atherosclerotic plaque homogenate, but not collagen, at arterial shear rates.3.5In addition to looking at thrombus size, we also assessed the level of platelet activation within thrombi formed on atherosclerotic plaque Figure . The lowProcoagulant platelet formation, characterized by membrane phosphatidylserine (PS) expression, on atherosclerotic plaque was completely abolished by low and high concentrations of ibrutinib Figure . Low conBtk inhibition of procoagulant activity of platelets was further assessed using a thrombin generation assay Figure . ETP and4In this study we demonstrate that highly selective Btk inhibition blocks platelet responses to atherosclerotic plaque. Platelet adhesion to collagen, which is important for physiological hemostasis, remains intact with highly selective LH34, but not ibrutinib, which may be due to off‐target inhibition of Src. We also show that Btk inhibition blocks the procoagulant functions of platelets induced by atherosclerotic plaque.Platelet responses to atherosclerotic plaque have been shown to be mediated almost exclusively by GPVI,Profound effects of Btk inhibition were observed to atherosclerotic plaque, with aggregation and spreading, as well as both platelet adhesion and aggregate size, reduced as previously reported.−1) and concentration of ibrutinib (1 μM).Less effect of Btk inhibition was observed to collagen, reflecting the involvement of integrin α2β1 in its activation of platelets. The GPVI‐specific agonist CRP however showed very similar inhibition as plaque, providing indirect evidence that Btk inhibitors block atherosclerotic plaque–mediated platelet activation by blocking GPVI. Selective Btk inhibitors LH34 and evobrutinib did not affect platelet adhesion to collagen under flow, with only high concentration ibrutinib inhibiting platelet surface area coverage. Lack of effect by more selective inhibitors shows Btk has little role in integrin α2β1 function, which is critical for platelet arrest and aggregate formation on collagen, and signifies ibrutinib has off‐target effects on this pathway.2+ from intracellular stores and extracellular influx.q‐coupled protease‐activated receptors can also result in sustained calcium increase, but activation by secondary mediators ADP and thromboxane A2 alone result in weak generation of procoagulant platelets.1 and P2Y12 have only minimal effect on their formation.GPVI signaling is the major pathway for procoagulant platelet formation, which requires high sustained elevation of cytosolic CaAlthough not as potent as ibrutinib, LH34 still strongly inhibited plaque‐mediated platelet responses. Its high selectivity for Btk suggests it is less likely to result in bleeding, previously associated with off‐target inhibition of Src (and Tec) kinases in ibrutinib‐treated patients.A recent report has however shown an increase in platelet function analyzer 200 (PFA‐200) in vitro closure time with remibrutinib.The present study focused on in‐vitro experimental systems relating to platelet function. While this enables dissection of mechanistic differences, the in‐vitro data has clear limitations in prediction of clinically‐relevant risk and benefit. Ex‐vivo experiments looking at the effect of ibrutinib‐treated patients on GPVI‐mediated platelet function have been performed previouslyIn conclusion, the selective Btk inhibitor LH34 shows potent inhibition of atherosclerotic plaque–induced thrombus formation without the off‐target kinase inhibition observed with ibrutinib. Additionally, Btk inhibition can limit the platelet–coagulation interplay in response to atherosclerotic plaque. This suggests highly selective Btk inhibitors could be repurposed as promising new treatments for atherothrombosis with a reduced effect on bleeding.Christopher W. Smith, Maan H. Harbi, and Phillip L.R. Nicolson designed and performed experiments, analyzed data, and wrote and revised the manuscript. Lourdes Garcia‐Quintanilla, Helena Brown, Kieran Rookes, and Natalie S. Poulter performed experiments, analyzed data, and revised the manuscript. Steve P. Watson designed experiments and revised the manuscript. Mark R. Thomas designed experiments and wrote and revised the manuscript. The manuscript has been read and approved for submission to JTH by all authors.PLRN, MRT, and SPW have received research grants from Novartis, Principia, and Rigel Pharmaceuticals. PLRN has had honoraria from Bayer, Grifols, and Takeda. All other authors have no conflicts to report.This work was funded by Novartis.Table S1Figure S1Figure S2Figure S3Figure S4Click here for additional data file.


Competitive advantage and sustainability emerge as important factors for the success of an organization’s overall differentiation. This research aims to identify the relationship between internet banking and bank investment decision, as well as gaging the mediating effects of sustainability and competitive advantage as attributes of investment decisions. To achieve that, a questionnaire was administrated to banks’ employees and customers. To carry out the hypothesis testing, we have employed structural equation modeling through SPSS and SmartPLS. The findings suggest that internet banking, sustainability, and competitive advantage constructs are significant antecedents of banks because they highlight valuable attributes for banks to attain future benefits. This paper contributes to bank managers and scholars by providing a framework and supporting theories that help to identify relevant constructs and strategic resource characteristics. From the findings, we recommend conducting future studies in other countries or fields to generalize our results. Recent advancements in IT have contributed to the rapid growth of modern and creative financial services known as financial technology (Fintech). Investment decision is a growing challenge in many business enterprises in the world. Investors are focusing on new ways to invest in their business for better performance; however, it seems to face difficulty in making investment decisions. According to The investment decisions indicate “capital budgeting” in the language of financial management. The “investment decision-making and capital budgeting” in the finance industry are not deemed distinct acts. The concept “capital” is specifically perceived in the investment decision to relate to real assets that can take any form of construction, plants, equipment, raw materials, etc., while investment relates to any actual assets of this type. In other words, investment decisions must relate to issues of if today’s contributions to financial assets will rise tomorrow’s sales to cover expenses . InvestmIn nowadays era of globalization and the competitive marketplace, firms experience spectacular changes . TherefoInnovation and investment decisions have been discussed in several top management journals. However, empirical research on the outcomes of innovation and investment decisions is still rare . NeverthWe note that past works pointed to analyzing the impact of investment and financial decisions on a collection of the response variables, like financial performance, by reviewing relevant literature . In compThe next part examines the theoretical background and hypotheses development. Follows by section three, which deals with methodology and collection of data. Section four lays out the analysis of the results. In the last section, we present the discussion, limits and further investigation directions, and conclusion.Scholars have progressively arrived at the resolution that to get a competitive advantage, organizations have to invest, group, and convey assets (resources), amongst which human capital (employees) and customers are particularly significant . Using hThe empirical works of literature on this subject are broad since the study of corporate investment has been the focal point of financial literacy for the previous numerous periods. Although, these investigations are typically influenced by the impacts of cash flows on investment , the impParticularly, there are four major social consequences of technology-based financial intermediaries. To begin with, Fintech services have substantially improved the efficiency of financial intermediation by providing much cheap, speedier, and more comfortable intermediation via the mobile platforms or the Internet . SecondlThrough a variety of routes, digital technology can have both beneficial and unfavorable effects on financial sustainability. For instance, Innovation, either as processes or as outputs, could be defined as a scarce resource that is liable to work as the source of sustained competitive advantage . The maiThe world is now living in a time where, without any thought, the marginal advantages of innovation and technology nevertheless surpass the marginal expenses related to them. H1: Internet banking significantly and positively influences bank investment decisions.Sustainability is concerned with meeting today’s needs without jeopardizing future generations’ ability to meet their own. The ideology of sustainability is built on three pillars: economic, environmental, and social, commonly known as profit, planet, and people.Schumpeterian creativity theory suggests that businesses that are continually taking on new variations and creative destruction are more likely to perform better than their competitors . The wilLiterature suggests that companies that are unable to innovate run the risks of being side-lined by shifts in legislation, technology, products, competition, and consumer needs . InnovatH2: There is a positive significant relationship between internet banking and bank sustainability.It is possible to describe innovation as new or creative methods used by the organization or the new goods it produces. The development of new technologies and methods requires innovation. Innovation may be the most significant source of competitive advantage. Innovation attracts rivals. Since innovation offers a business unique benefit , it can be viewed as the primary source of competitive advantage. Innovation has always been a critical driver of long-term market growth since it leads substantially and positively to value generation, distribution, and sustainability, and assists businesses to identify new business opportunities . CompaniThere is a consensus in the literature that all types of innovation can contribute to competitive advantage . TherefoH3: Internet banking has a significant positive effect on bank competitive advantage.Banks are generally regarded as having an essential role to achieve the Sustainable Development Goals (SDG) .Our investigation is also linked to the literature exploring the impact of sustainability on the decision-making of investors. Financial services can encourage or impede (non-)sustainable activities of governments, businesses, and individuals because of their intermediary role, and can also activate systemic changes in society . TherefoH4: There is a significant relationship between bank sustainability and bank investment decision.Competitive pressures are present in almost every industry. These pressures lower returns on capital and could lower future investment returns. Finding companies with sustainable competitive advantages can aid investors in generating higher investment returns .An investigation by According to the above discussion, we hypothesized;H5: Bank Competitive Advantage has a positive significant relationship with Bank investment decisions.The profits of a firm, contrary to common belief, are not the outcome of the services and products it offers. In addition, when deciding whether to invest, successful investors do not only rely on an organization’s earnings. The profits of a company are the product of its sustainability and competitive benefits. Consequently, successful investment managers concentrate on whether the sustainability and competitive advantages of a firm would allow it to protect and increase profits . The invResource-Based View Theory (RBVT) explains that capital (resources) ownership is valuable, difficult to duplicate, rare, and irreplaceable . This suFrom the foregoing theoretical underpinnings and practical observations, as well as the fact that online banking services are a precondition for innovative technologies and bank investment decisions, bank sustainability and bank competitive advantage can help to evaluate bank investment decision-making, therefore we assume that BS and BCA would mediate the link between internet banking and BID. The resulting hypotheses will be tested based on this assumption:H6: Bank sustainability would mediate the relationship between internet banking and Bank investment decision.H7: bank competitive advantage can mediate the link between IB and Bank investment decisions.This research employs a methodological approach that emphasizes the use of quantitative data collection and analysis tools. The quantitative factors were measured using a questionnaire survey. The objective of this research is to assess the effects of online banking as well as bank sustainability and competitive advantage mediation on bank investment decisions.A suitable research design is the primary methodological concern when examining the theoretical model. The scholars considered employees’ and customers’ points of view in this research for several reasons. Firstly, employees are very important to organizational innovation as they are responsible for interacting, modifying, and developing ideas. Similarly, to be innovative, organizations must manage and improve the internal environment that supports the characteristics of employees’ innovative behavior . SecondlTo do this, we first secured senior management consent from 11 active commercial banks, after which we approached individual branch managers and asked for their help with the study. In total, 55 branches agreed to take part in the research. We targeted the collection of data on 1,200 clients and 600 workers from 11 licensed commercial banks in the country based on the information available from the banks. On one hand, the surveys were shared with 600 employees from 11 institutions in our sample, which included 55 branches. The 500 employee questionnaires with valid responses have been obtained during the data collection, resulting in an approximately 83 percent employee response rate. The study enlisted the participation of an average of nine (9) employees in each branch. On the other hand, we utilized proportionate stratified random sampling once more to verify that the number of consumers in the sample across the two major cities was proportional to the percentage of total customers in the 11 banks. We collected 1,000 survey questionnaire responses from clients out of 1,200 initial targets using this approach, resulting in an 83 percent overall customer response rate. The available banking industry archival data provided to us correspond to customer engagements  before and during the study period.To sum up, we collected 1,000 and 500 usable questionnaires from banks’ customers and workers, respectively. Numerous rules of thumb recommend that a “50/20” or a “100/10” percentage is satisfactory . In our The data was collected from the distinct work units of 11 banking institutions that are now operating in Brazzaville and Pointe-Noire, Republic of Congo, the two cities that have the majority of the banks’ branches. The completed survey is comprised of close-ended questions. To ensure correct interpretation and transmission of the items’ intended implications, the survey instruments were first translated into French and then retranslated into English. The data were collected from November 2019 to June 2020. Additionally, the study used a “Likert scale” ranging from 1 to 5, with 1 indicating “strongly disagree” and 5 indicating “strongly agree,” as well as pertinent questionnaires from other scholars who conducted similar studies.The measurements employed in this research were derived from earlier studies and tailored for this study. The items of internet banking have been undertaken by via diagrams. SEM has also been used to analyze the model’s fit using approaches like path analysis, factor analysis, and regression analysis  is a method that concurrently solves worldwide various social sciences problems and examines factors’ relationships analysis . This apanalysis . SEM as analysis .Partial least square-structural equation modeling and CB SEM are logically different . For theThe composite reliability and Cronbach’s alpha coefficients were calculated to assess the constructs’ reliability.Cronbach’s alpha is a metric for determining the consistency or reliability of a set of scale or test items. As per the findings, Cronbach’s Alpha (CA) ranges from 0.863 to 0.875 which is above the 0.7 acceptancy value. In addition, all the lower bounds of the Cronbach’s Alpha confidence interval are greater than 0.7, indicating that internal consistency is verified . The intThe values of the average variance extracted (AVE) have been examined to ensure convergence validity. The results demonstrate that the AVE’s values for this study range from 0.509 to 0.663, which is acceptable and higher than the minimum required degree of 0.5. We assessed descriptive statistics of the items that comprised means, standard deviations, kurtosis, skewness, and the VIF. The results in The other results revealed both kurtosis and skewness values are relatively small and negative. Particularly, the negative values of the skewness imply that the data are skewed left while the kurtosis negative values indicate a “light-tailed” distribution. We also evaluated the correlation of the indicator in our research. The findings indicated a substantial and positive correlation between the study’s items, with values, ranging from 0.164 to 0.789. As both dependent and independent factors were quantified using the same questionnaires, it is critical to conduct a common technique of bias test . The stuThe fit indices of the measurement model, such as the SRMR and NFI have been also assessed, and the values are 0.064 and 0.971. This indicates that the fit indices were within acceptable limits. In addition, the item loadings were further examined, and the values are varying from 0.628 to 1.014 see , which aThe questionnaires comprise the respondents’ profiles: gender, age, educational level, and Income per month. The results from F = 0004; P = 0.951], this implies that the test of equality of variances was not violated. The Brown-Forsythe Test, on the other hand, with values of Statistic = 0.003; P = 0.953 for the BID validated the robustness of Levene’s test , e.g., are not significantly distinct. The Test of Homogeneity of Variance, often known as Levene’s test was used to determine whether the population included in the sample frame is homogenous . Accordie’s test . Also, tWe employed consistent partial least square (PLSc) to conduct the SEM, as recommended by t = 3.69; p < 0.01), IB - > BS  and IB - > BCA , respectively. Therefore, the hypotheses  were supported.To evaluate direct effect of internet banking (IB), bank sustainability (BS), and bank competitive advantage (BCA) on bank investment decision, we employed a consistent PLS structural equation modeling. Internet banking was found to have standardized coefficients see  that wert = 9.734; p < 0.01) and BCA - > BID . Conclusively, H4 and H5 are also accepted. The mediation analysis is used to test the other hypotheses.The outcomes in The term “mediation” refers to the study of how a third variable influences the relationship between other two different variables. A mediation analysis was performed as part of this study.t = 8.11; p < 0.01). Likewise, the mediating effect of bank competitive advantage on the link between internet banking and bank investment decision is as IB - > BCA - > BID . Along the same lines, a significant statistical mediation effect may be determined, implying that hypotheses (“H6 and H7”) are also partially supported. Comparably, BCA depicts greater significant direct and indirect effects on BID than BS  value can be used to assess the strength of the mediation analysis. Also, as suggested by Bank sustainability and BCA are the intermediates in this study, as per the literature and postulated model. In the instance of PLS-SEM, the variance accounted for (VAF) approach was also utilized to investigate the mediating effects, since it is one of the greatest ways of examining the mediation effects . The VAFFull mediation is defined as a VAF value greater than 0.8; partial mediation is defined as a VAF value ranging from 0.2 to 0.8; no mediation is defined as a VAF value is below 0.2 . Table 7In this study, we notice that constituents of innovation, sustainability, and competitive advantage affect the bank investment decisions. Our findings confirm the argument appointed by previous studies that investment decision depends on various factors . In addiSecondly, our results affirm the mediating roles of sustainability and competitive advantage in the IB-BID relationship, like this, reinforcing the view that multiple factors can mediate the effect of internet banking on bank investments decision . This fiThis study has some limits, which can be discussed by further studies. First, data have been gathered in the Republic of Congo, thus providing difficulties to generalize the context. Further works should test whether the constructs employed in the present research may also be examined in other countries. Second, information about how techniques emerge is not covered by the data. Future research may explore the changes in trends. Third, this investigation is not illustrative with concern to the distribution of banks in Congo because its foundation in data is limited only to two main cities namely Pointe-Noire and Brazzaville. Therefore, it is an attractive issue for scholars to shed light on in all cities in Congo or other countries in the World. Such information would allow an investigation of the examined determinants of firm innovation, sustainability, competitive advantage, and investment decisions and an analysis of proposed relationships. Particularly, banks’ patterns for innovation services, competition, sustainability, investment decisions, and the ways in which the decision process is taken, may play a vital role in sustainable benefits. Additional research directions comprise the absence of moderator constructs such as internet security, gender, perceived risk, and investment risk in this investigation. In order to strengthen this research results, further study may analyze our conceptual framework with a moderation effect. Scholars may also examine the reasons financial institutions do not consider the significance of environmental and social aspects in maintainable growth and thus, forgo sustainable practices implementations. In addition, the sustainability strategies typology assists in better measurements of the specific effects of sustainability practices on behaviors and clients’ attitudes. The examination of this association between clients’ responses and corporate sustainability also unlocks possibilities for future investigations. Another road for future research is that we have not hypothesized the relationship between sustainability and competitive advantage, and the links between sustainability, competitive advantage, and financial performance. Therefore, future studies may adopt our research framework and include the above-mentioned relationships.In this paper, we have built a theoretical framework based on Resource-Based View Theory (RBVT), Decision Theory (DT), and Theory of Planned Behavior (TPB) that explains the mediating effects of sustainability and competitive advantage on the relationship between internet banking and BID, and established the hypotheses by evaluating data collected from distinct banks in Congo. The findings show that an element of innovation, namely internet banking, is significantly linked with sustainability and competitive advantage that contribute substantially to bank investment decisions. Both sustainability and competitive advantage partially mitigate the effect of internet banking on banks’ investment decisions (BID). The resource-based view theory is regarded as one of the most important theories in financial literature. The current study aims to apply the concept of resource-based view theory to the financial management and investment decisions of firms. Only a few authors have examined resource-based view theory and decision theory to firms’ financial management and investment decisions in the literature. Another point to note is that previous research has primarily focused on the role of innovative activities as investment predictors. According to the resource-based view theory, financial innovations or techniques influence investment decisions through performance, sustainability, competitive advantage, and stakeholder satisfaction. The current study looked at the role of bank sustainability and competitive advantage, which had previously been overlooked by academics.The majority of innovation research is focused on the individual or contextual factors that contribute to its improvement, as few businesses can remain viable, prosper, and perform effectively in the competitive real world without innovations and technologies . WhereasThis research has important implications for organizational managers, and it adds to the body of knowledge by expanding on previous research. As twenty first-century businesses are operating in an unexpected and difficult business environment, the rate of growth is anticipated to accelerate. Therefore, the employees’ capabilities will grow because of gaining more knowledge and exchanging it with others. It reinforces a sense of commitment to the assigned tasks. The present investigation makes an important contribution to the Resource-Based View Theory and Theory of Planned Behavior (TPB). The usage of innovation to evaluate the investment decision is directly linked to how Resource-Based View Theory and TPB are comprehended. Investment decisions are affected by the Fintech services, sustainability, competitive advantage, and mutual coordination between managers, employees, and customers inside the company. This enables the consideration of both customers and employees in the decision-making process for the survival of the corporation. The effects of BS and BCA on BID were included in the study model. These findings demonstrate that the nexus between IB and BID can be bettered by enhancing both mediators. To this end, this research adds the contingence viewpoint to the managerial investigation.Apart from theoretical implications, this research offers clear guidance to a number of banking practitioners, including human resource practitioners, team leaders, decision-making managers, and management information systems managers. Increased investment and promotion of effective innovation tend to increase competitive advantage and long-term viability. HR professionals concentrate on enhancing the company’s image. Fintech is an effectual practice that assists organizations to gain a competitive advantage and strengthen their sustainability, which ultimately improves investment decisions. Proper implementation of innovative technology leads to BID. Moreover, the outcomes of this paper show that banks’ managers should concentrate on innovation as well as other factors that increase performance, competitive advantage, and long-term viability, all of which would benefit BID as a whole. This research also aids the researchers in clarifying the importance of IB, BS, and BCA in strengthening the BID. In their studies, the researchers can also use this empirical evidence framework to see if there are any other contributory factors that fit into this model.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JP-A: conceptualization, methodology, software, visualization, investigation, writing—reviewing and editing, data curation, and writing—original draft preparation. MW: conceptualization, supervision, validation, investigation, and reviewing and editing. Both authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this review, we will discuss how Nrxns and their ligands sculpt synaptic inhibition in a brain-region, cell-type and synapse-specific manner.Since the discovery of neurexins (Nrxns) as essential and evolutionarily conserved synaptic adhesion molecules, focus has largely centered on their functional contributions to glutamatergic synapses. Recently, significant advances to our understanding of neurexin function at GABAergic synapses have revealed that neurexins can play pleiotropic roles in regulating inhibitory synapse maintenance and function in a brain-region and synapse-specific manner. GABAergic neurons are incredibly diverse, exhibiting distinct synaptic properties, sites of innervation, neuromodulation, and plasticity. Different classes of GABAergic neurons often express distinct repertoires of Nrxn isoforms that exhibit differential alternative exon usage. Further, Nrxn ligands can be differentially expressed and can display synapse-specific localization patterns, which may contribute to the formation of a complex  Vertebrate neurexins (Nrxns) are essential and evolutionarily conserved presynaptic cell adhesion molecules (CAMs) that organize and critically regulate synaptic transmission of excitatory and inhibitory synapses through pleiotropic functions. Nrxns are encoded by three genes, Nrxns 1–3. Each gene encodes a longer α and shorter β isoform, and in the case of Nrxn1, a highly truncated γ isoform from independent promoters . α and βSince the discovery of Nrxns in 1992, the field has focused extensively on elucidating Nrxn function at excitatory synapses. Studies examining Nrxn function at inhibitory synapses, however, are only just emerging. Recent transcriptomics studies reveal that Nrxn isoform expression profiles in inhibitory cells differ drastically from those of excitatory cells. Moreover, different classes of GABAergic neurons exhibit distinct Nrxn isoform expression and/or alternative splicing profiles . SimilarGABAergic neurons are classified by a combination of properties which includes their protein and peptide expression, receptor expression, electrophysiological properties, and morphological properties. Their defining morphological properties include features like spines, such as those found on medium spiny neurons (MSNs) of striatum, or details of their axon targeting locations which include soma, proximal and distal dendrites, and axons. The most prominent GABAergic interneurons in forebrain are the perisomatically targeting parvalbumin-expressing (PV) and cholecystokinin-expressing (CCK) neurons and the dendritically targeting somatostatin-expressing (SOM) neurons . These iParvalbumin neurons are fast-spiking interneurons that synapse onto the perisomatic regions of their post-synaptic partners. PV neurons can be further classified as basket cells, defined by their synaptic contacts with soma and proximal dendrites, or chandelier cells, defined by their synaptic contacts with the axon initial segment. Unique from SOM or CCK neurons, PV neurons express μ-opioid receptors and P/Q-type calcium channels . The othvia in-situ hybridization, single-cell quantitative reverse transcriptase (qRT)-PCR techniques -derived interneurons (such as PV and SOM) and caudal ganglionic eminence (CGE)-derived interneurons (such as CCK and VIP)  are embrThe molecular mechanisms that regulate the selective cell-type specific usage of the alternative SS4 exon are being examined. Convincing evidence has implicated three members of the signal transduction and activation of RNA (STAR) domain family of RNA binding proteins—SAM68, SLM1, and SLM2—in regulating splicing at Nrxn SS4. SAM68, SLM1, and SLM2 possess a single central RNA-binding KH domain and their expression in neurons promotes the skipping of the SS4 exon to produce SS4- Nrxns . Of the The functional relevance of the other Nrxn splice sites is not as well characterized as SS4, even at excitatory synapses. However, the other splice sites could play significant roles in inhibitory synaptic function, as their expression profiles are highly region-and cell-type specific. Notably, the alternative SS2 insert appears to be excluded (SS2-) from Nrxns 1 and 3 of both MGE and CGE-derived interneurons, but is included in Nrxn1 in pyramidal neurons . AccordiIn addition to alternative splicing, post-translational modifications further add to the diversity of Nrxns. While all Nrxns are N-glycosylated, the sequences between LNS6 and the transmembrane region are subject to O-linked glycosylation . More reUnique to Nrxn3 is the presence of an in-frame stop codon encoded in the SS5 exon (Nrxn3 SS5+), that results in the production of Nrxn3 without transmembrane and intracellular domains . This trMeasuring the expression and localization of Nrxn protein is no easy task due to the lack of antibodies that detect individual Nrxns . Currentin vitro studies show that Nrxns are synaptogenic, facilitate synapse specialization, and enable neurotransmission), studying Nrxns in intact neural circuits reveals that different brain regions, presynaptic cell-types, and post-synaptic cell-types display remarkable diversity in their functional requirement and utilization of neurexins  was found using conditional knock-out of all three Nrxns (triple KO). The triple KO of Nrxns reduced synaptic inhibition mediated by PV and SOM interneurons onto layer 5 pyramidal neurons in medial prefrontal cortex (mPFC), however, the synaptic properties impacted by the deletion of all Nrxns differed significantly. Triple KO in PV interneurons reduced the density of synapses made onto pyramidal neurons, while triple KO in SOM neurons impaired action potential-induced presynaptic calcium influx and significantly reduced presynaptic release . FurtherIn a separate study, the impact of Nrxn3 KO in SOM neurons that synapse onto pyramidal neurons in layer 2/3 of anterior cingulate cortex (ACC), a region of prefrontal cortex, was investigated. The KO of Nrxn3 from SOM neurons impaired presynaptic release probability and reduced SOM-mediated inhibitory post-synaptic current (IPSC) amplitudes at SOM-pyramidal neuron synapses . ImportaRecent work from our lab demonstrated that in subiculum of ventral hippocampus, Nrxn3 plays an essential role in PV-mediated synaptic transmission. Similar to the triple Nrxn KO in mPFC, PV synapse numbers, post-synaptic strength, and PV-IPSC amplitudes were drastically reduced following the ablation of Nrxn3 from PV neurons (PV-Nrxn3 KO) in males . Howevervia an epitope tag inserted into endogenous Nrxn3 SS5) is present in PV, CCK, and SOM-expressing interneurons, however, it exhibits selective presynaptic enrichment only in synapses made by SOM and CCK neurons  , FAM19A1-4, GABA(IgSF21) , are far(IgSF21) . The modariables . CA10/11les . Nlgns were quickly appreciated for their powerful synaptogenic properties- when expressed in non-neuronal cells or coated onto beads, these molecules rapidly induced the recruitment of presynaptic specializations . Howeverof Nlgn2 . Splice-of Nlgn2 .AR activity and then recruits gephyrin, the central scaffolding molecule at inhibitory synapses, and activates collybistin to recruit and organize inhibitory post-synaptic proteins, including GABAARs . PTPδ is a member of the type-II receptor protein tyrosine phosphatases, which, like Nrxns, are important presynaptic hubs for synaptic organization . Overexpression of Nlgn3 lacking splice-site A (Nlgn3Δ) or only including splice-site A2 enhances IPSCs, whereas inclusion of just A1 or both A1A2 decreases IPSCs . FurtherBeyond cortex and hippocampus, Nlgn3 also mediates cerebellar and striatal inhibitory synapses in a cell-type and input-specific manner. In cerebellum, Nlgn3 is localized at a subset of inhibitory synapses, including at molecular layer interneuron-Purkinje cell synapses and at synapses in the inner granular layer . In nucltrans-synaptic organization, synapse maintenance and synaptic plasticity independent of ion flux through their pore (Only α and β neurexins with an insert at splice site 4 (SS4+) bind to secreted cerebellins (Cblns)1, 2, or 4 and form a tripartite complex with post-synaptic ionotropic glutamate delta receptors, GluD1 or GluD2 . Cerebeleir pore . The funThe expression patterns of Cbln1-4 are developmentally regulated and vary dramatically by brain region and cell type. Cbln1, 2, and 4 are expressed abundantly throughout the brain while Cbln3 expression is largely restricted to neurons in cerebellum . Triple via Cbln/GluD complexes are prime binding candidates to regulate inhibitory synapse function because Nrxns predominantly express the alternative SS4 exon, which is required to bind to Cblns to generate a synaptic Nrxn-Cbln-GluD tripartite complex. Indeed, several studies have shown that GluD1 and Cblns are critical for inhibitory synapse formation and specification. GluD1 expressed in HEK cells primarily induced inhibitory presynaptic differentiation when co-cultured with cortical neurons when Cbln1 or 2 were added to the media . Nxph genes are highly conserved in vertebrates but are absent in invertebrates . Intriguthalamus . Nxph1 wthalamus . Recentlthalamus . FurtherCalsyntenins are post-synaptic transmembrane proteins encoded by three evolutionarily conserved genes (Cstn1-3). The extracellular sequences of Cstns contain two cadherin domains and an LNS domain . AlthougThere are conflicting reports regarding the binding of Cstn3 with Nrxns. A series of elegant cell binding and biochemical and electron tomography assays identified direct interactions between Cstn3 and α-Nrxns, but not β-Nrxns, which occurred independent of SS4 . This inARs to impair the maturation of inhibitory synapses  was recently identified as a Nrxn2α-specific interacting protein . IgSF21 f Nrxn2α . IgSF21 The expression and function of neurexins and their ligands at inhibitory synapses is an emerging area of study, having been historically overlooked in favor of investigation of excitatory synapses. It is clear from several decades of work that neurexins specify multiple key aspects of neurotransmission at glutamatergic synapses. Accumulating evidence indicates these synaptic adhesion molecules play critical roles at inhibitory synapses as well by mediating pre- and post-synaptic properties of synaptic transmission and maintaining connectivity. A significant challenge to understanding the functional role of neurexins at inhibitory synapses has been accounting for the profound diversity of inhibitory neuron classes in the brain. Thus far, many studies have focused on defining Nrxn and Nrxn-ligand function in the three prominent inhibitory classes in cortex and hippocampus, PV, SOM, and CCK GABAergic interneurons. Together these studies demonstrate that neurexin expression and function is cell-type and region-specific. This has been most extensively demonstrated in studies of Nrxn3, which mediates different synaptic properties depending on the brain region, identity of the pre- and post-synaptic neuron, and sex of the animal.trans-synaptically via neurexin ligands. Indeed, as we discuss here, Nrxn ligands also exhibit brain-region and synapse-specific localization and function at inhibitory synapses. Thus, in addition to continuing to explore individual neurexin function in other classes of inhibitory neurons and in other brain regions, future studies should also aim to clarify these possible mechanisms as new tools to interrogate Nrxn localization become available. Finally, a major gap in the literature is whether neurexins control synaptic plasticity at inhibitory synapses, like they do at excitatory synapses. Similarly, several studies have revealed dynamic, circuit-specific changes in individual neurexin expression following behavior and experience, but it is largely unknown if these changes are occurring in inhibitory neurons, and if they are, how this may be associated with inhibitory synaptic plasticity. Neurexins are consistently implicated in several neuropsychiatric and developmental disorders that are thought to be in part driven by deficits in inhibitory function and dysregulation of E/I balance, such as ASDs, schizophrenia, epilepsy, substance use disorders, and stress. Continued investigation of the expression, localization, and function of neurexins in inhibitory transmission at a synapse-specific and activity-dependent level will be crucial to elucidate how these molecules contribute to brain function in both healthy and disease states.Due to current technical limitations, one major difficulty has been determining the precise mechanisms by which these diverse functions are manifested, as cell-type specific differences in neurexin isoform and alternative splicing mRNA expression profiles do not fully explain all cases of functional diversity. Heterogeneity could also be mediated by synapse-specific expression or localization of neurexin proteins as well as synapse-specific signaling, both intracellularly and EB and JA wrote the article. Both authors contributed to the article and approved the submitted version.