Natural killer (NK)cells, an important part of the innate immunesystem, can clear a wide variety of pathological challenges, includingtumor, senescent, and virally infected cells. They express variousactivating and inhibitory receptors on their surface, and the balanceof interactions between them and specific ligands displayed on thesurface of target cells is critical for NK cell cytolytic functionand target cell protection. The CD94/NKG2A heterodimer is one of theinhibitory receptors that interacts with its trimeric ligand consistingof HLA-E, β2m, and a nonameric peptide. Here, multi-microsecond-longall-atom molecular dynamics simulations of eight immune complexeselucidate the subtleties of receptor (NKG2A/CD94)–ligand (HLA-E/β2m/peptide)molecular recognition that mediate the NK cell protection from a geometricand energetic perspective. We identify key differences in the interactionsbetween the receptor and ligand complexes, which are via an entanglednetwork of hydrogen bonds fine-tuned by the ligand-specific nonamericpeptide. We further reveal that the receptor protein NKG2A regulatesthe NK cell activity, while its CD94 partner forms the majority ofthe energetically important interactions with the ligand. This knowledgerationalizes the atomistic details of the fundamental NK cell protectionmechanism and may enable a variety of opportunities in rational-baseddrug discovery for diverse pathologies including viral infectionsand cancer and elimination of senescent cells associated with potentialtreatment of many age-related diseases. Externally, they present various inhibitory or activating  receptors4 thatallow them to directly recognize ligands (proteins) expressed on thetarget cells and determine their fate. Healthy cells tend to presentMHC class I molecules, which are ligands for the inhibitory receptorsand mediate protection from the NK cell clearance activity. On theother hand, cellular stress  leads to the upregulationof expression of ligands for activating receptors  or downregulation of normally present MHC class I molecules,shifting the balance in favor of NK cell activation and eliminationof compromised cells.5Naturalkiller (NK) cells are a subpopulation of lymphocytes, animportant part of the innate immune system, that respond quickly withoutpriming or preactivation to a wide variety of pathological challengessuch as virally infected and tumor cells.2 HLA-E is encoded by MHC HLA loci and is exhibiting a low level ofpolymorphic occurrences. Two nearly equally presented6 functional HLA-E alleles, HLA-ER (HLA-E*01:01)and HLA-EG (HLA-E*01:03),7 which differin one amino acid at the position 107, Arg or Gly, respectively, accountfor almost 100% of HLA-E in the human population. Both alleles canbe beneficial in certain settings as *01:01 allele is more favorablein cancer diseases and *01:03 is more favorable in bacterial infections,bone marrow transplantation, and periodic abortions.6 For cell surface expression of HLA-E, a trimeric complexcomposed of HLA-E, its light chain beta-2 microglobulin (β2m),and the specific nonameric peptide derived from signal sequences ofother HLA class I molecules  must beestablished.9 The array of suitable nonamericpeptides is restricted, and only a handful of them allow effectiverecognition of the HLA-E complex expressed on the cell surface bythe receptor presented by the NK cells. This leads to either the subsequentdeath of the target cell or its protection from elimination.16 Of all the peptides studied to date, the HLA-G leader sequence provedto be the most effective mediator.13Several receptor familiesfor the MHC class I molecules presentedon the NK cells have been identified, including killer cell Ig-likereceptors, immunoglobulin-like transcripts, and the C-type lectinfamily, which consists of a heterodimer of the CD94 protein and arepresentative of the NKG2 family molecules , except NKG2D, which exists as a homodimer. While the ligandsfor the former two receptor families are (Ia) HLA class I moleculesand the non-classical HLA-G, the ligand for most CD94/NKG2 heterodimersis the non-classical (Ib) glycoprotein HLA-E.13Amongthe various NK cell receptors, the inhibitory CD94/NKG2Areceptor complex, which upon activation enables cell protection, isthe one with the highest micromolar affinity for the HLA-E complex.In fact, NKG2A holds a sixfold higher affinity for HLA-E comparedto the activating receptor containing NKG2C.17 idiopathic pulmonary fibrosis,18 and cancer.19 Cellularsenescence, a state of irreversible cell cycle arrest in responseto cellular damage, has been identified as one of the key featuresof human aging that can be targeted to reduce the negative healtheffects of aging.21 At least three ligands for eitheractivating or inhibitory NK cell receptors have been reported to bepresent on the senescent cells. With increasing age, also, more abundantexpression of the NKG2A receptor ligand HLA-E on senescent cells derivedfrom human skin was reported. Blocking the interaction of HLA-E andNKG2A has been shown to enhance the immune response against senescentcells in vitro, suggesting a potentially new strategyto eliminate senescent cells,22 therebyreducing their adverse health effects. Increased expression of theinhibitory NKG2A receptor associated with NK cell exhaustion was alsodetected in patients with severe COVID-19.23The implication of NK cells and their activating/inhibitoryreceptorshas also recently been linked to aging, a process that affects thevast majority of living organisms and appears to be the major riskfactor for the development of several age-related diseases, includingdiabetes,26 and mutagenesis studies27 have clarified the molecular details of the receptor–ligandinteractions, while binding and cytotoxicity assays28 revealed the role of specific nonamericpeptide residues. However, their mechanistic details and the intricaciesof the small peptide-mediated NK cell action still remain elusive.Several crystal structures of various compositions of HLA-E/β2m/peptide/NKG2A/CD94immune complex systemsHere, we performed all-atom molecular dynamics (MD) simulationsof several immune complexes of the HLA-E/β2m/NKG2A/CD94 systemwith nonameric peptides to elucidate the mechanistic details guidingthe receptor NKG2A/CD94 molecular recognition of the ligand HLA-E/β2m/peptideat the atomistic level. We identified a hydrogen bonding network betweenCD94 and NKG2A that occurred more frequently in complexes with nonamericpeptides, which effectively mediates the receptor–ligand recognition,allowing successful protection from NK cell elimination. In addition,we coupled these results with the analysis of the system internaldynamics and free energy calculations to provide an extensive pictureof this complex molecular recognition event.24 In our simulations, we took into account only the resolvedextracellular domains of proteins CD94 (residues 57–179), NKG2A(residues 113–232), HLA-E (2–274), and β2m deprivedof its signal peptide (residues 21–119), for which structuraldata are available. The CD94, NKG2A, and HLA-E proteins further consistof cytoplasmic and transmembrane domains, which were not consideredin this study. The first model (i) comprises HLA-E, β2m, NKG2A,and CD94 proteins and the G nonameric peptide (sequence: VMAPRTLFL),hereafter referred to as +GCOM (Supporting Information files COMg_structure.pdband parameter file COMg_structure.top). The second model (ii) alleleCOM+G represents theallelic variant HLA-E*01:03, with the 107R > G (residue 107 coordinatestaken from PDB entry 6GH1).25Eight different models were builtbased on the crystal structure of the extracellular domains of humanCD94/NKG2A in complex with the extracellular domain of HLA-E [light(β2m) and heavy chain ]and the leader peptide of the HLA class I histocompatibility antigen,alpha chain G, solved at a resolution of 3.4 Å (PDB entry: 3CDG).+GCOM in the sequence of the nonamericpeptide, a key element for a successful recognition of HLA-E by NKG2A/CD94receptors located on NK cells. Namely, the complex with the HLA-B7peptide (VMAPRTVLL) represents the third model (iii) +COMB7 (peptide taken from PDBentry 1KPR).7 Next, the model containing the Hsp60sp signalpeptide (sequence: QMRPVSRVL), referred to as (iv) –COMHsp60sp, was obtained by introducingseveral mutations into peptide G of model +GCOM [sequence: (V > Q)M(A > R)P(R > V) (T >S) (L > R)(F > V)L]. Mutation R5V of peptide B7 [sequence: VMAP(R > V)TVLL]introduced in model +COMB7 resulted in model (v) COM–B7_R5V. Two additional models (vi) COM∼B27 and (vii) COM∼Cw7 with experimentally inconclusive NK cell protection were built,with model COM∼B27 containing the leader sequence of HLA-B27 (peptidetaken from PDB entry 1KTL)7 and model COM∼Cw7 of HLA-Cw7 (peptidetaken from PDB entry 3BZF).16 Finally, model (viii) apoCOM was generated by removing the nonameric peptidefrom the model +GCOM (Movie S1). The simulated eight models along withthe additional data are shown in +), models lacking NK cell protection (−), and models in which NK cell protection is inconclusive(∼). All mutations were built using the tleap module of Ambertools 18.29Next, models differ from 29 and the AMBER-ff14SBforce field (FF) was used for proteins.30 Protonation states of ionizable residues were determined using thePDB2PQR web tool under a neutral pH condition of 7.31 Carboxylic amino acids were found in their common deprotonatedstates, whereas histidines were protonated at Nε, Nδ,or both positions. The system was embedded in a 10 Å layer ofTIP3P water molecules,32 resulting in abox of 126.653 × 134.686 × 123.583 Å3, togetherwith 19 Na+ counterions, and water molecules counted upto 206 859 atoms. All disulfide bonds were built using the tleap module of Ambertools 18,29 which was also used to prepare the topologies of the models.Classical moleculardynamics simulations (MD) were performed using the Amber 18 PMEMDsoftware package,2 on the heavy atoms were used, respectively. Next, the restraintswere removed, and 10 ns of isothermal–isobaric ensemble (NPT) function was performed, where pressure control (1 bar)was achieved using a Berendsen barostat.33 Productive MD was conducted for the canonical ensemble (NVT) using periodic boundary conditions for 1.2 μsfor each model, resulting in a total simulation time of ∼10μs. During the MD simulations, temperature control (303 K) wasperformed using the Langevin thermostat34 with a collision frequency of 1 ps–1. The SHAKEalgorithm35 was used to constrain bondsof hydrogens, and the particle mesh Ewald method36 with a cutoff of 10 Å was used to account for long-rangeelectrostatic interactions. An integration time step of 2 fs was setduring all MD runs.After the initial minimization, the system was gradually heated to303 K in two sequential steps—0–100 K over 5 ps in thefirst step and 100–303 K over the next 120 ps in the secondstep. Positional restraints of 200 and 100 kcal/mol Å37 and PyMol38 software tools were used for visualization andinspection of trajectories,respectively. MD trajectory analyses, including root-mean-square fluctuations(RMSF), and calculation of the cross-correlation matrices were performedwith the cpptraj module of Ambertools 1829 and with the Gromacs 201639 suite on the stripped trajectories without water and counterions,considering only the equilibrated part of the trajectories .VMDcpptraj moduleof Ambertools 18.29 A distance cutoff of3.0 Å and an angle cutoff of 135° were used as geometricconsiderations to account for a formed hydrogen bond. Structural populationswere settled with a cluster analysis of the trajectories using the cpptraj module of Ambertools 18.29 Here, a hierarchical agglomerative approach, a distance cutoff of∼2 Å, and a distance metric of the mass-weighted rmsdof atoms were employed.40H-bonds were determined using the CCij), quantify correlated and anti-correlatedmotions between pair residues along the MD trajectory. CCij values can range from −1, indicating completely anti-correlatedmotion between two residues, to +1, indicating correlated motion;meanwhile, 0 indicates no correlation.Thecross-correlation matrices, based on Pearson’s correlationcoefficients were calculated from the covariance matrices with the aid of the cpptraj module of Ambertools 18.29 In order to make the relationships between individual proteins anddomains of the immune complex immediately clear, the correlationsfor each protein/domain pair were assessed by summing the correlationscores (CSs) between each protein/domain and all others. Next, a correlationdensity for each area was obtained by summing the CSs of the protein/domainpair, which was then divided by the product of the number of residuesbelonging to that pair of proteins/domains. This approach eventuallyled to a simplified variant of the CCij matrices.45First, the covariancematrices were built from the atom position vectors. To capture onlythe internal dynamics of the complex, a RMS-fit to a reference structure(an averaged structure from the MD run) was performed, removing therotational and translational motions as previously described.46 and Amber18 code29 with pairwise and per-residue decomposition. The value of the igbflag was set to 5, and a salt concentration of 0.1 M was used. MM-GBSAcalculations were performed for 100 equally distant frames from eachMD trajectory in the production simulation time interval between 600and 900 ns. The conformational entropic contribution of free energywas not included in the calculations because it was previously suggestedthat this term does not improve the quality of the results when usingMM-GBSA.46 The interaction energies betweenHLA-E/β2m/NKG2A/CD94 and the corresponding nonameric peptidewere also calculated using the gmx energy module of the Gromacs201639 software package.The binding free energies between the complexes HLA-E/β2m/NKG2A/CD94and the corresponding nonameric peptide and between NKG2A/CD94 (receptor)and HLA-E/β2m/peptide (ligand) were calculated using the molecularmechanics/generalized born surface area (MM-GBSA) methodAll modelssimulated in this study were firmly based on the availablecrystal structure of the extracellular domains of the human CD94/NKG2Ain complex with HLA-E (PDB entry: 3CDG). Our models include all resolved componentsconsisting of NKG2A, CD94, HLA-E, β2m, and nonameric peptidenested between the α1 and α2 domains of the HLA-E 1.+GCOM), (ii) complex of allelic variant *01:03 of HLA-E, (iii) complex with peptide derived from HLA-B7 (COM+B7), (iv) complexwith Hsp60 peptide (COM–Hsp60sp), (v) complex with B7 R5V signalpeptide (–COMB7_R5V), (vi) complex with B27 peptide (∼COMB27), (vii) complex with Cw7 peptide(∼COMCw7),and (viii) complex without peptide (COMapo). Models +GCOM, allele+GCOM, and +COMB7 correspond to theNK cell protection scenario; in models –COMHsp60sp and –COMB7_R5V, protection is absent, andin models ∼COMB27 and ∼COMCw7, experiments revealed inconclusive results. The model of allelicvariant  serves as a replica of a basic model (+GCOM), whereas the model without peptide (COMapo) represents a negative control (Movie S1).In our simulations, we evaluated structural features and dynamicbehavior of eight models of immune complexes, which are presentedin 13 where peptide G, present in complexes +GCOM and allele+GCOM, possesses the highest affinity for the NKG2A/CD94receptor when incorporated with HLA-E and provides protection againstthe cytolytic activity of NK cells.14 The peptideB7, present in +COMB7 model, also provides protection against cytolysis by NK cells.12 Meanwhile, in cases of Hsp60sp (–COMHsp60sp) and the mutatedform of B7 (–COMB7_R5V), such protection is missing  and derivedtheir simplified versions for a clearer presentation . This observation couldbe linked to the ability of the nonameric peptides incorporated intothese complexes to promote a conformation responsible for mediatingsuccessful protection. Contrarily, in other investigated complexes, thecross-correlation matrices display a less-pronounced averaged (anti)correlationrelations.Analysis of the simplified cross-correlation matricesof all subjectedmodels pointed toward similar patterns. This observation suggeststhat protein–protein correlations are generally preserved regardlessof the productive or non-productive receptor–ligand recognition,leading to NK cell inhibition or lack thereof. Furthermore, the cross-correlationmap obtained for the NK cells 2 and S1.+GCOM, allele+GCOM, and +COMB7 appear to have a stronger correlation with HLA-E α2 comparedto peptides present in the investigated –COMHsp60sp and –COMB7_R5V models (S1). Thissuggests that close cooperation between the nonameric peptide andHLA-E α2 region on the target cell is necessary for successfulrecognition by the NK receptor NKG2A/CD94 and protection against NKkilling.The cross-correlation matrices also revealed thatnonameric peptidesG and B7 present in models V models 2 and S1.+GCOM, allele+GCOM, and +COMB7 models mediating NK cellprotectiongenerally have lower rmsd values . The average rmsd values obtained for complexes +GCOM, allele+GCOM, and +COMB7 were 1.8 ± 0.3, 1.9 ± 0.4, and 1.8± 0.3 Å, respectively. On the other hand, in models –COMHsp60sp and –COMB7_R5V, theaverage rmsd values 2.8 ± 0.3 and 2.2 ± 0.3 Å, respectively,were calculated . It is importantto mention that the observed higher rmsd values could in part be alsorelated with the changes introduced into the initial peptide sequence.According to the rmsd trajectory analysis, the peptidesin S4). We furtherquantitatively confirmed this observation by measuring the distancesbetween the Cα atom of the P5 nonameric peptide and the centerof the alpha helix between residues 151 and 162 of the HLA-E α2domain in the most representative clusters. The distances for themodels containing the peptides G, B7, B27, and Cw7 were around 9 Å,whereas in models with the peptides Hsp60sp and B7 R5V, they were12 Å and 11 Å, respectively. This observation is most likelyassociated with the absence of the Glu152HLA-E–ArgP5 salt bridge in the models –COMHsp60sp and –COMB7_R5V.After visual inspection of the most representative clusters extractedfrom the MD simulation trajectories for each simulated model, we noticedevident positional differences of the amino acid at the P5 positionamong the simulated nonameric peptides G, B7, B27, Cw7, Hsp60sp, andB7 R5V relative to the HLA-E α2 domain 3a, S4. WS5 and S6). Furthermore,we also observed at least comparable flexibility of the nonamericpeptide, although the HLA-E α2 domain may even be more flexiblethan the bound peptides . The most flexible part of the peptide is mostly located at itsC-terminal side, while the N-terminus is more rigid. This later observationmight be connected with the higher flexibility of the HLA-E α2domain that is in close contact with the C-terminal side of the peptide.Visualization and RMSF analysis of local flexibilitywithin oureight simulated immune complexes revealed that the α1 domainof the HLA-E heavy chain is more rigid in the regions closer to thenonameric peptide compared to its α2 domain, which is somewhatmore flexible, especially at the C-terminal side of the peptide, betweenthe residues 144 and 151 , while in the models with Hsp60sp or B7 R5V  withabsent NK cell protection, this interaction was absent, which is inaccordance with the literature data.28After a close-up inspection, we noticed notabledifferences in the intermolecular interactions between the nonamericpeptide, HLA-E, CD94, and NKG2A proteins. To begin with, the Glu152CD94 and the backbone oxygen of the ThrP6 (AlaP6 for ∼COMCw7) in all models except –COMHsp60sp, where instead, a quite analogous H-bond betweenGln112CD94 and the backbone nitrogen of the ValP8 is sparsely present. The H-bond interaction between Lys146HLA-E and the backbone oxygen of the highly conserved LeuP9 residue located at the C-terminus of the nonameric peptide was presentin the most representative clusters extracted from the MD simulationtrajectory of all peptide-enclosing models except –COMHsp60sp and ∼COMB27, where the interaction was missing.Furthermore, the Lys135NKG2A–Asp106CD94 salt bridge and Lys135NKG2A–Ser109CD94 H-bond were present in models +GCOM, alleleCOM+G, and +COMB7, enabling NK cellprotection, and were not noticeable in the most representative clustersof all other models studied by MD . It should also be noted that no particular hydrophobicinteractions important for peptide binding were observed during theanalysis of MD trajectories.Additionally, we observed an H-bond between Gln112HLA-E–ArgP5 salt bridgeand Gln112CD94–ThrP6 (AlaP6 for ∼Cw7COM), Lys135NKG2A–Asp106CD94, and Lys135NKG2A–Ser109CD94 H-bond interactions, as presented in Local interaction analysis of our simulations revealed that theGlu152Gb) were calculated between thenonameric peptide and the HLA-E/β2m/NKG2A/CD94 complex and betweenthe NKG2A/CD94 (receptor) and HLA-E/β2m/peptide (ligand) entitiesusing the MM-GBSA method.46 With the pairwisedecomposition of ΔGb, we identify at the atomic levelthe energetically most important interactions, and with the per-residuedecomposition, we calculated the energy contribution of each singleresidue by summing its interactions over all residues in the systems.Residues with the highest per-residue contribution can also be referredto as hotspots.48Binding free energies. The same observation was made whenwe performed the interaction energy calculations using the gmx energymodule of Gromacs201639 (Table S4). Here, the value of the ∼COMCw7 interaction energy is locatedbetween the values determined for the +COMB7 and –COMHsp60sp models; meanwhile, the inconclusivemodel ∼COMB27 is positioned between the –COMHsp60sp and –COMB7_R5V models in terms of interaction energy.The latter also correlates with our findings from the interactionanalysis of the ∼Cw7COM modelgeometric properties, showing that the ∼COMCw7 system exhibits more similar featuresto the models with reported successful ligand–receptor recognitionand NK cell protection, whereas model ∼COMB27 traits are more similar to themodels –COMHsp60sp and –COMB7_R5V.The nonameric peptidecomplex energies correlate well with the reported NK cell protectionability. The studied models b between the peptide and the remaining HLA-E/β2m/NKG2A/CD94immune complex (S2 and S5). This is in line withthe interactions of the immune complex HLA-E/β2m/peptide G/CD94/NKG2Areported in the literature.24 Interestingly,the H-bond interactions, namely, Glu152HLA-E–P5,Lys146HLA-E–LeuP9, and Gln112CD94–P6, are among those that have the largest contributionsto the ΔGb of the nonameric peptide,further reinforcing the observed correlations between the geometricand energetic traits of the studied system.Obtained results of the pairwiseenergy decomposition revealedthat P1, P5, P6, P8, and P9 residues of the nonameric peptide interactionsseem to contribute the most to the binding free energy ΔGb between HLA-E/β2m/NKG2A/CD94and the corresponding nonameric peptide  among all nine peptide residues. This is consistent with previousreports that P2 and P9 are considered the dominant peptide anchorpositions28 and that P8 and P6  residues form the majority of contacts of the peptide with theNKG2A/CD94 receptor.28On the other hand,the per-residue free energy decomposition pinpointsthe P2, P8, and P9 residues of the nonameric peptide to generallycontribute most to the ΔGGb, in themodels +GCOM, alleleCOM+G, and +COMB7, exhibiting successful protection againstthe NK cells, which are only partially present in –COMHsp60sp and –COMB7_R5V models with absent recognition.These interactive pairs are Asp69HLA-E–Arg171CD94, Arg75HLA-E–Asp163CD94, Asp162HLA-E–Lys199NKG2A, Gln72HLA-E–Glu164CD94, Arg68HLA-E–Asp168CD94, Asp162HLA-E–Arg215NKG2A, and ThrP6–Gln112CD94 (S28 and Tables S6 and S7).Next, pairwisefree energy decomposition between the receptor–ligandentities identifies seven consistent interactions with the greatestcontribution to the Δ+GCOM, alleleCOM+G, and +COMB7 models.These residues were Asp162HLA-E, Asp69HLA-E, Gln72HLA-E, Arg75HLA-E, Phe114CD94, Asp163CD94, Leu162CD94, and Pro171NKG2A. All eight residues are also present in the model –COMHsp60sp, whiletwo of them are missing in the model –COMB7_R5V. Energy decomposition also showedthat the residues at P6 and P8 positions of the peptide are amongthe top 10 ligand residues with the greatest contribution to the ΔGb in all models except +COMB7 and –COMHsp60sp (S28 and Table S8).Lastly, the per-residue free energydecomposition between the receptor–ligandentities unraveled consistent occurrence of eight residues contributingmost to their productive binding in Gb obtainedby the per-residue free energy decomposition analysis of all models,they could be considered as potential hotspots due to their largecontribution to the binding free energy.48 It was gratifying to observe that the ligand–receptor recognitionof binding affinity was also previously attributed to all but oneof the proposed hotspots. Indeed, mutations of Asp162HLA-E, Arg75HLA-E,27 Phe114CD94, and Leu162CD9426 to Ala were found to abolish successful molecular recognition betweenthe HLA-E/2m/peptide and NKG2A/CD94 binding partners. Meanwhile, mutationsof Gln72HLA-E, Asp69HLA-E,27 and Asp163CD9426 to alanineresulted in impaired binding.Based on the consistency of contributions of the aforementionedeight residues to the ΔThe free energy calculations reproducedseveral experimentallydiscovered hotspots and showed that the CD94 protein forms the majorityof the energetically important interactions with the HLA-E/β2m/peptideligand, primarily with the HLA-E α1, thus being the core ofmolecular recognition and successful complex formation 5.Inour study, we generated eight complexes of the HLA-E/β2m/NKG2A/CD94system with nonameric peptides in an attempt to elucidate the mechanisticdetails guiding the receptor (NKG2A/CD94) molecular recognition ofthe ligand (HLA-E/β2m/peptide) at the atomistic level.Multi-microsecond-long all-atom MD simulations based on the availableexperimental (NKG2A/CD94)–(HLA-E/β2m/peptide) complexcrystal structures showed that no major conformational changes occurduring the molecular recognition event taking place between the receptorand ligand. Furthermore, no specific interactions between the NKG2Aprotein and nonameric peptide residues could be detected. The analysisof the MD-generated conformations of all simulated systems furtherpinpointed that the CD94 receptor protein forms most of the energeticallyimportant contacts with the ligand, which underlines its core anchoringrole in the receptor–ligand recognition. Given the observedHLA E α2-flexibility in the region near the C-terminus of thepeptide, it is possible that the interaction between the nonamericpeptide and NKG2A protein occurs via the α2-domain of HLA-E6.P5,24 which forms an H-bond with Glu152HLA-E, and theimportance of Gln112CD94,26 which formsan H-bond with the peptide’s P6 residue  Glu152HLA-E–ArgP5 H-bond formation drives the placement ofthe HLA-E α2 domain and thus enables the interaction betweenthe peptide and NKG2A protein. It seems likely that (iii) the LeuP9–Lys146HLA-E interaction also influencesthe HLA-E α2 positioning. Furthermore, (iv) HLA-E α2 affectsthe position of the Arg137NKG2A, which is located in itsvicinity , which subsequentlycontacts Lys135NKG2A, ultimately allowing (v) the formationof key interactions between the NKG2A and CD94 proteins (Asp106CD94–Lys135NKG2A and Ser109CD94–Lys135NKG2A). Indeed, Arg137NKG2A frequentlyforms interactions with the α2-residues Asn148HLA-E and Asp149HLA-E in the +GCOM, alleleCOM+G, and +COMB7 models. Energetic analysis further corroborates this proposed networkand shows the importance of Gln112CD94–P6, Glu152HLA-E–ArgP5, and LeuP9–Lys146HLA-E interactions.Although the simulations do notprovide the time series of theevents, we hypothesized an intricate key network for the Asp106to occur 6 compris∼COMB27 and ∼COMCw7, containing peptides,for which inconclusive information could be found in the literatureregarding their ability to successfully facilitate receptor–ligandrecognition and subsequent NK cell protection. Our simulations showedthat the ∼COMB27 model containing peptide B27 behaved similarly to the –COMHsp60sp and –COMB7_R5V models withoutNK cell protection, while the COM–Cw7 model containing peptide Cw7 bearsmore resemblance to +GCOM, alleleCOM+G, and +COMB7 models providing the NKcell protection. The latter is consistent with previous reports thatan inefficient receptor–ligand interaction may eventually reacha threshold sufficient to counteract the NK cell activation.47Our computational studiesalso explored two models, COM+G model, where alleleCOM+G can be consideredas its replica, a single replica per system was run. Nevertheless,in eight long MD simulations performed here, three models  acted as positive controls and other two of them (–COMHsp60sp and –COMB7_R5V) served as negativecontrols. The obtained good correlation of the observed behavior ofall these systems with the experimental data suggests that such asetup of molecular systems can at least in part serve as an alternativeto the replica approach.It should be mentioned that with the exceptionof the In summary, our multi-microsecond-longmolecular dynamics (MD)simulations comprehensively contribute to the understanding of interactionsand their complex interrelations that are critical for a successfulligand (HLA-E/β2m/peptide)–receptor (NKG2A/CD94) recognitionat the atomistic level in the fundamental immune process event ofthe natural killer (NK) cell action. The knowledge obtained here couldform the basis for the targeted design of new biochemical and structuralstudies that may pave the way toward unraveling the complex mechanismsthat are part of the innate immune system. Moreover, understandingthis fundamental mechanism of NK cell protection may further enablerational structure-based design of interventions, for example, inthe form of different ligands and small molecules that would affectHLA-E/β2m/peptide and CD94/NKG2A interactions, which are overexpressedunder some pathological conditions and in senescent cell accumulation.


N,N-dimethyltryptamines, mescaline, psilocybin/psilocin, and 2,5-dimethoxy-4-methamphetamine (DOM), which can evoke intense visual and emotional experiences. We are witnessing a renaissance of research interest in hallucinogens, driven by increasing awareness of their psychotherapeutic potential. As such, we now present a narrative review of the literature on hallucinogen binding in vitro and ex vivo, and the various molecular imaging studies with positron emission tomography (PET) or single photon emission computer tomography (SPECT). In general, molecular imaging can depict the uptake and binding distribution of labelled hallucinogenic compounds or their congeners in the brain, as was shown in an early PET study with N1-([11C]-methyl)-2-bromo-LSD ([11C]-MBL); displacement with the non-radioactive competitor ketanserin confirmed that the majority of [11C]-MBL specific binding was to serotonin 5-HT2A receptors. However, interactions at serotonin 5HT1A and other classes of receptors and pleotropic effects on second messenger pathways may contribute to the particular experiential phenomenologies of LSD and other hallucinogenic compounds. Other salient aspects of hallucinogen action include permeability to the blood–brain barrier, the rates of metabolism and elimination, and the formation of active metabolites. Despite the maturation of radiochemistry and molecular imaging in recent years, there has been only a handful of PET or SPECT studies of radiolabeled hallucinogens, most recently using the 5-HT2A/2C agonist N-(2[11CH3O]-methoxybenzyl)-2,5-dimethoxy- 4-bromophenethylamine ([11C]Cimbi-36). In addition to PET studies of target engagement at neuroreceptors and transporters, there is a small number of studies on the effects of hallucinogenic compounds on cerebral perfusion ([15O]-water) or metabolism ([18F]-fluorodeoxyglucose/FDG). There remains considerable scope for basic imaging research on the sites of interaction of hallucinogens and their cerebrometabolic effects; we expect that hybrid imaging with PET in conjunction with functional magnetic resonance imaging (fMRI) should provide especially useful for the next phase of this research.Hallucinogens are a loosely defined group of compounds including LSD,  Introduction2.1.The Nature of Agonist-Receptor Interactions2.2.Affinities of LSD at Neuroreceptors in Vitro2.3.Affinities of Hallucinogenic Phenylethylamines in Vitro2.4.Affinities of Hallucinogenic Tryptamines in Vitro2.5.The Strange Case of IbogaineBinding Sites of Hallucinogens in Vitro3.1.LSD Derivatives3.2.Phenyethylamine DerivativesEx vivo/In vitro Binding Studies with Hallucinogens4.1.LSD Derivatives4.2.Phenylethylamine Derivatives4.3.Tryptamine Derivatives4.4.Competition from Hallucinogens at Dopamine Receptors in Vivo4.5.Competition from Hallucinogens at Serotonin Receptors in VivoMolecular Imaging Studies in Vivo with Hallucinogens4.1.LSD5.2.Phenylethylamine Derivatives5.3.Tryptamine DerivativesMetabolism of Hallucinogenic Compounds and TracersAyahuasca and Pharmahuasca7.1.Cerebral Glucose Metabolic Rate7.2.Cerebral Blood FlowEffects of Hallucinogens on Energy Metabolism and PerfusionGeneral ConclusionsContents:References1) towards more clinical applications, especially involving psilocybin (2) -guanosine-5-O-(3-thio)-triphosphate in membranes reveals agonist interactions. Indeed, that assay may serve as a forensic tool for operationally predicting the hallucinogenic properties of members of a series of tryptamine derivatives , and bufotenin (10)) inhibit serotonin neuron activity with a potency following the rank order of their potency as hallucinogens ketanserin, 0.43 nM at 5HT1A sites, and 6.6 nM at 5HT1B sites -ketanserin (mainly 5HT2A) binding sites in vitro. That series of compounds had 10-fold lower affinity at 5HT2C sites and 1000-fold lower affinity at 5HT1A and 5HT1B sites, indicating much greater selectivity than LSD (1). A newer phenethylamine derivative 25CN-NBOH (13) had even higher 5HT2A selectivity over 5HT2C and 5HT2B receptors than other reported compounds -ketanserin), alpha2 (1.4 µM) and TAAR1 (3.3 µM) receptors  dihydrogen phosphate) is a naturally occurring hallucinogen from mushrooms of Psylocybe and other genera, which was first isolated in 1959 by the Swiss chemist Albert Hoffman. Initially distributed in the 1960s for use in psychotherapy, psilocybin (2) is again finding this application after a hiatus of four decades. Strictly speaking, psilocybin (2) is a prodrug, which undergoes rapidly hydrolysis in the gut and liver to yield the active and brain-penetrating metabolite psilocin (8). In vitro binding studies indicate that psilocin (8) is an agonist with moderate affinity (Ki) of 49 nM at 5HT2A sites, 94 nM at 5HT2C sites and 123 nM at 5HT1A sites, but >µM affinity at dopamine and adrenergic receptors [2) at 5HT2B sites [123I]-DOI indicated ten-fold higher affinity at 5HT2A sites [2/3 receptors [Psilocybin  had a Ki of 0.5 µM [2A/C sites labelled with [3H]-ketanserin, indicating rather low affinity in vitro. Other displacement studies against [3H]-ketanserin likewise showed µM Ki at 5HT2A/C sites and insensitivity to addition of GTP, suggesting an antagonist interaction -ketanserin displacement studies in vitro seems at odds with its much greater potency in functional assays, which showed a 38 nM EC50 for calcium mobilization in vitro [15) against [123I]-DOI (16)-labelled 5HT2A agonist sites as compared to [3H]-ketanserin-labelled 5HT2A antagonist sites [1A sites labeled with the agonist [3H]8-hydroxy-DPAT and a clear shift to the right in the displacement curve in the presence of GTP, indicating agonism. Others reported that DMT (9) had Kis of 0.25 µM at 5HT2A sites, 0.4 µM at 5HT2C sites, and only 0.1 µM at 5HT1A sites -R-(-)DOI (16) MK801 from caudate membranes with an IC50 of 5 µM -iodo-lysergic acid diethylamide  was developed as a presumably non-hallucinogenic ligand for the molecular imaging of serotonin receptors, whereby -Nmethylation of [125I]-LSD (25) was intended to impart greater selectivity and sensitivity for 5HT2 receptors [125I]-MIL (24) had an apparent KD of 0.14 nM at 5HT2A receptors. However, [125I]-MIL (24) also showed a KD of 0.4 nM for 5HT2C receptors in vitro. The specific binding of [125I]-MIL (24) in mouse brain peaked at 45 min post injection, when the binding ratio relative to cerebellum was 4 in frontal cortex and 2 in striatum; lesser cortical binding and more rapid washout from frontal cortex was seen in corresponding studies with [125I]-LSD (25). In other ex vivo studies, ketanserin (7) evoked a dose-dependent displacement of [125I]-MIL (24) in mouse striatum and cortex, suggesting an IC50 of about 30 µg/kg ketanserin (7). Rat autoradiographic studies with [125I]-MIL (24) revealed that repeated doses with the non-hallucinogenic psychostimulant MDMA evoked a substantial down-regulation of 5HT2-like receptors iodo-lysergic acid diethylamide  was developed as a ligand for molecular imaging of serotonin receptors. It had very high affinity for 5HT2A receptors in rat cerebral cortex, with a dissociation constant (KD) of 0.2 nM -EIL (23) is likely to be an antagonist. Ex vivo studies indicated an extraordinary persistence of its specific binding in mouse brain relative to cerebellum, whereby the binding ratio was 9 at 16 h post injection. Ketanserin (7) displaced the cerebral binding, but dopamine D2 or adrenergic antagonists were without effect, consistent with a main interaction of [125I]-EIL (23) at 5HT2A sites. However, the authors predicted that [125I]-EIL (23) might also bind to 5HT2C receptors of the choroid plexus.f 0.2 nM . FollowiD-enantiomer of LSD (1) had 1000-fold higher affinity for serotonin receptors than the L-enantiomer [D-[125I]-LSD (25) (200 pM) showed abundant binding in the extended striatum and the cerebral cortex, and nearly complete displacement of the cortical binding be co-incubation with R-(-)-DOB ((-)12, 500 pM), but only 50% displacement in striatum, consistent with the ambivalence of LSD (1) for dopamine and serotonin receptors [R-[125I]-DOI (16) showed an abundance of LSD-displaceable binding in the deep layers of the cerebral cortex and in the claustrum. However, there was only sparse binding in striatum, consistent with the ligand’s considerable specificity for serotonin receptors [D-[125I]-LSD (25) binding in rat brain indicated a single population of binding sites (KD 170 pM) in cerebral cortex, where the Bmax was about 4 pmol/g wet weight -LSD (25) revealed a significant ketanserin (7) displaceable component in rat striatum sections, ranging from 30% in rostral parts to 74% in the caudal regions [The active antiomer . Autoradeceptors . Other aeceptors . More det weight . The bin14C]-psilocin at a dose of 10 mg/kg [Autoradiographic analysis of the rat brain revealed the time-dependent distribution of radioactivity at various times after intravenous injection of [10 mg/kg . There w14C]-mescaline in cat (25 mg/kg) showed a plasma half-life of two hours after intravenous injection, very rapid uptake into brain giving a brain/plasma ratio of 3 at one hour after injection, and persistent retention in brain, with only slight washout at six hours. Autoradiographic studies with [3H]-mescaline in the brain of marmoset monkeys  showed preferential accumulation in the hippocampus, amygdala, lateral geniculate and anterior cingulate cortex, persisting even 18 h after administration  [3H]-2,3,4-trimethoxy-β-phenylethylamine (4 mg) showed a more homogeneous pattern of binding -DOI (16) can be used to study the functional desensitization of serotonin 5HT2-likereceptors after repeated doses of DOI or other agonists. Thus, chronic treatment with (+/−)-DOI (1 mg/kg daily for a week) significantly reduced the binding of [3H]ketanserin, [125I]-LSD (25), and R-[125I]-DOI (16) as measured at single ligand concentrations in rat cortical homogenates [max values for [3H]-ketanserin and R-[125I]-DOI (16) without altering the KD values. Repeated treatment of rats with (+/−)-DOI  resulted in an attenuation of the DOI-induced release of the hypothalamic hormone oxytocin, which is indicative of receptor desensitization [R-[125I]-DOI (16) in the paraventricular nucleus, despite increased 5HT2A immunoreactivity to western blot analysis, suggesting an internalization process or altered coupling with intracellular G-proteins.2,5-Dimethoxy-4-iodoamphetamine (DOI) is a prototypical phenylethylamine 5-HTivatives . Autoradogenates . Saturattization . This fuN-(2-methoxybenzyl)ethanamine  and 2-[[2- ethylamino] methyl] phenol  -2,5-dimethoxy-4-cyanophenylethylamine) had >52-fold Ki2C/Ki2A selectivity ratio and a 37-fold Ki2B/Ki2A ratio, and likewise showed substantial 5HT2A selectivity in functional assays of IP turnover. Various isomers of 25H-NBOMe in which the two methoxy-groups are in different positions  were compared with respect to their efficacy in activating signaling pathways, in conjunction with molecular docking studies at the 5HT2A receptor binding pocket -methyl)-2-bromo-LSD  as a non-hallucinogenic ligand for serotonin 5-HT2A receptors -MBL (33) in baboon (0.1 µg/kg body weight) indicated relatively high binding throughout cerebral cortex, with a peak binding ratio of 1.4 at 40 min that tended to decline in the following 45 min; this binding was displaced by ketanserin (7) pretreatment. Similar results were seen in an awake human, with a maximal binding ratio of about 2:1 prevailing in cerebral cortex during 20–60 min post-injection.One of the very first receptor PET molecular imaging studies employed N1-(-iodoamphetamine -propane-2-amin]) of high specific activity showed high uptake in thalamus, cortex, and striatum, and lesser accumulation in the midbrain and cerebellum in baboon methoxybenzyl) ethanamine  was investigated as a potential PET tracer in rats ex vivo and in pigs in vivo -Cimbi-5 (34) binding. Others conducted a comparative PET study of [11C]-Cimbi-5 (34) and the antagonist [11C]-MDL100907 (35) in living non-human primates, finding that the antagonist ligand had two or three-fold higher BPND than [11C]-Cimbi-5 (34) in most brain regions [11C]-Cimbi-5 (34) binding in all brain regions, indicating somewhat incomplete selectivity.The phenylethylamine 5-HT in vivo . It had  regions ; pretrea3H]-MDL100907 ranging from 0.2 nM  to 47 nM ; their receptor agonism was assessed from PI hydrolysis in GF62 cells overexpressing the 5-HT2A receptor -Cimbi-36 (38). In brain of living rat, [11C]-Cimbi-36 shows preferential uptake in frontal cortical regions and relatively little uptake in the striatum, diencephalon and cerebellum  in healthy volunteers showed BPNDs, which ranged from 0.25 in striatum to 1.7 in broad cortical regions, and showing a high degree of covariance between repeat measures in the same subjects [11C]-Cimbi-36 (38) and [18F]-altanserin (39) BPND estimates in most brain regions, but significantly greater binding of [11C]-Cimbi-36 (38) in hippocampus and choroid plexus. The authors interpreted this to indicate the composite of 5HT2A and 5HT2C receptors in that structure; whereas [11C]-Cimbi-36 is nearly equipotent at 5HT2A and 5HT2C receptors, altanserin has nearly 100-fold selectivity for 5HT2A binding sites [11C]-Cimbi-36 (38) and [11C]-MDL100907 (35) binding in most brain areas, and substantial displaceability by ketanserin (7) in all regions but cerebellum [11C]-Cimbi-36 (38) binding in hippocampus and choroid plexus by treatment with the 5HT2C antagonist SB 242084 (40), again consistent with a mixed signal in those regions. The metabolism of [11C]-Cimbi-36 (38) in humans is characterized by loss of the [11C]-methoxy label  or demethylation of the other methoxygroup, yielding a relatively hydrophobic radiometabolite iodo-DMT [131I]-iodo-tryptamine showed no comparable retention in brain. They authors did not undertake any displacement studies to confirm specific binding, nor did they pretreat the rabbits with a monoamine oxidase (MAO) inhibitor, arguing that the intravenous route of administration avoided first pass metabolism in the liver.DMT , first in a placebo baseline, and again after administering a psychoactive dose of psilocybin (2) (0.25 mg/kg p.o.) [11C]-raclopride (41) binding in the striatum, which is similar to the displacements evoked by amphetamine or other strong releasers of dopamine. Furthermore, the decline in the ventral striatum correlated with individual ratings for euphoria and depersonalization. In formal terms, the reduced [11C]-raclopride (41) binding could be indicative of competition from psilocin (8) at the D2/3 receptors, or enhanced release of endogenous dopamine. Rat microdialysis studies showed that psilocin (8) (10 mg/kg) evoked a transient 40% increase in interstitial dopamine levels -raclopride (41) PET study of anesthetized pigs [11C]-raclopride (41) BPND, but there was a 19% decline at four hours after the challenge, which seemed consistent with a high affinity component of the displacement of [3H]-raclopride binding by LSD (1) in pig brain sections. Furthermore, their rat microdialysis studies did not indicate any effect of LSD (1) on dopamine release per se, suggesting that the [11C]-raclopride (41) binding reduction must be due to direct competition from LSD. However, the time delay to a main effect suggests that some other pharmacological mechanism or methodological factor may have been at play. The pharmacokinetics of oral LSD (1) (100 and 200 µg) in human subjects indicated a mean plasma half-life of 2–6 h and a persistently close relationship between the individual LSD (1) concentration in plasma and the magnitude of subjective response within subjects, with moderate counterclockwise hysteresis during the 12 h after dosing [1) at [11C]-raclopride binding sites in pig brain in the first post LSD (1) scan, rather than with a delay of several hours.The effects of intravenous challenge with acute LSD  (11C]-raclopride PET study in rats with MAO inhibition [18F]-fluoroethylharmol (49) as an alternative PET tracer for MAO-A in brain -altanserin (39) are indicative of increased release of endogenous serotonin after challenge with dexfenfluramine [18F]-altanserin (39) binding was measured at baseline, and (two weeks later) again at 75 min after administration of psilocybin (2) (20 mg). The drug treatment provoked widespread 50% decreases in the uptake (DV’) of the tracer in the dorsolateral prefrontal cortex, orbitofrontal cortex, medial temporal cortex, and other cortical regions. Voxelwise regression analysis showed that the binding reductions in the anterior cingulate spreading to the dorsomedial prefrontal cortex correlated with higher scores in the Altered States of Consciousness Rating Scale (5D-ASC)  .8) at serotonin 5HT2A receptors in brain of human volunteers relative to plasma drug concentrations after administration of psilocybin (2) at doses in the range of 3 to 30 mg [2A receptor availability was measured using the agonist ligand [11C]-Cimbi-36 (38). Results indicated a dose-dependent brain receptor occupancy as high as 76%, which occurred when the plasma psilocin (8) concentration was about 100 nM. Furthermore, the intensity of the psychedelic experience correlated with receptor occupancy, and with the plasma psilocin (8) concentration, assuming a single site model. These calculations assumed steady states in the drug concentration and intensity during the PET measurement, which the authors showed to be approximately true on the time scale of about two hours. Their data also showed a rough relationship between the duration of the hallucinogenic experience (60–360 min) and the interval in which the plasma psilocin (8) concentration exceeding approximately 10 nM. Interestingly, the occupancy by psilocin (8) at [11C]-Cimbi-36 (38) agonist binding sites (circa 60%) was quite similar to the displacement of [18F]-altanserin (39) by a similar dose of psilocybin (2) reported by Hasler et al. [2A sites labelled with [11C]-Cimbi-36 (38) by endogenous serotonin in anesthetized pig increased from 17% at baseline to 44% after fenfluramine treatment, which transiently increased the interstitial serotonin content in cortex ten-fold according to microdialysis [11C]-Cimbi-36 (38) binding to fenfluramine challenge seemed to exceed that reported earlier for [18F]-altanersin (39) in healthy human volunteers, albeit at a lower relatively lower dose of fenfluramine in human -Cimbi-36 (38) -Cimbi-36 (38) binding [2) at a presumably hallucinogenic dose  reduced by up to 50% the cortical abundance of 5HT2A binding sites labelled in vitro with [3H]-MDL100907 or [3H]-Cimbi-36 [3H]-UCB-J showed a significant 5–10% increase in synaptic density in hippocampus, occurring in association with the down-regulation of serotonin receptors. This phenomenon suggests that the benefits of psilocybin (2) in a therapeutic setting may be due to an enhancement of synaptic density and or plasticity; it might be more correct to suggest that the treatment reinstates the synaptic overshoot that occurs early in post-natal brain development, which may set the stage for plastic changes in synaptic plasticity.At baseline in healthy individuals, at least, there was no significant association between the trait for openness to experience with 5HT-36 (38) . However binding . Recent Cimbi-36 . Other e2A receptors -Cimbi-36 are metabolized in pigs and humans by demethylation, usually in the 5-position, and subsequently glucuronation [11C]-Cimbi-36 showed that labelling on the 5-position yielded a lower specific binding in pig brain, apparently due to the formation of [11C]-formaldehyde or other brain-penetrating labelled metabolites MeO-DMT (47), instead of labelled DMT (9). There was rapid uptake of 5-[11C]MeO-DMT (47) in brain of anesthetized pigs, but with little evidence of heterogenous distribution matching that expected for a 5HT2A ligand; indeed, efforts to increase its uptake in pig brain by pretreatment with the MAO inhibitor pargyline (7) were unsuccessful. We were unable to account for the negative results, but suppose that the Bmax/KD ratio may simply have been too low to impart a measurable BPND. This pharmacological identity of the “hotspot” in pig ventral striatum remains unknown.We have attempted to investigate the basis of Ayahuasca polypharmacology in PET studies using the hallucinogen 5-[argyline  or displ14C]deoxyglucose autoradiographic method gives qualitative or quantitative estimates of the local rate of cerebral glucose consumption (CMRglc) based on the semi-irreversible metabolic trapping of the tracer in living cells. Administration to awake (but immobilized) rats of LSD (1)  evoked widespread 10–30% reductions in CMRglc, notably throughout neocortex, and in thalamus, lateral geniculate, the basal ganglia, and the raphé nuclei, but with little effect in cerebellum -deoxyglucose trapping in cerebellum and motor cortex, and decreases in hippocampus, whereas the 5HT2B agonist also evoked large increases in the basal ganglia -fluorodeoxyglucose (FDG) PET study, in which post-drug findings were compared with the volunteers’ own baseline PET recordings -psilocin (8) confirmed that hallucinogenic compounds rapidly enter the brain, being relatively unhindered at the blood–brain barrier. This is not always the case; the low octanol:water partition coefficient for bufotenine (10) and the predicted low permeability to the blood–brain barrier, has been invoked to explain its relatively low hallucinogenic potency, despite moderate activity in serotonin receptor functional assays -methyl)-2-bromo-LSD , there have been relatively few molecular imaging studies of radiolabeled hallucinogen analogues, either in humans or experimental animals. More commonly, hallucinogens serve as a pharmacological challenge to determine indirect effects on availability of dopamine D2 receptors labelled with [11C]-raclopride (41), or occupancy at serotonin receptors labelled with some other radiotracer. A very few PET studies have examined the effects of treatment with a hallucinogen on cerebral metabolism to FDG-PET or cerebral blood flow; the limited available data indicate hypermetabolism despite hypoperfusion, which implies that hallucinogens may alter flow-metabolism coupling in brain. Thus, there is an enormous scope to undertake PET and SPECT studies of hallucinogen binding at various receptor sites, and to examine the effects of acute and chronic exposure on cerebral metabolism and synaptic density markers.Despite early success with


Rett syndrome is a neurodevelopmental disorder caused by mutations of the methyl-CpG binding protein 2 gene. Abnormal physiological functions of glial cells contribute to pathogenesis of Rett syndrome. Semaphorin 4D (SEMA4D) regulates processes central to neuroinflammation and neurodegeneration including cytoskeletal structures required for process extension, communication, and migration of glial cells. Blocking SEMA4D-induced gliosis may preserve normal glial and neuronal function and rescue neurological dysfunction in Rett syndrome. We evaluated the pre-clinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody in the Rett syndrome Mecp2T158A transgenic mouse model and investigated the contribution of glial cells as a proposed mechanism of action in treated mice and in primary glial cultures isolated from Mecp2T158A/y mutant mice. SEMA4D is upregulated in neurons while glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1-positive cells are upregulated in Mecp2T158A/y mice. Anti-SEMA4D treatment ameliorates Rett syndrome-specific symptoms and improves behavioural functions in both pre-symptomatic and symptomatic cohorts of hemizygous Mecp2T158A/y male mice. Anti-SEMA4D also reduces astrocyte and microglia activation in vivo. In vitro experiments demonstrate an abnormal cytoskeletal structure in mutant astrocytes in the presence of SEMA4D, while anti-SEMA4D antibody treatment blocks SEMA4D–Plexin B1 signaling and mitigates these abnormalities. These results suggest that anti-SEMA4D immunotherapy may be an effective treatment option to alleviate symptoms and improve cognitive and motor function in Rett syndrome. MECP2). Rett syndrome is the second most common cause of severe intellectual disability after Down syndrome in females, affecting one out of every 10,000 live births [Rett syndrome is a rare neurodevelopmental disorder, caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 , also known as CD100, initially identified as an axonal guidance molecule during neuronal development, has also recently been found to regulate the assembly of the cytoskeleton required for the normal functions and migration of glial precursor cells, which give rise to astrocytes and oligodendrocytes, and activation of microglia ,14. SEMAT158AMecp2 Rett syndrome mouse model. Here, we demonstrate reversal of key features of the Rett syndrome phenotype following antibody blockade of SEMA4D as well as upregulation of SEMA4D expression during disease progression and the effects of SEMA4D on reactive transformation of mutant glial cells expressing the Plexin B1 receptor. This study expands mechanistic insight into Rett syndrome pathology and suggests the potential of anti-SEMA4D antibody therapy in this paediatric neurological disorder.Anti-SEMA4D monoclonal antibody therapy has been shown to inhibit activation of glial cells and ameliorate neurological disorders in several in vivo experimental models, including a transient middle cerebral artery occlusion mouse model of stroke , lysolecT158A/yMecp2 mice compared to WT mice (p = 0.048), but no difference between the treatment groups. All mice were weighed weekly, and their body weights increased over the 10-week trial with no significant differences between groups in the pre-symptomatic and symptomatic cohorts  and 8-week (symptomatic) anti-SEMA4D antibody-treated Rett syndrome cohorts showed significant improvements in the phenotypic clinical scores relative to the isotype control antibody groups.T158A/yMecp2 mice climbed to the maximum of 1.1 in week 3 and was reduced to 0.7 in week 5 where it plateaued for the duration of the study. The hind limb clasping score of the anti-SEMA4D antibody-treated T158A/yMecp2 mice was significantly lower than that of the T158A/yMecp2 mice receiving placebo between week 5 and 10 (p < 0.001). Similar to the 4-week cohort, the hindlimb clasping in the symptomatic mice that received isotype control antibody progressively worsened throughout the 10-week study, reaching a score of ~1.8 in week 10 . The wild-type mice showed no hindlimb clasping throughout the study, as expected.Hind limb clasping is a hallmark feature of many Rett syndrome mouse models and a behaviour that is reminiscent of the hand movements seen in Rett syndrome girls. Hind limb clasping in the pre-symptomatic mice that received isotype control antibody progressively worsened throughout the 10-week study, reaching a top mean score of ~1.8 in week 10 A. In con week 10 B. The hiT158A/yMecp2 mice that received isotype antibody control progressively increased to the maximum of 1.8 in week 8, dropping slightly to 1.6 in week 10 . As in the pre-symptomatic cohort, the symptomatic cohort tremor scores in the placebo group progressively increased over the 10-week period and reached a top score of 1.9 in week 10 (p < 0.05) to week 10 (p < 0.001). The wild-type mice showed no tremors throughout the 10-week trials.Tremors are another common feature of Rett syndrome mice models, resulting from a loss of Mecp2 in excitatory neurons. In the pre-symptomatic cohort, the tremor score of  week 10 C. Tremor week 10 D. SimilaT158A/yMecp2 mice receiving isotype antibody progressively decreased over the 10-week trial and was significantly lower than that of wild-type mice from week 3 onwards (p < 0.001). The anti-SEMA4D antibody-treated T158A/yMecp2 mice gradually increased the latency to fall from the rod during the trial, which was significantly higher than that of T158A/yMecp2 mice receiving isotype control antibody between week 3 (p < 0.01) and week 9 (p < 0.001). In the symptomatic cohort, the latency to fall in the placebo Rett syndrome group progressively declined during the trial and was significantly lower than that of the wild-type group from week 1 (p < 0.01) to week 9 (p < 0.001) (T158A/yMecp2 mice receiving anti-SEMA4D antibody continuously improved over time and was significantly higher than that of T158A/yMecp2 mice with isotype control antibody from week 3 to week 9 (p < 0.001). Surprisingly, the anti-SEMA4D antibody-treated mice appeared to stay significantly longer on the rotating rod compared with the wild-type mice in week 7 (p < 0.05).Latency to fall from the acceleratingly rotating rod in the rotarod test was used to assess the coordination function in the treated mice, such that longer latency indicates better coordination function. In the pre-symptomatic cohort, the latency to fall of the  onwards E (p < 0.< 0.001) F. The laT158A/yMecp2 mice spent less time in the open arms and made fewer open-arm entries over the 10-week pre-clinical trial. In contrast, the T158A/yMecp2 mice receiving placebo spent more time in the open arms and made more entries into the open arms over the 10-week course. The percentage of time in the open arms and percentage of open-arm entries in the placebo group were significantly higher than those in the other two groups from week 5 to 9 (p < 0.001). Similarly, the wild-type and anti-SEMA4D antibody-treated T158A/yMecp2 mice in the symptomatic cohort spent less time in the open arms and made fewer entries into the open arms over the 10-week trial  and higher than the anti-SEMA4D antibody-treated T158A/yMecp2 mice between week 5 and week 9 (p < 0.01). The T158A/yMecp2 mice with isotype control antibody treatment made significantly more open-arm entries compared with the wild-type mice at all time points assessed (p < 0.05) and more entries than the T158A/yMecp2 mice with anti-SEMA4D antibody treatment between week 5 and week 9 (p < 0.01).The elevated plus maze test was employed to evaluate the cognitive functional effects of anti-SEMA4D antibody treatment, based on the general aversion of mice to open spaces. Animals with impaired cognition are less able to recognise the aversive properties of the open arms, detected by spending longer time in the open arms and making more open-arm entries during the test. In the pre-symptomatic cohort, mice from all groups did not demonstrate any changes in cognitive function in the first 3 weeks of the trial G,I. Bothek trial H,J. The T158A/yMecp2 mice receiving isotype control antibody treatment showed a significantly lower mean velocity in the total area compared to the wild-type and anti-SEMA4D antibody-treated mice between week 5 and 9 (p < 0.05) (p < 0.05)  and the mice in the anti-SEMA4D treatment group showed a lower number of central area entries than wild-type mice in week 7 (p < 0.05) (T158A/yMecp2 mice receiving isotype control antibody demonstrated fewer entries into the boundary area compared with the wild-type mice in week 7 (p < 0.01) . In the pre-symptomatic cohort, the  < 0.05) A. The di < 0.05) C. Compar < 0.05) E. The Me < 0.01) G.T158A/yMecp2 mice treated with isotype control antibody showed a significantly lower mean velocity and total distance travelled compared to the wild-type mice throughout the 10-week trial (p < 0.05) (T158A/yMecp2 mice compared to the isotype control antibody in week 3 and 5 (p < 0.05) but not week 7 and 9. Similarly, in the central and boundary areas, the isotype antibody-treated T158A/yMecp2 mice demonstrated significantly fewer entries than wild-type mice throughout the 10-week trial period (p < 0.05), and the anti-SEMA4D antibody treatment significantly improved the number of entries into the central and boundary areas in the T158A/yMecp2 mice compared with the isotype control antibody in week 3 (p < 0.05) but not in week 5–9  B,D. The week 5–9 F,H. AddiIn the pre-symptomatic cohort, effects of anti-SEMA4D antibody were sustained throughout the 10-week treatment period, while in symptomatic mice, activity in the open field test appeared to be reduced after week 5, which coincided with a reduced dose from twice weekly to once weekly of anti-SEMA4D antibody in this cohort. To determine a potential dose-dependent effect, serum antibody levels were assessed at the endpoint. In previous pre-clinical studies, it was determined that target saturation requires at least 5 μg/mL of antibody and corresponds with the minimal effective dose in mice ,21. AssuT158A/yMecp2 mice treated with anti-SEMA4D antibody showed a respiratory pattern similar to that of the wild-type mice in all parameters measured, including inspiratory and expiratory time  and higher peak inspiratory and expiratory flow from week 3 to week 9 (p < 0.05), compared with the wild-type mice. The mice in the placebo group were also found to have higher tidal and expired volume than the wild-type mice in week 5 and week 9 (p < 0.05). Interestingly, the end-expiratory pause (EEP), an indicator of apnoea, of the isotype control antibody-treated T158A/yMecp2 mice progressively increased over the 10-week trial and was significantly higher than that of the wild-type and anti-SEMA4D-treated mice from week 3 to week 9 (p < 0.001).In the pre-symptomatic cohort, the ory time A,B, peakory time E,F, tidaory time I,J, and ory time M,N. In cT158A/yMecp2 mice treated with isotype control antibody demonstrated an irregular pattern of respiration throughout the trial, with reduced inspiratory and expiratory time (p < 0.001) (p < 0.001)  and decreased peak inspiratory and expiratory flow between week 3 and week 7 (p < 0.05), compared with the isotype control antibody-treated mice. The T158A/yMecp2 mice in the placebo group showed a higher tidal and expired volume than the wild-type mice in week 1 (p < 0.05), week 5 (p < 0.01), week 7 (p < 0.05), and week 9 (p < 0.001) (p < 0.05). There were no differences in the end-inspiratory pause of any groups throughout the course (T158A/yMecp2 mice was significantly lower than that of the placebo treated T158A/yMecp2 mice from week 3 (p < 0.05) to week 9 (p < 0.01)  C,D and i< 0.001) G,H compa< 0.001) K,L. The e course O. The en < 0.01) P. Data fT158A/yMecp2 mice, we observed a dramatic increase in SEMA4D colocalising with neuronal marker NeuN, suggestive of the neuronal origin of SEMA4D in the diseased brain (T158A/+Mecp2 mice (data not shown). The mean SEMA4D intensity in NeuN+ neuronal cells was significantly elevated in the cortex and striatum . In T158A/yMecp2 mice treated with anti-SEMA4D antibody, the mean SEMA4D intensity in NeuN+ neuronal cells was slightly reduced relative to T158A/yMecp2 mice treated with isotype control in the cortex and striatum in both pre-symptomatic and symptomatic cohorts (p < 0.05), as well as in the pons in the symptomatic cohort (p < 0.01). It appears that antibody binding promotes only limited internalisation of SEMA4D in neurons, as SEMA4D levels in treated mice, however, remained significantly elevated relative to wild-type mice [In the male ed brain A. In constriatum B,C, the striatum D,E, and striatum F,G of thype mice .T158A/yMecp2 mice appear to increase in number and exhibit an amoeboid shape indicative of activation, as shown in representative high-magnification images from the striatum . Antibody treatment significantly reduced levels of Iba1+ cells in the striatum (p < 0.01) and outer layer of the cortex (p < 0.05), and a trend of reduced Iba1+ cells following treatment was observed in the pons . Morphometric changes, as evidenced by increased soma size and reduced hypertrophy of astrocyte processes and significant reduction in GFAP, are indicative of astrocyte activation [T158A/yMecp2 mice in the cortex and striatum (p < 0.05), the pons (p < 0.01), and the cerebellum (p < 0.05) in both cohorts.Astrocytes play an important role in cross talk with neurons and microglia to propagate neuroinflammation that can exacerbate degenerative pathology in the setting of chronic stimulation. Astrocytes were stained for GFAP, a predominantly astrocytic marker which is upregulated in reactive astrocytes. The mean GFAP intensity was significantly increased in A/y mice A, predomA/y mice B,C, the A/y mice D,E, and A/y mice F,G compativation . Treatmep < 0.001 compared with the PBS groups), which was rescued by anti-SEMA4D antibody treatment   in GFAP-positive astrocytes. GFAP-positive astrocytes express PLXNB1 receptor for SEMA4D. Representative images of immunofluorescent staining of α-tubulin (red) with nuclear counterstain of Hoechst (blue) in GFAP-positive astrocytes (staining not shown) are presented in ntibody) B.Here, we have demonstrated the therapeutic efficacy of anti-SEMA4D antibody treatment in alleviating phenotypic symptoms and functional deficits in a transgenic mouse model of Rett syndrome. We show that anti-SEMA4D antibody therapy not only prevents, but reverses, many Rett syndrome-specific phenotypes, including coordination, cognition, locomotion, and respiratory deficits in both pre-symptomatic and symptomatic mice. The beneficial effects of anti-SEMA4D antibody therapy observed in the pre-clinical trial may be a result of antibody blockade of SEMA4D-mediated Plexin B1 signal transduction in glial cells, as previously suggested .T158A/yMecp2 mice receiving isotype control antibody started developing classic Rett syndrome-specific phenotypes at 5 weeks of age which persisted to the end of trial, in line with previous studies [Rett syndrome mice share several symptoms with Rett syndrome patients, such as stereotypic movements and body tremor ,25. Hind studies ,28,29. OT158A/yMecp2 treated and untreated mice cohorts. Rett syndrome predominantly affects girls where individuals have the potential for prolonged survival with approximately 60% surviving to early middle age while Rett syndrome is rare in males and they usually succumb to death before birth or in early infancy owing to only having one X chromosome [T158A/yMecp2 model due to its robust phenotype which includes a significantly shorter survival rate which is not observed in females with Rett syndrome. Rett syndrome patients experience deterioration of motor coordination skills that impact sensation, movement, and communication in early childhood. In this study, we demonstrate the prevention and reversal of motor coordination and cognitive deficits (as observed by the rotarod test and the elevated plus maze test) in anti-SEMA4D-treated T158A/yMecp2 mice, whereas T158A/yMecp2 mice treated with control antibody exhibited deficits as early as 6 weeks of age which progressively worsened throughout the trial. Surprisingly, the treated mice in the symptomatic cohort stayed longer on the rotarod than the WT mice in week 7, suggesting a potential improvement in motor learning ability. This may be further assessed by conducting the accelerating rotarod test in one session per day for 5 consecutive days, then comparing the performance on the fifth day to that on the first [No significant differences were observed in the survival of romosome . In thishe first .T158A/yMecp2 mice in both the pre-symptomatic and symptomatic cohorts were consistent with the locomotor impairments reported in previous studies of the T158AMecp2 mouse model [The locomotor deficits (open field test) observed in the isotype control antibody-treated se model ,28. The se model ,33,34,35se model . Anti-SE2) chemosensitivity of astrocytes [T158A/yMecp2 mice treated with isotype control antibody. Our results indicate that anti-SEMA4D treatment prevents and improves arrhythmic breathing in pre-symptomatic and symptomatic mice. Interestingly, the respiratory improvements in the symptomatic cohort follow the same dose-dependent pattern found in the area-specific analysis of the open field test.Rett syndrome patients suffer from respiratory symptoms including hyperventilation, apnoea, forced deep breathing, apneustic breathing, and severely arrhythmic breathing, profoundly affecting quality of life . These rtrocytes ,39. In tT158A/yMecp2 mice. SEMA4D was shown to be upregulated in neurons of T158A/yMecp2 mice, perhaps in response to stress induced by aberrant Mecp2 expression. The interaction of SEMA4D molecules and Plexin B1 receptors on glial cells has been reported to activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), Ras homolog family member A (RhoA), and Ras related protein (R-Ras) pathways, leading to cytoskeletal restructuring in astrocytes and inflammatory responses in microglia via the NF-κB, RhoA, and R-Ras signalling pathways [T158A/yMecp2 mice, which is reduced following treatment with SEMA4D blocking antibody. This suggests a model whereby MECP2 mutations induce neuronal stress to trigger neuronal expression of SEMA4D, which then binds to cognate Plexin B1 receptors to trigger astrocyte reactivity and microglial activation. By blocking SEMA4D–Plexin B1 binding, the anti-SEMA4D antibody may alleviate the cellular and inflammatory responses in glial cells, manifested as symptomatic relief and functional improvement in the T158A/yMecp2 mice.The upregulation of SEMA4D/Plexin B1 signalling and the importance of neuroglia interactions driving the pathology of Rett syndrome provide a mechanistic rationale for the amelioration of Rett syndrome-specific symptoms and the improvement of behavioural functions in anti-SEMA4D antibody-treated pathways ,40. As sT158A/yMecp2 mice treated with placebo compared to wild-type littermate controls and significant lowering of activated astrocytes and microglia with SEMA4D antibody treatment. These observations are highly pertinent to Rett neuropathology, as Mecp2 mutations are associated with impairments in locomotion and motor skill learning, which directly relates to the functionality of striatal motor control. Similarly, Mecp2 mutant mice show respiratory problems, including dysrhythmia and apnea, which are attributed to the malfunctioning/hyperexcitability of the pons [T158A/yMecp2 mice following treatment. One possibility is that antibody treatment reduced stress and restored health of neurons which reduced SEMA4D expression. Another possibility is that antibody binding to SEMA4D on neurons resulted in internalisation of antigen–antibody complexes, as reported previously [T158A/yMecp2 mice [Mecp2 mutations affect Plexin B1 expression in glial cells, and effects of SEMA4D on neuronal function and phenotype, as well as the impact on peripheral immune responses that can also influence neuropathology of Rett syndrome.We found marked activation of glial cells in the brain, including the striatum and pons regions, which registered a significant increase in cells expressing GFAP and Iba1 in the pons ,42. Inteeviously , and theeviously , howeverMecp2 deficiency or malfunction in microglia and astrocytes in various regions of the brain [The behavioural impairment observed in Rett syndrome is believed to be heavily affected by he brain ,44,45,46To our knowledge, the current study is the first to report the beneficial effects of anti-SEMA4D antibody in preventing and improving the symptoms and deficits in a transgenic mouse model of Rett syndrome. The pre-clinical trial results suggest that anti-SEMA4D immunotherapy is a potentially promising therapeutic strategy to improve phenotype, coordination, cognition, locomotion, and respiration in Rett syndrome.All experimental procedures were carried out in strict accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, National Health and Medical Research Council, and approved at the Children’s Medical Research Institute and the Children’s Hospital by Westmead Joint Animal Ethics Committee .T158AMecp2 knock-in Rett syndrome mouse model harbouring a p.Thr158Ala variation resulting in a reduction in Mecp2 binding to methylated DNA [These studies used wild-type (C57BL/6) and the ated DNA . The thrated DNA . Althougated DNA ,48,49.T158AMecp2 mice recapitulate many phenotypic features of Rett syndrome patients, including hypoactivity, motor dysfunction, neurodevelopmental regression, abnormal anxiety levels, and learning and memory deficits. Male hemizygous mice (T158A/yMecp2) usually start showing symptoms at 4–6 weeks old, while female heterozygous mice (T158A/+Mecp2) only show symptoms after 17 weeks. T158A/yMecp2 mice show characteristic Rett-like phenotypes, including hindlimb clasping, dysregulated weight, and seizure activity. In addition, they spend significantly less time on the rotarod compared to WT littermates, significantly less time in the closed arm and significantly more time in the open arm of the elevated plus maze compared to their WT littermates, with a significant reduction in locomotor activity and distance travelled.T158A/yMecp2 and wild-type C57BL/6 male mice were treated with either anti-SEMA4D (Mab 67) or mouse immunoglobulin G1 (IgG1) isotype control monoclonal antibody at 4 weeks old (when the T158A/yMecp2 mice were pre-symptomatic) or 8 weeks old (when the T158A/yMecp2 mice were symptomatic) (Hemizygous tomatic) . In the Phenotypic scoring was performed on a weekly basis for the severity of Rett syndrome-specific symptoms, including tremor and hindlimb clasping, according to the criteria in Elevated Plus Maze. Each mouse was placed in the cross-section of the elevated plus maze at 75 cm height for 5 min as previously published . The timAccelerating Rotarod. Mice were placed on an accelerating rotarod apparatus for 3 trials with at least 15 min of rest between trials. Each trial lasted for a maximum 3 min, during which the rod accelerated 5 rpm every 15 s from 5 to 60 rpm. The latency to fall from the rod was recorded for each trial.Open Field. At the beginning of each trial, the mouse was placed at the centre of the open field apparatus  for 15 min. The subject’s behaviour was tracked and recorded by ActiTrack software  for the duration of each trial.Whole Body Plethysmography. Each mouse was placed individually in a chamber of the whole-body plethysmography apparatus  in a dark room for 3 h without disturbance. The breathing pattern of each animal was recorded and analysed by EMKA iox software .Brains were coronally dissected and formalin fixed and paraffin embedded (FFPE). Left brain hemispheres were immersion fixed in formalin followed with paraffin embedding for immunohistochemistry. Three consecutive mouse brain sagittal serial sections (10 µm), 25 µm apart, from 6 mice per cohort, were evaluated. Fluorescent immunohistochemistry staining was performed for mouse semaphorin 4D , NeuN , Iba1 , and GFAP , in accordance with manufacturer-recommended concentrations combining host-dependent secondary Alexa Fluor antibodies. For the same host species primary antibodies, monovalent Fab fragment antibodies  were used for blocking along with sequential staining. Imaging was performed on an AxioObserver7 Automated Inverted Microscope with Plan-Apochromat 40×/0.95 objective and Axiocam 702 Monochrome Camera.http://imagej.nih.gov/ij/, version 1.53C, accessed on 30 August 2021). Cell densities were calculated as the mean from three consecutive sections from a sum of the numbers for each image divided by the area of the region selected. For Iba1, Z-stack images from three consecutive mouse brain sagittal serial sections (10–25 µm apart) were used for cell density calculations.For analysis of immunofluorescence intensity and cell numbers, whole tissue sections were mounted on a slide, with three consecutive slices per sample, and images of stained samples were analysed with Fiji/ImageJ software . Serum pools were diluted 1:900 in 1× PBS buffer, 0.5% bovine serum albumin , 0.02% Tween-20 . Plates were coated overnight with rSEMA4D  at 1.7 µg/mL in PBS, blocked for 1 h, and incubated with titrated serum samples for 2 h. Drug was detected using goat anti-mouse IgG1-HRP  and developed with TMB substrate . Plates were read at 450 nm using a BioTek™ Powerwave™ 340 plate reader, and drug concentrations were calculated based on the standard curve.v/v) foetal bovine serum , and 100 units/mL penicillin–streptomycin . The brain tissue was then triturated through a 1 mL pipette tip until a homogeneous suspension was obtained, pelleted by centrifugation at 170× g for 5 min and resuspended in DF medium. The cell suspension was then added to poly-D-lysine -coated 75 cm2 cell culture flasks (T75) containing DF medium. Cells were incubated at 37 °C in an atmosphere of 5% CO2, with medium being changed on the following day and then every 3 days. Once confluent, cells were dissociated by TrypLE Express Enzyme  and split at a ratio of 1:6 in poly-D-lysine-coated T75 cell culture flasks.Mixed glial cell cultures were prepared from post-natal day 3 (P3) male mice. Brains were removed and placed in modified Eagle’s medium  containing 25 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid . After removal of the meninges, cerebral cortices were chopped and incubated with 1 mL MEM-HEPES solution containing 1 mg papain , 240 μg L-cysteine , and 1140 units of DNase I type IV  at 37 °C for 1 h. Supernatant was removed, and the tissue was washed in 1 mL of medium consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with L-glutamine , 10%  or PBS and treated with anti-SEMA4D or isotype control antibody for 1 h. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilised for 20 min with 1% Triton X-100  in PBS and blocked for 1 h in blocking solution containing 0.5% bovine serum albumin  and 0.01% Tween 20  in PBS. Cells were incubated with primary anti-α-tubulin antibody , anti-glial fibrillary acidic protein , and anti-Plexin B1 antibody  overnight at 4 °C, followed by appropriate species-specific secondary antibodies  for 2 h at room temperature in combination with Hoechst nuclear stain  and washed in PBS for 3 × 5 min. Microscopic images were taken by an inverted microscope  and relative intensities per cell were determined using ImageJ software .Mixed mouse glial cells from t-test was used to determine differences between 2 groups. One-way analyses of variance with Bonferroni post hoc tests were used to determine significant differences for more than 2 groups. Two-way analyses of variance with repeated measures of time followed by Bonferroni post hoc tests were used to determine significant differences in all behavioural assessments. A Kaplan–Meier estimator was used to compare the differences between animal survival of any treatment groups during the pre-clinical trial. Differences were considered statistically significant at p < 0.05. Analysis of immunohistochemistry data was performed by IBM SPSS software (version 1.0.0.1327). Group differences among treatment conditions were assessed through one-way ANOVA. All error bars in the figures represent the standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.Analysis of all behavioural and in vitro data was performed using Prism 7 . A two-tailed, unpaired Student’s A patent application for the USE OF SEMAPHORIN-4D BINDING MOLECULES FOR THE TREATMENT OF RETT SYNDROME has been lodged .


S- and R-glycidol. The homochiral dendrimers bind to HaCaT and SCC-15 cell membranes with an R/S glycidol enantioselectivity ratio of 1.5:1, as was quantitatively determined by fluorescence microscopy and visualized by confocal microscopy. Fully substituted G2 and G3 dendrimers were equipped with 32 and 64 N-propyl residues and showed effectively radial symmetry for homochiral derivatives in 13C NMR spectrum in contrary to analogs obtained by reaction with rac-glycidol. The sub-stoichiometric derivatives of G2 and G3 were also obtained in order to characterize them spectroscopically. The homochiral dendrimers were labeled with two different fluorescent labels, fluorescein, and rhodamine B, using their isothiocyanates to react with G2 and G3 followed by the addition of S- and R-glycidol. Obtained fluorescent derivatives were deficiently filled with N-propyl substituents due to steric hindrance imposed by the attached label. Nevertheless, these derivatives were used to determine their ability to bind to the cell membrane of human keratinocytes (HaCaT) and squamous carcinoma cells (SCC-15). Confocal microscopy images obtained from cells treated with variously labeled conjugates and fluorescence analysis with fluorescence reader allowed us to conclude that R-glycidol derivatives were bound and entered the cells preferentially, with higher accumulation in cancer cells. The G3 polyamidoamine (PAMAM)-based dendrimers were taken up more efficiently than G2 derivatives. Moreover, S- and R-glycidol furnished dendrimers were highly biocompatible with no toxicity up to 300 µM concentrations, in contrast to the amine-terminated PAMAM analogs.The generation 2 and 3 poly(amidoamine) dendrimers (PAMAM G2 and G3) were converted into N-propyl derivatives by the addition of enantiomerically pure  Polyamidoamine (PAMAM) dendrimers were synthesized in 1985 by Tomalia et al. . Full geThe example of class 1 dendrimers are BINAP-cored and polyphenylether-expanded dendrimers, which were shown to provide catalytic space for hydrogenation of various substrates, i.e., alkenes, such as 2-(4-isobutylphenyl)acrylic acid or acetamidocinnamic acid, and acetophenones, which were reduced into corresponding chiral products with high chemoselectivity and >90% enantiomeric excess (ee) in presence of catalytically active Ru(I) and Rh(II) organometallic complexes .The example of class 2 chiral dendrimers constructed of chiral vicinal diol subunits linked and branched by phenyl groups are polyhydroxylated dendrimers obtained in 2000 by McElhanon and McGrath  and PAMA4 with high yield (>90%) and enantiomeric excess (ee), depending on dendrimer generation and solvent (THF or H2O) -1,2-cyclohexanediamine, chiral salen type ligand via imidazolyl linkers. Dimeric Ti(IV)-(µ-O)yl group .R- or 2-S, 3-dihydroxypropyl substituents, but the next arm of the dendrimer is involved in symmetry recognition.Chiral space inside the dendrimer voids is currently exploited for catalytic purposes. As exemplified above, the chiral substituents on the periphery of dendrimers can operate as asymmetry inductors and be recognized by biological membrane receptors, especially if chiral inductors are enantiomerically pure amino acids or specific carbohydrates. The simplest chiral substituent, readily introduced into peripheral amine groups of PAMAM dendrimers is 2, 3-dihydroxypropyl residue, as it was demonstrated earlier . PAMAM drac-glycidol-covered core G3 was able to bind up to 12 G0, and this megamer was demonstrated as an efficient DDS for nimesulide, the non-steroidal anti-inflammatory drug  for G2 and SglG2  were observed at 3.23 and 2.71 ppm, respectively, with [32H] integral intensity both. Inner sphere c and d resonances showed overlapping triplet from c1,c0 at 3.29 ppm (intensity [24H]) and d1,d0  for G2  for G2 A. Upon a1H S-gl resonances of protons g and f of integral intensity corresponding to [32H] and [64H] are unresolved multiplets and AB-type spectrum of gg’ diatereotopic pair, respectively (The ectively B.13 C NMR spectra of G2 and SglG2 are shown in 13C resonances were assigned based upon standard HSQC and HMBC experiments (the combined HSQC and HMBC spectrum of SglG2 is presented in 2 (−4.1 ppm) and c2 (+14.5 ppm) resonances occur upon substitution of terminal NH2 groups with two S-gl substituents, with d1d0 and c1c0 resonances almost unaltered. Three 13C resonances of S-gl substituents indicate that all terminal amine groups were uniformly substituted with two gl-S each. Analogous 1H and 13C NMR spectra were obtained for RglG2. Similar 1H and 13C NMR spectral pattern was obtained for higher generation PAMAM dendrimers, namely, G3, G4, and G5, which were substituted with 64, 128, and 256 enantiomerically pure R- or S-gl, respectively. In comparison with the spectra of SglG2 or RglG2, the only differences observed were due to the integral intensity of resonances.The aliphatic regions of 13C NMR spectra of PAMAM G2 and G3 dendrimers substituted with enantiomerically pure S-gl or R-gl, which show only one set of gl resonances for Sgl32G2 or Rgl32G2 and corresponding fully converted G3–G5 (spectra not shown) indicates that enantiomeric purity of S-gl is retained in conversion to formally R-chiral n-2(R), 3-dihydroxypropyl substituents. Unfortunately, the chirality checked with CD showed the Cotton effect on the almost background level, as it was observed also for other chiral dendrimers of radial symmetry . Three G2Sngl species were obtained, where average Sgl molecules added (n) was 9, 14, and 22 (see Sgl32G2 (AB = 0.11 ppm) with geminal coupling constant Jgem = 11.7 Hz and different vicinal coupling constants J(gf) = 4.2 Hz and J(g’f) = 5.9 Hz.Stepwise addition of  G232Sgl E is viciS-gl was used the Sgl9G2 was obtained, containing some unsubstituted amine groups, mostly mono-substituted amine groups with 2, 3-dihydroxypropyl, and a minor amount of double substituted amine groups. The AB-type spectrum of gg’ protons on single substituted amine groups showed slightly lower magnetic nonequivalence , coupled geminally with the same Jgem = 11.7 Hz, while vicinal constants were larger than for double-substituted arms, namely, J(gf) = 4.6 Hz and J(g’f) = 6.7 Hz .When an under-stoichiometric amount of 7 Hz see , trace B3.24 ppm , trace A G232Sgl , trace E13C NMR spectra of G2Sngl  are shown in 13C resonances were assigned based upon standard HSQC and HMBC experiments. We found that PAMAM inner shell resonances remained unaffected upon terminal amine group substitution, while d2 and c2 proton resonances shifted from 42.3 and 40.5 in amine-terminated G2 into 39.3 and 48.1 upon mono- and 37.7 and 54.2 ppm upon disubstitution with glS, respectively. The resonances of S-glycidol-derived 2(R), 3-dihydroxypropyl substituents for single-substituted arms are located at 51.3, 64.7, and 71.0 ppm for e, f, and g carbon nuclei  derivatives with the same protocol, using 20% molar excess of enantiomerically pure S- and R-gl in relation to available amine groups . Obtained derivatives were not fully substituted. We obtained series of derivatives, namely, RglF16G2, SglF19G2, RglF19G3, SglF35G3, RglR19G2, SglR30G2, RglR45G3, and SglR35G3. The average stoichiometry of compounds was determined by 1H NMR spectroscopy (spectra not shown). These compounds were then tested as single dendrimers and in homochiral pairs labeled with two different fluorescent labels on human cell cultures (vide infra).The fluorescent labels, FITC and RBTC were carefully attached to G2 and G3 dendrimers. Single-labeled G2Sgl9G2 and Sgl22G2 and characterize them by NMR spectroscopy (vide supra).Fully gl-substituted PAMAM dendrimers require a temperature regime to avoid polyhydroxylation, which starts already at a temperature above 50 °C. When 20% molar excess of gl is used, the number of glycidol residues attached to PAMAM becomes higher than twice the number of terminal amine groups of PAMAM dendrimer as determined by the integral intensity of glycidol-derived multiplets of protons f. On the other hand, the substoichiometric amount of gl results in the formation of dendrimers with double- and single-substituted arms. Thus, we could obtain Sgl23Rgl9G2 and Sgl10Rgl22G2. The corresponding 1H NMR spectra enabled us to identify these species as fully substituted, although they did not illustrate the symmetry of mixed-isomer derivatives. Therefore, we examined the 13C NMR, which are presented for starting Sgl9G2 and Sgl22G2 . There are essentially two sets of gl resonances observed in the 13C NMR spectra of the products, which were assigned to the chiral arm and meso arm. Two sets of carbon f resonances can be identified at ca 70 ppm , which is obtained by one portion addition of rac-gl into G2 dendrimer. These puzzling symmetry arms might origin from elongated polyhydroxy substituents because on the one hand the intensity of gl 1H NMR resonances clearly indicates that the number of attached gl residues is 32; on the other hand, in the 13C NMR spectrum, the resonance from the single-substituted arm is still present at ca 51 ppm .These two species were then used to convert remaining free amine groups and mono-substituted ones into bis-substituted ones. Thus, we obtained the average substituted  G222Sgl A,E and ma 51 ppm , trace C and squamous carcinoma cells (SCC-15)— both belonging to epithelial tissue. SCC-15 is a model of cancer cells used as a target for the synthesized PAMAM conjugates occurring as a drug delivery system, while HaCaTs comparatively as non-tumorigenic cells. We used immortalized, non-tumorigenic keratinocytes, compared to normal ones, since the latter from skin biopsies indicate high variations between passages, short culture lifetime, and donor variability . To asseS or R glycidol  were highly biocompatible and did not indicate cytotoxic action against both immortalized human keratinocytes (HaCaT) and human squamous carcinoma cells (SCC-15) up to 300 µM concentration  is targeting specific sites or cell populations ,35. In aRhizopus javanicus enantioselectively catalyzed esterification of R-(+)-gl with n-butyric acid. However, the highest enantioselectivity was achieved after pretreatment of silica gel templated lipase with R-(-)-2-octanol [It has been observed before, that gel-entrapped lipase from -octanol .The enzymes catalyzing water addition to epoxide with concomitant ring-opening show a variety of stereoselectivity of diol products, with retention or inversion of configuration, depending on substituents on gl and enzyme origin . The invThe 2,3-hydroxypropyl substituents are formed upon food treatment, they covalently attach into Val and were found as N-2, 3-dihydroxypropyl Val residues in hemoglobin. This derivative was considered as a marker in food examination . NeitherR-gl enantiomer-derived G3 dendrimer conjugates (RglFG3) were always significantly stronger accumulated than S-gl enantiomer-derived ones (RglFG3) after 0.1–4 h incubation with 1µM concentrations. An analogous effect was observed also in the case of G2 dendrimers, but only in cancer cells and from already 1 h incubation , while G2 conjugates are much more scattered in the area of the cytoplasm, with visible slightly lower accumulation in intracellular vesicles  derivatives. Fully substituted homochiral derivatives had effective radial symmetry, which was illustrated by one set of 2, 3-dihydroxypropyl carbon resonances in 13C NMR spectra. Deficient substitution of G2 or G3 with enantiomerically pure glycidol enabled us to identify the mono-substituted arms of dendrimer along with bis-substituted ones by 13C NMR spectroscopy. The single-fluorescent labeled homochiral G2 and G3 dendrimers were obtained in order to test their ability to bind with the cell membrane and enter into different human cells in vitro.PAMAM G2 and G3 dendrimers were quantitatively converted by the addition of enantiomerically pure S- and R-glycidol furnished dendrimers were highly biocompatible with no toxicity up to 300 µM concentrations in contrast to the pristine PAMAM dendrimers, for which significant toxicity was observed already within 10–50 µM concentrations. The R and S glycidol derivatives of G3 PAMAM dendrimer bind to the cellular membrane and enter into the cells with higher accumulation in cancer cells. The R enantiomer conjugate quantity in studied cells was always significantly higher than S ones. The G3gl dendrimers were taken up more efficiently than G2gl derivatives; however, G2gl derivatives penetrated also nuclei of studied cells.Biological studies with human non-tumorigenic keratinocytes (HaCaT) and squamous carcinoma cells (SCC-15) indicated that