We report the expression, purification, crystallization and 2.10 Å X-ray structure of 3CLpro from PEDV. Analysis of the PEDV 3CLpro structure and comparison to other coronaviral 3CLpro’s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CLpro’s are conserved, with the exception of a loop that comprises the protease S2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro, (R)-16, to have inhibitor activity against PEDV 3CLpro, despite that SARS-3CLpro and PEDV 3CLpro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CLpro are conserved in PEDV-3CLpro; however, the sequence variation and positional difference in the loop forming the S2 pocket may account for large observed difference in IC50 values. This work advances our understanding of the subtle, but important, differences in coronaviral 3CLpro architecture and contributes to the broader structural knowledge of coronaviral 3CLpro’s.Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CL The causative virus of porcine epidemic diarrhea in pigs was first described in England in 197834pro) cleaves the polyprotein at 3 sites, whereas the 3C-like protease (3CLpro), also known as the main-protease (Mpro) or nsp5, cleaves the polyprotein at 11 sites. The function of both proteases is essential for viral replication and therefore they are attractive targets for the development of anti-coronaviral therapeutics. In this study, we report the expression, purification, crystallization, and X-ray structure of PEDV-3CLpro in its unbound state to 2.10 Å. We tested PEDV-3CLpro for inhibition by (R)-16, a compound known to inhibit severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro, and found (R)-16 to inhibit PEDV-3CLpro with an IC50 of 25.4 ± 1.4 μM. The structural information provided by the PEDV-3CLpro X-ray crystal structure adds new details to our general understanding of the subtle differences between phylogenetically related 3CLpro’s and provides key insights that can be used to inform and guide the design of inhibitors against PEDV-3CLpro.Coronaviruses are enveloped, single-stranded, positive-sense zoonotic RNA viruses with genomes ranging from 27 to 32 kilobases in length, where the genes encoding for the structural proteins are located at the 3′ end and the replicase genes are located at the 5′ end of the genome. Two long ORFs encode for the replicase proteins, ORF1a and ORF1b, which are connected by a ribosomal frame-shift and produce the coronaviral polyproteins pp1a and pp1ab upon translationpro crystallized in space group P212121 as a dimer in the asymmetric unit  and 2-methyl-2,4-pentanediol (MPD) (PEDV-3CLric unit . As repo monomer 1112. TheI and II . Domain I and II 1213. In one side . Interesol (MPD) . The prepro and the unbound form of human coronavirus 229E 3CLpro (PDB entry 1P9S)pro and PEDV-3CLpro shows that the overall architectures of both the 229E-3CLpro and PEDV-3CLpro active sites in their unbound states are structurally very similar, and the active site residues of the catalytic dyad, residues Cys144 and His41 in both PEDV-3CLpro and 229E-3CLpro, are located in almost identical structural space within the active site cavity, which is solvent exposed on one side  are found in the active site (Least-squares (LSQ) superposition of PEDV-3CLone side . In the ive site .pro active site that are important in inhibitor and substrate binding, we generated an LSQ superposition of PEDV-3CLpro and an inhibitor-bound form of feline infectious peritonitis virus 3CLpro , which belongs to the same alpha-coronavirus lineage as PEDV-3CLpro and has 61.9% sequence identity  in nearly identical orientations despite Cys144 of FIPV-3CLpro being covalently modified by the inhibitor, compound 6  with PEDV-3CLpro; however, we reasoned that the similar tertiary structure and conserved active site architecture of 3CLpro’s would allow for inhibition by the same molecule. We therefore tested the inhibition of PEDV-3CLpro by (R)-16, which was developed as a non-covalent inhibitor against SARS-3CLpro with potential broad-spectrum activity (R)-16 to inhibit PEDV-3CLpro with an IC50 value of 25.4 ± 1.4 μM, where the representative curve for (R)-16 inhibition of PEDV-3CLpro is shown in R)-16 inhibition of SARS-3CLpro has been previously published50 of (R)-16 against SARS-3CLpro is 1.5 ± 0.3 μMR)-16 with PEDV-3CLpro. The inhibition of PEDV-3CLpro by (R)-16, though weak, is significant because it indicates that the development of non-covalent broad-spectrum inhibitors of 3CLpro’s may be possible.Our observations of the overall conserved structural features surrounding the PEDV-3CLactivity 15. We foR)-16 may bind to PEDV-3CLpro, a structural alignment of the X-ray structure of SARS-3CLpro:(R)-16 complex with PEDV-3CLpro was generated (pro and inhibitor-bound SARS-3CLpro (PDB entry 3V3M)pro is from the alpha-coronavirus phylogenetic group and SARS-3CLpro is from the beta-coronavirus phylogenetic subgroup 2b, resulted in an all-atom RMSD value of 5.18 Å and a C-alpha RMSD value of 4.98 Å. The structural alignment shows that, similarly to 229E-3CLpro and FIPV-3CLpro, the overall active site architectures of SARS-3CLpro and PEDV-3CLpro are largely similar despite their lower sequence identity (R)-16 via hydrogen-bonding interactions are all conserved  or Cys145 (for SARS-3CLpro), located in almost identical structural space (R)-16 similarly occupies the S2-S1′ subpockets in each of the 3CLpro active sites, where the tert-butyl amide resides in the channel leading to the active site, the tert-butylanilido group (P2) sits in the S2 pocket, the 3-pyridyl group (P1) resides in the S1 region, and the tetrahydrofuran (P1′) occupies the S1′ subsite -16 acts as a hydrogen-bond acceptor for SARS-3CLpro His163, with a distance of 2.8 Å between heteroatoms (pro by (R)-16 as His162 of PEDV-3CLpro is in an almost identical structural location as that of SARS-3CLpro His163. Additionally, the bifurcated interaction between the furan ring oxygen and the amide carbonyl oxygen of (R)-16 and the backbone amide NH of SARS-3CLpro Gly143 is mimicked by PEDV-3CLpro Gly142 -16 apparently binds in the same orientation in the PEDV-3CLpro active site as it does in the SARS-3CLpro active site, and likely utilizes the same hydrogen-bonding interactions, the IC50 of (R)-16 against PEDV-3CLpro is 17-fold higher than against SARS-3CLpro. We were therefore curious if the difference in position of the S2 loop between PEDV- and SARS-3CLpro may be important for (R)-16 binding. A structural alignment of the ligand-bound structures of FIPV- and SARS-3CLpro  shows that compounds 6 and (R)-16 bind to the respective 3CLpro’s by positioning their sterically bulky, hydrophobic groups in the S2 subsite , and human coronaviruses NL63 and 229E12131415161718192 loops of FIPV-, TGEV-, NL63-, 229E-, PEDV-, and SARS-3CLpro shows that the SARS-3CLpro S2 loop is one residue longer than those from the alpha-coronaviruses and shares no sequence identity with FIPV-, TGEV-, NL63-, 229E- and PEDV-3CLpro -16 against PEDV-3CLpro as compared to SARS-3CLpro . Furthermore, the combination of the increase in length and charge properties of the S2 loop of SARS-3CLpro relative to that of the alpha-coronavirus 3CLpro’s likely changes the size and shape of the S2 binding pocket and therefore may allow for increased variability at the P2 site of inhibitor and substrate molecules.We then analyzed the reported X-ray structures and sequences for other alpha-coronavirus 3CLV-3CLpro . Additio2-subsite and relationship to inhibitor and substrate binding, we examined the recognition cleavage sequences in polyprotein 1ab of both PEDV- and SARS-3CLpro -16, allowing the bulky t-butylanilido group to take full advantage of hydrophobic interactions within the S2 subsite and position (R)-16 to optimize hydrogen-bonding with SARS-3CLpro Gly143 and His163.To further investigate the variation in the SS-3CLpro 2122. Thepro to 2.10 Å resolution. We found the structure of PEDV-3CLpro to be similar to the X-ray structure of the unbound human coronavirus 229E 3CLpro, which belongs to the same coronaviral phylogenetic subgroup as PEDV-3CLpro. To investigate the role of inhibitor binding on the overall architecture of the catalytic site, we generated a superimposition of PEDV-3CLpro with FIPV-3CLpro (PDB entry 4zro), which belongs to the same alpha-coronavirus phylogenetic group as PEDV-3CLpro and shares 61.9% sequence identity, in complex with the covalent inhibitor 6. We observed little difference between the active site architectures of unbound PEDV-3CLpro and the inhibitor-bound form of FIPV-3CLpro, except for differences in the position and composition of a loop comprising the S2 subsite, which was also observed in comparison to the 229E-3CLpro structure.In this study, we have determined the X-ray crystal structure of an unbound form of PEDV-3CLpro for inhibition by a known SARS-3CLpro inhibitor, compound (R)-16 and found (R)-16 to be capable of inhibiting PEDV-3CLpro although the IC50 value was roughly 17-fold higher than the reported IC50 for (R)-16 against SARS-3CLpro. Structural comparison of SARS-3CLpro bound with (R)-16 and PEDV-3CLpro in its unbound form revealed that the residues that directly interact with (R)-16 in SARS-3CLpro are conserved in PEDV-3CLpro. Additional analysis of the S2 loop across alpha-coronavirus 3CLpro’s and SARS-3CLpro proved that the sequence identity of the S2 loop is not conserved across alpha- and beta-coronaviral 3CLpro’s and the SARS-3CLpro S2 loop is one residue longer than that of the alpha-coronaviral 3CLpro’s, which likely increases the effective volume of the SARS-3CLpro S2 protease subsite relative to the alpha-coronaviral 3CLpro S2 subsites. These findings provide a potential explanation for the roughly 17-fold weaker inhibition of PEDV-3CLpro by (R)-16 compared to SARS-3CLpro.We tested PEDV-3CLpro’s. Small structural changes have been shown to be essential in enzymatic catalysis and an understanding of these such differences is also very pertinent for the design of both broad-spectrum and selective coronaviral 3CLpro inhibitors for the treatment of coronaviral infectionThis work advances our understanding of the subtle, but important, structural differences between 3C-like proteases from different coronaviral phylogenetic groups and contributes to the broader structural knowledge of coronaviral 3CLpro of PEDV (residues 2998–3299 in the Porcine epidemic diarrhea virus polyprotein AHA38151.1)E. coli and cloned into pET-11a expression vector with an N-terminal (His)6-tag followed by nsp4-/5 auto-cleavage site by BioBasic Inc. This pET-11a PEDV-3CLpro construct was used because it results in the expression of PEDV-3CLpro without an N-terminal or C-terminal extension.The gene encoding the 3CL. coli BL21(DE3) cells were transformed with the pET-11a PEDV-3CLpro plasmid and then grown at 25 °C for 24 hours in 500 mL Super LB media  that was also supplemented with 1 mL 100 mg mL−1 carbenicillin, 25 mL 8% lactose, 10 mL 60% glycerol, and 5 mL of 10% glucose per 1 L of expression culture. The cells were harvested by centrifugation  to yield 14.5 g L−1 of cells. The cell pellet was then re-suspended in Buffer A  containing 1 mg mL−1 lysozyme, where 5 mL of Buffer A was used per 1 g cell pellet. After the cells were homogenized, they were lysed via sonication for a total of 10 minutes with 10 s pulses and 20 s delays at 50% amplitude using a Branson Digital Sonifier.Eg, 4 °C, 20 minutes).The cell lysate was clarified by pelleting the cell debris via centrifugation  column equilibrated with Buffer B. Protein was eluted with a linear gradient to 50% Buffer C  over five column volumes (300 mL) collecting 5 mL fractions. Fractions containing pure PEDV-3CLpro, as judged by SDS-PAGE and specific activity, were pooled, dialyzed into storage buffer , and concentrated using a spin concentrator (Millipore) with a 10 kDa molecular weight cutoff membrane. The protein was flash-frozen in liquid nitrogen and then stored at −80 °C in a freezer until further use  and loaded onto a 24 mL Superdex 75  size exclusion chromatography column equilibrated with 25 mM HEPES pH 7.5 and 2.5 mM DTT. Peak fractions containing PEDV-3CLpro, as determined via visualization of SDS-PAGE and confirmed by protein activity assay, were pooled and concentrated to protein concentrations of 1.5, 2.8, and 4.0 mg mL−1 for crystallization.The resultant supernatant was loaded onto a 60 mL Phenyl Sepharose 6 Fast Flow HiSub  column equilibrated with Buffer A. Protein was eluted with a linear gradient to 100% Buffer B  over five column volumes (300 mL) collecting 5 mL fractions. Fractions containing PEDV-3CLther use . Prior tpro crystallization conditions. Crystallization trials  were set up in a 96-well sitting drop tray (Intelli-Plate 96) using a TTP LabTech Mosquito® liquid robotics system at PEDV-3CLpro concentrations of 1.5, 2.8, and 4.0 mg mL−1 in buffer containing 25 mM HEPES pH 7.5 and 2.5 mM DTT. An initial hit of clusters of needle-like crystals was obtained at 20 °C from the PEGS II Suite condition No. 50 consisting of 10% (w/v) PEG-4000 and 20% (w/v) isopropanol. Crystals were also obtained at each of the other PEDV-3CLpro concentrations. Optimization of this condition was achieved by varying the concentration of both isopropanol and PEG-4000 between 15–30% and 7–22% respectively, where diffraction quality crystals grew at a variety of conditions from this round of optimization. The best crystals grew at 20 °C in 25% isopropanol and 22% PEG-4000 at a protein concentration of 1.5 mg mL−1 and appeared after 24–72 hours (The PEGS II Suite (Qiagen) sparse-matrix screen was used to screen for initial PEDV-3CL72 hours . CrystalHKL-2000PHENIXpro in complex with the peptidomimetic compound EPDTC  at Argonne National Laboratory, Argonne, Illinois, USA. Data were indexed, integrated and scaled using R)-16 was synthesized according to Jacob et al.pro by (R)-16 at a concentration of 100 μM was first tested in an enzymatic assay containing the following buffer . The assays were carried out in triplicate using Costar 3694 EIA/RIA 96-Well half-area, flat bottom, black polystyrene plates from Corning Incorporated. 1 μL of 100X inhibitor stock in DMSO was added to 79 μL of enzyme in assay buffer and the enzyme-inhibitor mixture was incubated for 10 minutes. The reaction was initiated by the addition of 20 μL of 10 μM UIVT3 substrate, a custom synthesized Förster resonance energy transfer peptide substrate with the following sequence: HilyteFluorTM488-ESARLQSGLRKAK-QXL520TM-NH2, producing final concentrations of 100 nM for the 3CLpro enzyme and 2 μM UIVT3 substrate. The increase in fluorescence intensity of the reaction was then measured over a period of 10 minutes as relative fluorescence units (RFUt). An excitation wavelength of 485 (bandwidth of 20 nm) and an emission wavelength at 528 (bandwidth of 20 nm) was used to monitor the reactions using a BioTek Synergy H1 multimode microplate reader. The percent inhibition of the PEDV-3CLpro was determined using the following The compound -16 against PEDV-3CLpro was determined at ambient temperature using 100 μL assays conducted in the following buffer: 50 mM HEPES, 0.1 mg/mL BSA, 0.01% TritonX-100, 1 mM DTT. Kinetic assays were carried out in triplicate and in Costar 3694 EIA/RIA 96-Well Half Area, flat bottom, black polystyrene plates from Corning Incorporated. (R)-16 was tested at concentrations of 0.313, 0.652, 1.25, 2.5, 5.0, 10, 20, 40, 60, 80, 100 and 120 μM by adding 1 μL of 100X inhibitor stock in DMSO to 79 μL of enzyme in assay buffer and then incubating the enzyme-inhibitor mixture for 10 minutes. The reaction was initiated by the addition of 20 μL of 10 μM UIVT3 substrate, producing final concentrations of 100 nM for 3CLpro and 2 μM for the UIVT3 substrate. The increase in fluorescence intensity over time during the reaction was then measured.The ICpro was then calculated from triplicate data, and the final averaged data with standard deviation were then plotted as a function of inhibitor concentration. The data were fit to the following 50:The average percent inhibition of PEDV-3CLmax is the percent maximum inhibition of 3CLpro. The errors in the IC50 and %Imax values are the errors in the fitted parameters resulting from fitting of the equations to the data.where %IAccession codes: Model coordinates and structure factor amplitudes for the X-ray crystal structure of unbound PEDV-3CLpro were deposited in the Protein Data Bank (acc. #: 5HYO).How to cite this article: St. John, S. E. et al. X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus. Sci. Rep.6, 25961; doi: 10.1038/srep25961 (2016).


S. cerevisiae kinesin Kip2 stabilises astral microtubules (MTs) and facilitates spindle positioning through transport of MT-associated proteins, such as the yeast CLIP-170 homologue Bik1, dynein and the adenomatous-polyposis-coli-related protein Kar9 to the plus ends of astral MTs. Here, we show that Kip2 associates with its processivity factor Bim1, the yeast homologue of the plus-end-tracking protein EB1. This interaction requires an EB1-binding motif in the N-terminal extension of Kip2 and is negatively regulated by phosphorylation through Mck1, the yeast glycogen synthase kinase 3. In addition, Mck1-dependent phosphorylation decreases the intrinsic MT affinity of Kip2. Reduction in Kip2 phosphorylation leads to stabilisation of astral MTs, and accumulation of Kip2, dynein and Kar9 at MT plus ends, whereas loss of Mck1 function leads to defects in spindle positioning. Furthermore, we provide evidence that a subpopulation of Mck1 at the bud-cortex phosphorylates Kip2. We propose that yeast GSK-3 spatially controls astral MT dynamics and the loading of dynein and Kar9 on astral MT plus ends by regulating Kip2 interactions with Bim1 and MTs.The  Summary: The yeast GSK-3 kinase controls astral microtubule functions by regulating the interaction of the microtubule-stabilising kinesin Kip2 with microtubules and its processivity factor Bim1/EB1. The microtubule (MT) plus end is a site of remarkable versatility: owing to its dynamicity it easily explores cellular space, but it can also mediate and maintain stable interactions with other cytoskeletal factors. Functionality of microtubule plus ends can be modified by a set of specialised proteins termed MT plus-end tracking proteins (+TIPs) that are able to stably associate with the growing end of the MT . Among +S. cerevisiae. Spindle positioning in budding yeast ensures that the spindle elongates along the mother-bud axis during anaphase and depends on two redundant pathways, the dynein pathway and the Kar9 pathway. The plus-end-directed kinesin motor Kip2 participates in both pathways by transporting +TIPs to the plus ends of astral microtubules (aMTs). In the Kar9 pathway, Kip2 is required for efficient accumulation of the +TIP Kar9 at aMT plus ends (Yeast homologues of EB1 (Bim1) and CLIP-170 (Bik1) are both involved in spindle positioning during mitosis of lus ends . Similarlus ends . As partlus ends . Dynein lus ends .KIP2 results in extremely short aMTs, whereas KIP2 overexpression leads to cells with abnormally long aMTs. Stabilisation of aMTs by Kip2 seems to be coupled to the transport of Bik1 to aMT plus ends  is a highly conserved kinase with a key role in signalling during development  displayed a complex migration pattern that collapsed after treatment with alkaline phosphatase . This suggested that part of Kip2 is present within cells as a number of phosphoisoforms that display different electrophoretic mobilities on SDS PAGE. Furthermore, replacing S at position 63 with A (Kip2-AT13myc) largely abrogated Kip2 phosphorylation . The single T275A replacement did not show any significant effect in vivo , whereas combination of S63A and T275A mutations (Kip2-AA13myc) displayed similar reductions when compared with Kip2-AT13myc . We next tested whether Cdc28 phosphorylates Kip2 in vivo by inhibiting Cdc28 over time using the cdc28-as1 strain . In addition, pairwise deletion of the yeast cyclins Clb1–6, which are essential for Cdc28 activity did not significantly decrease Kip2 phosphorylation . Hence, we conclude that Cdc28 is not the main kinase involved in Kip2 phosphorylation.Mitotic Cdc28 (the budding yeast Cdk1) phosphorylates Kip2 in vitro . In linein vitro A. Indeed1 strain . In this1 strain . Whereas13myc phosphorylation in cells with mutations in the yeast Polo kinase . Phosphorylation of Kip2 in rim11Δ cells is only marginally reduced . Moreover, phosphorylation of Kip2 in vivo decreased upon downregulation of MCK1 (expressed under the regulatable GAL1-10P promoter) and hyperphosphorylation was restored upon overexpression of the kinase – the latter effect was absent upon introduction of the S63A mutation  phosphorylated by a priming kinase . Such a clusters C. Since in vitro . Deletion of the clusters A and B (Δ37-Kip213myc) severely reduced phosphorylation, whereas Kip2 deleted for the whole N-terminus before aa residue 70 (Δ70-Kip213myc) did not exhibit any detectable phosphorylation  followed by GFP  and Mck1TAP from yeast cells. In line with the requirement for a priming-phosphorylation by GSK-3 kinases, Mck1TAP alone was not able to phosphorylate MBPKip2ΔC . Furthermore, S63 is part of cluster C but is not the most downstream Ser within that cluster. We, therefore, reasoned that S69 or S72 might act as a priming site in vivo.Although our 13myc phosphorylation upon introduction of S69A and the highest reduction upon introduction of the S72A mutation, suggesting that these sites control phosphorylation of the upstream cluster in vivo (http:/elm.eu.org/). However, we did not observe a significant reduction in Kip213myc phosphorylation in yeast stt1-1 (PKC) or yck1Δ tsyck2-2 (CKI) mutants . Therefore, the (priming) kinases that phosphorylate these sites remain unknown.Indeed, we observed a clear reduction of Kip2 in vivo A. A consdbf20Δ-null mutants as well as in dbf2Δ strains . Dbf20 is a close homologue of the LATS-related Dbf2 kinase in yeast. Dbf2 kinase is a part of the mitotic exit network that is required in metaphase for correct spindle orientation . Phosphorylation levels of Kip213myc were lowest during metaphase, followed by increase in Kip213myc phosphorylation immediately after. Therefore, the pattern of Kip2 phosphorylation fits well with the timing of principle Dbf2 activation. Although some phosphorylation was still evident, the increase of phosphorylated Kip213myc upon anaphase entry was less pronounced in the dbf2-2 dbf20Δ mutant (and the mck1Δ mutant) when compared to wild type , in agreement with the hypothesis that Dbf2 acts on Kip2 as a priming kinase for Mck1.However, during the analysis of the yeast-kinase deletion strains, we observed that Kip2 phosphorylation is diminished in entation  and is fentation . Upon an reduced B. FurtheFig. S1H and 13myc phosphorylation pattern. We refrained from mutating more R residues in the N-terminal Dbf2 consensus sites because this would have drastically changed the pI of this domain and might have perturbed Kip2-MT interactions (see below). Our interpretation of these data so far is that priming for Mck1 phosphorylation is mediated by Dbf2 and/or Dbf20 within the Kip2 N-terminus, and possibly by additional kinases that act at S69 and S72.Upon closer inspection of the Kip2 sequence, we noticed that residues S13, S63 and S69 within the N-terminal clusters are preceded by an R and, thus, match the preferred sequence for the Dbf2 kinases [RxxS/T] ; Fig. 1Akip2Δ cells that genomically express different Kip2 variants under the control of the native KIP2 promoter. Interestingly, cells that express Kip2 variants that are defective in Mck1 phosphorylation (Kip2-AT) displayed an increased number as well as abnormally long astral MTs . Consistently, reduction of Kip2 phosphorylation through deletion of MCK1 also results in aMT stabilisation and increase in aMT number . Importantly, we also observed the same aMT phenotypes in dbf2-2 dbf20Δ cells in all phases of the cell cycle . Furthermore, to mimic a constitutively phosphorylated Kip2, we mutated all S or T residues to D within the N-terminal cluster of our MBPKip2ΔCGFP construct  and repeated the co-sedimentation experiments. Again, wild-type MBPKip2ΔCGFP co-sedimented with increasing tubulin concentrations, the majority of allD-MBPKip2ΔCGFP remained in the supernatant  and is reminiscent of the Ndc80 N-terminal extension that contributes to binding of Ndc80 to MTs . Interesernatant C. Consis. The SNIP1 Bim1 binding site at aa 21–24 is of particular interest because Bim1  act as a processivity factor for Kip2 , in addition to its interaction with MTs.During sequence analysis of the N-terminal extension, we also identified a putative EB1/Bim1-binding (SxIP) motif  at aa 21for Kip2 . Phosphofor Kip2 . In thisin vivo. Indeed, HABim1 co-immunoprecipitated Kip213myc from cell extracts  or the entire N-terminal Kip2 extension (Δ70-Kip213myc) almost completely lost interaction with HABim1  and this interaction was abrogated after deletion of the N-terminal sequences that included the SxIP1 motif  or KAR9  also resulted in increased amounts of dynein at aMT ends, whereas Mck1 overexpression led to the opposite effect .To further clarify this, we asked whether aMTs are required for Kip2 phosphorylation. After depolymerisation of aMTs with nocodazole, phosphorylation of Kip2 was lost within 20 min E. Loss oIn vitro, replacing GSK-3β sites with D or E residues supports the phosphorylation of S/T residues that are located further upstream . Second, cellular Kip2 levels did not increase upon Mck1 downregulation or decrease upon Mck1 overexpression  or BY4743 (for details see Table S1) and grown on standard medium.All strains were derivates of S288C (http://www.cbs.dtu.dk/services/NetPhosK/) and the Eukaryotic Linear Motif resource (http:/elm.eu.org/). All Kip2 variants were generated by site-directed mutagenesis. Untagged versions as well as Venus-YFP-tagged Kip2 variants were cloned into the integrative pRS305 plasmid under control of the endogenous KIP2 promoter and integrated into the LEU2 locus. 13×Myc-tagged (13myc) Kip2 variants expressed under control of the KIP2 promoter were sub-cloned into the pRS314 plasmid. For full plasmid list see Table S2.Sequence analysis to identify phosphorylation sites was performed by NetPhosK  in the relevant focal plane of a nine-image z-stack after background subtraction. Only bud aMTs of metaphase cells were examined. This restriction takes into account the temporal and spatial localisation pattern of Mck1 and Rim11 . MTs were visualised by integrating a GFP–Tub1- or CFP–Tub1-encoding plasmid into the im11 see D. Mck1–Gt-test of unequal variance. The number of measurements was large enough to allow generation of histograms. aMT lengths are stochastic and expected to follow a normal distribution.Statistical comparison of datasets was made by using a two-tailed 2, 0.5 mM ATP, 5% glycerol, 0.1% Tween-20, 15 mM sodium pyrophosphate plus protease inhibitors. Purification was carried out in a two steps by using Blue Sepharose (6 Fast Flow) and Ni2+-Sepharose .All proteins were expressed in SF9 cells from a pFastBacM13 vector integrated in its corresponding Bacmid system carried by DH10Bac cells  according to Yeast cells expressing Mck1-TAP expressed from its genomic locus were lysed in 50 mM Tris pH 7.5, 300 mM NaCl, 1.5 mM MgCl, 0.5 mM DTT, 0.1 mM ATP, 10% glycerol, 0.01% NP-40, complete protease inhibitor and 1 mM β-phosphoglycerate. Bait protein was allowed to bind to IgG-Sepharose  and bound protein was eluted by incubating with TEV-protease.2, 1 mM EGTA, 0.01% NP-40 plus protease inhibitors  and purified using standard conditions. After elution, the proteins were dialysed against the lysis buffer, aliquoted and snap-frozen in liquid nitrogen.Cells expressing the corresponding proteins from the pET28 vector in the Rosetta host strain  were lysed in 50 mM Tris pH 7.5, 200 mM NaCl, 1 mM MgCl2, protease- and phosphatase-inhibitors and 0.1% NP-40 (better resolution of phosphospecies). For visualisation of Kip2 phosphoisoforms cell lysates were analyzed by SDS-PAGE (6%) and western blotting. Antibodies used for western blots were primary antibodies anti-Myc  anti-HA , anti-GFP , anti-Arc1 , anti-His , rabbit PAP, polyclonal  and anti-tubulin , and secondary antibodies anti-mouse IgG peroxidase (Fc-part) and anti-rabbit IgG peroxidase (entire molecule) . For cycloheximide-chase cells were grown in selective medium, protein translation was stopped by addition of 500 µg/ml cycloheximide and samples were analysed by western blotting. For CIP-treatment lysates were treated with 0.5 u/µg protein CIP or phosphatase inhibitor cocktail II (Sigma-Aldrich). The reaction-mix was incubated for 30 min at 37°C.Protein extraction was performed as described in  or with GAL1P-dependent expression was induced at OD0.5 for 4 h by adding galactose to 2% final concentration. Cells were lysed in 50 mM Tris pH 7.6, 150 mM NaCl, 1 mM MgCl2, protease- and phosphatase-inhibitors and 0.1% NP-40 and proteins precipitated overnight with 2 µg anti-haemagglutinin (HA) antibody  and protein-A–Sepharose.Infected SF9 cells were harvested, washed twice with PBS and fixed with 4% paraformaldehyde. After washing with PBS, permeabilisation with 1% Triton X-100 in PBS for 10 min and additional washing with PBS, cells were blocked in PBS+0.1% Tween+1% BSA for 1 h at 4°C. Cells were stained overnight at 4°C with anti-tubulin antibody , washed three times for 10 min with PBS and incubated for 1 h at 4°C with secondary antibody . Cells were again washed three times with PBS and mounted for microscopy.0.5 in low phosphate YPD; 50 ml were harvested and re-suspended in 1 ml low-phosphate YPD with 4% glucose. Cells were then grown for an additional hour in the presence of 500 µCi/ml inorganic 32P phosphate. After cell lysis, Kip213myc was immunoprecipitated (see above) using anti-Myc  antibody for 2 h at 4°C and analyzed by western blot.Cells were grown to an OD6-tag of eGFP–Kip2 was cleaved off by His6-TEV protease. Uncleaved His6–eGFP as well as the TEV-protease itself was removed with metal affinity beads . Six pmol of purified eGFP–Kip2 were incubated with 2.5 pmol purified Mck1-CBP for 30 min at 30°C in kinase-buffer . For the subsequent in vitro binding assay, an excess of His6mCherry–Bik1 (120 pmol) or His6Bim1 (300 pmol) was added and the buffer adjusted to 150 mM NaCl (plus complete protease inhibitor). Proteins were allowed to bind to metal affinity beads for 1 h at 4°C and beads were washed five times with IP-Buffer (see above). Supernatants and beads were analyzed by SDS-Page followed by western blotting or autoradiography.The Hisg at 28°C through a 40% glycerol cushion. His6eGFP–Kip2 was cleared for 15 min at 67,000 g and 28°C. 7 pmol Kip2 and ∼40 nmol tubulin were incubated for 20 min at 28°C. Bound Kip2 was again separated by centrifugation through a glycerol cushion. Supernatants and MT-containing pellets were analyzed by SDS-PAGE and western blotting as described above.MT polymerisation of 200 mM tubulin was induced by 5% glycerol and 1 mM GTP for 30 min at 37°C. Tubulin was diluted to 10 mM final concentration in 50 mM Tris pH 7.0, 150 mM KCl supplemented with 10 mM Taxol or 30 µM nocodazole. Polymerised and non-polymerised tubulin was separated by centrifugation at 67,000


Apolipoprotein E Receptor 2 (ApoER2) and the tyrosine kinase Fyn are both members of the Reelin pathway, a signaling pathway essential for the laminar formation of the cortex during development and proper dendritic spine density and long-term potential (LTP) in the adult brain. In the presence of extracellular Reelin, ApoER2 binds the intracellular protein Dab1, an adaptor protein that is phosphorylated by Fyn. However, direct interactions between ApoER2 and Fyn are not well defined. Here, we show that total levels of ApoER2 and surface levels of ApoER2 are increased by active Fyn. Via a separate mechanism, ApoER2 is also phosphorylated by Fyn, an event that peaks in the postnatal cortex at day 5 and can occur at multiple ApoER2 tyrosine residues. Dab1 is also involved in this phosphorylation, promoting the phosphorylation of ApoER2 by Fyn when it is itself phosphorylated. These results elucidate some of the intracellular mechanisms that give rise to a functional Reelin pathway. The Reelin pathway helps to establish the laminar organization of the cortex, hippocampus, and cerebellum during brain development, and has a role in dendritic spine density and long-term potentiation (LTP) in the adult brain ApoER2 is a type I transmembrane protein with a large extracellular tail (705 amino acids) and a shorter intracellular tail (115 amino acids) Fyn and Src are very similar to Reelin knockouts, but Fyn seems to be the more crucial kinase as Fyn knockouts exhibit an intermediate phenotype and Src knockouts appear normal Fyn is a non-receptor tyrosine kinase of the Src family kinases that is myristoylated and associated with the cell surface in vitro and in vivo. Fyn also increases the interaction between ApoER2 and Dab1, and the presence of phosphorylated Dab1 increases Fyn’s ability to phosphorylate ApoER2. Together these results clarify interactions between ApoER2 and Fyn and reveal a novel detail–ApoER2 phosphorylation–of the Reelin pathway.By binding Reelin extracellularly ApoER2-myc and mutant ApoER2 constructs were created using the QuikChange Lightning Site-Directed Mutagenesis kit (Stratagene), according to the manufacturer’s protocol. Double-tagged ApoER2 (HA-ApoER2-myc) was created by inserting an HA tag after the endogenous ApoER2 signal peptide of the ApoER2-myc construct. ApoER2 lacking the intracellular domain (ΔICD) was created by inserting a stop codon into HA-ApoER2-Myc after W730, leaving only three intracellular amino acids intact. Untagged ApoER2 containing exon 19 and ApoER2 lacking exon 19 were kind gifts from Dr. Joachim Herz (University of Texas Southwestern). Tyrosine mutants to both the double tagged ApoER2 and the untagged ApoER2 were created sequentially, with single point mutations that changed each tyrosine to a phenylalanine. Whenever possible, experiments were performed on both sets of constructs (tagged and untagged) and representative blots are shown for one set for simplicity. VLDLR-myc was used as previously described in vivo immunoprecipitation blots, for which anti-ApoER2 was used at 1∶500.We used antibodies anti-ApoER2 , anti-Fyn (Upstate), anti-tubulin (Sigma), anti-β-actin (Sigma), 4G10 (Millipore), anti-GFP (Invitrogen), anti-myc (abcam), and anti-HA (Abcam). All antibodies were used at 1∶1000 dilutions, except for 2. Cells were transiently transfected with 0.67–2 µg of total plasmid DNA using FuGENE 6 (Roche) according to manufacturer’s protocol. After twenty hours, cells were collected in RIPA Lysis Buffer  or IP buffer (see below). For PP2 and cycloheximide treatments, media was changed to serum free media the day after transfections. PP2  or DMSO was added to the media  for 6 hours, after which cells were collected in RIPA buffer or IP buffer. Cycloheximide  was added to the media  for 0, 15, 30, 60, 120, and 360 minutes after which cells were collected in RIPA buffer.COS7 cells  supplemented with 2% B-27 (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 0.25% 100X glutamine and 0.1% of 10 mM glutamate (Invitrogen). After 5 days Cells were co-transfected with ApoER2 and either Fyn or empty vector. A six-well plate was used for each condition. After twenty hours in culture, the media was aspirated from all wells and phosphate-buffered saline (PBS) with or without Sulfo-NHS-SS-Biotin  was added to 5 of the 6 wells.  After 30 minutes, cells were treated with Quenching Solution (Pierce), washed in Tris-buffered saline, collected in Lysis Buffer (Pierce), and sonicated. Lysates were added to a column with immobilized NeutrAvidin gel (Pierce) for 1 hour at room temperature. Columns were washed four times and incubated with SDS-PAGE sample buffer containing dithiothreitol. Surface proteins were then analyzed by Western blot. Briefly, the samples were separated by SDS-PAGE on polyacrylamide gels, transferred electrophoretically to nitrocellulose, and blocked with 5% nonfat dry milk before overnight incubation with primary antibodies.Transfected COS7 cells were collected in immunoprecipitation (IP) buffer: 50 mM Tris-HCl (pH 8.0), 4.5 mM NaCl, 1% Nonidet P-40, with phosphatase inhibitors (Sigma), and protease inhibitors (Roche). Lysates were pre-cleared with sepharose beads for 30 minutes at 4°C. Protein-G or protein-A sepharose beads  were incubated with antibodies for 1 hour at 4°C. Beads were collected by centrifugation at 4000×rpm for 5 minutes. The beads bound to the antibodies were then incubated with the pre-cleared lysate overnight. Bead-protein complexes were washed 5 times with IP buffer and resuspended in SDS sample buffer containing dithiothreitol. The samples were separated by SDS-PAGE. Samples defined as “Input” were lysates not incubated with beads or antibody; the percentage refers to the amount of sample loaded onto the gel compared to the amount added to beads.Fyn knockout mice were obtained from Jackson Laboratory. Controls were wild-type C57BL6. For comparisons of ApoER2 levels between knockout and wild-type mice, brains from three-week-old mice were homogenized in tissue homogenization buffer containing 250 mM sucrose, 20 mM Tris base, 1 mM EDTA, 1 mM EGTA, and protease inhibitors. Samples were further extracted in RIPA buffer, as described previously All animal experiments were conducted in compliance with the rules and regulations of the Institutional Animal Care and Use Committee at Georgetown University under animal protocol number 12-044. p<0.05. All experiments were performed a minimum of three times.All data were analyzed using analysis of variance (ANOVA) with Graphpad Prism 4 software. Bonferroni’s multiple comparisons test was used for post hoc analyses and significance determined as in vivo, comparing young wild-type and Fyn knockout mice. ApoER2 levels in brains of Fyn knockout animals were significantly lower by half compared to wild-type animals (in vitro. COS7 cells were co-transfected with myc-tagged ApoER2 and either empty vector (as a control) or constitutively-active Fyn. In the presence of Fyn, ApoER2 levels were significantly increased by 100% , or kinase-dead Fyn. All constructs were expressed in these cells. As above, ApoER2 levels were significantly increased in the presence of constitutively-active Fyn . HoweverTo test whether the kinase activity of Fyn affects endogenous ApoER2, we treated cortical primary neurons with PP2. First, we tested the PP2 treatment was working in these cells by running a 4G10 blot for total phosphorylated proteins. Phosphorylated proteins decreased in the presence of PP2, as expected . The difMurine ApoER2 has three intracellular tyrosines  that could be phosphorylated by Fyn. Y745 and Y769 are contained in exon 18, while Y776 is in the alternatively spliced exon 19. Since Fyn’s kinase activity contributes to the increase in ApoER2 levels, we hypothesized that the intracellular ApoER2 tyrosines were necessary for this effect of Fyn. We created ApoER2 constructs containing point mutations that changed the tyrosines to phenylalanines. Using a double mutant with both exon 18 tyrosines (Y745 and Y769) mutated or a construct lacking exon 19 (and Y776), we found that Fyn still increased ApoER2 levels (data not shown). Next we created a construct lacking the intracellular domain of ApoER2 (ΔICD), and thus all the intracellular tyrosines . In thisWith the ApoER2 constructs we created lacking intracellular tyrosines, we were able to ask if Fyn phosphorylated ApoER2. To assay tyrosine phosphorylation of ApoER2, we transfected cells with untagged wild-type ApoER2 and empty vector or Fyn, as above. Samples were immunoprecipitated with the phosphotyrosine antibody 4G10 and probed for ApoER2. In the presence of Fyn, but not empty vector, tyrosine-phosphorylated ApoER2 was detected . By compWe next investigated which tyrosine or tyrosines in ApoER2 are phosphorylated. We mutated each intracellular tyrosine to a phenylalanine. Both single mutants  and a triple mutant with all three tyrosines mutated (tYF) were created using the double-tagged ApoER2 as a template . Cells wAs above, wild-type ApoER2 was phosphorylated in the presence of Fyn . HoweverEach of the single mutants  was also phosphorylated in the presence of Fyn , indicatThe phosphotyrosine binding domain of Dab1 interacts with ApoER2 in vitro system, we transfected COS7 cells with Dab1 and either empty vector or Fyn  is removed. Therefore, Fyn has two separate effects on ApoER2 via different mechanisms.ApoER2 is enriched in caveolae Fyn’s effect on ApoER2 levels is likely not direct, but could be due in part to an as yet unknown interacting protein that is phosphorylated by Fyn and interacts with ApoER2 extracellularly. It should be noted that, when making the ApoER2 ΔICD construct, we kept the first three intracellular amino acids intact to ensure the construct would be correctly inserted into the membrane. It is possible that interactions with Fyn–a protein present at the membrane Degradation of Dab1 has already been identified as one mechanism for termination of the Reelin signal in vivo is controversial We observed decreased degradation of ApoER2 in the presence of Fyn. Two independent pathways are responsible for ApoER2 degradation: ubiquitination by the E3 ligase IDOL (inducible degrader of Low Density Lipoprotein Receptor) and binding by PCSK9 (proprotein convertase subtilisin/kexin type 9) In vivo we found that total ApoER2 levels in brain decline steadily with age. Others have shown that both ApoER2 and Reelin levels are highest in the early postnatal cortex and decline rapidly thereafter Fyn’s increase of ApoER2 levels could act as a positive feedback loop, promoting Reelin signaling. Although endocytosis via ApoER2 is slow, Reelin increases endocytosis in vitro findings that Fyn phosphorylates ApoER2. However, evidence for direct phosphorylation of ApoER2 by Fyn will depend on experiments using purified components of this system.As opposed to total ApoER2, which decreased consistently with aging, phosphorylated ApoER2 peaked at postnatal day 5 in the cortex, a time point that also had a peak in Fyn levels. Compared to the cortex, the hippocampus had both lower Fyn levels and undetectable levels of ApoER2 phosphorylation. Although these data are only correlative, the alignment between levels of Fyn and levels of phospho-ApoER2 in these regions at the time points examined  supports our Src family kinases (SFKs) such as Fyn can be activated in a number of ways. In the inactive, closed conformation, the SH2 and SH3 domains of SFKs interact with and repress the catalytic domain We found that phosphorylation of ApoER2 occurs at multiple tyrosines, as phosphorylation was lost only when all three tyrosines were mutated. It may be that only two tyrosines are phosphorylation sites and future studies using double mutants could address this question. One of the tyrosines, Y745, is within the NPXY domain, a well-characterized motif that signals internationalization of extracellular ligands Reelin increases the interaction between ApoER2 and the phosphotyrosine binding domain of Dab1 Alternatively, while Dab1 interacts with the NPXY domain, Fyn could interact nearby at Y769. The ability of ApoER2 to interact with both molecules simultaneously and bring them together could lead to downstream signaling. Finally, the third tyrosine, Y776, is within exon 19, an alternatively spliced exon that could allow for another level of regulation. However, Y776 is not conserved in humans (NCBI Accession #004631.4) and there is no homologous site in VLDLR (NCBI Accession #013703.2), making it unlikely to be a tyrosine of critical importance for signal regulation.In this study we focused on the effects of Fyn levels on various signaling molecules. In addition, we have found that in primary neurons Reelin treatment increases the amount of phosphorylated ApoER2 (data not shown). Together with our results that inhibition of Fyn activity blocks its effects on ApoER2 levels, these findings support the hypothesis that alterations in Fyn activation produce our observed effects.ApoER2 knockout mice and Fyn knockout mice have reduced spine density in the cortex The ultimate functional downstream effects of ApoER2 phosphorylation remain to be elucidated. ApoER2 phosphorylation could turn off Reelin signaling (possibly by leading to the disruption of Dab1 binding) or promote further downstream signaling. The phosphorylation observed here occurs after the cortical layers are established Based on these findings, we propose the following mechanism for ApoER2 phosphorylation. Extracellular binding of Reelin induces clustering of ApoER2 and activates Fyn In conclusion, our results clarify interactions between ApoER2 and the kinase Fyn. Through two separate mechanisms, Fyn increases both surface levels of ApoER2 and phosphorylation of ApoER2, and phosphorylation of ApoER2 is further promoted by Fyn phosphorylation of Dab1. ApoER2 phosphorylation occurs early in post-natal life, revealing a novel mechanism in Reelin signaling.


The transporters for norepinephrine and dopamine  constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs. Transporters for the biogenic monoamine neurotransmitters norepinephrine, dopamine and serotonin  are integral membrane proteins that regulate monoaminergic signalling in the brain by performing sodium- and chloride-coupled uptake of neurotransmitters from the extracellular space into neurons467Drosophila melanogaster DAT (dDAT)101112141513171920212224252624282930313233Current structural understanding of human MATs is based on x-ray crystal structures of bacterial and invertebrate homologs, which include the bacterial amino acid transporters LeuT and MhsT and the en route to the binding site by residues in distal permeation sites close to the receptor surface45Resolving the molecular differences among NET, DAT and SERT that control selective inhibitor binding is important for structure-based design of MAT inhibitors with fine-tuned selectivity profiles. Within the S1 site, non-conserved residues can confer important differences among MATs in the shape and physicochemical properties of the inhibitor binding pocket, and importantly change the number and strength of direct interactions with the bound inhibitor molecule. The central location of the S1 site means that inhibitors must traverse from the extracellular space through a funnel-like extracellular entrance pathway, that include the S2 site, to reach the S1 site . Thus, n103621293338394041The human NET and DAT  share a particular high homology with almost 80% overall identity in their amino acid sequence , and bot48505142In the present study, we systematically examine the role of non-conserved residues in hNET and hDAT that are located within the S1 site and in the extracellular permeation pathway, including the S2 site and EL4, for selective inhibitor recognition. We find that non-conserved S1 residues in hNET are primary determinants for inhibitor selectivity. Surprisingly, the corresponding residues in hDAT do not hold an equivalent important role for inhibitor selectivity. This indicates that selective inhibition in these two closely related MATs is achieved by distinct mechanisms. Our findings provide new insight into the molecular basis for inhibitor selectivity between hNET and hDAT, which may be used in the rational design and development of novel therapeutic agents targeting hNET and hDAT with tailor-made selectivity profiles.In previous studies, we examined the role of non-conserved residues within the S1 site of human SERT (hSERT) and hNET for inhibitor selectivity by mutating all non-conserved residues to the corresponding residues in the other transporter, and thereby in principle transferred the S1 site from hNET into hSERT and vice versa. Hereby, we demonstrated that non-conserved S1 residues are key determinants for the hSERT/hNET selectivity of the two prototypical SERT selective inhibitors, escitalopram and fluoxetine293H]dopamine  hNET and hDAT in COS-7 cells and assessed for their ability to transport [dopamine . Four ofdopamine . For thedopamine . We also5I]β-CIT . Further5I]β-CIT . In summ3H]dopamine uptake inhibition assays  and DAT-(NET S2) . As prevng assay . Furtherng assay , suggestKi compared to the shifts for the corresponding DAT-(NET S1) mutant for all inhibitors except rimcazole  of binding. Recently, differences in kon for some inhibitors, including methylphenidate and desipramine, was shown to be a key determinant for their ability to discriminate between hDAT and hSERTOur finding that the pharmacological profile of ten MAT inhibitors at hDAT is largely unaffected by mutation of non-conserved S1 residues, suggest that residues outside the inhibitor binding site are controlling inhibitor selectivity in hDAT. Interestingly, molecular dynamic simulations have revealed that high affinity binding for certain ligands in G protein-coupled receptors is regulated in a distal entry site located at the receptor surface45en route to their high affinity binding site, including transient transporter-inhibitor interactions in the extracellular entry pathway and conformational transition states, that contribute to MAT inhibitor selectivity.The location and identity of the determinants outside the high affinity site that control inhibitor affinity in MATs (for example by influencing the association rate of inhibitor binding) remains enigmatic. The extracellular entrance pathway is an obvious candidate, and in the present project we focused on the role of non-conserved residues in the S2 and EL4 regions of the extracellular entry pathway. For S2, we found that non-conserved residues within this region are minor determinants for inhibitor selectivity . Unfortu54In summary, our findings demonstrate that non-conserved residues within the primary binding site in hNET and hDAT have differential roles for selective inhibitor recognition, and thus highlight an emerging pattern of a highly complex molecular pharmacology among closely related neurotransmitter transporters. Our data provide new insight into the molecular basis for selectivity of therapeutic and recreational drugs towards hNET and hDAT, which we believe will be important for future design of novel ligands with tailor-made selectivity towards MATs.3H]dopamine (30–90 Ci/mmol) and [125I]β-CIT  was purchased from PerkinElmer . Talopram, nisoxetine, imipramine, atomoxetine, GBR 12,909, bupropion and rimzazole were available from the H. Lundbeck A/S compound collection. Reboxetine and cocaine were purchased from Sigma-Aldrich , JHW 007 from Tocris Bioscience  and β-CIT from ABX . and Asn341 to Thr432 in hDAT [in which the sequence encoding the EL4 region (Leu368 to Trp407) was substituted for the corresponding hNET sequence] was purchased from GeneArt. The synthetic DNA was digested with MfeI and AgeI and then ligated into MfeI/AgeI digested pCI-IRES-neo-NET and pCI-IRES-neo-DAT using the Rapid DNA ligation kit . The sequence integrity was verified by restriction digests and DNA sequencing .For transferring the EL4 region between hNET and hDAT, a unique and silent 2 environment. Cells were transfected using TransIT LT-1 DNA transfection reagent by following the procedure supplied by the manufacturer, and dispensed into either poly-d-lysine coated white 96-well plates (for uptake assays) or 150 mm tissue culture Petri plates (for membrane preparations).COS-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified 5% CO2HPO4, 1.4 mM KH2PO4, 0.1 mM CaCl2 and 0.5 mM MgCl2) on an ELx50 automated microplate strip washer . In all uptake experiments, the PBSCM buffer was supplemented with 1 mM ascorbic acid and 1 μM of the catechol-O-methyltransferase inhibitor Ro 41–0960 (Sigma-Aldrich) and pH was adjusted to 7.4.Uptake assays were performed 40–48 h after transfection. Transiently transfected COS-7 cells growing in 96-well plates were washed twice with PBSCM buffer , cells were preincubated with 40 μL of PBSCM per well with no or increasing concentrations of inhibitor for 30 min before addition of 10 μL of PBSCM containing [3H]dopamine . Uptake was allowed to proceed for 5 min at room temperature, before uptake was terminated by washing three times with PBSCM. For determination of KM values, cells were incubated with increasing concentrations of unlabelled dopamine in PBSCM and 25–50 nM [3H]dopamine at room temperature for 5 min. Uptake was terminated as described above. The amount of accumulated [3H]dopamine was determined by solubilising cells in scintillant cocktail (MicroScint-20) followed by counting of plates in a Packard TopCounter. Non-specific uptake was determined as uptake in non-transfected cells and total uptake was determined in the presence of PBSCM buffer alone. Assays were carried out in triplicate and repeated at least three times.In inhibition studies (ICg) for 5 min, and the cell pellet was suspended in cold H2O and frozen at −20 °C for at least 1 h. The suspension was thawed on ice and subjected to 10–15 passages through a 21 gauge needle to disrupt cells. Homogenate was transferred to 2 mL tubes and centrifuged at 18.000 × g for 30 min at 4 °C. Supernatant was aspirated, and the pellet was suspended in PBSCM buffer. Protein concentration was determined by the BCA method using Pierce BCA Protein Assay. Membranes were used directly for binding assays or stored at −80 °C until use.Cell membranes were prepared 40–48 h after transfection. Transiently transfected COS-7 cells growing in 150 mm tissue culture Petri plates were washed with PBS supplemented with 1 mM EDTA to detach from the plate. Cell suspension was centrifuged  preincubated with 0.1% polyethyleneimine (30 μL per well) by using a Packard Bell cell harvester and washed four times with water. Nonspecific binding was determined in parallel at membranes from nontransfected COS-7 cells. Filter plates were dried and soaked in scintillant cocktail (MicroScint-0) followed by counting of plates in a Packard TopCounter. For competition binding assays, 2–30 μg total membrane protein was incubated with 0.2 nM [125I]β-CIT in the presence of no or increasing concentration of inhibitor in PBSCM by using the same protocol as for saturation binding experiments. Binding assays were carried out in duplicate and repeated at least three times.For saturation binding studies, increasing concentrations of [Ki) for inhibitors were calculated from IC50 values determined in functional uptake assays by using the equation: Ki = IC50/(1 + ([L]/KM)), where [L] is the concentration of [3H]dopamine and KM is the Michaelis-Menten constant for dopamine. The binding affinity (Ki) for inhibitors were calculated from IC50 values determined in competition binding assays by using the equation: Ki = IC50/(1 + ([L]/KD)), where [L] is the concentration of [125I]β-CIT and KD is the dissociation constant for β-CIT. All data are expressed as mean ± s.e.m. from at least three independent experiments carried out in triplicate (for uptake assays) or duplicate (for binding assays). Statistical analyses were performed by using one-way analysis of variance (ANOVA). Values of p < 0.05 were considered statistically significant.All data analysis was performed using GraphPad Prism version 6.0 . The inhibitory potency .


Left ventricular non-compaction (LVNC) is an unclassified cardiomyopathy and there is no consensus in the diagnosis of LVNC. The aims of this study were to establish quantitative method to diagnose isolated LVNC (INC) using cardiac magnetic resonance (CMR) imaging and to suggest novel qualitative method to diagnose isolated LVNC.This retrospective study included 145 subjects with moderate to severe trabeculation of LV myocardium . LVNC patients had to fulfill Petersen's CMR criteria. LV ejection fraction, global LV volume, trabeculated LV volume, and number of segments with late gadolinium enhancement were measured. And most prominent noncompacted (NC), compacted (C), normal mid-septum, normal mid-lateral wall and apical trabeculation thickness on the end-diastolic frames of each long-axis slice was also measured.Results In the patients with isolated LVNC, the percentage of trabeculated LV volume was 1.4 times higher (42.6 ± 13.8) than in DCM [YHC1], and 1.7 times higher than in controls . However, there were no significant differences between INC and DCMNC . And a value of percentage of trabeculated LV volume above 32% was predictive of isolated LVNC with a specificity of 90.3%  and sensitivity of 79.2% . A value of NC/septum over 1.1 was considered predictive for isolated LVNC with a specificity of 80.6%  and sensitivity of 95.8% . And a value of apex/C above 3.1 was considered predictive of isolated LVNC with a specificity of 93.5%  and sensitivity of 87.5% .As a quantitative approach, a trabeculated LV volume above 32% of the total LV volume is diagnostic for isolated LVNC with high sensitivity and specificity. And as a qualitative approach, apex/C and NC/septum ratio could be useful for supplemental diagnostic criteria.None.


Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 μM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs.Human parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive  Human parainfluenza viruses (hPIVs) are the second most prevalent cause of acute respiratory tract infection in infants, after human respiratory syncytial virus. Among parainfluenza viruses, hPIV-3 is the principal causative agent of disease in infants1245N-acetylneuraminic acid, Neu5Ac) residues71011121314161718hPIV-3 has two surface envelope glycoproteins, a haemagglutinin-neuraminidase (HN) and a fusion protein (F). HN plays three roles in the life-cycle of the virus namely, 1) attachment of the virus to host cell sialylglycoconjugate receptors that contain α2-3 or α2-6-linked terminal sialic acids , one of the first inhibitors of a broad range of neuraminidases 2117231–4, Most of the compounds that have been investigated as anti-hPIV-3 agents are competitive inhibitors of HN. They are primarily 5, In an effort to contribute to the discovery of potent inhibitors of hPIV-3 infection, we have recently published a series of studies focused on the hPIV-3 HN protein. We demonstrated, using Molecular Dynamics simulations, that the protein possesses a flexible loop in the vicinity of the active site around Asp216. The cavity formed by an open 216 loop can accommodate more of the receptor than just the terminal Neu5Ac moiety of a sialylglycoconjugate that may provide broader receptor specificity and contribute to host cell tropismN-acylneuraminic acids, that may inhibit hPIV-3 HN haemagglutination and neuraminidase functions. Such drugs could provide an alternative approach for hPIV-3 infection blockade and have an advantage for potential repositioning. Through this approach, we have successfully identified suramin (7), a trypanocidal drug commonly prescribed for the treatment of sleeping sickness in Africa2728in vitro antiviral potency and acts synergistically when combined with competitive inhibitors of HN. Our study shows that compounds other than N-acylneuraminic acid derivatives can indeed inhibit neuraminidases by binding, in the case of the hPIV-3 HN, in a region of importance in close proximity to its active site.In the present study we look into another strategy to block HN-mediated hPIV-3 infection, by investigating approved drugs, not related to N-acetylneuraminide  α-D- MUN, 6, . The dru50 above 100 μM. Out of 1280 drugs, three inhibitors of the hPIV-3 neuraminidase activity were identified and confirmed in a secondary screening (data not shown): suramin  as an inhibitor of hPIV-3 HN neuraminidase activity was further characterised by dose-response HN neuraminidase activity inhibition (NI) experiments. The NI assay was carried out following the conditions of the primary screening, using serial dilutions of drug. Suramin (7) was found to inhibit HN neuraminidase in a dose-dependent manner, with an IC50 of 13.5 μM , we challenged the capacity of HN to agglutinate guinea pig red blood cells (gpRBC) in the presence of suramin (7). We qualitatively observed the agglutination of gpRBC after 1 h 30 min of incubation with the virus and the drug at 4 °C. The dose required for suramin (7) to inhibit 50% of the HN haemagglutinin activity was ~200 μM, compared with 2.7 μM for compound 5 As the HN neuraminidase active site is also an 7) has been shown to inhibit some neuraminidases, its mechanism of action remains unknown31327) and the substrate MUN (6). In the setup of the neuraminidase activity inhibition experiment, we evaluated the initial velocity vi of the neuraminidase at several substrate concentrations ([S]). At each of these [S], we challenged the enzyme with a dilution range of inhibitor concentrations ([I]). Linear regressions of data points from Lineweaver-Burk plots using 7, M) value, which approximately corresponds to the HN affinity for the substrate, was calculated from this point of convergence and found to be of 30.3 ± 1.02 mM. The inhibition constant (Ki) of suramin (7) was equal to 5.06 ± 0.62 μM. In contrast, the enzyme’s maximum velocity (Vmax) was 0.068 mmol/sec and was reduced when suramin (7) was present in the reaction with apparent maximum velocities  on hPIV-3-infected LLC-MK2 cells by immunostained focus reduction assay. We evaluated the drug at binding stage stage at 4 °C and adsorption stage at 37 °C for an hour to evaluate the effect on virus binding (4 °C) as well as early events of infection including fusion (37 °C). In another experiment the drug was added post-virus adsorption at 37 °C to evaluate post-internalisation effects. Interestingly, suramin (7) had the most potent antiviral effect during virus binding at 4 °C with an IC50 value of 3.1 μM, showing that the drug efficiently blocked hPIV-3 HN receptor binding site and prevented entry (7) also inhibited infection during adsorption at 37 °C (binding and fusion events) but with a lesser efficacy (IC50 = 26 μM), and post-adsorption with an IC50 value of 35 μM. As shown on 7) could be accurately measured using automated high-resolution image treatments. While the number of foci remained constant, their size could be reduced down to the size of single infected cells.We evaluated the dose-dependent antiviral potency of suramin (ed entry . Suramin7) is a non-competitive inhibitor of HN showing antiviral potency in vitro, it may be expected to have a synergistic effect when combined with a competitive inhibitor, as both compounds would not bind the same region of the protein. Furthermore, as suramin (7) is an approved drug, we tested this hypothesis with the approved anti-influenza drug and competitive hPIV-3 HN inhibitor, zanamivir (3) to see if lower concentrations of both drugs could be used to block infection in vitro. We also evaluated combinations with compound 5, a recently designed hPIV-3 HN inhibitor, to observe if a synergy could also be observed with a better competitive inhibitor. We used the method of Chou and Talalay to design the experiments and assess the various combinations343550 value that was determined for each experiment  had an average IC50 value of 74.5 μM, compared with 61.5 μM for zanamivir (3) and 2.2 μM for compound 5. The slope m was also determined from a linear regression of median-effect plots  and compound 5 act in synergy with suramin (7) to efficiently block hPIV-3 infection. The CI-effect plot also allows the visualisation of combination effects, based on the combination index CI calculated using 7) and zanamivir (3) or compound 5.In this particular assay, suramin —zanamivir (3) or suramin (7)—compound 5 combinations to inhibit in vitro hPIV-3 infection, the stronger the effect, the higher the DRI. Although this result is as expected, it does not translate to high synergism as the DRI is calculated for individual drugs at a given effect in combination. Based on the DRI-effect plot, 100% inhibition of infection in combination is reached when the concentration of a compound can be reduced more than 10-fold as compared with the concentration needed to reach the same effect, individually. In addition, as shown in 50 value of suramin (7) and zanamivir (3), and 2-times the IC50 value of suramin (7) and compound 5 used in combination are sufficient to inhibit 100% of in vitro hPIV-3 infection, compared with 4-times the IC50 value for any drug used individually.The fact that the compounds show synergism means that their concentration in combination yields an effect that is stronger than individual compounds at a similar or higher concentration. This property of synergistic inhibitors can be evaluated via a calculation of the drug reduction index DRI, , for eac7) and competitive inhibitors of HN work in cooperativity to block in vitro infection, and because HN possesses a unique, dual-function active site, we hypothesised that suramin (7) may bind in close proximity to the active site. To verify this hypothesis, and to confirm the non-competitive inhibition of suramin (7), we followed another approach using a competition-Saturation Transfer Difference (STD)-NMR experiment in presence of the approved drug zanamivir (3). STD NMR spectroscopy is a dynamic technique that enables the determination of how ligands engage proteins in solution193) to purified hPIV-3 particles upon addition of suramin (7), as well as the change in binding of suramin (7) upon addition of zanamivir (3).Because suramin  were higher than those of zanamivir (3), mainly due to the perfect symmetry of the molecule and to the consequent amplification of the signal of each proton.As expected, their ensities  and STD-ensities . The STDversus c). The STD-NMR intensities of suramin (7) remained unchanged upon addition of zanamivir , however, were increased by ~30% upon addition of suramin  was added before zanamivir (3), which suggests that suramin (7) binding to HN modifies the architecture of the primary binding site, thus influencing the zanamivir (3) binding event.The sequential order in which the drugs were added to the biological sample did not influence the final STD-NMR spectra of the drug combinations versus cmivir 3, . The STDramin 7, . Similar7) on HN, we performed some initial in silico docking simulations using available hPIV-3 HN structures 7) and reduced the drug’s structure to leave a terminal R-NHCONH2 moiety with R being the polycyclic half of the molecule  from the available structure 1V3E was re-docked into the HN active site. As shown in 3). The simulated yellow and orange zanamivir structures were in opposite orientations when compared to zanamivir  bound in the original crystal structure, however the simulated magenta zanamivir (3) structure was positioned very close to the original zanamivir -bound structure with an RMSD = 0.46 Å (RMSD: root mean square deviation).To establish a putative binding site for suramin (h (8) molecule on hPIV-3 HN using Autodock Vina on 36 overlapping search spaces covering an entire monomer (h (8) binding conformations were generated for each of the 36 search spaces. In the apo-structure, the lowest energy structure of suraminh (8) was positioned in the HN active site -bound structure the 2 lowest energy complexes with 8 were identified near the HN active site where 8 covered zanamivir (3)  as a probe, that suramin (7) is most likely to bind in the vicinity of the HN active site. To confirm our prediction of the orientation of suramin (7) to the HN active site region, dockings were performed on the zanamivir (3)-bound structure using the full suramin structure 7. right, shows a dominant cluster of 8 conformations of 7. In this cluster, the naphthalene trisulfonate groups engage basic residues within the HN active site, while the rest of the molecule fills the space in front of the HN binding site. Further refined simulations of 7 and 8 on the apo structure failed to yield significant clustering of the naphthalene trisulfonate moieties, although binding was restricted to the active site region.We then designed a blind-docking simulation experiment to probe the potential binding site of the suramin monomer . A totalive site . The expivir (3) . A refin7) as a novel inhibitor of hPIV-3 HN. We found that it acts in synergy with competitive inhibitors of HN to block hPIV-3 in vitro infection and thus is a non-competitive inhibitor for the enzyme.Our approach in this study was to discover novel scaffolds of hPIV-3 HN neuraminidase activity inhibitors that could be repositionable drugs and work either synergistically with other inhibitors (or drugs), or act as a tool to better understand the mechanism by which the protein’s functions are regulated. Following a target-based drug discovery strategy, we identified the approved drug suramin (7) by enzyme-based semi-high throughput screening of approved drugs on the neuraminidase function of HN. This is, to our knowledge, the first such study conducted on human parainfluenza virus neuraminidase. The drug, not related to the sialic acid family, was found to have activity both on the neuraminidase and the haemagglutinin functions of HN.We determined the anti-hPIV-3 neuraminidase activity of suramin (7) showed that the drug did not compete with the substrate. While the apparent maximum velocity of the enzyme changed significantly in presence of drug, its constant of affinity for the substrate remained unaffected. The binding of suramin (7) and the MUN (6) substrate to HN are therefore unaffected by one another, meaning that they bind to distinct sites on the protein.The kinetic analysis we conducted on the hPIV-3 sialidase with suramin (7) would inhibit both entry and release in vitro if it bound closely to the sialic acid recognition site. We found suramin (7) to be the most potent at binding stage in vitro, although it is also active post-adsorption. This indicates that suramin (7) prevents the binding of HN to sialic acid-containing receptors, as well as their cleavage. The lack of molecular and cell dynamics in a 4 °C environment could be responsible for a longer-lasting binding event between suramin (7) and the virus and cells. Most importantly, unlike at 37 °C, at 4 °C the hPIV-3 fusion protein would not be engaged upon binding of the virus12Given that the HN protein possesses a single primary binding domain, suramin (N-acetylneuraminic acid-based drug zanamivir (3) was shown to have in vitro activity in several studies against hPIV-3, although its potency is in the high micromolar range15217), but we have shown that the two drugs can act in synergy to inhibit hPIV-3 infection in vitro. As a result, we showed that the dose of both drugs can be significantly reduced to reach high levels of inhibition, as compared to the individual dose that would otherwise be needed to reach a similar effect. In this study we also demonstrated that this feature of suramin (7) can be applied to better competitive inhibitors of hPIV-3 HN, such as compound 5. Similar levels of synergy as with zanamivir (3) can be achieved in combination with compound 5, but as it is more potent than zanamivir (3) even lower concentrations of drugs are needed. This data also confirms the non-competitive inhibition nature of suramin (7). Indeed, if suramin (7) bound to the active site, either suramin (7) or the competitive inhibitor would occupy the identical binding pocket and no synergism would be observed. However, suramin (7) must bind in proximity to the HN binding site in order to block the protein’s functions.The 7), we conducted competition STD-NMR experiments with suramin (7) and zanamivir (3) using purified virus particles. Suramin (7) appeared to bind more strongly than zanamivir (3) on HN, as determined by the intensities of STD-NMR signals. Suramin (7) and zanamivir (3) were both found to bind HN, whether they were in combination or not, confirming the non-competitivity of suramin (7) towards HN. The STD-NMR signals of zanamivir (3) however were enhanced by about 30% upon addition of an equimolar ratio of suramin (7). Similar STD-NMR signals of zanamivir (3) were found when suramin was added first. To-date, only a closed-216 loop structure of hPIV-3 HN has been observed in complex with zanamivir (3)253) in the presence of suramin (7) and hPIV-3 HN, it suggests that a faster exchange of zanamivir (3) from bound- to free-form occurs7) induces the 216 loop to open enabling this faster exchange.To further investigate this property of suramin (7) is a non-competitive inhibitor of HN, our results indicate that suramin (7) binds most likely in the vicinity of the active site. The extensive in silico analysis that we conducted in the present study substantiate this hypothesis. We probed the binding site of a truncated variant of suramin, suraminh (8), by blind docking simulations on 36 overlapping search spaces covering an entire HN monomer. In both the apo-form and the zanamivir (3)-bound forms of HN, the dockings of lowest energies were located around the active site. In a cluster of low energy structures, suraminh (8) was shown to bind in the vicinity of the zanamivir (3)-bound active site. We have also shown that a full-suramin (7) molecule orients towards the HN active site in a zanamivir (3)-bound structure, as supported by a dominant conformational cluster. This suramin (3) binding mode extends out of the binding pocket of the protein and may alter the zanamivir binding site as well as prevent binding of longer sialylglycoconjugates. Together, these results confirm what was observed in STD-NMR experiments and in vitro combination assays, and suggest that both drugs act in synergy to block hPIV-3 infection.Although suramin  and zanamivir (3) are approved drugs. Furthermore, we show that such a method has the potential to be applied using suramin (7) in conjunction with more specific hPIV-3 HN inhibitors. Suramin (7), originally a trypanocidal drug, has been found to be a promising clinical candidate for the treatment of enterovirus 71 infection in vitro and in vivoThis study presents an 2 in DMEM  supplemented with 10% FBS and penicillin/streptomycin. The human parainfluenza type-3 virus (strain C-243) was obtained from the American Type Culture Collection  and propagated in LLC-MK2 cells in un-supplemented DMEM. Virus stocks were prepared by infecting cells at a low multiplicity of infection (MOI) of 0.1. After 3 days of incubation, the virus-containing culture supernatant was clarified by centrifugation at 2000 × g for 15 min at 4 °C, aliquoted and stored at −80 °C for subsequent titration by focus forming assay.LLC-MK2 cells were provided by Institut Pasteur Shanghai’s Pathogen Diagnosis Centre and maintained at 37 °C; 5% CO5 and zanamivir (3) were synthesised in the Institute for Glycomics, as previously described19N-acetyl-2,3-didehydro-2-deoxyneuraminic acid, 2) was purchased from J & K Chemical , and the neuraminidase substrate MUN (2′-(4-methylumbelliferyl) α-D-N-acetylneuraminic acid, 6) was purchased from Sigma Aldrich .Compound g at 4 °C and the supernatants were discarded. The pellets were soaked at 4 °C overnight with 1/100th of the initial volume of GNTE buffer . They were resuspended on the next day by gentle up-and-down pipetting. The precipitated virus was pooled and kept at 4 °C for later use in neuraminidase assay.For neuraminidase activity inhibition assays (NI) and STD-NMR experiments, hPIV-3 prepared by propagation in LLC-MK2 cells at a MOI of 1 were precipitated by treating clarified virus-containing supernatant of infection with a 5X solution of sterile 40%PEG-6000. Tubes were kept at 4 °C for 2 h under gentle agitation, centrifuged for 15 min at 3000 × For STD-NMR experiments, the hPIV-3 particles were purified on a 30–60% non-linear sucrose gradient in GNTE bufferN-acetyl-2,3-didehydro-2-deoxyneuraminic acid, 2) was used as a positive control of inhibition at a final concentration of 10 mM (2 mM IC50 for hPIV-3’s HN50) of the hits was further determined by dose-response experiments.The USA-approved and International-approved drug libraries (1280 compounds) were purchased from MicroSource Discovery System, CT. The drugs were diluted in reaction buffer prior to testing in NI assay in duplicates, at a concentration of 100 μM. The compound Neu5Ac2en . Into the wells of black 96-well plates was introduced 7 μL of reaction buffer, 1 μL of compound, and 1 μL of virus. The plates were pre-incubated for 30 min at 37 °C before 1 μL of the fluorogenic neuraminidase substrate MUN (2′-(4-methylumbelliferyl) α-D-N-acetylneuraminic acid, 6) was added to the mixture at a final concentration of 2 mM. The plates were incubated at 37 °C for 30 min at 1000 rpm. The reaction was stopped in each well by addition of 150 μL of carbonated buffer (pH 10.8). The plates were read at an excitation wavelength of 335 nm and an emission wavelength of 460 nm in a Varioskan Flash Multimode Reader (Thermo Scientific). The Relative Fluorescence Units (RFU) values for each measurement were normalised to the blank (reaction stopped immediately after addition of substrate) and expressed as percentage of inhibition of hPIV-3 HN neuraminidase activity. The IC50 for NI assay has been defined as the average inhibitory concentration of a compound leading to a 50% reduction of hPIV-3 HN neuraminidase activity.The anti-neuraminidase activity of compounds was evaluated by NI assay. They were performed as described previouslyet al.et al.et al.6) [S]: 2, 4, 8, 10 and 20 mM, and 3 concentrations of inhibitor [I]: 0, 10 and 16 μM. The initial velocity vi of the enzyme for each [S]—[I] combination was determined automatically by the software provided with the Varioskan Flash Multimode Reader. A Lineweaver-Burke plot was represented for all [I], by plottingThe inhibition mechanism of hit compounds was determined by enzyme kinetics experiments on the hPIV-3 HN protein using a modified protocol of Ryu M of the enzyme for the substrate, as the intercept when Y = 0 equals Vmax of the enzyme without inhibitor as well as the apparent maximum velocities i of the inhibitor for the enzyme was calculated for each [I].and applying a linear regression using the software GraphPad Prism version 5 . The point of convergence of the 4 linear regressions helped determine the nature of the compound inhibition , and allowed the calculation of the Michaelis-Menten constant K50 for HI assay was defined as the compound concentration that results in a similar agglutination as the one observed in a control well containing only 0.5 HAU of hPIV-3 and gpRBC.Guinea pig red blood cells (gpRBC) were used for the haemagglutination inhibition (HI) assay. The HN inhibitors were assessed in duplicate as reported previouslyin vitro, dose-dependent potency of compounds was determined by immuno-stained focus reduction assay, using confluent cells in 96 or 48-well plates infected with 20–100 focus forming units (ffu) of hPIV-3 per well. To test inhibition of virus binding or adsorption (binding and fusion), compounds and virus in un-supplemented DMEM were applied to cells for 1 h at 4 °C or 37 °C, respectively. The inoculum was removed, the cells washed with PBS and overlaid with un-supplemented DMEM containing 0.75% carboxymethyl cellulose (CMC). The cells were further incubated for 72 h at 37 °C, 5% CO2. To assess inhibition of virus propagation, compounds were applied after 1 h of virus adsorption at 37 °C, for 72 h at 37 °C 5% CO2 in DMEM containing 0.75% CMC. The immuno-staining was performed by fixing cells for 20 min with 4% PFA in PBS at room temperature, followed by a 30 min incubation at 37 °C with a primary anti-hPIV-3 HN monoclonal antibody  diluted 1:2000 in PBS-5% skim milk, and a 30 min incubation at 37 °C with a 1:6000 dilution of secondary HRP-conjugated polyclonal antibody (goat anti-mouse IgG (H + L) – Bethyl, Montgomery, TX) in PBS-5% skim milk. Virus-infected cells were stained by addition of TrueBlue Peroxidase Substrate  until appearance of dark blue foci. The concentration of compound resulting in 50% inhibition (IC50) of virus binding (4 °C) or adsorption (37 °C) was determined by foci counting, while the IC50 for virus replication (post-adsorption) was determined by measurement of foci size. IC50 values were determined using non-linear regression analysis with the software GraphPad Prism .The Foci counts and size measurements were done using the software FijiThe toxicity of compounds towards LLC-MK2 cells was evaluated using a CellTiter-Glo Luminescent Cell Viability Assay , following the manufacturer’s instructions in the conditions of the focus reduction assay.In vitro chemical combinations were performed to assess the synergism or the antagonism of suramin (6) in presence of a competitive inhibitor of hPIV-3 HN to block infection. Infections were performed in a focus reduction assay format, in quadruplicates in 48-well plates with ~30 focus-forming units of hPIV-3 per well, and the compounds were added post-virus adsorption to mimic post-exposure treatment. The experiments were done following the method of Chou et al.50in vitro: 0.25-, 0.5-, 1-, 2-, and 4-fold of IC50. The synergism, antagonism, or additive effect of each of the combinations was assessed by calculating the combination index value (CI). According to Chou et al.361 and D2 are the doses of compound 1 and 2 respectively responsible for an effect x in combination, while D1x and D2x are the doses of compound 1 and 2 respectively responsible for the same effect individually. If CI < 1 the compounds have a synergistic effect, if CI > 1 they are antagonistic, and if CI = 1 additive effect is observed. A normalised isobologram was created by plotting the normalised concentrations x%, and the numerators represent the respective doses of compound 1 and compound 2 reducing viral load by x% in combination. The normalised concentrations in where DDm1 is the IC50 of compound 1 in vitro, fa is the fraction affected (or (% effect) ÷100), and m1 is the slope of linear regressions from median effect plots using the functionwhere x alone (D1x), to the concentration of the same compound resulting in an effect x in combination (D1):The CI-Effect graph representing the CI as a function of the associated antiviral effect was also plotted, as well as the log(DRI)-effect plot representing the log of the dose reduction index (DRI) as a function of the associated antiviral effect. The DRI is the ratio of the concentration of a compound resulting in an effect 2 5 mM, pD 4.6, following the manufacturer’s instructions. For the single-compound STD-NMR, the virus was resuspended into exchange buffer to a final volume of 200 μL and suramin (7) or the competitive neuraminidase inhibitor zanamivir (3) dissolved in D2O was added at a final concentration of 5 mM. A control STD-NMR spectrum was acquired for each compound, in absence of virus. For competition STD-NMR experiments, the sample was reprocessed after receiving an equimolar concentration of the other inhibitor. The 1D proton (1H) NMR and STD-NMR spectra were acquired at 600 MHz and 283 K, as described previouslyPurified hPIV-3 was firstly inactivated by exposure to UV light for 10 min. It was then loaded onto a 0.5 mL 30 kD cut-off concentration column  and the virus purification buffer was exchanged at least 4 times with deuterated NaOAc 50 mM, CaClapo and zanamivir (3)-bound structures of HN were taken from the Protein Data Bank, accession numbers 1V3B and 1V3E, respectively. Water molecules were removed, as well as B-chains to keep only one monomer. The half-suramin molecule ), truncated after the urea moiety (NHCONH2-term), and suramin (7) were optimised through the PRODRG2 server3) was extracted directly from the crystal structure 1V3E. Blind docking simulations were performed on the apo and zanamivir (3)-bound structures, in a 30 × 30 × 30 Å grid box . A total of 36 grids were designed to overlap by 15 Å in all three x, y and z directions, to reconstitute a global search space of 60 × 60 × 75 Å  containing an entire HN monomer. The refined dockings of suramin (7) and suraminh (8) were performed in a 40 × 40 × 40 Å grid box  positioned in the vicinity of the HN active site, using the apo or the zanamivir-bound structures. Simulations were run on the Griffith University High Performance Computing Cluster “Gowonda” with exhaustiveness parameters of 16 and 512 for the blind and refined dockings, respectively. Up to 20 conformations were generated for each simulation.Simulations were carried out using the software Autodock VinaHow to cite this article: Bailly, B. et al. A dual drug regimen synergistically blocks human parainfluenza virus infection. Sci. Rep.6, 24138; doi: 10.1038/srep24138 (2016).


Early phase II studies of mono-mTOR inhibition have not shown promise. However, studies have shown that combined inhibition of multiple steps along the mTOR signaling pathway may lead to sustained responses by targeting mechanisms of tumor resistance. Coordinated inhibition of mTOR along with specific KRAS-dependent mutations in molecularly defined PDAC subpopulations may offer a viable alternative for treatment in the future.Treatment options for advanced pancreatic ductal adenocarcinoma (PDAC) are limited; however, new therapies targeting specific tumor-related molecular characteristics may help certain patient cohorts. Emerging preclinical data have shown that inhibition of mammalian target of rapamycin (mTOR) in specific KRAS-dependent PDAC subtypes leads to inhibition of tumorigenesis  Pancreatic ductal adenocarcinoma (PDAC) remains the fourth leading cause of cancer-related mortality throughout the United States, with 12.3 new cases reported per 100,000 men and women annually and 10.9 deaths reported per 100,000 men and women annually  therapy with the intention of reducing locoregional and metastatic recurrence has been shown to improve survival compared to postoperative observation alone . The ChaStage III disease is divided into borderline resectable disease  . NeoadjuCurative resection is not recommended in stage IV PDAC, and treatment focuses on palliation. The use of FOLFIRINOX or gemcitabine + nab-paclitaxel has been shown to extend survival by at least 2 years in at least 10% of the patients, survival numbers that were rarely seen before and now represent standard first-line options, particularly in patients with good performance status [ECOG 0–1 ]. OveralThe investigation of genetic and molecular characteristics of PDAC remains a focus of current innovations in an effort to identify potential therapeutic targets. Genomic analysis of PDAC has revealed complex mutational patterns including near ubiquitious activation of KRAS, inactivation of >50% of major genetic pathways, such as TP53, SMAD4, and CDKN2A, involvement of 10% of genes in chromatin modification and DNA damage repair, and a collection of infrequently mutated genes that contribute to heterogeneity and create challenges in the development of targeted therapies . AffecteThe KRAS proto-oncogene is mutated in 90% of PDAC, with somatic alterations and locally rearranged, focal amplifications being quite common , 9, 10. While wild-type KRAS has been described as a predictive marker for treatment success of EGFR, inhibitors such as erlotinib or cetuximab and panitumumab in metastatic non-small cell lung and colorectal cancer, its predictive and prognostic value in PDAC has not been clearly established . InhibitAdditional downstream players of KRAS include phosphatidylinositol 3-kinase (PI3K) and AKT, which link ligation of growth factor receptors to the phosphorylation and activation of the serine/threonine kinase, mammalian target of rapamycin (mTOR), further downstream , 15. ThiThe mTOR complex is also closely related to the insulin/IGF-1 pathway. Decrease of adenosine triphosphate (ATP) production by metformin, for example, leads to AMPK activation and disruption of insulin/IGF-1 signaling through inhibition of mammalian target of rapamycin , 16. InhUpstream of mTOR, the PI3K/AKT pathway is influenced by PTEN, the negative regulator of PI3K signaling, which decreases its expression in many cancers including pancreatic, and may be downregulated through several mechanisms including mutation, deletion, and methylation .Clinically investigated mTOR inhibitors include rapamycin (sirolimus) and its analogs, such as temsirolimus, everolimus, and ridaforolimus. These analogs combine with mTOR accessory protein FKBP12 forming complexes that bind to mTOR and inhibit mTORC1 downstream signaling, preventing S6K1 and 4EBP1 phosphorylation . While FIn vitro studies have demonstrated diverse effects of mTOR inhibition on cell cycle arrest, autophagy, decreased desmoplastic inflammation, and inhibited epithelial-to-mesenchymal transition in preclinical studies of pancreatic cancer  did not respond to rapamycin treatment, which has other distinct pathways that are mTOR independent  leads to proliferative inhibition in PANC-1 xenografts . Use of in vivo .in vitro (Table First and second generation mTOR inhibitors both act to sensitize PDAC to radiation therapy ro Table . PC-2 anro Table . PSN1 cero Table .via AMPK-mediated (via insulin-like growth factor-1 receptor (IGF-1R)–AKT signaling by improving insulin tissue sensitivity  in 15% of the cases . Even siin vivo . Biopsie or mFOLFOX6 (without 5-FU bolus) every 2 weeks. The most common toxicities, including rash, mucositis, dehydration, and fatigue, were observed in 34 patients in the MS arm compared to hematologic toxicities, fatigue, nausea, and vomiting observed in 19 patients in the mFOLFOX arm. MS did not improve overall survival, and shorter survival was observed compared to mFOLFOX  .Recently, combination therapy with capecitabine (5-FU prodrug) and everolimus in a phase II trial of the first-line and second-line treatment of PDAC demonstrated modest benefit to combined therapy over monotherapy . Median Limitations of the aforementioned trials include the lack of characterization of the molecular pathology underlying PDAC tumorigenesis, which preclinical data suggest would predict treatment response. The modest improvement noted in the combined therapy everolimus/capecitabine phase II study supports the notion that coordinated inhibition of downstream KRAS signaling may improve antitumor efficacy of therapy .Although initial trials targeting mTOR inhibition have generally failed to demonstrate treatment efficacy, specifically targeting therapy to the diverse mutations underlying KRAS-dependent PDAC in specific subpopulations offer potential for coordinated inhibition of synergistic trophic mechanisms and the resistance-related feedback mechanisms underlying tumorigenesis. Targeting these mutations offers the advantage of improved tumor treatment with the potential for less systemic toxicity. Inhibition of patient-specific mTOR activity has strong preclinical data. Although many initial clinical trials with mTOR inhibition have been negative, the coordinated inhibition of multiple steps along the mTOR pathway may offer a viable alternative form of treatment for genetically defined PDAC in the future. Results from further late-phase studies involving combined mTOR pathway inhibition are eagerly awaited.SI and SA are co-first authors of this paper as they contributed to concept, initial drafting, and literature review. All authors otherwise contributed to this paper through literature review, drafting, critical revision, editing, and final approval of the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To assess the proportion of children with specific IgE to Der p 10 in children with sensitization to house dust mites (HDM).®, Thermo Fisher Scientific) to Der p 1, Der p 2 and Der p 10 simultaneously in children with symptoms of asthma and/or rhinoconjunctivitis who had positive skin tests to HDM. Values <0.35 kUA/L were considered negative.We determined specific IgE (ImmunoCAPWe studied 232 children (147 M/85 F) aged 1.25 - 16.7 years. Negative results were found in 50 (21.6%) with Der p 1, 53 (22.8%) with Der p 2, 208 (89.7%) with Der p 10, and 29 with all three (12.5%). There were 24 patients with positive IgE to Der p 10: three of these had IgE values of 6.1 to 11 kUA/L to Der p 10 and negative results to Der p 1 and 2 (0.1 to 0.18), three had much higher IgE to Der p 10 (79.9 to >100) than to Der p 1 or 2 , and one slightly higher IgE to Der p 10 (2.8) than to Der p 1 or 2 (0.8 and 1.19). The rest of the patients had clearly lower IgE to Der p 10.About 10% of patients sensitized to HDM had specific IgE to Der p 10. Sensitization to Der p 10 without sensitization to major allergens Der p 1 and Der p 2 appeared in 1.3% of patients, and serologically more relevant sensitization to Der p 10 in an additional 1.3% of patients. This should be considered when prescribing immunotherapy, as Der p 10 is not standardized in current vaccines.