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SmartScreen Technology for Building a Better Assay


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A lipid derived nanoparticle the recreates the cellular membrane in solution based assays. See increased enzymatic activity, identify more relevant biological substrates, find novel hits from the compound library.

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SmartScreen Technology for Building a Better Assay

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  2. 2. There are several classes of membrane proteins: single- and multi- passtransmembrane proteins, proteins which are associated with the membrane via lipidanchors (such as myrisoylation, palmitoylation or GPI anchors) or electrostaticinteractions, and proteins which are normally cytosolic but form complexes withmembrane proteins. TDA 2.0™ is not suitable for use with multi-pass transmembraneproteins in general, however, all other membrane proteins which have distinctdomains on one side of the membrane would work with TDA 2.0™, regardless ofwhich subcellular membrane, or face of the membrane, the protein is associatedwith. 2
  3. 3. [LEFT PANEL] Typically membrane proteins are assayed by expressing recombinantfragments, often containing only the active site of the enzyme, and are interrogatedin a high-throughput solution-based assay. While cost-effective and expeditious, thisformat ignores any organization, structure and topology imparted by membrane.[RIGHT PANEL] An alternative assay is to examine endogenous or over-expressedenzymes in a living cellular system. While this system faithfully replicates themembrane environment and contains the full compliment of every other relevantanimal protein, it is slow, very expensive, and not readily adaptable to high-throughout testing of a chemical library.Importantly, efficacy of compounds identified in a solution assay tends to correlatepoorly with the efficacy in cellular assays.*MIDDLE PANEL+ TDA 2.0™ is an enabling technology which bridges the gap betweenthese two formats, providing the context afforded by a biological membrane in aplatform fully compatible with HTS and all detection formats. 3
  4. 4. How does template directed assembly work? Engineered recombinant HIS-taggedproteins are produced such that the HIS-tag is on the correct terminus of the proteinto reflect the polarity of the enzyme with respect to the membrane (for example,ecto-domains of a receptor would be C-terminally tagged while intracellular domainsare N-terminally tagged). TDA 2.0™ is a soluble stable liposome which is made fromderivitized lipids which have Ni-NTA covalently attached to the lipid head group. Thisallows the HIS-tagged proteins to bind to the liposome creating an environment muchlike a cellular membrane. Unlike a sepharose or agarose bead where HIS-taggedproteins must detach and reattach to translate across the surface, the lipids are fullyfluid within the 2-dimensional surface of the liposome, so associated proteins cantranslate and rotate freely. The spatial and relational organization provided by themembrane surface, combined with this fluidity, promotes the formation of higher-order structures, such as homo- or hetero- dimers or multimers, and allowsrecruitment of accessory factors. 4
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  6. 6. Insulin receptor is activated by dimerization which leads to trans-phosphorylation onseveral tyrosines. The phosphorylated dimer is the active RTK. Typically, in order tosee activation in solution, manganese is included in the reaction, which creates anon-physiological environment. Above, the left panel shows a radiometric filterbinding assay for autophosphorylation, while the right panel showsautophosphorylation and phosphorylation of an IRS1-derived peptide substrate. Asyou can see, addition of 10mM MnCl2 increases autophosphorylation as well assubstrate phosphorylation. However, addition of TDA 2.0™ in a physiological relevantbuffer without MnCl2 shows robust activation. We interpret this data to showenhanced functional dimerization of InR on TDA 2.0™ in a physiologic buffer. 6
  7. 7. Zap-70 is a T-Cell receptor effector that is normally activated by recruitment to thephosphorylated ITAM domains of CD3-zeta. In vitro, recombinant Zap-70 is difficultto screen as it has been reported to have a very long and variable lag phase. We seethis same effect as shown in the top three panels where after two hours we seedramatically different activity of Zap-70 in three different experiments. However,addition of TDA 2.0™ reduces the lag time significantly and increases thepredictability of activation, creating a very robust assay for Zap-70. This data wasgenerated using a Caliper EZ reader. 7
  8. 8. Many enzymes show enhanced activity when assayed in the presence of TDA 2.0™.While secondary in importance to the effects TDA 2.0™ has on improving the biologyof an enzyme, this is nonetheless another very beneficial feature of TDA 2.0™. 8
  9. 9. When looking at the ability of an enzyme to phosphorylate a peptide substrate,researchers often use synthetically derived peptides, such as poly-Glu(4)-Tyr. Wenoticed that when examining activity of ErbB4, a receptor tyrosine kinase (RTK),towards PolyGlu, there was no improvement in activity in the presence of TDA 2.0™.However, when examining activity towards peptides derived from natural substratesof ErbB4, such as Abl and Src, a considerable enhancement in activity is noted in thepresence of TDA 2.0™. We interpret this as an indication that the substratepreference of the enzyme is altered when in the membrane context, perhapsselecting more biologically relevant substrates. 9
  10. 10. To investigate that systematically, we used a different RTK, TrkB, and contractedMolecular Devices to screen a library of peptides in the absence (red) and presence(blue) of TDA 2.0™. The first observation is that the best substrates of TrkB arecompletely different in the presence and absence of TDA 2.0™. This indicates to usthat a key biological property of the enzyme, namely substrate selection, issignificantly altered by TDA 2.0™. The second observation from this data set comesfrom analysis of the sequences of the peptides substrates above. In the absence ofTDA 2.0™, comparison of substrate sequence to the non-redundant protein databasereveals these substrates either fail to match anything in the database (they aresynthetic peptides) or they match viral proteins, which are not likely to be naturalsubstrates of TrkB. In the presence of TDA 2.0™ many synthetic or non-relevantsubstrates are also identified, however, peptides derived from IRS1 and EGFR, knownsubstrates of TrkB, are identified as substrates. This indicates that presentation ofTrkB in the context of TDA 2.0™ biases the substrate selectivity towards relevantsubstrates of the enzyme. 10
  11. 11. Initial rates were measured and Km for ATP determined for various enzymes in thepresence and absence of TDA 2.0™ using the Caliper EZ reader platform. All enzymesexamined to date show significantly lower Km ATP in the presence of TDA 2.0™. 11
  12. 12. Data presented by Dr. Kathleen Seyb (Marcie Glicksman’s group at HarvardNeuroscience/Brigham and Women’s) at the Society for Biomolecular Sciences annualmeeting. They had a grant-driven project to screen Lyn kinase through their library of75,000 compounds. In order to get high enough signal in their solution-based assay,high concentrations of enzyme (>200 nM) had to be used which made the screenfinancially impossible. Addition of TDA 2.0™ reduced the amount of enzyme requiredfor a good signal over 25-fold and reduced the cost per well by 50%, leading tosuccessful completion of the screen. 12
  13. 13. This graph shows a subset of the compounds which were screened both in thepresence and absence of TDA 2.0™. It’s not a direct comparison as the enzymeconcentration required to get data in the absence of TDA 2.0™ is not the same, andthe signal was much lower, but is instructive nevertheless. The graph plots %inhibition in the absence of TDA 2.0™ along the X-axis and in the presence along theY. Noticeably, TDA 2.0™ alters the pharmacology of Lyn as there are hits unique toeach condition. We’re in the process now of examining these compounds in follow-up cellular assays to try to determine if using TDA 2.0™ leads to better quality leadcompounds and is more predictive of cell-based assays. Regardless, this data showsthat TDA 2.0™ reveals differences in compound SAR (structure-activity relationship), 13
  14. 14. Insulin signal in the cell initiates upon receptor dimerization and activation bytransphosphorylation on specific tyrosines. The phosphotyrosines serve as bindingsites for PI3k which is recruited and propagates the signal by converting PIP2 to PIP3,in turn recruiting PDK1 and AKT to the membrane. Activation of Akt by PDK1 andmTOR leads to phosphorylation of many AKT-substrates when ultimately lead tobiological effects such as lipolysis, glucose update, growth or proliferation. We aredeveloping an assay which replicates many of these steps in a chemically definedsystem. 14
  15. 15. The first step we’re replicating is the phosphorylation of AKT by PDK1. We’ve made aHIS tagged construct of AKT, and well as a HIS-tagged form of PDK1 to deliver theseenzymes to the membrane without PIP3. To extend the utility of this assay format, weincluded GST-tagged mTOR which phosphorylates AKT on S473. When AKT isphosphorylated on both S473 and T308, AKT kinase activity is increased severalorders of magnitude. 15
  16. 16. This figure shows Western blots of reactions containing different combinations ofPDK, mTOR and AKT with and without TDA 2.0™.The top panels are anti-GST Westerns showing consistent loading of GST-taggedmTOR.The second set of panels show consistent loading of AKT (lower band) and PDK1(upper band) using an anti-HIS tag antibody.The third set of panels employs a phospho-specific anti-AKT-pS473 antibody to showphosphorylation of AKT by GST-tagged mTOR.The lower set of panels employs a phospho-specific anti-AKT-pT308 antibody to showphosphorylation of AKT by HIS-tagged PDK1.In the presence of TDA 2.0™, when AKT and PDK are combined, phosphorylation ofAKT increases 2-3 fold (compare lanes 5 to 6). This is not solely due to co-localizationof AKT and PDK as the same result is obtained using a FLAG-tagged version of PDK1(data not shown). Further, when AKT and mTOR are combined in the presence ofTDA 2.0™, phosphorylation on S473 increases 4-5 fold (compare lanes 7 to 8). SincemTOR is GST-tagged and not co-localized, this confirms our result with FLAG-taggedPDK1 and indicates that AKT is a better substrate for it’s upstream activators whenassociated with a membrane such as TDA 2.0™. 16
  17. 17. Finally, when examining the ability of AKT to phosphorylate CROSSTide™, only whenAKT, PDK1 and mTOR are combined in the presence of TDA 2.0™ do we see robustkinase activity. 17
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