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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Smith, Mathew Wayne
Languages: English
Types: Doctoral thesis
Subjects:
Phage display, a powerful polypeptide display technology, affords the rapid identification of peptides and proteins that interact with a target of interest The aims of the project were the phage display identification of peptides that interact with a druggable target in a brain disorder (glioblastoma multiforme) and the identification of peptides that serve as targeting vectors for brain delivery. Validation studies were undertaken to qualify the use of a cyclic 7-mer peptide phage library against targets including streptavidin and paracetamol chosen as examples of a large complex and small simple molecule, respectively. With the aim of identifying peptide phages that bind to the luminal surface of brain micro vasculature, a primary in-vitro porcine model of the blood-brain barrier (BBB) comprising primary brain capillary endothelial cells was established and characterised. An in-vivo phage display was undertaken in the rat with the aim of identifying peptide sequences that mediated translocation across the BBB into brain grey matter. A 7-mer cyclic peptide was identified with sequence AC-SYTSSTM-CGGGS that enhanced the uptake of phages into brain grey matter by 4-fold compared to control wild-type phages. This peptide may serve as a novel targeting vector for the delivery of a therapeutic cargo to the brain. Caveolin-1 was identified as a potential new therapeutic target in in-vitro models of grade IV astrocytomas (glioblastoma multiforme), with siRNA knockdown of caveolin-1 associated with reduced glioma cell proliferation and invasiveness. With the caveolin-1 scaffolding domain (aa 81-101 in the caveolin-1 protein) as a target, an in-vitro peptide phage selection was undertaken and identified a series of peptides that bind the scaffolding domain with high affinity. These peptides will serve as a template for the development of low molecular weight peptidomimetics that inhibit caveolin-1 function. In conclusion, the studies in this thesis have demonstrated the utility of phage display in experimental therapeutics of brain disorders.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • Chapter 1 Phage display and experim ental brain therapeutics ___ 1 1.1 CNSdisorders------------------------------------------------------------------------------ 2 12 Astrocytomas-------------------------------------------------------------------------------3 13 TheBlood-Brain barrier_______________________________________ 6 1.4 Caveolaeand caveolin________________________________________ 7 1.5 Polypeptide display systems___________________________ .________ 9 1.5.1 Phage Display__________________________________________ 10 1.6 Phage Biology---------------------------------------------------------------------------- 11 1.6.1 Phage Structure------------------------------------------------------------------- 11 1.6.2 Phage Genome..................... 14 1.7 Phage Lifetyde--------------------------- 16 1.7.1 Infection.......................................... 16 1.7.3 Assembly______________________________________________19
    • 1.8 Coatproteins usedfo r display__________________________________ 21 1.8.1 p ill and pVIII display ................................... 22
    • 1.9 Wild-typephage vectorM13KE -------------- 24
    • 1.10 Applications o fphage display._________________________________25 1.10.1 Applications of phage display in disease therapeutics----------------- 26 1.10.2 Applications of phage display in drug delivery-------------------------- 28 1.10.3 Phage therapy---------------- 32
    • 1.11 Scopeo fthesis____________________________________________ 35
    • Chapter 2 Phage display method developm ent -------------------------49 2.1 Introduction_________ ._____________________________________ 50 2.1.1 General principles of phage display panning studies-------------------- 50 2.1.2 Affinity selection-------------------- 52 2.1.3 Target presentation______________________________________53 2.13.1 Elution____________________________________________________55 2.1.4 Technology validation------------- 56 2.1.4.1 Streptavidin_______________________________________________ 56 2.1.42 4-AAP____________________________________________________ 57 2.1.5 Objectives..................... 59 22 Materials & Methods - Phage Display------------------- 60 2.2.1 Materials........................ 60 2.2.2 Phage library__________________________________________60 2.2.3 Phage display media and solutions---------------------------------- ------ 61 2.2.4 Maintenance of the bacterial host used for phage propagation..........62 2.2.5 Phage amplification and purification--------------------------------------- 62 2.2.6 Phage plaque formation assay......................... 63 2.2.7 Gene sequencing ........................................................................... 64 2.2.7.1 Phage plaque amplification and DNA extraction ........................... 64 22.72 PCR_______________________________________________ 65 2.2.8 Statistical analysis............................................. 66
    • 23 Streptavidin Panning________________________________________ 66 2.3.1 Materials & Methods................................. 66
    • 3.5 Discussion......................... 161
    • 43 Results:______________________________________ _______ ___203 4.3.1 Analysis of in-vivo phage recovery and peptide sequences_____ 203 4.3.2 Phage amplification errors______________________________ 214 4.3.3 Ex-vivo stability of SYTSSTM-M13 and insertless-M13________ 215 4.3.4 Effects of perfusion on phage numbers____________________ 216 4.3.5 Fifteen minute brain uptake o fselected peptide-phage clones____ 217 4.3.6 In-vivo tissue distribution o fSYTSSTM-M13__________________218 4.3.7 Pharmacokinetic simulations o fbrain uptake________________ 226
    • 4.4 rilSCUSSMm________________________ ------------------------------------- 233
    • Figure 1.1 Polypeptide display technologies......................................................9
    • Figure 12 Schematic representation o fthe F fbacteriophage virion..............12
    • Figure 13 Schematic o fthe phage DNA replication, protein synthesis and
    • protein location o fthe F ffilamentous bacteriophage.................................. 17
    • Figure 2.1 Schematic o fa generic phage display panning strategy..............51
    • Figure 2 2 Structure o f4-acetamidophenol and its isomers 2-
    • acetamidophenol and 3-acetamidophenol.................................................... 58
    • Figure 2 3 Phage recovered in glycine elutionsfrom streptavidin coated
    • plastic support orfrom SA-PMPs .................................................................. 70
    • Figure 2.4 ELISA confirmation o fpeptide sequence AC-GSYWHPQ-CGGGS
    • binding to streptavidin................... 72
    • Figure 2.5 Schematic o f4-AAPphage panning experiments.......................... 74
    • Figure 2.6 Phage recovered in 4-AAP panning isolations............................... 82
    • Figure 2.7 Permeability o f4-AAP through a nucleopore membrane in the
    • presence o fa 4-AAP binding peptide phage.................................................. 87
    • Figure 2.8 Mitochondrial dehydrogenase activity ofHep3B cells exposed to 4-
    • AAP. .................................. 88
    • Figure 2.9 Mitochondrial dehydrogenase activity in Hep3B cells exposed to 3-
    • AAP ............................... 90
    • Figure 2.10 Mitochondrial dehydrogenase activity o f Hep3B cells exposed to
    • 4-AAPor 3-AAP in the presence o fa 4-AAP binding peptide phage............... 92
    • Figure 3.1 Schematic representation o f the cellular architecture at the blood-
    • brain barrier....................... 104
    • Figure 32 Schematic representation o f the biopanning procedure used to
    • endothelia............................. 137
    • Figure 33 Ultrastructural morphology o fPBMVECs....................................141
    • Figure 3.4 Alkaline phosphatase activity o f PBMVECs................................. 143
    • Figure 3.5 Western blot analysis o fcaveolin-1 brain tissue and PBMVECs.. 145
    • Figure 3.6 Western blot analysis o fcaveolin-1 in PBMVECS........................ 146
    • Figure 3.7 mRNA expression o f tigh tjunctional elements and transporters in
    • PBMVECs........................... 148
    • Figure 3.8 Functional P-gp activity in PBMVEC cells................................... 150
    • Figure 3.9 Permeability o fPBMVECs paracellular and transcellular probes.
    • Table 1.1 World Health Organization classification o fastrocytomas............. 4
    • Table 12 Filamentous phagegenes and protein products.............................12
    • Table 1.3 Classification o fphage display systems_________________ 23
    • Table 2.1 Example immobilization matrices usedfo r target presentation in
    • phage binding selections ....................................................................54
    • Table 3.4 Reverse transcription mixture....................................................... 131
    • Table 3.5 Thermocycling program................................................................ 131
    • Table 3.6 RT-PCRfo r tightjunction elements; carrier mediated transporters
    • and hormone receptors in PBMVECs............................................................133
    • Table 3.7 RT-PCRfo r carrier mediated transporters RBMVECs...................134
    • Table 3.8 Polymerase chain reaction m ixture.............................................. 135
    • Table 3.9 Thermocycling program................................................................ 135
    • Table 3.10 Permeability coefficientsfo r sucrose and diazepam across
    • PBMVECmonolayers .......................................................................158
    • Table 3.11 Amino acid sequences o f brain microvascular endothelial cell
    • surface associatedphage clones...................................................................160
    • Table 3.12 Permeability coefficientsfo r sucrose and propranolol across
    • PBMVECmonolayers cultured on large inserts (4.7 cm2) ............................168
    • Table 4.1 Density o fendothelial vesicles in capillariesfrom various tissues 187
    • Table 42 Selection o fkey studies that highlight the use ofRMT to deliver a
    • payload into the brain.................................................................................. 189
    • Table 43 Ratphysiological parameters used in whole body PBPK model... 201
    • Table 4.4 Amino acid sequences o fbrain homing peptides isolated in multiple
    • copy numberfrom strategies 1 and 2........................................................... 207
    • Table 4.5 Physicochemical properties o fhigh frequency clones identified in
    • isolation studies. ........................................................................................212
    • Table 4.6 Amplification defects o fpeptide phages....................................... 215
    • Table 4.7 Brain homing capacity o fselect peptide phage clones................. 218
    • Table 4.8 Blood pharmacokinetic parameters o fSYTSSTM-M13 and
    • insertless-M13 ............................................................................................ 223
    • Table 4.9 AUCs calculatedfrom experimentally determined tissue
    • distributions ofSYTSSTM-M13 and insertless-M13......................................224
    • Table 5.1 Carcinomas in which raised caveolin-1 expression correlates with
    • poor clinical prognosis............... 254
    • Table 5.2 Composition o f12 % SDSpolyacrylamide running gel solution... 264
    • Table 5.3 Composition o f4 % SDSpolyacrylamide stacking gel solution 264
    • Table 5.4 Composition o floading buffer.......................................................264
    • Table 5.5 Effects ofsiRNA mediated down-regulation o fcaveolin-1 on cellular
    • proliferation............................ 270
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  • Discovered through pilot similarity algorithms. Send us your feedback.

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