Part I: Measles virus entry into cells and the inhibition offusion and replicationPart II: The nature of cyclostreptin's interaction withmicrotubules Öffentlichkeit
Prussia, Andrew James (2009)
Published
Abstract
In recent years, resurgence in measles virus infections has occurred in the developed world due to decreased vaccination coverage. To counter this virus and related viruses in the paramyxovirus family, two key functions of the viral lifecycle have been targeted: fusion of the viral and cellular membranes, mediated by viral attachment and fusion glycoproteins; and the replication of the viral genome, mediated by the viral RNA-dependent RNA polymerase complex (RdRp). Using a structural model of the measles virus fusion protein, a series of fusion inhibitors were developed, yielding a compound with 600-3000 nM activity depending on the viral strain. Viral resistance to these inhibitors was demonstrated by mutations that were located on the fusion protein but at positions distal to the predicted binding site. Through models of both the prefusion and postfusion conformations of the fusion protein, a structural proposal consistent with the experimental fusion activities was developed for both the mechanism of action and the occurrence of resistance. Use of these models also facilitated the prediction of a fusion protein containing an engineered disulfide bond that inhibits fusion by stabilization of the prefusion form, but also has restored fusion activity in reducing conditions. Additionally, a model for the interaction of the fusion and attachment proteins was proposed based on available structural information and secondary structure predictions that is fully consistent with experimental results. Finally, a quantitative structure activity relationship was developed for the newly discovered, potent inhibitors of measles virus RdRp.
Cyclostreptin is a recently discovered natural product with cytotoxic activity caused by microtubule stabilization. It is the only known microtubule-stabilizing agent (MSA) which covalently binds to tubulin, but also exhibits the fast binding kinetics seen for other MSAs. Buey et al. found that Thr220, in the type I microtubule pore, is labeled by cyclostreptin, leading the authors to propose Thr220 resides at the previously predicted low-affinity site. Using structural considerations of the microtubule pore and tubulin dimer, an alternative explanation is proposed viewing the microtubule pore as a structural entity that presents a kinetic barrier to ligand passage to the known taxane binding site.
Table of Contents
List of Figures.................................................................................................................. xii List of Tables.................................................................................................................... xv Part 1: Measles virus entry into cells and the inhibition of fusion and replication.............. 1 Chapter 1: Viruses and their entry into cells...................................................................... 2 1.1 Classes of viral fusion proteins.................................................................................... 4 1.1.1 Class 1 fusion.............................................................................................................. 4 1.1.2 Class 2 fusion............................................................................................................ 13 1.1.3 Class 3 Fusion........................................................................................................... 18 1.2 Inhibition of viral fusion............................................................................................. 21 1.2.1 HIV fusion inhibition................................................................................................... 22 1.2.3 Respiratory Syncytial Virus fusion inhibition................................................................... 28 1.3 The unique challenge of measles virus entry.............................................................. 30 Chapter 2: Early model of the measles virus fusion protein and the discovery of fusion inhibitors......................................................................................................................... 32 2.1 Model based on Newcastle Disease Virus fusion protein............................................ 32 2.2 Discovery of fusion inhibitors of measles virus.......................................................... 36 2.2.1 Measles virus fusion protein is confirmed as the target for OX-1....................................... 38 2.2.2 Engineered mutations in MeV F induce resistance to OX-1............................................... 38 2.2.3 Docking model of OX-1 to the Val94 microdomain.......................................................... 39 2.3 Design of a more potent fusion inhibitor.................................................................... 41 Chapter 3: Structure-based design of fusion inhibitors.................................................... 43 3.1 AM-4 analogs............................................................................................................. 43 3.1.1 SAR for compounds with a mono-substituted acetanilide phenyl ring................................. 45 3.1.2 SAR for compounds with a disubstituted acetanilide phenyl ring....................................... 47 3.1.3 SAR for compounds with modifications on the distal phenyl ring and the intermediate linker region...................................................................................................................... 50 3.2 Balance between the polar interactions and the desolvation penalty......................... 52 3.3 AS-48 as a model fusion inhibitor.............................................................................. 53 Chapter 4: Resistance to AS-48 fusion inhibition............................................................. 55 4.1 Adaptation study of MeV to AS-48............................................................................. 55 4.2 Resistance-conferring mutations located in a model of the MeV F 6HB...................... 58 4.2.1 Homology model of the MeV F 6HB.............................................................................. 58 4.2.2 Models of the MeV F 6HB with resistance-conferring mutations........................................ 61 4.3 Destabilizing effect of the mutations........................................................................ 62 4.3.1 Peptide inhibition with wild-type and mutated HR-B domains.......................................... 63 4.3.2 Molecular dynamics studies of wild-type and mutated 6HB structures.............................. 64 4.3.3 Mutation of the HR-A residue interacting with Asn462.................................................... 66 4.3.4 Coimmunoprecipitation efficiency of MeV F with a Flag-tagged variant of the HR-B derived peptide................................................................................................................ 66 4.4 Mutations affect the fusion activation of MeV F........................................................ 67 4.4.1 Transport competence of mutant MeV F constructs restored at 30ºC................................ 67 4.4.2 AS-48 stabilizes a transport-competent prefusion conformation...................................... 69 4.5 Dual roles of the MeV F residue 462......................................................................... 70 Chapter 5: Conformational changes in MeV F during fusion........................................... 72 5.1 Limitations of the NDV based model of MeV F.......................................................... 72 5.2 New model of the postfusion form of MeV F............................................................. 73 5.3 Prefusion model of MeV F......................................................................................... 75 5.4 Structural features and changes in the prefusion and postfusion MeV F models...... 75 5.4.1 Pre- and postfusion MeV F models imply large domain movements during fusion................ 76 5.4.2 The hydrophobic character of the HR-A and HR-B domains is consistent with the proposed model of fusion.................................................................................................. 80 5.4.3 The large, water-filled cavity in the prefusion protein is a metastable feature that provides MeV F with a source of potential energy.................................................................. 81 5.4.4 The Val94 microdomain is rearranged and occluded in the prefusion model...................... 85 5.5 The nature of the AS-48 binding to the Val94 microdomain..................................... 88 5.5.1 Defined antisera against the Val94 microdomain.......................................................... 88 5.5.2 Conformational state of MeV F targeted by AS-48........................................................ 91 5.6 Structural proposal for the development of AS-48 resistance.................................. 94 5.6.1 MeV F residues 462 and 367 are located at the critical interface between the HR-B and DI domains in the prefusion conformation of F............................................................... 94 5.6.2 Mutations at MeV F residues 462 and 367 disrupt the hydrophobic interactions holding the HR-B and HR-linker to the DI domain................................................................. 95 5.6.3 Mutations at position 462 enhance fusion activity at lower temperatures........................ 100 5.7 Limitations of the AS-48 molecular scaffold............................................................ 101 Chapter 6: Fusion inhibition through stabilization of prefusion MeV F........................... 104 6.1 An engineered disulfide bridge in prefusion MeV F.................................................. 104 6.1.1 Molecular modeling of disulfide bonds in prefusion MeV F.............................................. 104 6.1.2 Expression and fusion activity of F candidates with engineered disulfide bonds................ 107 6.1.3 Oligomerization of F variants with engineered disulfide bonds....................................... 108 6.1.4 Reactivation of fusion activity by DTT-treatment confirms proper folding of F-Edm 452C/460C and arrest in a prefusion conformation.................................................... 110 6.2 Screening for small molecule inhibitors of prefusion MeV F.................................... 113 6.2.1 Computational identification of the target site in the prefusion MeV F model................... 113 6.2.2 Further refinement of the prefusion MeV F structure.................................................... 114 6.2.3 Virtual screen at the HR-B / head domain interface..................................................... 117 6.2.4 Biological testing of the compounds selected by virtual screening.................................. 119 Chapter 7: Functional interaction between paramyxovirus fusion and attachment proteins..................................................................................................... 122 7.1 Differential activation of F and H in morbilliviruses................................................ 124 7.1.1 MeV H efficiently triggers CDV F-ODP but not CDV F-Lederle......................................... 124 7.1.2 Minimal domain required for heterotypic triggering of CDV F-ODP.................................. 126 7.1.3 Four point mutations disrupt productive interaction of F-ODP with MeV H....................... 127 7.1.4 Location of the four point mutations in a structural model of CDV F................................ 128 7.1.5 Changes in the strength of H and F interaction due to mutations................................... 129 7.1.6 Assay reversal demonstrates the H stalk domain determines productive interaction of MeV H with CDV F-Lederle........................................................................................... 130 7.1.7 A five-residue fragment in the MeV H stalk determines specificity for F-Lederle.............. 131 7.1.8 Co-expression of F and H chimeras reveals interdependence of the identified residues in productive H-F interaction............................................................................... 132 7.1.9 Role of identified F residues in homotypic fusion......................................................... 133 7.2 Structural model of morbillivirus H-F interaction................................................... 135 7.2.1 Structural prediction of the MeV H stalk domain......................................................... 136 7.2.2 Structural model for the entire MeV H ectodomain...................................................... 138 7.2.3 Hypothetical MeV H - CDV F interaction model........................................................... 138 7.3 Probing the spatial organization of H-F complexes................................................ 142 7.3.1 Effect of engineering N-glycosylation sites into the H stalk.......................................... 143 7.3.2 H residues 111 and 114 are determinants for F triggering........................................... 147 7.3.3 Only stalk elongation downstream of residues 111 and 114 is compatible with F triggering.................................................................................................................. 150 7.3.4 Extensive stalk insertions argue against specific contacts between H and F head domains ...................................................................................................................... 154 7.3.5 Overall consistency of the H-F interaction model with experimental evidence................ 156 7.4 H-F interaction model with tetrameric H.............................................................. 158 7.4.1 Predicting the tetrameric MeV H structure............................................................... 160 7.4.2 Compatibility of the MeV H tetramer model with natural glycosylation sites.................. 165 7.4.3 Incorporating tetrameric MeV H in the H-F interaction model...................................... 166 Chapter 8: RNA-dependent RNA polymerase inhibitors.............................................. 170 8.1 Qualitative SAR for compound 1 analogs.............................................................. 171 8.2 Quantitative SAR for compound 1 analogs............................................................ 175 8.3 SAR conclusions for compound 1 analogs............................................................. 180 Chapter 9: Conclusions and future directions............................................................. 181 9.1 MeV fusion inhibition............................................................................................ 181 9.2 H-F interaction..................................................................................................... 182 9.3 RdRp inhibition.................................................................................................... 182 Part 2: The nature of cyclostreptin's interaction with microtubules........................... 184 1.1 The binding of microtubule stabilizing agents...................................................... 185 1.2 Docking of cyclostreptin to the microtubule......................................................... 189 1.2.1 No defined MSA binding site is present in the microtubule pore................................... 189 1.2.2 CS binding most favorable at the taxane binding site................................................. 191 1.3 Constrained molecular dynamics of MSAs in the microtubule pore....................... 196 1.3.1 MSA diffusion across the microtubule pore............................................................... 196 1.3.2 Blockage of MSA diffusion by cyclostreptin............................................................... 199 1.3.3 CS labels Thr220 due to its exposure in the microtubule pore..................................... 201 1.4 Molecular dynamics simulation of the apo-tubulin dimer...................................... 203 1.4.1 CS binding at the taxane site is blocked by the M-loop............................................... 203 1.4.2 Thr220 remains exposed in apo- and MSA-bound tubulin dimer................................... 206 1.5 Conclusions on the nature of MSA binding to microtubules................................... 206 1.5.1 Weak microtubule polymerization by CS is explained by its limited effect on the M-loop .................................................................................................................. 206 1.5.2 CS labeling of Asn228 could occur through migration during MS................................. 207 1.5.3 Two activation energies for CS binding to tubulin are unlikely...................................... 212 1.5.4 Implications for the design of new MSAs.................................................................. 213 References ................................................................................................................ 214
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