Chemical Biology Core FacilityPublications
Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein.
Bartonova, V., Igonet, S., Sticht, J., Glass, B., Habermann, A., Vaney, M.C., Sehr, P., Lewis, J.D., Rey, F.A. & Krausslich, H.G.
J Biol Chem. 2008 Nov 14;283(46):32024-33. Epub 2008 Sep 4.
Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.
The structural basis for cap binding by influenza virus polymerase subunit PB2.
Guilligay, D., Tarendeau, F., Resa-Infante, P., Coloma, R., Crepin, T., Sehr, P., Lewis, J.D., Ruigrok, R.W., Ortin, J., Hart, D.J. & Cusack, S.
Nat Struct Mol Biol. 2008 May;15(5):500-6. Epub 2008 May 4.
Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.
Thanatop: a novel 5-nitrofuran that is a highly active, cell-permeable inhibitor of topoisomerase II.
Polycarpou-Schwarz, M., Muller, K., Denger, S., Riddell, A., Lewis, J.D., Gannon, F. & Reid, G.
Cancer Res. 2007 May 1;67(9):4451-8.
A series of nitrofuran-based compounds were identified as inhibitors of estrogen signaling in a cell-based, high-throughput screen of a diverse library of small molecules. These highly related compounds were subsequently found to inhibit topoisomerase II in vitro at concentrations similar to that required for the inhibition of estrogen signaling in cells. The most potent nitrofuran discovered is approximately 10-fold more active than etoposide phosphate, a topoisomerase II inhibitor in clinical use. The nitrofurans also inhibit topoisomerase I activity, with approximately 20-fold less activity. Moreover, the nitrofurans, in contrast to etoposide, induce a profound cell cycle arrest in the G(0)-G(1) phase of the cell cycle, do not induce double-stranded DNA breaks, are not substrates for multidrug resistance protein-1 export from the cell, and are amenable to synthetic development. In addition, the nitrofurans synergize with etoposide phosphate in cell killing. Clonogenic assays done on a panel of human tumors maintained ex vivo in nude mice show that the most active compound identified in the screen is selective against tumors compared with normal hematopoietic stem cells. However, this compound had only moderate activity in a mouse xenograft model. This novel class of topoisomerase II inhibitor may provide additional chemotherapeutic strategies for the development of cytotoxic agents with proven clinical utility.
Evaluation of Different Glutathione S-Transferase-Tagged Protein Captures for Screening E6/E6AP Interaction Inhibitors Using AlphaScreen(R).
Sehr P, Pawlita M, Lewis, J.D.
J Biomol Screen 2007/05