Human Topoisomerase I
Our long-term objective for the human
topoisomerase I (Top1) project is to define the binding orientation of CPT and derivatives in the
Top1/dsDNA active-site so that novel CPT derivatives can be rationally designed for the treatment of
drug-resistant cancers and cancers which are not currently sensitive to chemotherapy. The specific
hypothesis is that Top1 rotates the +1 nucleoside, of the bound dsDNA, out of the helix and in so
doing generates a binding cavity for CPT and derivatives. That hypothesis is based on the following
observations: first, when Top1 is in covalent-complex with dsDNA the +1 nucleoside is oxidized by
cobalt, which specifically oxidizes extra helical bases (1); and second, the Top1 Asn356Ala mutant
is as sensitive to CPT as the more potent 10-OH CPT derivative (2); this places the 10-OH CPT A-ring
near Asn356. Based on these observations, this project is focused on further defining the Top1/dsDNA
active-site and its interaction with substrate and inhibitor, and the design of CPT derivatives
which make additional interactions with as yet un-utilized active site residues.
Figure 1: Top1 backbone (blue) in covalent complex with dsDNA (white backbone), and
bound inhibitor (red). +1 guanine (green) rotated out of the helix, left of bound inhibitor.
Human T-cell Leukemia Virus Type-1 Protease
Human T cell
leukemia virus type 1 (HTLV-1) has infected ~30 million individuals worldwide, of which 1-5% will
develop an aggressive and terminal adult T cell leukemia for which there is currently no effective
treatment. The HTLV-1 protease is resistant to all commercial HIV-1 protease inhibitors. The X-ray
crystal structure of HTLV-1 protease with a bound peptidomimetic inhibitor was solved (3). Using
this three dimensional structure, wild-type and mutant HTLV-1 proteases were modeled in
silico and their interactions with commercial HIV-1 protease inhibitors were analyzed using
computational chemistry software. The wild-type and mutant HTLV-1 proteases, and HIV-1 protease,
were expressed in E. coli and then purified for in vitro assays utilizing a
fluorescent substrate and commercial HIV-1 protease inhibitors. This work is directed at
understanding how HTLV-1 protease interacts with HIV-1 protease inhibitors, so that effective
therapies can be developed to control HTLV-1 infections.
Figure 2: HTLV-1 protease dimer (A monomer blue, B monomer teal) shown as ribbon
with bound peptide shown in CPK (hydrogen, white; carbon, grey; oxygen, red; nitrogen, blue).
General Laboratory Research Methods
Dry laboratory in
silico research is carried out using computational chemistry software and includes docking and
minimization of substrates and inhibitors into the active sites of enzymes, followed by the
calculation of the interaction energies between either the substrate, or inhibitor, and the enzyme.
Novel substrates and derivatives of inhibitors are also constructed and tested in silico.
Wet laboratory research methods include PCR based mutagenesis of genes, cloning of the
DNA, and expression of the recombinant enzymes in either E. coli, or in insect cells using a
recombinant baculovirus, followed by purification of the enzymes. Enzyme activity and inhibition are
monitored using fluorescent substrates and a JASCO spectrofluorometer.
1) Laco, G. S., Du, W., Kohlhagen, G., Sayer, J. M., Jerina,
D. M., Burke, T. G., Curran, D. P., and Pommier, Y. (2004) Analysis of human topoisomerase I
inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and
molecular modeling studies. Bioorg Med Chem 12, 5225-35.
2) Laco, G. S., Collins, J. R., Luke,
B. T., Kroth, K., Sayer, J. M., Jerina, D. M., and Pommier, Y. (2002) Human Topoisomerase I
Inhibition: Docking Camptothecin and Derivatives into a Structure-Based Active Site Model.
Biochemistry 41, 1428-1435.
3) Li, M., Laco, G. S., Jaskolski, M., Rozycki, J., Alexandratos,
J., Wlodawer, A., and Gustchina, A. (2005) Crystal structure of human T cell leukemia virus
protease, a novel target for anticancer drug design. Proc Natl Acad Sci U S A 102, 18332-7.