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Theodore E Carver,
Brian Bordeau,
Maxwell D Cummings, Eugene C Petrella,
Michael J Pucci,
Laura E Zawadzke,
Brian A Dougherty,
Jeffrey A Tredup,
James W Bryson,
Joseph Yanchunas, [......],
Mark R Witmer,
Marina I Nelen,
Renee L DesJarlais,
Edward P Jaeger,
Heather Devine,
Eric D Asel,
Barry A Springer,
Roger Bone,
F Raymond Salemme,
Matthew J Todd
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ABSTRACT: The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
Journal of Biological Chemistry 04/2005; 280(12):11704-12. · 4.77 Impact Factor
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Bruce L Grasberger,
Tianbao Lu,
Carsten Schubert,
Daniel J Parks,
Theodore E Carver,
Holly K Koblish,
Maxwell D Cummings,
Louis V LaFrance,
Karen L Milkiewicz,
Raul R Calvo, [......],
Marie Zhang,
Carl L Manthey, Eugene C Petrella,
Michael W Pantoliano,
Ingrid C Deckman,
John C Spurlino,
Anna C Maroney,
Bruce E Tomczuk,
Christopher J Molloy,
Roger F Bone
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ABSTRACT: HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.
Journal of Medicinal Chemistry 03/2005; 48(4):909-12. · 5.25 Impact Factor
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Daniel J Parks,
Louis V Lafrance,
Raul R Calvo,
Karen L Milkiewicz,
Varsha Gupta,
Jennifer Lattanze,
Kannan Ramachandren,
Theodore E Carver, Eugene C Petrella,
Maxwell D Cummings,
Diane Maguire,
Bruce L Grasberger,
Tianbao Lu
[show abstract]
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ABSTRACT: A library of 1,4-benzodiazepine-2,5-diones was screened for binding to the p53-binding domain of HDM2 using Thermofluor, a miniaturized thermal denaturation assay. The hits obtained were shown to bind to HDM2 in the p53-binding pocket using a fluorescence polarization (FP) peptide displacement assay. The potency of the series was optimized, leading to sub-micromolar antagonists of the p53-HDM2 interaction.
Bioorganic & Medicinal Chemistry Letters 03/2005; 15(3):765-70. · 2.55 Impact Factor
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Lei Jin,
Scott Pluskey, Eugene C Petrella,
Susan M Cantin,
Joan C Gorga,
Michael J Rynkiewicz,
Pramod Pandey,
James E Strickler,
Robert E Babine,
David T Weaver,
Katherine J Seidl
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ABSTRACT: The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the zeta subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 A resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N- and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.
Journal of Biological Chemistry 11/2004; 279(41):42818-25. · 4.77 Impact Factor
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Lei Jin,
Scott Pluskey, Eugene C. Petrella,
Susan M. Cantin,
Joan C. Gorga,
Michael J. Rynkiewicz,
Pramod Pandey,
James E. Strickler,
Robert E. Babine,
David T. Weaver,
Katherine J. Seidl
[show abstract]
[hide abstract]
ABSTRACT: The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target
for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex
with a peptide derived from the ζ subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green,
J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L.
(1995) Nature 377, 32–38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional
structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 Å resolution, utilizing an active kinase
domain containing residues 327–606 identified by systematic N- and C-terminal truncations. The crystal structure shows that
this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation
loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will
be useful in the design of ZAP-70-selective inhibitors.
Journal of Biological Chemistry 10/2004; 279(41):42818-42825. · 4.77 Impact Factor
