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Fragment screening of infectious disease targets in a structural genomics environment.

ABSTRACT: Structural genomics efforts have traditionally focused on generating single protein structures of unique and diverse targets. However, a lone structure for a given target is often insufficient to firmly assign function or to drive drug discovery. As part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we seek to expand the focus of structural genomics by elucidating ensembles of structures that examine small molecule-protein interactions for selected infectious disease targets. In this chapter, we discuss two applications for small molecule libraries in structural genomics: unbiased fragment screening, to provide inspiration for lead development, and targeted, knowledge-based screening, to confirm or correct the functional annotation of a given gene product. This shift in emphasis results in a structural genomics effort that is more engaged with the infectious disease research community, and one that produces structures of greater utility to researchers interested in both protein function and inhibitor development. We also describe specific methods for conducting high-throughput fragment screening in a structural genomics context by X-ray crystallography.

SUBMITTER: Begley DW

PROVIDER: S-EPMC4472360 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Fragment screening of infectious disease targets in a structural genomics environment.

Begley Darren W DW Davies Douglas R DR Hartley Robert C RC Edwards Thomas E TE Staker Bart L BL Van Voorhis Wesley C WC Myler Peter J PJ Stewart Lance J LJ

Methods in enzymology 20110101

Structural genomics efforts have traditionally focused on generating single protein structures of unique and diverse targets. However, a lone structure for a given target is often insufficient to firmly assign function or to drive drug discovery. As part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we seek to expand the focus of structural genomics by elucidating ensembles of structures that examine small molecule-protein interactions for selected infectious disease ...[more]

PMID: 21371605

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Project description:Burkitt lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL (eBL) in developing countries, necessitating new strategies. The normal germinal center B cell is the presumed cell of origin for both BL and diffuse large B cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may utilize different oncogenic pathways. BL is subdivided into a sporadic subtype (sBL) that is diagnosed in developed countries, the EBV-associated eBL subtype, and an HIV-associated subtype (hivBL), but it is unclear whether these subtypes employ similar or divergent oncogenic mechanisms. Here we used high throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways that cooperate with MYC, the defining oncogene of this cancer. In 70% of sBL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival PI(3) kinase pathway in BL, in part by augmenting tonic B cell receptor signaling. In 38% of sBL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL. Gene expression was analyzed using Agilent human 4X44K oligo gene expression arrays. Cell lines (Namalwa, BL41, Daudi, Defauw, and THOMAS) were infected with control (empty vector, Cy3) paired with ID3 (Cy5) or control (shControl, Cy3) paired with shTCF3 (Cy5) constructs, and changes in gene expression were monitored over time after induction of the constructs with doxycyclin. In Namalwa (n=8) and THOMAS (n=4) cell lines, a four timepoint series (24, 48, 72, 96 hours) of construct induction was analyzed, for a total of 12 arrays. In BL41 (n=4), Daudi (n=4) and Defauw (n=4) cell lines, a two timepoint series (24 and 48 hours) of construct induction was analyzed, for a total of 12 arrays. In addition, two cell lines (Daudi and THOMAS) were treated with Rapamycin (100 pM) and paired with a DMSO control in a four timepoint series (3, 6, 12 and 24 hours) for a total of 8 arrays.

Dataset Information

Fragment screening of infectious disease targets in a structural genomics environment.

Publications

Fragment screening of infectious disease targets in a structural genomics environment.

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