Koehler Group: Projects

• Small-molecule probes of oncogenic transcription factors
• Structurally and mechanistically novel probes of deacetylase enzymes
• Small-molecule microarrays

Small-molecule probes of oncogenic transcription factors

Our team is developing a general approach to small-molecule probe discovery for transcription factors by coupling a high-throughput binding assay involving small-molecule microarrays (SMMs) with functional assays involving transcriptional and cellular readouts. Representative projects are described below:

• c-Myc activation is one of the most common oncogenic events in human maligancies. In normal cells, the Myc family of transcription factors regulates a diverse set of cellular processes including cell growth, proliferation, apoptosis, metabolism, differentiation, self-renewal, and angiogenesis. It is estimated that c-Myc regulates expression of more than 15% of all genes and is therefore considered to be a master regulator. c-Myc deregulation may result in uncontrolled cell proliferation, alterations in the apoptotic pathway, genomic instability, escape from immune surveillance, growth factor independence, and immortalization. c-Myc is one of the only oncogenes for which true oncogene addiction has be demonstrated in vivo. Inactivation of c-Myc using small molecules that directly target the protein or that disrupt Myc-associated proteins may be an effective approach to treating a broad swath of human cancers in which the oncoprotein is aberrantly active and a driver of the malignant phenotype. We have identified several small molecules that directly bind to c-Myc or heterodimer partner Max and inhibit Myc-driven transcription in cells. Our group is engaged in a variety of biophysical and cellular studies aimed at understanding the mechanism of action for these compounds. We have also undertaken chemistry efforts aimed at probe optimization. We collaborate with the laboratory of Professor Benjamin Ebert of Brigham and Women’s Hospital on this project.

• ETS transcription factors are a ubiquitous class of factors that regulate numerous cellular processes including differentiation, cell cycle control, proliferation, migration, apoptosis, and angiogenesis. The family is defined by a highly conserved winged helix-turn-helix DNA-binding domain. Several ETS factors have been implicated in human malignancies and are commonly involved in oncogenic chromosomal translocations (e.g. TMPRSS2-ERG in prostate cancer, EWS-FLI1 in Ewing sarcoma). We recently screened several ETS family members including ETS1, ERG, and ETV1 for direct small-molecule binders using SMMs. We are working collaboratively with the laboratories of Professor Todd Golub, Professor Levi Garraway, and Professor Bill Hahn at Dana-Farber Cancer Institute to characterize these direct ligands in a broad swath of biophysical and phenotypic studies, including high throughput gene expression assays. Finally, we have also extended the SMM approach to screen for small molecules that selectively target oncogenic fusion proteins involving ETS factors over the non-oncogenic unfused counterparts.

• Latent cytoplasmic transcription factors reside in the cytoplasm in an inactive form until activation is triggered by a cell surface receptor-ligand interaction. Upon activation, these transcription factors translocate into the nucleus where they interact with other transcription factors to regulate transcription. Several latent cytoplasmic transcription factors such as the STATs and NF-κB have increased activity in most human cancers. Our group has identified natural products and synthetic compounds that directly bind to various members of the STAT or NF-κB families and inhibit transcription in cells. We are engaged in a variety of biophysical and cellular studies to understand the mechanism for these compounds and we have undertaken synthetic chemistry to elucidate structure-activity relationships.

Structurally and mechanistically novel probes of deacetylase enzymes

Histone deacetylases (HDACs) have emerged as valuable targets for small-molecule probes and drugs due to their fundamental role in transcriptional regulation and implication in several diseases. Much effort has been placed on structure-aided design for inhibitors of the HDAC active site, though limited progress has been made towards developing inhibitors selective for individual isoforms. High-throughput and unbiased screens to discover novel molecular scaffolds will be critical for developing a collection of isoform-selective small molecules, which would enable investigators to probe the function of individual isoforms in different cellular and medical contexts. For example, our group has an interest in elucidating the relevant cellular functions and substrates for HDAC3. HDAC3 forms multiprotein complexes with co-repressors such as SMRT and NCoR to regulate the transcription of several genes important in development and disease. HDAC3 also deacetylates several non-histone substrates, suggesting the protein may have cellular roles beyond transcriptional repression. Recent studies suggest novel functions for HDAC3 independent of deacetylase activity. Small molecules that selectively modulate HDAC3 over other HDACs should be useful probes to understand the novel roles of HDAC3 in a cellular context. These probes may also be used to understand emerging associations between HDAC3 and leukemia or ovarian cancer. Our group combines direct binding assays with biochemical and cellular assays to identify HDAC modulators with novel chemical structures, novel selectivity patterns, or novel mechanisms unrelated to deacetylase activity.

Small-molecule microarrays

Over the last decade, small-molecule microarrays (SMMs) have proven to be a general, robust and scalable screening platform for discovering protein-small molecule interactions that lead to modulators of protein function. SMMs are manufactured by robotically arraying stock solutions of compounds onto functionalized substrates such as glass microscope slides. The arrays are then incubated with proteins of interest and putative protein-small molecule interactions are typically detected using fluorescently labeled antibodies and a standard fluorescence slide scanner. The high-throughput and miniaturized nature of the microarray-based binding assay allows for screening of large panels of proteins against tens of thousands of compounds in a relatively short time frame. Our group has developed multiple approaches to manufacturing SMMs including fluorous-based homogeneous display and isocyanate-based covalent attachment for heterogeneous display. The isocyanate approach allows us to make SMMs that contain compounds not intentionally synthesized or modified for immobilization onto a solid substrate allowing us to make arrays containing bioactive small molecules, including FDA-approved drugs, synthetic drug-like compounds, natural products, and products of diversity-oriented synthesis on a single slide. Based on an in silico evaluation of more than 400,000 compounds coming from a variety of these sources, we estimate that 77% of the compounds are compatible with immobilization using the isocyanate strategy and we routinely prepare SMMs containing compounds from Broad Institute screening collection. Our group and other groups have used SMMs to identify small molecules that target proteases, kinases, histone deacetylases, and transcription factors. Notably, our group recently used this approach to identify a direct binder to the ‘undruggable’ extracellular growth factor Sonic hedgehog (Shh) that inhibits the Shh signaling pathway in human cell lines and primary keratinocytes. Projects are underway to expand the utility of the SMM platform to other applications including target identification and diagnostics.