Research Background and Accomplishments

Transcription factors (TFs) are the master regulators of cellular identity. These critical biological molecules specifically recognize stretches of DNA and act as genetic switches, turning sets of genes on and off in concert with one another in the cell. Owing to their broad control over the genetic landscape, if and when transcription factor function is altered, disease often follows. This is particularly true in cancer, where genetic insults frequently lead to inappropriate activation of oncogenic TFs. Despite their critical role in cancer, TFs have proven to be the most difficult class of biological targets for the development of small molecule drugs. A prime example is the MYC transcription factor. Aberrant activation of the MYC gene occurs in nearly all cases of Burkitt’s Lymphoma and is implicated in a majority of other cancers. Despite their known role in cancer and other diseases, nearly all transcription factors like MYC are considered “undruggable” due to the difficulty in developing small molecules to effectively bind their molecular surfaces. Therefore, there is an urgent need for new classes of molecules capable of blocking the oncogenic activity of MYC, and other TFs like it. The research proposed herein represents a novel approach to develop inhibitors that combine the properties of typical small molecule drugs with newer protein-based drugs, which we term “synthetic biologics,” to mimic the DNA binding domain of transcription factors themselves. The prestigious support of the Cancer Research Foundation for this ambitious project has enabled the development of Myc inhibitors in cancers driven by its overactive signaling, as well as set the stage for the broad development of synthetic gene regulatory agents in cancer and other diseases.

Support from the Cancer Research Foundation has enabled us to launch this inhibitor platform. Specific areas of progress include:

1. We have identified key design elements to retain the binding affinity and specificity afforded by full-length proteins (~60 kD in size) in our much smaller (~6 kD) synthetic biologics. We have provided evidence that these structural stabilization elements must encompass both secondary structure and tertiary structural stabilization motifs. We have also found that these design elements are general in nature, and can be applied to mimic other protein domains from oncogenic proteins. These data indicate that we should be able to develop new transcriptional antagonists beyond Myc.

2. We have establish that optimized compounds are inherently cell permeable through an active macropinocytosis uptake mechanism that is ubiquitous in cancer cells. Molecular imaging, proteomics and gene expression studies have shown that these compounds access both the cytosol and nucleus of cancer cells and antagonize Myc-dependent transcriptional activation at known target genes.

3. We have demonstrated that several optimized synthetic biologics can directly interfere with DNA binding by full length Myc/Max both in vitro as well as in live cells. These results validate our platform as the first to directly mimic the structure and function of these proteins.

Together, these initial findings represent an excellent foundation for the development of novel chemical probes target gene expression in cancer. The funding from CRF has been essential for our lab to pursue this high-risk, high-reward project, and has additionally provided a great source of initial funding to help establish my research program.

Research Program Accomplishments

Publications During the Funding Period.

1) Yang H, Swartz AM, Ellis-Guardiola K, Park HJ, Upp DM, Lee G, Zhang C, Srivastava P, Moellering RE & Lewis JC. “Evolving artificial metalloenzymes via iterative random mutagenesis.” Nat. Chem., 2018; 10(3): 318-24.

2) Li G, Montogomery JE, Eckert MA, Chang JW, Tienda SM, Lengyel E & Moellering RE*, “An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells.” Nat. Commun., 2017; 8(1): 1775. • Research Highlight – Nature Methods. 2018; 15(2): 99.

3) KleinJan A, Nimwegen MV, Tindemans I, Bruijn M, Bergen I, Montgomery JE, Moellering RE, Hoogsteden H, Boon L, Amsen A & Hendriks RW. “The Notch pathway inhibitor SAHM1 abrogates the hallmarks of asthma.” J. Allergy Clin. Immunol., ASAP, DOI: 10.1016/j.jaci.2017.08.042.

4) Mitra S, Montgomery JE, Kolar MJ, Li Gang, Verdine GL, Mills GB & Moellering RE*. “Stapled peptide inhibitors of RAB25 target context-specific roles in cancer.” Nat. Commun., 2017; 8(1): 660.

5) Chang JW, Lee G, Coukos J & Moellering RE*. “Profiling reactive metabolites via chemical trapping and targeted mass spectrometry.” Anal. Chem., 2016; 88(13): 6658-61.

6) Raymond E. Moellering & Sean Shangguan. “Synthetic DNA binding domain peptides and uses thereof.” The University of Chicago, U.S. patent application: 62/329,497.

Five additional manuscripts are in review or in revision.

New Grants During the Funding Period (leveraging CRF support).

1) American Cancer Society, Research Scholar Grant 01/01/18 – 12/31/21 PI: Moellering (no Co-PI/co-I) Title: Mimicking Myc: Targeting transcription with synthetic biologics

2) NIH/NIGMS (DP2GM128199-01) 09/30/17 – 05/31/22 PI: Moellering (no co-PI/co-I) Title: Targeting transcription with synthetic biologics Scientific Presentations During Funding Period (selected) 1) Monoclonal Antibodies in Medicine. Konnos Bay, Cyprus, June 18-21, 2018. 2) NIH High-Risk/High-Reward Research Symposium. Bethesda, MD, June 6-8, 2018.

3) V Scholars Summit. Fairfax, VA, April 25-26, 2018.

4) University of Illinois, Chicago, Department of Medicinal Chemistry. Chicago, IL, March 2, 2018.

5) Chicago Biomedical Consortium Symposium – “Small Molecule Discovery,” (plenary). Chicago, IL, November 17, 2017.

6) AbbVie/University of Chicago Chemistry Symposium. North Chicago, IL, September 20, 2017.

7) Molecular Therapeutics Symposium, University of Chicago, (co-organizer, talk). Chicago, IL, June 16, 2017.

8) 244th American Chemical Society National Meeting, “Peptides, proteins, and amino acids.” San Francisco, CA, April 5, 2017.

Research Overview.

Transcription factors (TFs) are the master regulators of cellular identity. These critical biological molecules specifically recognize stretches of DNA and act as genetic switches, turning sets of genes on and off in concert with one another in the cell. Owing to their broad control over the genetic landscape, if and when transcription factor function is altered, disease often follows. This is particularly true in cancer, where genetic insults frequently lead to inappropriate activation of oncogenic TFs. Despite their critical role in cancer, TFs have proven to be the most difficult class of biological targets for the development of small molecule drugs. A prime example is the MYC transcription factor. Aberrant activation of the MYC gene occurs in nearly all cases of Burkitt’s Lymphoma and is implicated in a majority of other cancers. Despite their known role in cancer and other diseases, nearly all transcription factors like MYC are considered “undruggable” due to the difficulty in developing small molecules to effectively bind their molecular surfaces. Therefore, there is an urgent need for new classes of molecules capable of blocking the oncogenic activity of MYC, and other TFs like it. The research proposed herein represents a novel approach to develop inhibitors that combine the properties of typical small molecule drugs with newer protein-based drugs, which we term “synthetic biologics,” to mimic the DNA binding domain of transcription factors themselves.

The prestigious support of an Cancer Research Foundation for this high-risk, high-reward project will enable the development of Myc inhibitors in cancers driven by its overactive signaling, as well as set the stage for the broad development of synthetic gene regulatory agents in cancer and other diseases. Project Update. We have made significant progress toward the development of synthetic biologics that are capable of specifically blocking oncogenic transcription factor function in cancer cells.

Our progress in the first year focused on three areas:

1. We have identified key design elements to retain the binding affinity and specificity afforded by full-length proteins (~60 kD in size) in our much smaller (~6 kD) synthetic biologics. We have provided evidence that these structural stabilization elements must encompass both secondary structure and tertiary structural motifs. We have also found that these elements are general in nature, and can be applied to mimic other protein domains with the same function.

2. We have shown that optimized compounds are inherently cell permeable through an active macropinocytosis uptake mechanism that ubiquitous in cancer cells. Focused imaging and proteomics studies have shown that these compounds access both the cytosol and nucleus of cancer cells.

3. We have demonstrated that several optimized synthetic biologics can directly interfere with DNA binding by full length Myc/Max both in vitro as well as in live cells. These results validate our platform as the first to directly mimic the structure and function of these proteins in live cells. Together these initial findings represent an excellent foundation for the development of novel chemical probes target gene expression in cancer. The funding from CRF has been essential for our lab to pursue this project and has additionally provided a great source of initial funding to help establish my lab.