William C. K. Pomerantz

Specific Acetylation Patterns of H2A.Z Form Transient Interactions with the BPTF Bromodomain

Co-reporter:Gabriella T. Perell, Neeraj K. Mishra, Babu Sudhamalla, Peter D. Ycas, Kabirul Islam, and William C. K. Pomerantz

Biochemistry September 5, 2017 Volume 56(Issue 35) pp:4607-4607

Publication Date(Web):August 3, 2017

DOI:10.1021/acs.biochem.7b00648

Post-translational lysine acetylation of histone tails affects both chromatin accessibility and recruitment of multifunctional bromodomain-containing proteins for modulating transcription. The bromodomain- and PHD finger-containing transcription factor (BPTF) regulates transcription but has also been implicated in high gene expression levels in a variety of cancers. In this report, the histone variant H2A.Z, which replaces H2A in chromatin, is evaluated for its affinity for BPTF with a specific recognition pattern of acetylated lysine residues of the N-terminal tail region. Although BPTF immunoprecipitates H2A.Z-containing nucleosomes, a direct interaction with its bromodomain has not been reported. Using protein-observed fluorine nuclear magnetic resonance (PrOF NMR) spectroscopy, we identified a diacetylation of H2A.Z on lysine residues 4 and 11, with the highest affinity for BPTF with a Kd of 780 μM. A combination of subsequent 1H NMR Carr–Purcell–Meiboom–Gill experiments and photo-cross-linking further confirmed the specificity of the diacetylation pattern at lysines 4 and 11. Because of an adjacent PHD domain, this transient interaction may contribute to a higher-affinity bivalent interaction. Further evaluation of specificity toward a set of bromodomains, including two BET bromodomains (Brd4 and BrdT) and two Plasmodium falciparum bromodomains, resulted in one midmicromolar affinity binder, PfGCN5 (Kd = 650 μM). With these biochemical experiments, we have identified a direct interaction of histone H2A.Z with bromodomains with a specific acetylation pattern that further supports the role of H2A.Z in epigenetic regulation.

Synthesis of Intrinsically Disordered Fluorinated Peptides for Modular Design of High-Signal 19F MRI Agents

Co-reporter:Steven E. Kirberger;Sofia D. Maltseva;Joseph C. Manulik;Dr. Samuel A. Einstein;Dr. Bradley P. Weegman; Dr. Michael Garwood; Dr. William C. K. Pomerantz

Angewandte Chemie International Edition 2017 Volume 56(Issue 23) pp:6440-6444

Publication Date(Web):2017/06/01

DOI:10.1002/anie.201700426

Abstract19F MRI is valuable for in vivo imaging due to the only trace amounts of fluorine in biological systems. Because of the low sensitivity of MRI however, designing new fluorochemicals remains a significant challenge for achieving sufficient 19F signal. Here, we describe a new class of high-signal, water-soluble fluorochemicals as 19F MRI imaging agents. A polyamide backbone is used for tuning the proteolytic stability to avoid retention within the body, which is a limitation of current state-of-the-art perfluorochemicals. We show that unstructured peptides containing alternating N-ϵ-trifluoroacetyllysine and lysine provide a degenerate 19F NMR signal. 19F MRI phantom images provide sufficient contrast at micromolar concentrations, showing promise for eventual clinical applications. Finally, the degenerate high signal characteristics were retained when conjugated to a large protein, indicating potential for in vivo targeting applications, including molecular imaging and cell tracking.

Synthesis of Intrinsically Disordered Fluorinated Peptides for Modular Design of High-Signal 19F MRI Agents

Co-reporter:Steven E. Kirberger;Sofia D. Maltseva;Joseph C. Manulik;Dr. Samuel A. Einstein;Dr. Bradley P. Weegman; Dr. Michael Garwood; Dr. William C. K. Pomerantz

Angewandte Chemie 2017 Volume 129(Issue 23) pp:6540-6544

Publication Date(Web):2017/06/01

DOI:10.1002/ange.201700426

Abstract19F MRI is valuable for in vivo imaging due to the only trace amounts of fluorine in biological systems. Because of the low sensitivity of MRI however, designing new fluorochemicals remains a significant challenge for achieving sufficient 19F signal. Here, we describe a new class of high-signal, water-soluble fluorochemicals as 19F MRI imaging agents. A polyamide backbone is used for tuning the proteolytic stability to avoid retention within the body, which is a limitation of current state-of-the-art perfluorochemicals. We show that unstructured peptides containing alternating N-ϵ-trifluoroacetyllysine and lysine provide a degenerate 19F NMR signal. 19F MRI phantom images provide sufficient contrast at micromolar concentrations, showing promise for eventual clinical applications. Finally, the degenerate high signal characteristics were retained when conjugated to a large protein, indicating potential for in vivo targeting applications, including molecular imaging and cell tracking.

Protein-Observed Fluorine NMR: A Bioorthogonal Approach for Small Molecule Discovery

Co-reporter:Keith E. Arntson

Journal of Medicinal Chemistry 2016 Volume 59(Issue 11) pp:5158-5171

Publication Date(Web):November 24, 2015

DOI:10.1021/acs.jmedchem.5b01447

The 19F isotope is 100% naturally abundant and is the second most sensitive and stable NMR-active nucleus. Unlike the ubiquitous hydrogen atom, fluorine is nearly absent in biological systems, making it a unique bioorthogonal atom for probing molecular interactions in biology. Over 73 fluorinated proteins have been studied by 19F NMR since the seminal studies of Hull and Sykes in 1974. With advances in cryoprobe production and fluorinated amino acid incorporation strategies, protein-based 19F NMR offers opportunities to the medicinal chemist for characterizing and ultimately discovering new small molecule protein ligands. This review will highlight new advances using 19F NMR for characterizing small molecule interactions with both small and large proteins as well as detailing NMR resonance assignment challenges and amino acid incorporation approaches.

Protein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to 1H CPMG Ligand-Observed NMR

Co-reporter:Andrew K. Urick, Luis Pablo Calle, Juan F. Espinosa, Haitao Hu, and William C. K. Pomerantz

ACS Chemical Biology 2016 Volume 11(Issue 11) pp:3154

Publication Date(Web):September 14, 2016

DOI:10.1021/acschembio.6b00730

To evaluate its potential as a ligand discovery tool, we compare a newly developed 1D protein-observed fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We compare the two methods’ hit sensitivity, triaging ability, experiment speed, material consumption, and the potential for false positives and negatives. To this end, we screened 930 fragment molecules against Brd4 in mixtures of five and followed up these studies with mixture deconvolution and affinity characterization of the top hits. In selected examples, we also compare the environmental responsiveness of the 19F chemical shift to 1H in 1D-protein observed 1H NMR experiments. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titration calorimetry. We conclude that for the protein understudy here, PrOF NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observed 1H NMR may also be effective; however, the 19F chemical shift remains significantly more responsive.

Prediction of 19F NMR Chemical Shifts in Labeled Proteins: Computational Protocol and Case Study

Co-reporter:William C. IsleyIII; Andrew K. Urick; William C. K. Pomerantz;Christopher J. Cramer

Molecular Pharmaceutics 2016 Volume 13(Issue 7) pp:2376-2386

Publication Date(Web):May 24, 2016

DOI:10.1021/acs.molpharmaceut.6b00137

The structural analysis of ligand complexation in biomolecular systems is important in the design of new medicinal therapeutic agents; however, monitoring subtle structural changes in a protein’s microenvironment is a challenging and complex problem. In this regard, the use of protein-based 19F NMR for screening low-molecular-weight molecules (i.e., fragments) can be an especially powerful tool to aid in drug design. Resonance assignment of the protein’s 19F NMR spectrum is necessary for structural analysis. Here, a quantum chemical method has been developed as an initial approach to facilitate the assignment of a fluorinated protein’s 19F NMR spectrum. The epigenetic “reader” domain of protein Brd4 was taken as a case study to assess the strengths and limitations of the method. The overall modeling protocol predicts chemical shifts for residues in rigid proteins with good accuracy; proper accounting for explicit solvation of fluorinated residues by water is critical.

Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules

Co-reporter:Andrew K. Urick, Laura M. L. Hawk, Melissa K. Cassel, Neeraj K. Mishra, Shuai Liu, Neeta Adhikari, Wei Zhang, Camila O. dos Santos, Jennifer L. Hall, and William C. K. Pomerantz

ACS Chemical Biology 2015 Volume 10(Issue 10) pp:2246

Publication Date(Web):July 9, 2015

DOI:10.1021/acschembio.5b00483

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a 19F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of 19F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 μM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed 19F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

Fragment Screening and Druggability Assessment for the CBP/p300 KIX Domain through Protein-Observed 19FNMR Spectroscopy

Co-reporter:Clifford T. Gee;Edward J. Koleski ; William C. K. Pomerantz

Angewandte Chemie 2015 Volume 127( Issue 12) pp:3806-3810

Publication Date(Web):

DOI:10.1002/ange.201411658

Abstract

¹⁹F NMR spectroscopy of labeled proteins is a sensitive method for characterizing structure, conformational dynamics, higher-order assembly, and ligand binding. Fluorination of aromatic side chains has been suggested as a labeling strategy for small-molecule ligand discovery for protein–protein interaction interfaces. Using a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX, we report the first full small-molecule screen using protein-observed ¹⁹F NMR spectroscopy. Screening of 508 compounds and validation by ¹H–¹⁵N HSQC NMR spectroscopy led to the identification of a minimal pharmacaphore for the MLL-KIX interaction site. Hit rate analysis for the CREB-KIX and MLL-KIX sites provided a metric to assess the ligandability or “druggability” of each interface informing future medicinal chemistry efforts. The structural information from the simplified spectra and data collection speed, affords a new screening tool for analysis of protein interfaces and discovery of small molecules.

Fragment Screening and Druggability Assessment for the CBP/p300 KIX Domain through Protein-Observed 19FNMR Spectroscopy

Co-reporter:Clifford T. Gee;Edward J. Koleski ; William C. K. Pomerantz

Angewandte Chemie International Edition 2015 Volume 54( Issue 12) pp:3735-3739

Publication Date(Web):

DOI:10.1002/anie.201411658

Abstract

¹⁹F NMR spectroscopy of labeled proteins is a sensitive method for characterizing structure, conformational dynamics, higher-order assembly, and ligand binding. Fluorination of aromatic side chains has been suggested as a labeling strategy for small-molecule ligand discovery for protein–protein interaction interfaces. Using a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX, we report the first full small-molecule screen using protein-observed ¹⁹F NMR spectroscopy. Screening of 508 compounds and validation by ¹H–¹⁵N HSQC NMR spectroscopy led to the identification of a minimal pharmacaphore for the MLL-KIX interaction site. Hit rate analysis for the CREB-KIX and MLL-KIX sites provided a metric to assess the ligandability or “druggability” of each interface informing future medicinal chemistry efforts. The structural information from the simplified spectra and data collection speed, affords a new screening tool for analysis of protein interfaces and discovery of small molecules.

Fluorinated Aromatic Amino Acids Are Sensitive 19F NMR Probes for Bromodomain-Ligand Interactions

Co-reporter:Neeraj K. Mishra, Andrew K. Urick, Stuart W. J. Ember, Ernst Schönbrunn, and William C. Pomerantz

ACS Chemical Biology 2014 Volume 9(Issue 12) pp:2755

Publication Date(Web):October 7, 2014

DOI:10.1021/cb5007344

We describe a 19F NMR method for detecting bromodomain–ligand interactions using fluorine-labeled aromatic amino acids due to the conservation of aromatic residues in the bromodomain binding site. We test the sensitivity, accuracy, and speed of this method with small molecule ligands (+)-JQ1, BI2536, Dinaciclib, TG101348, and acetaminophen using three bromodomains Brd4, BrdT, and BPTF. Simplified 19F NMR spectra allowed for simultaneous testing of multiple bromodomains to assess selectivity and identification of a new BPTF ligand. Fluorine labeling only modestly affected the Brd4 structure and function assessed by isothermal titration calorimetry, circular dichroism, and X-ray crystallography. The speed, ease of interpretation, and low concentration of protein needed for binding experiments affords a new method to discover and characterize both native and new ligands.