William C. Pomerantz

A rationally designed aldolase foldamer.

Current strategies for creating enzyme-like catalysts range from rational[1] and computational design[2] to evolutionary searches of large molecular libraries.[3] Sequence-specific polymers are particularly attractive starting points for these efforts because of their ability to adopt three-dimensional structures that preorganize functional groups for catalysis. Although natural enzymes are constructed from α-amino acids, many other backbone structures can give rise to well-defined secondary and tertiary structures. Such non-natural oligomers, often referred to as “foldamers”, have the potential to display properties akin to those of proteins.[4–8]

Nanofibers and Lyotropic Liquid Crystals from a Class of Self‐Assembling β‐Peptides

Lyotropic liquid-crystalline (LC) phases, in other words, LC phases induced by the presence of a solvent, form through the assembly of large anisometric particles, such as rigid polymers, viruses, or inorganic rods, as a result of excluded volume interactions (Onsager theory). Small molecules can form lyotropic LC phases if they undergo self-assembly to generate anisometric nanostructures. Aqueous LC phases are commonly observed for small molecules that have clearly segregated hydrophilic and lipophilic segments, such as detergents and lipids. For such “globally amphiphilic” molecules it is believed that hydrophobically induced association of the lipophilic segments drives the assembly that underlies LC formation. Chromonic LCs are formed from a distinct class of small molecules that self-assemble in water but do not display a simple segregation of hydrophilic and lipophilic segments (they are non-globally amphiphilic). It is difficult to understand how the self-assembly of chromonic molecules (i.e., molecules that form chromonic LCs) is related to the nature and internal organization of lipophilic surfaces because these molecular properties cannot readily be altered in an incremental fashion. Here we describe a class of oligomers that, like the chromonic molecules, are not globally amphiphilic but nevertheless form LC phases in water. The intrinsic modularity of these oligomers allows rational and systematic modification of lipophilic or hydrophilic components, which enables us to define key features that are required for LC formation. Short oligomers of b-amino acids (b-peptides) are attractive for systematic study of assembly processes leading to LCs because b-peptides can display a diverse range of functionalized side chains, and these oligomers fold into compact and stable conformations that orient the side chains in predictable ways. The most widely studied b-peptide secondary structure is the 14-helix, which is defined by i, i 2 C=O···H N hydrogen bonds (14-membered ring) between backbone amides and contains approximately three residues per helical turn. The high stability of the 14-helix, in combination with ready manipulation of b-peptide sequence, permits considerable control over the nanopatterning of chemical functionality in three dimensions. Here we exploit these features by designing b-peptide oligomers that fold into helical nanostructures and are decorated with functional groups in patterns that either do or do not confer global amphiphilicity on the 14-helix. As described below, this sequence-based patterning strategy has led to the discovery of b-peptide oligomers that are non-globally amphiphilic and exhibit liquid crystallinity in aqueous solution. Our initial experiments focused on sequence isomers A and iso-A (Figure 1). The latter has a repeating triad motif containing trans-2-aminocyclohexanecarboxylic acid (ACHC), bhomophenylalanine (bhPhe), and bhLys residues; the lipophilic–lipophilic–hydrophilic ACHC-bhPhebhLys sequence repeat pattern in iso-A leads to a globally amphiphilic nanostructure. In contrast, the sequence of A does not lead to global segregation of lipophilic and hydrophilic side chains in the 14-helix, but rather to a distribution of lipophilic and hydrophilic side chains around the entire periphery of the helix (Figure 1). The helix from A is defined as non-globally amphiphilic. Since global amphiphilicity of bpeptides has previously been associated with liquid crystallinity, we expected iso-A but not A to form a LC phase in water. Surprisingly, however, strong birefringence was observed for aqueous solutions containing A 6.5 wt%, (36 mm, Figure 2), but no birefringence was detected for solutions of iso-A up to the solubility limit > 10 wt% (57 mm, Figure 2). Inspection of the birefringent domains at high magnification shows an absence of optical textures characteristic of higher ordered mesophases such as smectics and cholesterics; these observations lead us to conclude that the mesophase is likely nematic. The unexpected ability of the non-globally amphiphilic helix to form an LC phase is [*] V. M. Yuwono, Prof. J. D. Hartgerink Department of Chemistry and Bioengineering Rice University 6100 Main Street MS60, Houston, TX 77005 (USA) Fax: (+1)713-348-4201 E-mail: jdh@rice.edu

Effect of Sequence and Structural Properties on 14-Helical β-Peptide Activity against Candida albicans Planktonic Cells and Biofilms

Beta-peptides (beta-amino acid oligomers) that mimic the amphiphilic, helical, and cationic properties of natural antimicrobial peptides have previously been shown to display antifungal activity against planktonic Candida albicans cells. Beta-peptides offer several advantages over conventional peptides composed of alpha-amino acid residues, including conformational stability, resistance to proteases, and activity at physiological salt concentrations. We examined sequence-activity relationships toward both planktonic C. albicans cells and C. albicans biofilms, and the results suggest a toxicity mechanism involving membrane disruption. A strategy for fluorescently labeling a beta-peptide without diminishing antifungal activity was devised; labeled beta-peptides penetrated the cell membrane and accumulated in the cytoplasm of both planktonic and biofilm-associated cells. The labeled beta-peptide was detected only in metabolically inactive cells, which suggests that beta-peptide entry is correlated with cell death. The presence of a beta-peptide at a concentration near the minimum inhibitory concentration completely prevented planktonic C. albicans cells from forming a biofilm, suggesting that beta-peptides may be useful in preventing fungal colonization and biofilm formation.

Sekikaic acid and lobaric acid target a dynamic interface of the coactivator CBP/p300

Capturing a coactivator, naturally: the natural products sekikaic acid and lobaric acid, isolated after a high-throughput screen of a structurally diverse extract collection, effectively target the dynamic binding interfaces of the GACKIX domain of the coactivator CBP/p300. These molecules are the most effective inhibitors of the GACKIX domain yet described and are uniquely selective for this domain.

Lyotropic Liquid Crystals Formed from ACHC-Rich β-Peptides

We have examined the effect of β-peptide modifications on the propensity of these helical molecules to form lyotropic liquid crystalline (LC) phases in water. All of the β-peptides we have examined contain 10 residues. In each case, at least three residues are derived from trans-2-aminocyclohexanecarboxylic acid (ACHC), which strongly promotes folding to a 14-helical conformation. The structural features varied include the number of ACHC residues, the nature and spatial arrangement of charged side chains (cationic vs anionic), and the identity of groups at the β-peptide termini. We found that relatively small changes (e.g., swapping the positions of a cationic and an anionic side chain) could have large effects, such as abrogation of LC phase formation. The trends revealed by sequence-property studies led to the design of LC-forming β-peptides that bear biomolecular recognition groups (biotin or the tripeptide Arg-Gly-Asp). Structural analysis via circular dichroism and cryo-transmission electron microscopy revealed the existence of two different types of self-associated species, globular aggregates and nanofibers. Nanofibers are the predominant assembly formed at concentrations that lead to LC phase formation, and we conclude that these nanofibers are the functional mesogens. Overall, these studies show how the modularity of β-peptide oligomers enables elucidation of the relationship between molecular structure and large-scale self-assembly behavior.

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

The (19)F 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 (19)F NMR since the seminal studies of Hull and Sykes in 1974. With advances in cryoprobe production and fluorinated amino acid incorporation strategies, protein-based (19)F NMR offers opportunities to the medicinal chemist for characterizing and ultimately discovering new small molecule protein ligands. This review will highlight new advances using (19)F 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.

Profiling the Dynamic Interfaces of Fluorinated Transcription Complexes for Ligand Discovery and Characterization

The conformationally dynamic binding surfaces of transcription complexes present a particular challenge for ligand discovery and characterization. In the case of the KIX domain of the master coactivator CBP/p300, few small molecules have been reported that target its two allosterically regulated binding sites despite the important roles that KIX plays in processes ranging from memory formation to hematopoiesis. Taking advantage of the enrichment of aromatic amino acids at protein interfaces, here we show that the incorporation of six (19)F-labeled aromatic side chains within the KIX domain enables recapitulation of the differential binding footprints of three natural activator peptides (MLL, c-Myb, and pKID) in complex with KIX and effectively reports on allosteric changes upon binding using 1D NMR spectroscopy. Additionally, the examination of both the previously described KIX protein-protein interaction inhibitor Napthol-ASE-phosphate and newly discovered ligand 1-10 rapidly revealed both the binding sites and the affinities of these small molecules. Significantly, the utility of using fluorinated transcription factors for ligand discovery was demonstrated through a fragment screen leading to a new low molecular weight fragment ligand for CBP/p300, 1G7. Aromatic amino acids are enriched at protein-biomolecule interfaces; therefore, this quantitative and facile approach will be broadly useful for studying dynamic transcription complexes and screening campaigns complementing existing biophysical methods for studying these dynamic interfaces.

Ordering a Dynamic Protein Via a Small-Molecule Stabilizer

Like many coactivators, the GACKIX domain of the master coactivator CBP/p300 recognizes transcriptional activators of diverse sequence composition via dynamic binding surfaces. The conformational dynamics of GACKIX that underlie its function also render it especially challenging for structural characterization. We have found that the ligand discovery strategy of Tethering is an effective method for identifying small-molecule fragments that stabilize the GACKIX domain, enabling for the first time the crystallographic characterization of this important motif. The 2.0 Å resolution structure of GACKIX complexed to a small molecule was further analyzed by molecular dynamics simulations, which revealed the importance of specific side-chain motions that remodel the activator binding site in order to accommodate binding partners of distinct sequence and size. More broadly, these results suggest that Tethering can be a powerful strategy for identifying small-molecule stabilizers of conformationally malleable proteins, thus facilitating their structural characterization and accelerating the discovery of small-molecule modulators.

Comparison of Design Strategies for Promotion of β-Peptide 14-Helix Stability in Water

Many short β‐peptides adopt well‐defined conformations in organic solvents, but specialized stabilizing elements are required for folding to occur in aqueous solution. Several different strategies to stabilize the 14‐helical secondary structure in water have been developed, and here we provide a direct comparison of three such strategies. We have synthesized and characterized β‐peptide heptamers in which variously a salt bridge between side chains, a covalent link between side chains, or two cyclically constrained residues have been incorporated to promote 14‐helicity. The incorporation of a salt bridge does not generate significant 14‐helicity in water, according to CD and 2D NMR data. In contrast, incorporation either of a lactam bridge between side chains or of cyclic residues results in stable 14‐helices in water. The β‐peptides featuring trans‐2‐aminocyclohexanecarboxylic acid (ACHC) residues show the highest 14‐helical backbone stability, with hardly any sensitivity to pH or ionic strength. The β‐peptides featuring side‐chain‐to‐side‐chain cyclization show lower 14‐helical backbone stability and higher sensitivity to pH and ionic strength, but increased order between the side chains because of the cyclization.

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

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 (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F 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 (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.