Alanna Schepartz

Design and Evolution of a Miniature Bcl-2 Binding Protein

A major challenge for chemical biologists lies in the design of potent and selective ligands for protein surfaces. Here a protein grafting and evolution strategy is used to discover highly potent and specific miniature protein ligands for human Bcl-2 and Bcl-XL . Miniature proteins could be used to dissect, modulate, or analyze a single protein function, irrespective of the other functions the protein may regulate within the proteome.

Bridged β3-Peptide Inhibitors of p53-hDM2 Complexation–Correlation Between Affinity and Cell Permeability

Beta-peptides possess several features that are desirable in peptidomimetics; they are easily synthesized, fold into stable secondary structures in physiologic buffers, and resist proteolysis. They can also bind to a diverse array of proteins to inhibit their interactions with alpha-helical ligands. beta-peptides are usually not cell-permeable, however, and this feature limits their utility as research tools and potential therapeutics. Appending an Arg(8) sequence to a beta-peptide improves uptake but adds considerable mass. We previously reported that embedding a small cationic patch within a PPII, alpha-, or beta-peptide helix improves uptake without the addition of significant mass. In another mass-neutral strategy, Verdine, Walensky, and others have reported that insertion of a hydrocarbon bridge between the i and i + 4 positions of an alpha-helix also increases cell uptake. Here we describe a series of beta-peptides containing diether and hydrocarbon bridges and compare them on the basis of cell uptake and localization, affinities for hDM2, and 14-helix structure. Our results highlight the relative merits of the cationic-patch and hydrophobic-bridge strategies for improving beta-peptide uptake and identify a surprising correlation between uptake efficiency and hDM2 affinity.

Arginine Topology Controls Escape of Minimally Cationic Proteins from Early Endosomes to the Cytoplasm

Proteins represent an expanding class of therapeutics, but their actions are limited primarily to extracellular targets because most peptidic molecules fail to enter cells. Here we identified two small proteins, miniature protein 5.3 and zinc finger module ZF5.3, that enter cells to reach the cytosol through rapid internalization and escape from Rab5+ endosomes. The trafficking pathway mapped for these molecules differs from that of Tat and Arg(8), which require transport beyond Rab5+ endosomes to gain cytosolic access. Our results suggest that the ability of 5.3 and ZF5.3 to escape from early endosomes is a unique feature and imply the existence of distinct signals, encodable within short sequences, that favor early versus late endosomal release. Identifying these signals and understanding their mechanistic basis will illustrate how cells control the movement of endocytic cargo and may allow researchers to engineer molecules to follow a desired delivery pathway for rapid cytosolic access.

Minimally cationic, cell-permeable miniature proteins via alpha-helical arginine display

Protein therapeutics are a blossoming industry, with revenues exceeding

Miniature Protein Inhibitors of the p53–hDM2 Interaction

51 billion in 2005 and a growth rate nearly three times that of the overall pharmaceutical industry. Although it has been known for decades that cationic polymers can transport molecular cargos across the plasma membrane, inefficient cellular delivery continues to impede the development of protein drugs. Our lab recently reported that small, folded proteins containing a minimal cationic motif embedded within a type II polyproline (PPII) helix efficiently cross the plasma membrane of eukaryotic cells. Here we demonstrate that an even smaller cationic motif can be embedded within the α-helix of a small, folded protein to generate molecules that penetrate cells significantly more efficiently than arginine-rich sequences or Tat. Our results suggest that the function of cell permeability can be encoded by judicious placement of as few as 2−3 additional arginine residues on a protein α-helix.

Surveying Protein Structure and Function Using Bis-Arsenical Small Molecules

We have developed a strategy for the design of miniature proteins that bind DNA or protein surfaces with high affinity and selectivity. This strategy, which is often called protein grafting, involves dissecting a functional recognition epitope from its native a-helical or polyproline type II (PPII) helical context and presenting it on a small but structured protein scaffold (Figure 1A). Here we describe the development and characterization of miniature proteins that bind the human double-minute 2 oncoprotein (hDM2) in the nanomolar concentration range, and inhibit its interaction with a peptide (p53AD15–31) derived from the activation domain of p53 (p53AD). hDM2 is the principal cellular antagonist of the tumor-suppressor protein p53. Elevated hDM2 levels are found in many solid tumors that express wild-type p53 and there is considerable interest in hDM2 ligands that are capable of up-regulating p53 activity in vitro or in vivo. The high-resolution structure of the p53AD–hDM2 complex has revealed a recognition epitope that is composed primarily of three p53AD residues (F19, W23, and L26); these are located on one face of a short a-helix. Although the p53AD peptide possesses little a-helical structure in the absence of hDM2, augmenting the level of intrinsic a-helix structure in p53AD by using constrained, unnatural amino acids dramatically increases affinity for hDM2 in vitro and activity in vivo. In addition, several other scaffolds have been used to display the p53AD epitope, including large proteins, cyclic b-hairpin peptides, retro-inverso peptides, and b-peptides. The first highly active small-molecule inhibitors were reported in 2004 and had IC50 values for inhibiting the p53–hDM2 interaction in the 100–300 nM range. These molecules also resembled p53AD’s primary recognition epitope. Since all these inhibitors appear to preorganize the p53AD epitope to some degree, we reasoned that protein grafting would be a logical route to developing miniature protein hDM2 ligands. In contrast with the previously reported molecules, miniature protein-based inhibitors would be both synthetically tractable and genetically encodable. This would facilitate their use as in vitro and in vivo tools for probing the intricate p53/hDM2 pathway. Avian pancreatic polypeptide (aPP, Figure 1B) is a small, well-folded miniature protein that consists of an eight-residue PPII helix linked through a type I b-turn to an eighteen-residue a-helix. Structure-guided alignment of the a-helical segments of p53AD and aPP (Figure 1B) positions the three critical hDM2 contact residues (F22, W26, and L29 in the aPP-aligned sequence) and five residues important for aPP folding (L17,

A new strategy for directed protein cleavage

Exploration across the fields of biology, chemical biology, and medicine has led to an increasingly complex, albeit incomplete, view of the interactions that drive lifes processes. The ability to monitor and track the movement, activity, and interactions of biomolecules in living cells is an essential part of this investigation. In our laboratory, we have endeavored to develop tools that are capable not only of monitoring protein localization but also reporting on protein structure and function. Central to our efforts is a new strategy, bipartite tetracysteine display, that relies on the specific and high-affinity interaction between a fluorogenic, bis-arsenical small molecule and a unique protein sequence, conformation, or assembly. In 1998, a small-molecule analogue of fluorescein with two arsenic atoms, FlAsH, was shown by Tsien and coworkers to fluoresce upon binding to a linear amino acid sequence, Cys-Cys-Arg-Glu-Cys-Cys. Later work demonstrated that substituting Pro-Gly for Arg-Glu optimized both binding and fluorescence yield. Our strategy of bipartite tetracysteine display emanated from the idea that it would be possible to replace the intervening Pro-Gly dipeptide in this sequence with a protein or protein partnership, provided the assembled protein fold successfully reproduced the approximate placement of the two Cys-Cys pairs. In this Account, we describe our recent progress in this area, with an emphasis on the fundamental concepts that underlie the successful use of bis-arsenicals such as FlAsH and the related ReAsH for bipartite display experiments. In particular, we highlight studies that have explored how broadly bipartite tetracysteine display can be employed and that have navigated the conformational boundary conditions favoring success. To emphasize the utility of these principles, we outline two recently reported applications of bipartite tetracysteine display. The first is a novel, encodable, selective, Src kinase sensor that lacks fluorescent proteins but possesses a fluorescent readout exceeding that of most sensors based on Förster resonance energy transfer (FRET). The second is a unique method, called complex-edited electron microscopy (CE-EM), that facilitates visualization of protein-protein complexes with electron microscopy. Exciting as these applications may be, the continued development of small-molecule tools with improved utility in living cells, let alone in vivo, will demand a more nuanced understanding of the fundamental photophysics that lead to fluorogenicity, as well as creative approaches toward the synthesis and identification of new and orthogonal dye-tag pairs that can be applied facilely in tandem. We describe one example of a dye-sequence tag pair that is chemically distinct from bis-arsenical chemistry. Through further effort, we expect that that bipartite tetracysteine display will find successful use in the study of sophisticated biological questions that are essential to the fields of biochemistry and biology as well as to our progressive understanding of human disease.

In Silico Improvement of β3-Peptide Inhibitors of p53•hDM2 and p53•hDMX

Abstract Attachment of a Ni(II)-glyglyhis chelate to trifluoperazine results in a molecule capable of cleaving calmodulin at a single locus upon activation with a peracid.

β-Peptides with improved affinity for hDM2 and hDMX

There is great interest in molecules capable of inhibiting the interactions between p53 and its negative regulators hDM2 and hDMX, as these molecules have validated potential against cancers in which one or both oncoproteins are overexpressed. We reported previously that appropriately substituted beta(3)-peptides inhibit these interactions and, more recently, that minimally cationic beta(3)-peptides are sufficiently cell permeable to upregulate p53-dependent genes in live cells. These observations, coupled with the known stability of beta-peptides in a cellular environment, and the recently reported structures of hDM2 and hDMX, motivated us to exploit computational modeling to identify beta-peptides with improved potency and/or selectivity. This exercise successfully identified a new beta(3)-peptide, beta53-16, that possesses the highly desirable attribute of high affinity for both hDM2 and hDMX and identifies the 3,4-dichlorophenyl moiety as a novel determinant of hDMX affinity.

In Silico Improvement of beta(3)-Peptide Inhibitors of p53 center dot hDM2 and p53 center dot hDMX

We previously described a series of 3(14)-helical beta-peptides that bind the hDM2 protein and inhibit its interaction with a p53-derived peptide in vitro. Here we present a detailed characterization of the interaction of these peptides with hDM2 and report two new beta-peptides in which non-natural side chains have been substituted into the hDM2-recognition epitope. These peptides feature both improved affinity and inhibitory potency in fluorescence polarization and ELISA assays. Additionally, one of the new beta-peptides also binds the hDM2-related protein, hDMX, which has been identified as another key therapeutic target for activation of the p53 pathway in tumors.