Joshua A. Kritzer

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.

SUMMARY α-Synuclein (α-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because α-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson’s disease (PD). We previously created a yeast model of α-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to α-syn expression. We also uncovered a core group of proteins with diverse activities related to α-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of α-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress α-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of α-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced α-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of α-syn foci, re-established ER-to-Golgi trafficking and ameliorated α-syn-mediated damage to mitochondria. They also corrected the toxicity of α-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of α-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Getting in shape: controlling peptide bioactivity and bioavailability using conformational constraints.

Chemical biologists commonly seek out correlations between the physicochemical properties of molecules and their behavior in biological systems. However, a new paradigm is emerging for peptides in which conformation is recognized as the primary determinant of bioactivity and bioavailability. This review highlights an emerging body of work that directly addresses how a peptides conformation controls its biological effects, cell penetration, and intestinal absorption. Based on this work, the dream of mimicking the potency and bioavailability of natural product peptides is getting closer to reality.

Rapid selection of cyclic peptides that reduce alpha-synuclein toxicity in yeast and animal models.

Phage display has demonstrated the utility of cyclic peptides as general protein ligands, but cannot access proteins inside eukaryotic cells. Expanding a novel chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of α-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson’s disease. From a pool of five million transformants, we isolated two related cyclic peptide constructs which specifically reduce the toxicity of human α-synuclein. These expressed cyclic peptide constructs also prevent dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson’s model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease.

Comprehensive analysis of loops at protein-protein interfaces for macrocycle design

Inhibiting protein-protein interactions (PPIs) with synthetic molecules remains a frontier of chemical biology. Many PPIs have been successfully targeted by mimicking α-helices at interfaces, but most PPIs are mediated by non-helical, non-strand peptide loops. We sought to comprehensively identify and analyze these loop-mediated PPIs by writing and implementing LoopFinder, a customizable program that can identify loop-mediated PPIs within all protein-protein complexes in the Protein Data Bank. Comprehensive analysis of the entire set of 25,005 interface loops revealed common structural motifs and unique features that distinguish loop-mediated PPIs from other PPIs. “Hot loops,” named in analogy to protein hot spots, were identified as loops with favorable properties for mimicry using synthetic molecules. The hot loops and their binding partners represent new and promising PPIs for the development of macrocycle and constrained peptide inhibitors.

Miniature Protein Inhibitors of the p53–hDM2 Interaction

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,

Peptide Bicycles that Inhibit the Grb2 SH2 Domain

Developing short peptides into useful probes and therapeutic leads remains a difficult challenge. Structural rigidification is a proven method for improving the properties of short peptides. In this work, we report a strategy for stabilizing peptide macrocycles by introducing side‐chain‐to‐side‐chain staples to produce peptide bicycles with higher affinity, selectivity, and resistance to degradation. We have applied this strategy to G1, an 11‐residue peptide macrocycle that binds the Src homology 2 (SH2) domain of growth‐factor‐bound protein 2 (Grb2). Several homodetic peptide bicycles were synthesized entirely on‐resin with high yields. Two rounds of iterative design produced peptide bicycle BC1, which is 60 times more potent than G1 and 200 times more selective. Moreover, BC1 is completely intact after 24 hours in buffered human serum, conditions under which G1 is completely degraded. Our peptide‐bicycle approach holds promise for the development of selective inhibitors of SH2 domains and other phosophotyrosine (pTyr)‐binding proteins, as well as inhibitors of many other protein–protein interactions.

Stapled peptides: Magic bullets in nature's arsenal

Selectivity is a key obstacle in drug development. A new study describes how “peptide stapling,” a technique for making peptide α-helices more potent and cell permeable, allows the design of MCL-1 inhibitors with extraordinary selectivity.

Diversity-Oriented Stapling Yields Intrinsically Cell-Penetrant Inducers of Autophagy

Autophagy is an essential pathway by which cellular and foreign material are degraded and recycled in eukaryotic cells. Induction of autophagy is a promising approach for treating diverse human diseases, including neurodegenerative disorders and infectious diseases. Here, we report the use of a diversity-oriented stapling approach to produce autophagy-inducing peptides that are intrinsically cell-penetrant. These peptides induce autophagy at micromolar concentrations in vitro, have aggregate-clearing activity in a cellular model of Huntingtons disease, and induce autophagy in vivo. Unexpectedly, the solution structure of the most potent stapled peptide, DD5-o, revealed an α-helical conformation in methanol, stabilized by an unusual (i,i+3) staple which cross-links two d-amino acids. We also developed a novel assay for cell penetration that reports exclusively on cytosolic access and used it to quantitatively compare the cell penetration of DD5-o and other autophagy-inducing peptides. These new, cell-penetrant autophagy inducers and their molecular details are critical advances in the effort to understand and control autophagy. More broadly, diversity-oriented stapling may provide a promising alternative to polycationic sequences as a means for rendering peptides more cell-penetrant.

Macrocyclization of the ATCUN motif controls metal binding and catalysis.

We report the design, synthesis, and characterization of macrocyclic analogues of the amino-terminal copper and nickel binding (ATCUN) motif. These macrocycles have altered pH transitions for metal binding, and unlike linear ATCUN motifs, the optimal cyclic peptide 1 binds Cu(II) selectively over Ni(II) at physiological pH. UV-vis and EPR spectroscopy showed that cyclic peptide 1 can coordinate Cu(II) or Ni(II) in a square planar geometry. Metal binding titration and ESI-MS data revealed a 1:1 binding stoichiometry. Macrocyclization allows for coordination of Cu(II) or Ni(II) as in linear ATCUN motifs, but with enhanced DNA cleavage by the Cu(II)-1 complex relative to linear analogues. The Cu(II)-1 complex was also capable of producing diffusible hydroxyl radicals, which is unique among ATCUN motifs and most other common copper(II) chelators.

Metal-binding and redox properties of substituted linear and cyclic ATCUN motifs

The amino-terminal copper and nickel binding (ATCUN) motif is a short peptide sequence found in human serum albumin and other proteins. Synthetic ATCUN-metal complexes have been used to oxidatively cleave proteins and DNA, cross-link proteins, and damage cancer cells. The ATCUN motif consists of a tripeptide that coordinates Cu(II) and Ni(II) ions in a square planar geometry, anchored by chelation sites at the N-terminal amine, histidine imidazole and two backbone amides. Many studies have shown that the histidine is required for tight binding and square planar geometry. Previously, we showed that macrocyclization of the ATCUN motif can lead to high-affinity binding with altered metal ion selectivity and enhanced Cu(II)/Cu(III) redox cycling (Inorg. Chem. 2013, 52, 2729-2735). In this work, we synthesize and characterize several linear and cyclic ATCUN variants to explore how substitutions at the histidine alter the metal-binding and catalytic properties. UV-visible spectroscopy, EPR spectroscopy and mass spectrometry indicate that cyclization can promote the formation of ATCUN-like complexes even in the absence of imidazole. We also report several novel ATCUN-like complexes and quantify their redox properties. These findings further demonstrate the effects of conformational constraints on short, metal-binding peptides, and also provide novel redox-active metallopeptides suitable for testing as catalysts for stereoselective or regioselective oxidation reactions.