W. Seth Horne

Probing the bioactive conformation of an archetypal natural product HDAC inhibitor with conformationally homogeneous triazole-modified cyclic tetrapeptides.

Fooling enzymes with mock amides: Analogues of apicidin, a cyclic-tetrapeptide inhibitor of histone deacetylase (HDAC), were designed with a 1,4- or 1,5-disubstituted 1,2,3-triazole in place of a backbone amide bond to fix the bond in question in either a trans-like or a cis-like configuration. Thus, the binding affinity of distinct peptide conformations (see picture) could be probed. One analogue proved in some cases to be superior to apicidin as an HDAC inhibitor.

Sequence‐Based Design of α/β‐Peptide Foldamers That Mimic BH3 Domains

The design of molecules that bind tightly and selectively to a specific site on a protein constitutes a fundamental challenge in molecular recognition. Finding high-affinity ligands for protein surfaces that bind to other proteins has proven to be particularly difficult.[1] Foldamers, oligomers with discrete folding propensities,[2] represent an unconventional source of ligands for protein-recognition surfaces,[3] but realizing this potential requires that we learn how to design sequences that contain unnatural building blocks and effectively mimic one of the surfaces involved in a given protein-protein interaction. Here we show that systematic backbone modification throughout a natural protein-binding domain, a process we refer to as sequence-based design, can expeditiously generate foldamers that bind tightly and selectively to target protein surfaces.

Conformationally homogeneous heterocyclic pseudotetrapeptides as three-dimensional scaffolds for rational drug design: receptor-selective somatostatin analogues.

A would-be amide: A 1,4-disubstituted 1,2,3-triazole was used as a surrogate for a trans amide bond to create a library of 16 diastereomeric pseudotetrapeptides as beta-turn mimetics. High-resolution structural analysis indicated that these scaffolds adopt distinct, rigid, conformationally homogeneous beta-turn-like structures (see example), some of which bind somatostatin receptor subtypes selectively, and some of which show broad-spectrum activity.

Peptide and peptoid foldamers in medicinal chemistry

Introduction: Proteins and other biologics comprise emerging therapeutic class with efficacies against targets for which development of small-molecule antagonists has been unsuccessful. The biological function of a protein is intimately tied to its sequence-dependent folding. A variety of unnatural oligomer backbones show folding behavior analogous to proteins. Often termed ‘foldamers,’ these compounds have the potential to provide the benefits of existing protein therapeutics while overcoming some drawbacks, such as protease susceptibility. Areas covered: This review surveys work toward the development of foldamer therapeutics based on β-peptides, α-peptoids, β-peptoids and heterogeneous backbones composed of mixtures of these monomers with natural α-residues. Bioactivities targeted by foldamers are diverse but can be broadly divided into two categories: i) functions that require the simple separation of charged and hydrophobic functional groups and ii) functions that require a precise and complex three-dimensional display of side chains in the folded state. Expert opinion: A long-term goal in research on foldamers is to recreate the entire range of structure and function manifested by natural proteins on unnatural backbones. Successes in the development of bioactive foldamers not only show their promise, but also highlight the challenges associated with the invention of general and reliable design strategies. While there is still a long way to go to a clinically used foldamer drug, significant advances in recent years demonstrate the potential of such scaffolds for use in the discovery of new therapeutics.

β-Hairpin-Mediated Nucleation of Polyglutamine Amyloid Formation

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntingtons disease. Here, we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (tryptophan zipper, disulfide, d-Pro-Gly, Coulombic attraction, l-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n=4 for a simple, unbroken Q23 sequence to approximate unitary n values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of solid-state NMR (13)C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn values remain quite low (~10(-)(10)) and there is no evidence for significant enhancement of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.

Total synthesis and biological evaluation of pederin, psymberin, and highly potent analogs.

The potent cytotoxins pederin and psymberin have been prepared through concise synthetic routes (10 and 14 steps in the longest linear sequences, respectively) that proceed via a late-stage multicomponent approach to construct the N-acyl aminal linkages. This route allowed for the facile preparation of a number of analogs that were designed to explore the importance of the alkoxy group in the N-acyl aminal and functional groups in the two major subunits on biological activity. These analogs, including a pederin/psymberin chimera, were analyzed for their growth inhibitory effects, revealing several new potent cytotoxins and leading to postulates regarding the molecular conformational and hydrogen bonding patterns that are required for biological activity. Second generation analogs have been prepared based on the results of the initial assays and a structure-based model for the binding of these compounds to the ribosome. The growth inhibitory properties of these compounds are reported. These studies show the profound role that organic chemistry in general and specifically late-stage multicomponent reactions can play in the development of unique and potent effectors for biological responses.

Enhancement of α-Helix Mimicry by an α/β-Peptide Foldamer via Incorporation of a Dense Ionic Side-Chain Array

We report a new method for preorganization of α/β-peptide helices, based on the use of a dense array of acidic and basic side chains. Previously we have used cyclically constrained β residues to promote α/β-peptide helicity; here we show that an engineered ion pair array can be comparably effective, as indicated by mimicry of the CHR domain of HIV protein gp41. The new design is effective in biochemical and cell-based infectivity assays; however, the resulting α/β-peptide is susceptible to proteolysis. This susceptibility was addressed via introduction of a few cyclic β residues near the cleavage site, to produce the most stable, effective α/β-peptide gp41 CHR analogue identified. Crystal structures of an α- and α/β-peptide (each involved in a gp41-mimetic helix bundle) that contain the dense acid/base residue array manifest disorder in the ionic side chains, but there is little side-chain disorder in analogous α- and α/β-peptide structures with a sparser ionic side-chain array. These observations suggest that dense arrays of complementary acidic and basic residues can provide conformational stabilization via Coulombic attractions that do not require entropically costly ordering of side chains.

Structural basis of Bcl-xL recognition by a BH3-mimetic α/β-peptide generated by sequence-based design.

The crystal structure of a complex between the prosurvival protein Bcl‐xL and an α/β‐peptide 21‐mer is described. The α/β‐peptide contains six β‐amino acid residues distributed periodically throughout the sequence and adopts an α‐helix‐like conformation that mimics the bioactive shape of the Puma BH3 domain. The α/β‐peptide forms all of the noncovalent contacts that have previously been identified as necessary for recognition of the prosurvival protein by an authentic BH3 domain. Comparison of our α/β‐peptide:Bcl‐xL structure with structures of complexes between native BH3 domains and Bcl‐2 family proteins reveals how subtle adjustments, including variations in helix radius and helix bowing, allow the α/β‐peptide to engage Bcl‐xL with high affinity. Geometric comparisons of the BH3‐mimetic α/β‐peptide with α/β‐peptides in helix‐bundle assemblies provide insight on the conformational plasticity of backbones that contain combinations of α‐ and β‐amino acid residues. The BH3‐mimetic α/β‐peptide displays prosurvival protein‐binding preferences distinct from those of Puma BH3 itself, even though these two oligomers have identical side‐chain sequences. Our results suggest origins for this backbone‐dependent change in selectivity.

Hairpin Folding Behavior of Mixed α/β-Peptides in Aqueous Solution

The invention of new strategies for the design of protein-mimetic oligomers that manifest the folding encoded in natural amino acid sequences is a significant challenge. In contrast to the α-helix, mimicry of protein β-sheets is less understood. We report here the aqueous folding behavior of a prototype α-peptide hairpin model sequence varied at cross-strand positions by incorporation of 16 different β-amino acid monomers. Our results provide a folding propensity scale for β-residues in a protein β-sheet context as well as high-resolution structures of several mixed-backbone α/β-peptide hairpins in water.

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid, Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long-range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein-backbone structure. Herein we present the double-histidine (dHis) Cu(2+)-binding motif as a rigid spin probe for double electron-electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X-ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu(2+) DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein-backbone structure and flexibility.