Foldamers are sequence-specific oligomers akin to peptides, proteins and oligonucleotides that fold into well-defined three-dimensional structures. They offer the chemical biologist a broad pallet of building blocks for the construction of molecules that test and extend our understanding of protein folding and function. Foldamers also provide templates for presenting complex arrays of functional groups in virtually unlimited geometrical patterns, thereby presenting attractive opportunities for the design of molecules that bind in a sequence- and structure-specific manner to oligosaccharides, nucleic acids, membranes and proteins. We summarize recent advances and highlight the future applications and challenges of this rapidly expanding field.

The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics.antibiotic ͉ host defense peptide

Cracking the case: Aryl amide oligomers with amphiphilic secondary structure were designed that attack bacteria by lysing their membranes. A variety of groups were appended to the lead compound to adjust its overall charge, hydrophobicity, and hydrophobic moment. An Arg‐containing oligomer (see figure) was found to have good antimicrobial activity and low toxicity towards human erythrocytes.

Small molecules that bind to normally unoccupied thyroxine (T 4 ) binding sites within transthyretin (TTR) in the blood stabilize the tetrameric ground state of TTR relative to the dissociative transition state and dramatically slow tetramer dissociation, the rate-limiting step for the process of amyloid fibril formation linked to neurodegeneration and cell death. These so-called TTR kinetic stabilizers have been designed using structure-based principles and one of these has recently been shown to halt the progression of a human TTR amyloid disease in a clinical trial, providing the first pharmacologic evidence that the process of amyloid fibril formation is causative. Structure-based design has now progressed to the point where highly selective, high affinity TTR kinetic stabilizers that lack undesirable off-target activities can be produced with high frequency. Transthyretin aggregation appears to cause the transthyretin amyloidosesThe TTR amyloid diseases (amyloidoses) appear to be caused by extracellular TTR tetramer dissociation, monomer misfolding and misassembly into a variety of aggregate morphologies [1,2,3•] (Figure 1). The field has hypothesized that oligomers formed during the process of amyloidogenesis lead to cellular toxicity [4,5]. The displacement of tissue by extracellular amyloid is also thought to exacerbate pathology [6]. The TTR amyloidoses include senile systemic amyloidosis (SSA), familial amyloid cardiomyopathy (FAC), familial amyloid polyneuropathy (FAP), and central nervous system selective amyloidoses (CNSA) [3•]. Those amyloidoses impacting the most patients include SSA (10-25% of the elderly population) [7] and FAC (3-4% of African Americans), leading to congestive heart failure [8] (Figure 2). Compelling genetic evidence is supportive of the amyloid hypothesis in the TTR amyloidoses, where the notion is that the process of amyloidogenesis is causatively linked to disease pathology [9•,10].TTR is a homotetrameric protein, comprising 127-amino-acid, β-sheet-rich subunits [11,12], ( Figure 3A). Amyloidogenesis occurs when the thermodynamically linked equilibrium between tetramer, natively folded monomer and partially denatured monomer is shifted toward Figure 3C). This review focuses on recent developments, and outlines the current state of structure-based design of TTR ligands that kinetically stabilize TTR and prevent the process of amyloid fibril formation. Transthyretin's thyroxine binding sitesEach T 4 binding site is characterized by a series of subsites [15•] ( Figure 3D), an outer binding subsite, an inner binding subsite, and an intervening interface that are all composed of pairs of symmetric hydrophobic depressions referred to as halogen binding pockets (HBPs), wherein the iodine atoms of T 4 reside ( Figure 3B) [16]. HBPs 1 and 1′ in the outer binding subsite comprise the side chains of Ala108/108′, Thr106/106′, Met13/13′ and Lys15/15′ with the pocket lined by methyl and methylene groups of Lys15/15′, Ala108/108′ and Thr106/106′, whereas HBPs 2 and 2′, are made ...

A small molecule that could bind selectively to and then react chemoselectively with a non-enzyme protein in a complex biological fluid, such as blood, could have numerous practical applications. Herein, we report a family of designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin in preference to more than 4000 other human plasma proteins. They react chemoselectively with only one of eight Lys ε-amino groups within transthyretin. The crystal structure confirms the expected binding orientation of the stilbene substructure and the anticipated conjugating amide bond. These covalent transthyretin kinetic stabilizers exhibit superior amyloid inhibition potency, compared to their non-covalent counterparts, and prevent cytotoxicity associated with amyloidogenesis. While there are a few prodrugs that, upon metabolic activation, react with a Cys residue inactivating a specific non-enzyme, we are unaware of designed small molecules that react with one Lys ε-amine within a specific non-enzyme in a complex biological fluid.

We describe a non-fluorescent, second generation stilbene that very selectively binds to transthyretin in complex biological environments and remains dark until it chemoselectively reacts with the pKa perturbed Lys-15 ε-amino group of transthyretin to form a bright blue fluorescent conjugate. Stilbene A2 is mechanistically unusual in that it remains non-fluorescent in cell lysates lacking transthyretin, even though there is likely some proteome binding. Thus, it is especially useful for cellular imaging, as background fluorescence is undetectable until A2 reacts with transthyretin. The mechanistic basis for the effective lack of environment-sensitive fluorescence of A2 when bound to, but before reacting with, transthyretin is reported. Stilbene A2 exhibits sufficiently rapid transthyretin conjugation kinetics at 37 °C to enable pulse-chase experiments to be performed, in this case demonstrating that transthyretin is secreted from HeLa cells. As the chase compound, we employed C1-a cell permeable, highly selective, non-covalent, transthyretin binding dihydrostilbene that cannot become fluorescent. The progress reported is viewed as a first and necessary step toward our long-term goal of creating a one-chain, one-binding-site transthyretin tag, whose fluorescence can be regulated by adding A2, or an analogous molecule. Fusing proteins of interest to a one-chain, one-binding-site transthyretin tag regulated by A2 should be useful for studying folding, trafficking and degradation in the cellular secretory pathway, utilizing pulse-chase experiments. Immediate applications of A2 include utilizing its conjugate fluorescence to quantify transthyretin concentration in human plasma, reflecting nutritional status, and determining the binding stoichiometry of kinetic stabilizer drugs to transthyretin in plasma.

Transthyretin aggregation-associated proteotoxicity appears to cause several human amyloid diseases. Rate-limiting tetramer dissociation and monomer misfolding of transthyretin (TTR) occur before its aggregation into cross-β-sheet amyloid fibrils. Small molecule binding to and preferential stabilization of the tetrameric state of TTR over the dissociative transition state raises the kinetic barrier for dissociation, imposing kinetic stabilization on TTR and preventing aggregation. This is an effective strategy to halt neurodegeneration associated with polyneuropathy, according to recent placebo-controlled clinical trial results. In three recent papers, we systematically ranked possibilities for the three substructures composing a typical TTR kinetic stabilizer, using fibril inhibition potency and plasma TTR binding selectivity data. Herein, we have successfully employed a substructure combination strategy to use these data to develop potent and selective TTR kinetic stabilizers that rescue cells from the cytotoxic effects of TTR amyloidogenesis. Of the 92 stilbene and dihydrostilbene analogues synthesized, nearly all potently inhibit TTR fibril formation. Seventeen of these exhibit a binding stoichiometry of > 1.5 of a maximum of 2 to plasma TTR, while displaying minimal binding to the thyroid hormone receptor (< 20%). Six analogues were definitively categorized as kinetic stabilizers by evaluating dissociation time-courses. High resolution TTR•(kinetic stabilizer)2 crystal structures (1.31-1.70 Å) confirmed the anticipated binding orientation of the 3,5-dibromo-4-hydroxyphenyl substructure and revealed a strong preference of the isosteric 3,5-dibromo-4-aminophenyl substructure to bind to the inner thyroxine binding pocket.

Small arylamide foldamers designed to mimic the amphiphilic nature of antimicrobial peptides (AMPs) have shown potent bactericidal activity against both Gram-negative and Gram-positive strains without many of the drawbacks of natural AMPs. These foldamers were shown to cause large changes in the permeability of the outer membrane of Escherichia coli. They cause more limited permeabilization of the inner membrane which reaches critical levels corresponding with the time required to bring about bacterial cell death. Transcriptional profiling of E. coli treated with sublethal concentrations of the arylamides showed induction of genes related to membrane and oxidative stresses, with some overlap with the effects observed for polymyxin B. Protein secretion into the periplasm and the outer membrane is also compromised, possibly contributing to the lethality of the arylamide compounds. The induction of membrane stress response regulons such as rcs coupled with morphological changes at the membrane observed by electron microscopy suggests that the activity of the arylamides at the membrane represents a significant contribution to their mechanism of action.