The circulating antibody repertoire encodes a patient’s health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover “epitope surrogates” (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106 beads, 448 000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit’s occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens.

Chiral vinylogous β-amino acids (VBAA) were synthesized using enantioselective Mannich reactions of aldehydes with in situ generated N-carbamoyl imines followed by a Horner–Wadsworth–Emmons reaction. The efficiency with which these units could be incorporated into oligomers with different moieties on the C- and N-terminal sides was established, as was the feasibility of sequencing oligomers containing VBAAs by tandem mass spectrometry. The data show that VBAAs will be useful building blocks for the construction of combinatorial libraries of peptidomimetic compounds.

Methods to monitor and manipulate the immune system are of enormous clinical interest. For example, the development of vaccines represents one of the earliest and greatest accomplishments of the biomedical research enterprise. More recently, drugs capable of “reawakening” the immune system to cancer have generated enormous excitement. But, much remains to be done. All drugs available today that manipulate the immune system cannot distinguish between “good” and “bad” immune responses and thus drive general and systemic immune suppression or activation. Indeed, with the notable exception of vaccines, our ability to monitor and manipulate antigen-specific immune responses is in its infancy. Achieving this finer level of control would be highly desirable. For example, it might allow the pharmacological editing of pathogenic immune responses without restricting the ability of the immune system to defend against infection. On the diagnostic side, a method to comprehensively monitor the circulating, antigen-specific antibody population could provide a treasure trove of clinically useful biomarkers, since many diseases expose the immune system to characteristic molecules that are deemed foreign and elicit the production of antibodies against them. This Perspective will discuss the state-of-the-art of this area with a focus on what we consider seminal opportunities for the chemistry community to contribute to this important field.

There is great interest in the development of probe molecules and drug leads that would bind tightly and selectively to protein surfaces that are difficult to target with traditional molecules, such as those involved in protein–protein interactions. The currently available evidence suggests that this will require molecules that are larger and have quite different chemical properties than typical Lipinski-compliant molecules that target enzyme active sites. We describe here efforts to develop vast libraries of conformationally constrained oligomers as a potentially rich source of these molecules.

A potent and selective inhibitor of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is described. The compound was derived by improvement of a modest affinity primary hit isolated from the screening of a bead-displayed peptoid–azapeptoid hybrid library tethered to an oxadiazolone ‘warhead’. The oxadiazolone moiety of the inhibitors was found to react covalently with the active site serine residue of PAFAH1B2. This screening strategy may be useful for the identification of many selective, covalent inhibitors of serine hydrolases.

The proteasome is a multisubunit complex responsible for most nonlysosomal turnover of proteins in eukaryotic cells. Proteasome inhibitors are of great interest clinically, particularly for the treatment of multiple myeloma (MM). Unfortunately, resistance arises almost inevitably to these active site-targeted drugs. One strategy to overcome this resistance is to inhibit other steps in the protein turnover cascade mediated by the proteasome. Previously, Anchoori et al. identified Rpn13 as the target of an electrophilic compound (RA-190) that was selectively toxic to MM cells (Cancer Cell 2013, 24, 791–805), suggesting that this subunit of the proteasome is also a viable cancer drug target. Here we describe the discovery of the first highly selective, reversible Rpn13 ligands and show that they are also selectively toxic to MM cells. These data strongly support the hypothesis that Rpn13 is a viable target for the development of drugs to treat MM and other cancers.

A fundamental goal in understanding the mechanisms of autoimmune disease is the characterization of autoantigens that are targeted by autoreactive antibodies and T cells. Unfortunately, the identification of autoantigens is a difficult problem. We have begun to explore a novel route to the discovery of autoantibody/autoantigen pairs that involves comparative screening of combinatorial libraries of unnatural, synthetic molecules for compounds that bind antibodies present at much higher levels in the serum of individuals with a given autoimmune disease than in the serum of control individuals. We have shown that this approach can yield “antigen surrogates” capable of capturing disease-specific autoantibodies from serum. In this report, we demonstrate that the synthetic antigen surrogates can be used to affinity purify the autoantibodies from serum and that these antibodies can then be used to identify their cognate autoantigen in an appropriate tissue lysate. Specifically, we report the discovery of a peptoid able to bind autoantibodies present in about one-third of nonobese diabetic (NOD) mice. The peptoid-binding autoantibodies were highly enriched through peptoid affinity chromatography and employed to probe mouse pancreatic and brain lysates. This resulted in identification of murine GAD65 as the native autoantigen. GAD65 is a known humoral autoantigen in human type 1 diabetes mellitus (T1DM), but its existence in mice had been controversial. This study demonstrates the potential of this chemical approach for the unbiased identification of autoantigen/autoantibody complexes.

Solid phase synthesis of 1,3,4-oxadiazin-5(6R)-one and 1,3,4-oxadiazol-2-one scaffolds from resin-bound acyl hydrazides is described. We demonstrate here that the reactions of resin-bound aryl or hetero-aromatic acyl hydrazides with 2-substituted-2-bromoacetic acids and 4-nitrophenyl chloroformate and subsequent treatment with DIEA lead to intramolecular cyclization reactions to produce six-membered 1,3,4-oxadiazin-5(6R)-ones and five-membered 1,3,4-oxadiazol-2-ones, respectively. We also show that acyl hydrazide-derived 1,3,4-oxadiazol-2-ones may be useful serine hydrolase inhibitors.

On-bead screening of one-bead–one-compound (OBOC) libraries is a useful procedure for the identification of protein ligands. An important aspect of this experiment is the method by which beads that bind the target protein are separated from those that do not. Ideally, such a method would be rapid and convenient and result in the isolation of 100% of the “hits” with no false positives (beads that display compounds that are not good ligands for the target). We introduced a technique in which beads that have bound a labeled target protein can be magnetized, thus allowing their convenient isolation (Astleet al. Chem. Biol. 2010, 17, 38−45). However, recent work in our laboratory and others has shown that magnetic hit recovery can result in the isolation of large numbers of false positives and has also suggested that many true hit beads are missed. In this study, we employ a well-defined model system to examine the efficiency of various magnetic hit isolation protocols. We show that the choice of reagents and the particular operations employed are critical for optimal results.Keywords: antibody; magnetic recovery; one-bead−one-compound library; peptides; peptoids; screening

The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10 000 mixed peptoid-PTA oligomers.Keywords: combinatorial chemistry; peptide; peptoid; reductive amination; solid-phase synthesis

Primary hits that arise from screening one bead one compound (OBOC) libraries against a target of interest rarely have high potency. However, there has been little work focused on the development of an efficient workflow for primary hit improvement. In this study, we show that by characterizing the binding constants for all of the hits that arise from a screen, structure–activity relationship (SAR) data can be obtained to inform the design of “derivative libraries” of a primary hit that can then be screened under more demanding conditions to obtain improved compounds. Here, we demonstrate the rapid improvement of a primary hit against matrix metalloproteinase-14 using this approach.Keywords: iterative screening; one bead one compound (OBOC) libraries; rapid lead discovery; structure−activity relationship (SAR)

There has been much discussion of the potential desirability of macrocyclic molecules for the development of tool compounds and drug leads. But there is little experimental data comparing otherwise equivalent macrocyclic and linear compound libraries as a source of protein ligands. In this Letter, we probe this point in the context of peptoid libraries. Bead-displayed libraries of macrocyclic and linear peptoids containing four variable positions and 0–2 fixed residues, to vary the ring size, were screened against streptavidin and the affinity of every hit for the target was measured. The data show that macrocyclization is advantageous, but only when the ring contains 17 atoms, not 20 or 23 atoms. This technology will be useful for conducting direct comparisons between many different types of chemical libraries to determine their relative utility as a source of protein ligands.Keywords: combinatorial library; high-throughput screening; macrocycle; peptoid

Autoantibodies raised against β cell antigens are the most reliable preclinical biomarkers for predicting the imminent onset of type 1 diabetes mellitus (T1DM). The most current detection platforms are technically challenging or are run on clinically esoteric equipment. Here, we present a straightforward approach to detect autoantibody biomarkers that employs highly PEGylated microspheres onto which are mounted various capture agents that include affinity-tagged antigens or small molecule “antigen surrogates.” After incubation with small quantities of serum, the bound autoantibodies can be measured using a standard flow cytometer. By multiplexing this assay, we show that a panel of antigen and antigen surrogates reliably predicts hyperglycemia in a mouse model of diabetes without false positives.

Large combinatorial libraries of macrocyclic peptides are a useful source of bioactive compounds. However, peptides are not generally cell permeable, so there is great interest in the development of methods to create large libraries of modified peptides. In particular, N-alkylation of peptides is known to improve their bioavailability significantly. Incorporation of some level of N-methylated amino acids into peptide libraries has been accomplished with ribosome display or related methods, but the modest efficiency and the inability to employ more diverse N-alkylated amino acids in this type of system argue for the development of synthetic libraries. Here we present optimized procedures for synthesizing macrocyclic peptides containing multiple N-alkylated units and show that this chemistry is efficient enough for the creation of high quality combinatorial libraries by split and pool solid-phase synthesis.

The ability to monitor and manipulate antigen-specific immune responses would have a major impact on several areas of biology and medicine. In this perspective, I consider pharmacological methods to do this, with a focus on the development of abiological “antigen surrogates” capable of binding to the antigen-binding sites of antibodies and B cell receptors with high affinity and selectivity. I describe the application of combinatorial library screening to identify antigen surrogates for monoclonal antibodies of therapeutic interest using chronic lymphocytic leukemia as an example. Furthermore, I discuss the use of multiplexed assays for the quantification of antigen surrogate-antibody complexes as diagnostic tools and antigen surrogate discovery via serum screening. Although antigen surrogates are a fairly new concept, I argue that they will open new avenues for both basic and clinical research and that major advances can be expected over the next few years.Figure optionsDownload full-size imageDownload high-quality image (215 K)Download as PowerPoint slide

•A protocol is developed to identify ligands for antigen-specific B cell receptors•Dextran-ligand conjugates can bind to native, antigen-specific B cells•Pathogenic B cells from CLL patients can be targeted selectivelyIn patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates.Figure optionsDownload full-size imageDownload high-quality image (298 K)Download as PowerPoint slide

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors.

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule– and peptide–protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative composed of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships, and certain types of serological analyses.

Diketopiperazine (DKP) units are found in many bioactive small molecules. Here we report facile chemistry for incorporating diverse DKP units within peptoid and peptoid-like libraries made by solid-phase split and pool synthesis.

Large one-bead one-compound (OBOC) combinatorial libraries can be constructed relatively easily by solid-phase split and pool synthesis. The use of resins with hydrophilic surfaces, such as TentaGel, allows the beads to be used directly in screens for compounds that bind selectively to labeled proteins, nucleic acids, or other biomolecules. However, we have found that this method, while useful, has a high false positive rate. In other words, beads that are scored as hits often display compounds that prove to be poor ligands for the target of interest when they are resynthesized and carried through validation trials. This results in a significant waste of time and resources in cases where putative hits cannot be validated without resynthesis. Here, we report that this problem can be largely eliminated through the use of redundant OBOC libraries, where more than one bead displaying the same compound is present in the screen. We show that compounds isolated more than once are likely to be high quality ligands for the target of interest, whereas compounds isolated only once have a much higher likelihood of being poor ligands. While the use of redundant libraries does limit the number of unique compounds that can be screened at one time in this format, the overall savings in time, effort, and materials makes this a more efficient route to the isolation of useful ligands for biomolecules.Keywords: antibody screen; antigen surrogate; nonspecific binding; OBOC library; peptoids; redundant library; serum screen

Large combinatorial libraries of N-substituted peptides would be an attractive source of protein ligands, because these compounds are known to be conformationally constrained, whereas standard peptides or peptoids are conformationally mobile. Here, we report an efficient submonomer solid-phase synthetic route to these compounds and demonstrate that it can be used to create high quality libraries. A model screening experiment and analysis of the hits indicates that the rigidity afforded by the stereocenters is critical for high affinity binding.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (275 K)Download as PowerPoint slideHighlights► A convenient synthesis of libraries of conformationally-constrained N-substituted oligoalanines ► A screen of such a library provided much higher affinity hits than a library of peptoids lacking c-alpha substitution ► Analysis of the isolated protein ligands demonstrated that the stereochemistry of the chiral centers was important ► These libraries should be a useful source of high affinity protein ligands

Neuromyelitis optica (NMO) is an autoimmune inflammatory disorder of the central nervous system. In most NMO patients, autoantibodies to the water channel protein Aquaporin 4 (AQP4) are present at high levels and are thought to drive pathology by mediating complement-dependent destruction of astrocytes. Here, we apply recently developed chemical library screening technology to identify a synthetic peptoid that binds anti-AQP4 antibodies in the serum of NMO patients. This finding validates, in a well-defined human disease, that synthetic, unnatural ligands for the antigen-binding site of a disease-linked antibody can be isolated by high-throughput screening.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (81 K)Download as PowerPoint slideHighlights► Peptoid ligands for anti-AQP4 autoantibodies that drive NMO have been identified ► Some of these peptoids provide an accurate blood test for the disease

In this issue of Chemistry & Biology, Jahnsen and colleagues describe a multimodal mechanism of action of an unusual peptide-peptoid hybrid with broad-spectrum antibacterial activity. They demonstrate that the molecule has effects on many aspects of bacterial metabolism and also modulates host immune responses.

Peptoid libraries have been shown to be a useful source of protein-binding agents. However, simple linear peptoids lack conformational constraints, which may limit their binding affinity for proteins. Here we report facile chemistry for the assembly of 2-oxopiperazine rings into the main chain of peptoid-like oligomers, thus rigidifying the structure. This modified sub-monomer synthesis is suitable for the creation of high quality combinatorial libraries.

We recently reported efficient conditions for the synthesis of N-azapeptoid libraries via the typical submonomer strategy of peptoid synthesis but that substitutes N-acyl hydrazides for primary amines as submonomers. Unfortunately, this approach is not applicable to the synthesis of mixed azapeptoid–peptoid libraries. When an oligomer containing an N-terminal side chain derived from an acyl hydrazide is bromoacetylated and treated with a primary amine, a chain-terminating intramolecular ring-closure to form an oxadiazinone competes with the desired displacement of the bromide by the amine. Here we overcome this limitation and demonstrate that a hybrid peptoid–azapeptoid library derived from primary amines, acyl hydrazides, carbazates, and semicarbazides can be made efficiently using standard peptoid submonomer chemistry. We find that the unwanted, chain-terminating cyclization reaction is competitive with chain extension only when aryl acyl hydrazides are present. Alkyl or heteroaromatic acyl hydrazides do not cyclize under the conditions used for peptoid–azapeptoid synthesis. We also find that carbazates and semicarbazides work well for chain extension. Using primary amines, acyl hydrazides, carbazates, and semicarbazides as submonomers, a high-quality one bead one compound library of tetramers suitable for screening against protein targets was made by split and pool synthesis.Keywords: hybrid petoid−azapetoid library; one bead one compound; split and pool synthesis; submonomer

Combinatorial libraries of peptoids (oligo-N-substituted glycines) have proven to be useful sources of protein ligands. Each unit of the peptoid oligomer is derived from 2-haloacetic acid and a primary amine. To increase the chemical diversity available in peptoid libraries, we demonstrate here that heterocyclic halomethyl carboxylic acids can be employed as backbone building blocks in the synthesis of peptoid-based oligomers. Optimized conditions are reported that allow the creation of large, high quality combinatorial libraries containing these units.Keywords: heterocycles; one bead one compound libraries; peptidomimetics; peptoids; solid-phase synthesis

Many macrocyclic depsipeptides or related compounds have interesting medicinal properties and often display more favorable pharmacokinetic properties than linear analogues. Therefore, there is considerable interest in the development of large combinatorial libraries of macrocyclic peptidomimetic compounds. However, such molecules cannot be easily sequenced by tandem mass spectrometry, making it difficult to identify hits isolated from library screens using one bead one compound libraries. Here we report a strategy to solve this problem by placing a methionine in both the linker connecting the cyclic molecule to the bead as well as within the cycle itself. Treatment with CNBr both linearizes the molecule at a specific position and releases the molecule from the bead, making its characterization by tandem MALDI mass spectrometry straightforward.

The use of acyl hydrazides as peptoid sub-monomers is investigated. We demonstrate here that azapeptoids derived entirely from acyl hydrazides can be made conveniently and efficiently using standard peptoid sub-monomer chemistry. Structural studies reveal that the main chain amide bond in these molecules predominantly adopts a trans conformation. A high-quality one bead one compound library of tetramers was made by split and pool synthesis and we found that the identity of the molecule on a single bead could be determined by tandem MALDI mass spectrometry.

The dimerization of different PDZ domains suggests that these protein modules evolved to guide the assembly of multiprotein complexes. In this issue, Chang et al. (2011) describe a massively parallel approach for identifying all PDZ dimerization events that reveals the prevalence of this protein-protein interaction.

Synthetic equivalents of phosphoprotein-specific antibodies would be valuable reagents for biological research, since these antibodies can often be difficult to produce. Protein phosphorylation is thought to result in significant conformational changes in most substrate proteins. Therefore, one approach might be to simply screen combinatorial libraries for ligands to the phosphorylated state in the hope of isolating a ligand that binds to a pocket created by the conformational shift. In this study, we probe this strategy by screening a peptoid library for ligands to the phosphorylated form of the Brd4 chromatin adaptor and transcriptional coactivator protein. We find that peptoids with high selectivity for binding to the phosphorylation form of Brd4 can indeed be isolated in this screen. Moreover, these ligands do not bind promiscuously to other phospho-proteins. However, attempts to employ these reagents as antibody substitutes in an immunoaffinity purification-like application showed that they do not perform as well as bona fide antibodies and that significant optimization will be required. This study highlights the potential and current limitations of a naïve library screening strategy for phosphoprotein-specific antibody surrogates.A combinatorial peptoid library screen is shown to result in the isolation of ligands that have high selectivity for the phosphorylated form of a domain of the Brd4 protein.

The orexin neurohormones control a variety of important physiological processes by signaling through two related G protein-coupled receptors, including appetite and feeding, wakefulness and energy homeostasis. Pharmacological manipulation of orexin signaling is an important goal. Here we describe the isolation of orexin receptor ligands from a library of microarray-displayed peptoids via a novel two-color, cell-based screen. Functional analysis of derivatives of these “hits” resulted in the development of moderate potency, low molecular weight receptor antagonists. Moreover, further optimization efforts resulted in the fortuitous discovery of a compound that positively potentiates the activity of the receptor. This compound is the first small molecule reported to up-regulate orexin signaling.

Some therapeutic monoclonal antibodies function by focusing the killing power of the immune system on particular cellular targets, a process known as antibody-dependent cell-mediated cytotoxicity (ADCC). There is considerable interest in developing small-molecule therapeutics that utilize this mechanism of action and so act as a kind of antibody surrogate. Progress and challenges in this interesting field are reviewed here.

Several approaches have been developed for screening combinatorial libraries or collections of synthetic molecules for agonists or antagonists of protein function, each with its own advantages and limitations. In this report, we describe an experimental platform that seamlessly couples massively parallel bead-based screening of one-bead one-compound combinatorial libraries with microarray-based quantitative comparisons of the binding affinities of the many hits isolated from the bead library. Combined with other technical improvements, this technique allows the rapid identification of the best protein ligands in combinatorial libraries containing millions of compounds without the need for labor-intensive resynthesis of the hits.Highlights► Experimental platform couples parallel bead-based screening with microarray-based comparisons of hit compounds ► Beads displaying hit compounds are non-covalently magnetized for rapid isolation from millions of non-hit beads ► Selectively cleavable linkers allow direct attachment of compounds from hit beads to microarrays without re-synthesis ► Method decreases the time and effort required for isolating and characterizing protein ligands

The orexins are neurohormones that, in concert with their cognate receptors, regulate a number of important physiological processes, including feeding, sleep, reward seeking and energy homeostasis. The orexin receptors have recently emerged as important drug targets. This review provides an overview of recent development in deciphering the biology of orexin signaling as well as efforts to manipulate orexin signaling pharmacologically.

We report here a simple and rapid method by which to screen one bead one compound libraries for highly specific ligands to cell surfaceproteins such as G protein-coupled receptors. This protocol, which harvests “hits” in a cell-based binding screen magnetically, eliminates the most tedious aspects of previously published bead screening techniques and allows millions of different compounds to be screened rapidly and cheaply. The method is demonstrated using the orexin receptor 1, which resulted in the isolation of moderate potency antagonists.

Antigen-specific T cells play a major role in mediating the pathogenesis of a variety of autoimmune conditions as well as other diseases. In the context of experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis, we present here a general approach to the discovery of highly specific ligands for autoreactive cells. These ligands are obtained from a combinatorial library of hundreds of thousands of synthetic peptoids that is screened simultaneously against two populations of CD4+ T cells. Peptoids that recognize autoreactive T cells with extremely high specificity can be identified in the library. Since no specific knowledge is required regarding the nature of the native antigens recognized by the autoreactive T cells, this technology provides a powerful tool for the enrichment and inhibition of autoimmune cells in a variety of disease states.

Pyrrole−imidazole (Py-Im) polyamides are a group of chemicals that are able to bind specifically to DNA sequences in vitro and in mammalian cells. Using a cell based reporter assay, we investigated the size and linker affects on the cellular permeability of polyamides. We found that the conventional β-alanine-3,3′-diamino-N-methyldipropylamine (βDa) linker strongly limited the cellular permeability. We discovered that a short ethylene diamine (Et) linker displayed high cellular permeability. With the improved Et linker, we found that the cellular permeability of polyamides was size-dependent.

Chiral vinylogous β-amino acids (VBAA) were synthesized using enantioselective Mannich reactions of aldehydes with in situ generated N-carbamoyl imines followed by a Horner–Wadsworth–Emmons reaction. The efficiency with which these units could be incorporated into oligomers with different moieties on the C- and N-terminal sides was established, as was the feasibility of sequencing oligomers containing VBAAs by tandem mass spectrometry. The data show that VBAAs will be useful building blocks for the construction of combinatorial libraries of peptidomimetic compounds.

The orexin neurohormones control a variety of important physiological processes by signaling through two related G protein-coupled receptors, including appetite and feeding, wakefulness and energy homeostasis. Pharmacological manipulation of orexin signaling is an important goal. Here we describe the isolation of orexin receptor ligands from a library of microarray-displayed peptoids via a novel two-color, cell-based screen. Functional analysis of derivatives of these “hits” resulted in the development of moderate potency, low molecular weight receptor antagonists. Moreover, further optimization efforts resulted in the fortuitous discovery of a compound that positively potentiates the activity of the receptor. This compound is the first small molecule reported to up-regulate orexin signaling.

The use of acyl hydrazides as peptoid sub-monomers is investigated. We demonstrate here that azapeptoids derived entirely from acyl hydrazides can be made conveniently and efficiently using standard peptoid sub-monomer chemistry. Structural studies reveal that the main chain amide bond in these molecules predominantly adopts a trans conformation. A high-quality one bead one compound library of tetramers was made by split and pool synthesis and we found that the identity of the molecule on a single bead could be determined by tandem MALDI mass spectrometry.

Solid phase synthesis of 1,3,4-oxadiazin-5(6R)-one and 1,3,4-oxadiazol-2-one scaffolds from resin-bound acyl hydrazides is described. We demonstrate here that the reactions of resin-bound aryl or hetero-aromatic acyl hydrazides with 2-substituted-2-bromoacetic acids and 4-nitrophenyl chloroformate and subsequent treatment with DIEA lead to intramolecular cyclization reactions to produce six-membered 1,3,4-oxadiazin-5(6R)-ones and five-membered 1,3,4-oxadiazol-2-ones, respectively. We also show that acyl hydrazide-derived 1,3,4-oxadiazol-2-ones may be useful serine hydrolase inhibitors.

Diketopiperazine (DKP) units are found in many bioactive small molecules. Here we report facile chemistry for incorporating diverse DKP units within peptoid and peptoid-like libraries made by solid-phase split and pool synthesis.

Peptoid libraries have been shown to be a useful source of protein-binding agents. However, simple linear peptoids lack conformational constraints, which may limit their binding affinity for proteins. Here we report facile chemistry for the assembly of 2-oxopiperazine rings into the main chain of peptoid-like oligomers, thus rigidifying the structure. This modified sub-monomer synthesis is suitable for the creation of high quality combinatorial libraries.

There is great interest in the development of probe molecules and drug leads that would bind tightly and selectively to protein surfaces that are difficult to target with traditional molecules, such as those involved in protein–protein interactions. The currently available evidence suggests that this will require molecules that are larger and have quite different chemical properties than typical Lipinski-compliant molecules that target enzyme active sites. We describe here efforts to develop vast libraries of conformationally constrained oligomers as a potentially rich source of these molecules.