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The ability of antibodies to bind a wide variety of analytes with high specificity and high affinity makes them ideal candidates for therapeutic and diagnostic applications. However, the poor stability and high production cost of antibodies have prompted exploration of a variety of synthetic materials capable of specific molecular recognition. Unfortunately, it remains a fundamental challenge to create a chemically diverse population of protein-like, folded synthetic nanostructures with defined molecular conformations in water. Here we report the synthesis and screening of combinatorial libraries of sequence-defined peptoid polymers engineered to fold into ordered, supramolecular nanosheets displaying a high spatial density of diverse, conformationally constrained peptoid loops on their surface. These polyvalent, loop-functionalized nanosheets were screened using a homogeneous Förster resonance energy transfer (FRET) assay for binding to a variety of protein targets. Peptoid sequences were identified that bound to the heptameric protein, anthrax protective antigen, with high avidity and selectivity. These nanosheets were shown to be resistant to proteolytic degradation, and the binding was shown to be dependent on the loop display density. This work demonstrates that key aspects of antibody structure and function-the creation of multivalent, combinatorial chemical diversity within a well-defined folded structure-can be realized with completely synthetic materials. This approach enables the rapid discovery of biomimetic affinity reagents that combine the durability of synthetic materials with the specificity of biomolecular materials.

Glycoproteins adhered on the cellular membrane play a pivotal role in a wide range of cellular functions. Their importance is particularly relevant in the recognition process between infectious pathogens (such as viruses, bacteria, toxins) and their host cells. Multivalent interactions at the pathogen-cell interfaces govern binding events and can result in a strong and specific interaction. Here we report an approach to mimic the cell surface presentation of carbohydrate ligands by the multivalent display of sugars on the surface of peptoid nanosheets. The constructs provide a highly organized 2D platform for recognition of carbohydrate-binding proteins. The sugars were displayed using different linker lengths or within loops containing 2-6 hydrophilic peptoid monomers. Both the linkers and the loops contained one alkyne-bearing monomer, to which different saccharides were attached by copper-catalyzed azide-alkyne cycloaddition reactions. Peptoid nanosheets functionalized with different saccharide groups were able to selectively bind multivalent lectins, Concanavalin A and Wheat Germ Agglutinin, as observed by fluorescence microscopy and a homogeneous Förster resonance energy transfer (FRET)-based binding assay. To evaluate the potential of this system as sensor for threat agents, the ability of functionalized peptoid nanosheets to bind Shiga toxin was also studied. Peptoid nanosheets were functionalized with globotriose, the natural ligand of Shiga toxin, and the effective binding of the nanomaterial was verified by the FRET-based binding assay. In all cases, evidence for multivalent binding was observed by systematic variation of the ligand display density on the nanosheet surface. These cell surface mimetic nanomaterials may find utility in the inactivation of pathogens or as selective molecular recognition elements.

Development of resistance to antiandrogens for treating advanced prostate cancer is a growing concern and extends to recently developed therapeutics, including enzalutamide. Therefore, new strategies to block androgen receptor (AR) function in prostate cancer are required. Here, we report the characterization of a multivalent conjugate presenting two bioactive ethisterone ligands arrayed as spatially defined pendant groups on a peptoid oligomer. The conjugate, named Multivalent Peptoid Conjugate 6 (MPC6), suppressed the proliferation of multiple AR-expressing prostate cancer cell lines including those that failed to respond to enzalutamide and ARN509. The structure-activity relationships of MPC6 variants were evaluated, revealing that increased spacing between ethisterone moieties and changes in peptoid topology eliminated its antiproliferative effect, suggesting that both ethisterone ligand presentation and scaffold characteristics contribute to MPC6 activity. Mechanistically, MPC6 blocked AR coactivator-peptide interaction and prevented AR intermolecular interactions. Protease sensitivity assays suggested that the MPC6-bound AR induced a receptor conformation distinct from that of dihydrotestosterone- or enzalutamide-bound AR. Pharmacologic studies revealed that MPC6 was metabolically stable and displayed a low plasma clearance rate. Notably, MPC6 treatment reduced tumor growth and decreased Ki67 and AR expression in mouse xenograft models of enzalutamide-resistant LNCaP-abl cells. Thus, MPC6 represents a new class of compounds with the potential to combat treatment-resistant prostate cancer. Cancer Res; 76(17); 5124-32. ©2016 AACR., (©2016 American Association for Cancer Research.)

Molecules bearing one, two, three, or four copies of the tetrapeptide His-dPhe-Arg-Trp were attached to scaffolds based on ethylene glycol, glycerol, and d-mannitol by means of the copper-assisted azide-alkyne cyclization. The abilities of these compounds to block binding of a probe at the melanocortin 4 receptor were evaluated using a competitive binding assay. All of the multivalent molecules studied exhibited 30- to 40-fold higher apparent affinites when compared to a monovalent control. These results are consistent with divalent binding to receptor dimers. No evidence for tri- or tetravalent binding was obtained. Differences in the interligand spacing required for divalent binding, as opposed to tri- or tetravalent binding, may be responsible for these results.

Oligomers incorporating the tetrapeptide MSH4, the minimum active sequence of melanocyte stimulating hormone, were synthesized by an A 2 + B 2 strategy involving microwave-assisted copper-catalyzed azide-alkyne cycloaddition. A 2 contained an MSH4 core while B 2 contained a (Pro-Gly) 3 spacer. Soluble mixtures containing compounds with up to eight MSH4 units were obtained from oligomerizations at high monomer concentrations. The avidities of several oligomeric mixtures were evaluated by means of a competitive binding assay using HEK293 cells engineered to overexpress the melanocortin 4 receptor. When based on total MSH4 concentrations, avidities were only minimally enhanced compared with a monovalent control. The lack of variation in the effect of ligands on probe binding is consistent with high off rates for MSH4 in both monovalent and oligomeric constructs relative to that of the competing probe.

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported., (Copyright © 2013 Elsevier Ltd. All rights reserved.)