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Antigenic similarities between Zika virus (ZIKV) and other flaviviruses pose challenges to the development of virus-specific diagnostic tools and effective vaccines. Starting with a DNA-encoded one-bead-one-compound combinatorial library of 508,032 synthetic, non-natural oligomers, we selected and characterized small molecules that mimic ZIKV epitopes. High-throughput fluorescence-activated cell sorter-based bead screening was used to select molecules that bound IgG from ZIKV-immune but not from dengue-immune sera. Deep sequencing of the DNA from the "Zika-only" beads identified 40 candidate molecular structures. A lead candidate small molecule "CZV1-1" was selected that correctly identifies serum specimens from Zika-experienced patients with good sensitivity and specificity (85.3% and 98.4%, respectively). Binding competition studies of purified anti-CZV1-1 IgG against known ZIKV-specific monoclonal antibodies (mAbs) showed that CZV1-1 mimics a nonlinear, neutralizing conformational epitope in the domain III of the ZIKV envelope. Purified anti-CZV1-1 IgG neutralized infection of ZIKV in cell cultures with potencies comparable to highly specific ZIKV-neutralizing mAbs. This study demonstrates an innovative approach for identification of synthetic non-natural molecular mimics of conformational virus epitopes. Such molecular mimics may have value in the development of accurate diagnostic assays for Zika, as well as for other viruses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 104 molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries. [ABSTRACT FROM AUTHOR]

This article reports the design, synthesis, and evaluation of a novel class of molecules of intermediate size (approximately 7000 Da), which possess both the targeting and effector functions of antibodies. These compounds--called synthetic antibody mimics targeting prostate cancer (SyAM-Ps)--bind simultaneously to prostate-specific membrane antigen and Fc gamma receptor I, thus eliciting highly selective cancer cell phagocytosis. SyAMs have the potential to combine the advantages of both small-molecule and biologic therapies, and may address many drawbacks associated with available treatments for cancer and other diseases. [ABSTRACT FROM AUTHOR]

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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 × 10 6 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.

Synthetic immunology, the development of synthetic systems capable of modulating and/or manipulating immunological functions, represents an emerging field of research with manifold possibilities. One focus of this area has been to create low molecular weight synthetic species, called antibody-recruiting molecules (ARMs), which are capable of enhancing antibody binding to disease-relevant cells or viruses, thus leading to their immune-mediated clearance. This article provides a thorough discussion of contributions in this area, beginning with the history of small-molecule-based technologies for modulating antibody recognition, followed by a systematic review of the various applications of ARM-based strategies. Thus, we describe ARMs capable of targeting cancer, bacteria, and viral pathogens, along with some of the scientific discoveries that have resulted from their development. Research in this area underscores the many exciting possibilities at the interface of organic chemistry and immunobiology and is positioned to advance both basic and clinical science in the years to come.

Synthetic compounds for controlling or creating human immunity have the potential to revolutionize disease treatment. Motivated by challenges in this arena, we report herein a strategy to target metastatic cancer cells for immune-mediated destruction by targeting the urokinase-type plasminogen activator receptor (uPAR). Urokinase-type plasminogen activator (uPA) and uPAR are overexpressed on the surfaces of a wide range of invasive cancer cells and are believed to contribute substantially to the migratory propensities of these cells. The key component of our approach is an antibody-recruiting molecule that targets the urokinase receptor (ARM-U). This bifunctional construct is formed by selectively, covalently attaching an antibody-binding small molecule to the active site of the urokinase enzyme. We demonstrate that ARM-U is capable of directing antibodies to the surfaces of target cancer cells and mediating both antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) against multiple human cancer cell lines. We believe that the reported strategy has the potential to inform novel treatment options for a variety of deadly, invasive cancers.

Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen that has emerged as a major public health threat. Here we report that the cell wall of S. aureus can be covalently re-engineered to contain non-native small molecules. This process makes use of endogenous levels of the bacterial enzyme sortase A (SrtA), which ordinarily functions to incorporate proteins into the bacterial cell wall. Thus, incubation of wild-type bacteria with rationally designed SrtA substrates results in covalent incorporation of functional molecular handles (fluorescein, biotin, and azide) into cell wall peptidoglycan. These conclusions are supported by data obtained through a variety of experimental techniques (epifluorescence and electron microscopy, biochemical extraction, and mass spectrometry), and cell-wall-incorporated azide was exploited as a chemical handle to perform an azide-alkyne cycloaddition reaction on the bacterial cell surface. This report represents the first example of cell wall engineering of S. aureus or any other pathogenic Gram-positive bacteria and has the potential for widespread utility.