Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope.

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.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

DNA-Encoded Library Technology─A Catalyst for Covalent Ligand Discovery.

The identification of novel covalent ligands for therapeutic purposes has long depended on serendipity, with dedicated hit finding techniques emerging only in the early 2000s. Advances in chemoproteomics have enabled robust characterization of putative drugs to derisk the unique liabilities associated with covalent hit molecules, leading to a renewed interest in this targeting modality. DNA-encoded library (DEL) technology has similarly emerged over the past two decades as a highly efficient method to identify new chemical equity toward protein targets of interest. A number of commercial and academic groups have reported methods in covalent DEL synthesis and hit identification; however, it is evident that there is still much to be done to fully realize the power of this technology for covalent ligand discovery. This perspective will explore the current approaches in covalent DEL technology and reflect on the next steps to advance this field.

Mechanistic Studies of the Multiple Myeloma and Melanoma Cell-Selective Toxicity of the Rpn13-Binding Peptoid KDT-11.

We previously reported a peptoid ligand for the proteasomal ubiquitin receptor Rpn13 called KDT-11 and demonstrated that this compound is toxic to multiple myeloma cells, but not non-malignant cells. Here, we show that KDT-11 decreases the viability of a variety of cancer cell lines, especially melanomas and various blood cancers. The peptoid induces selective G1 cell-cycle arrest, resulting in eventual apoptosis. While KDT-11 does not antagonize any of the known protein-protein interactions involving Rpn13, the peptoid inhibits the ability of Rpn13 to stimulate the activity of an associated deubiquitylase Uch37/UCHL5 in vitro, suggesting a high level of Uch37 activity might be important for cancer cell proliferation. However, a variety of experiments in SK-MEL-5 melanoma cells suggest that KDT-11's cytotoxic effects are mediated by interactions with proteins other than Rpn13.

Physical and Functional Analysis of the Putative Rpn13 Inhibitor RA190.

Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue. However, chemical proteomics experiments in two different cell lines reveal that dozens of other proteins are heavily engaged by RA190. Finally, increasing or reducing the level of Rpn13 in HeLa and melanoma cells had no effect on the sensitivity of HeLa or melanoma cells to RA190. We conclude that Rpn13 is not the physiologically relevant target of RA190.

Beyond protein binding: recent advances in screening DNA-encoded libraries.

DNA-encoded library (DEL) screening has emerged as an important method for early stage drug and probe molecule discovery. The vast majority of screens using DELs have been relatively simple binding assays. The library is incubated with a target molecule, which is almost always a protein, and the DNAs that remain associated with the target after thorough washing are amplified and deep sequenced to reveal the chemical structures of the ligands they encode. Recently however, a number of different screening formats have been introduced that demand more than simple binding. These include a format that demands hits exhibit high selectivity for target vs. off-targets, a protocol to screen for enzyme inhibitors and another to identify organocatalysts in a DEL. These and other novel assay formats are reviewed in this article. We also consider some of the most significant remaining challenges in DEL assay development.

Synthesis and screening of bead-displayed combinatorial libraries.

The development of faster and less expensive methods to discover bioactive small molecules remains a high priority in chemical biology. This article discusses one alternative to traditional high-throughput screening: the synthesis and screening of one bead one compound (OBOC) libraries. Protocols are provided to create and screen libraries of peptoid displayed on TentaGel beads, which is a cheap and relatively straightforward process for the identification of selective protein ligands. However, peptoids bind to proteins with modest affinity in most cases. Therefore, we also describe protocols to create libraries of stiffer oligomers called PICCOs (peptoid-inspired, conformationally constrained oligomers) that have proven to be a superior source of high affinity ligands.

Chemical composition of DNA-encoded libraries, past present and future.

DNA-encoded libraries represent an exciting and powerful modality for high-throughput screening. In this article, we highlight recent important advances in this field and also suggest some important directions that would make the technology even more powerful.

Sweetening the Therapeutic Pot for Type 2 Diabetes with Long Noncoding RNAs.

Long noncoding RNAs (lncRNAs) have emerged as essential cellular regulators and potential disease targets. In this issue of Cell Chemical Biology, Shi et al. (2019) identify a cyclic peptide that results in restored glucose processing in type 2 diabetes mellitus (T2DM) patient derived adipocytes through stabilization of lncRNA GAS5.

DNA-Compatible Solid-Phase Combinatorial Synthesis of ß-Cyanoacrylamides and Related Electrophiles.

We demonstrate that the Knoevenagel condensation can be exploited in combinatorial synthesis on the solid phase. Condensation products from such reactions were structurally characterized, and their Michael reactivity with thiol and phosphine nucleophiles is described. Cyanoacrylamides were previously reported to react reversibly with thiols, and notably, we show that dilution into low pH buffer can trap covalent adducts, which are isolable via chromatography. Finally, we synthesized both traditional and DNA-encoded one-bead, one-compound libraries containing cyanoacrylamides as a source of cysteine-reactive reversibly covalent protein ligands.

Establishment of a suite of assays that support the discovery of proteasome stimulators.

BACKGROUND: The proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing. METHODS: The cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates. RESULTS: To demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold. CONCLUSION: Assays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides. GENERAL SIGNIFICANCE: A suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.

Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions.

Quantifying protein location and concentration is critical for understanding function in situ. Scaffold conjugated to environment-sensitive fluorophore (SuCESsFul) biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can, in principle, detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins, and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best-reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors.