DNA-encoded library-enabled discovery of proximity-inducing small molecules.

Small molecules that induce protein-protein associations represent powerful tools to modulate cell circuitry. We sought to develop a platform for the direct discovery of compounds able to induce association of any two preselected proteins, using the E3 ligase von Hippel-Lindau (VHL) and bromodomains as test systems. Leveraging the screening power of DNA-encoded libraries (DELs), we synthesized ~1 million DNA-encoded compounds that possess a VHL-targeting ligand, a variety of connectors and a diversity element generated by split-and-pool combinatorial chemistry. By screening our DEL against bromodomains in the presence and absence of VHL, we could identify VHL-bound molecules that simultaneously bind bromodomains. For highly barcode-enriched library members, ternary complex formation leading to bromodomain degradation was confirmed in cells. Furthermore, a ternary complex crystal structure was obtained for our most enriched library member with BRD4BD1 and a VHL complex. Our work provides a foundation for adapting DEL screening to the discovery of proximity-inducing small molecules.

Rational Screening for Cooperativity in Small-Molecule Inducers of Protein-Protein Associations.

The hallmark of a molecular glue is its ability to induce cooperative protein-protein interactions, leading to the formation of a ternary complex, despite weaker binding toward one or both individual proteins. Notably, the extent of cooperativity distinguishes molecular glues from bifunctional compounds, which constitute a second class of inducers of protein-protein interactions. However, apart from serendipitous discovery, there have been limited rational screening strategies for the high cooperativity exhibited by molecular glues. Here, we propose a binding-based screen of DNA-barcoded compounds on a target protein in the presence or absence of a presenter protein, using the "presenter ratio", the ratio of ternary enrichment to binary enrichment, as a predictive measure of cooperativity. Through this approach, we identified a range of cooperative, noncooperative, and uncooperative compounds in a single DNA-encoded library screen with bromodomain containing protein (BRD)9 and the VHL-elongin C-elongin B (VCB) complex. Our most cooperative hit compound, 13-7, exhibits micromolar binding affinity to BRD9 but nanomolar affinity for the ternary complex with BRD9 and VCB, with cooperativity comparable to classical molecular glues. This approach may enable the rational discovery of molecular glues for preselected proteins and thus facilitate the transition to a new paradigm of small-molecule therapeutics.

Machine Learning on DNA-Encoded Library Count Data Using an Uncertainty-Aware Probabilistic Loss Function.

DNA-encoded library (DEL) screening and quantitative structure-activity relationship (QSAR) modeling are two techniques used in drug discovery to find novel small molecules that bind a protein target. Applying QSAR modeling to DEL selection data can facilitate the selection of compounds for off-DNA synthesis and evaluation. Such a combined approach has been done recently by training binary classifiers to learn DEL enrichments of aggregated "disynthons" in order to accommodate the sparse and noisy nature of DEL data. However, a binary classification model cannot distinguish between different levels of enrichment, and information is potentially lost during disynthon aggregation. Here, we demonstrate a regression approach to learning DEL enrichments of individual molecules, using a custom negative-log-likelihood loss function that effectively denoises DEL data and introduces opportunities for visualization of learned structure-activity relationships. Our approach explicitly models the Poisson statistics of the sequencing process used in the DEL experimental workflow under a frequentist view. We illustrate this approach on a DEL dataset of 108,528 compounds screened against carbonic anhydrase (CAIX), and a dataset of 5,655,000 compounds screened against soluble epoxide hydrolase (sEH) and SIRT2. Due to the treatment of uncertainty in the data through the negative-log-likelihood loss used during training, the models can ignore low-confidence outliers. While our approach does not demonstrate a benefit for extrapolation to novel structures, we expect our denoising and visualization pipeline to be useful in identifying structure-activity trends and highly enriched pharmacophores in DEL data. Further, this approach to uncertainty-aware regression modeling is applicable to other sparse or noisy datasets where the nature of stochasticity is known or can be modeled; in particular, the Poisson enrichment ratio metric we use can apply to other settings that compare sequencing count data between two experimental conditions.

Water-Compatible Cycloadditions of Oligonucleotide-Conjugated Strained Allenes for DNA-Encoded Library Synthesis.

DNA-encoded libraries of small molecules are being explored extensively for the identification of binders in early drug-discovery efforts. Combinatorial syntheses of such libraries require water- and DNA-compatible reactions, and the paucity of these reactions currently limit the chemical features of resulting barcoded products. The present work introduces strain-promoted cycloadditions of cyclic allenes under mild conditions to DNA-encoded library synthesis. Owing to distinct cycloaddition modes of these reactive intermediates with activated olefins, 1,3-dipoles, and dienes, the process generates diverse molecular architectures from a single precursor. The resulting DNA-barcoded compounds exhibit unprecedented ring and topographic features, related to elements found to be powerful in phenotypic screening.

Stolonidiol: Synthesis, Target Identification, and Mechanism for Choline Acetyltransferase Activation.

Stolonidiol, a marine natural product, has been reported to potentiate the activity of choline acetyltransferase (ChAT), the enzyme that produces the neurotransmitter acetylcholine. Here we report the total synthesis of stolonidiol starting from (R)-(+)-limonene. To identify the mechanism by which ChAT activity is increased, we sought to identify the biological target of stolonidiol. We show that stolonidiol binds to the phorbol ester binding site of protein kinase C (PKC), induces translocation of PKC to the cell membrane, and activates kinase activity. Furthermore, we confirmed the increase in ChAT activity observed upon treatment of cells with stolonidiol and show that this effect is mediated by PKC. Collectively, our data strongly suggest that PKC activation by stolonidiol is responsible for the resulting potentiation of ChAT activity.

Rational screening for cooperativity in small-molecule inducers of protein-protein associations.

The hallmark of a molecular glue is its ability to induce cooperative protein-protein interactions, leading to the formation of a ternary complex, despite weaker binding towards one or both individual proteins. Notably, the extent of cooperativity distinguishes molecular glues from bifunctional compounds, a second class of inducers of protein-protein interactions. However, apart from serendipitous discovery, there have been limited rational screening strategies for the high cooperativity exhibited by molecular glues. Here, we propose a binding-based screen of DNA-barcoded compounds on a target protein in the presence and absence of a presenter protein, using the "presenter ratio", the ratio of ternary enrichment to binary enrichment, as a predictive measure of cooperativity. Through this approach, we identified a range of cooperative, noncooperative, and uncooperative compounds in a single DNA-encoded library screen with bromodomain (BRD)9 and the VHL-elongin C-elongin B (VCB) complex. Our most cooperative hit compound, 13-7 , exhibits micromolar binding affinity to BRD9 but nanomolar affinity for the ternary complex with BRD9 and VCB, with cooperativity comparable to classical molecular glues. This approach may enable the discovery of molecular glues for pre-selected proteins and thus facilitate the transition to a new paradigm of molecular therapeutics.

Diversity-oriented synthesis encoded by deoxyoligonucleotides.

Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface.

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.

Xanthates as synthetic equivalents of oxyacyl radicals: access to lactones under tin-free conditions.

In addition to their utility in Barton-McCombie deoxygenations, xanthates can engage in 5-exo-trig radical cyclizations to afford lactones after oxidative workup. In this paper, we describe a tin-free protocol that provides direct access to lactones via hydrolysis of labile thioketal intermediates. Analysis of several systems of varying complexity reveals that the reaction is most applicable for constrained systems in which the reacting center is prepositioned near the radical-accepting alkene.

The de-guanidinylated derivative of peramivir remains a potent inhibitor of influenza neuraminidase.

The guanidine function in the potent neuraminidase inhibitor peramivir was included early on in the drug design process, and examination of X-ray structural data for the enzyme-inhibitor complex would seem to indicate that the guanidine plays a critical role in promoting binding. However, this functional group may also contribute to the poor oral availability of the drug. Given that the relative stereochemistry on the guanidine-bearing carbon in peramivir is opposite to that in zanamivir (a related neuraminidase inhibitor, for which the guanidine function is known to contribute substantially to the potency), we sought to determine the importance of the guanidine group to peramivir's overall potency. Here we report that the de-guanidinylated analogue of peramivir is only ca. 1-order of magnitude less potent than peramivir itself in two in vitro inhibition assays. This suggests that next-generation inhibitors designed to improve on peramivir's properties might profitably dispense with the guanidine function.