Assessment of AI-Based Protein Structure Prediction for the NLRP3 Target.

The recent successes of AlphaFold and RoseTTAFold have demonstrated the value of AI methods in highly accurate protein structure prediction. Despite these advances, the role of these methods in the context of small-molecule drug discovery still needs to be thoroughly explored. In this study, we evaluated whether the AI-based models can reliably reproduce the three-dimensional structures of protein-ligand complexes. The structure we chose was NLRP3, a challenging protein target in terms of obtaining a three-dimensional model both experimentally and computationally. The conformation of the binding pockets generated by the AI models was carefully characterized and compared with experimental structures. Further molecular docking results indicated that AI-predicted protein structures combined with molecular dynamics simulations offers a promising approach in small-molecule drug discovery.

Triaging of DNA-Encoded Library Selection Results by High-Throughput Resynthesis of DNA-Conjugate and Affinity Selection Mass Spectrometry.

DNA encoded library (DEL) technology allows for rapid identification of novel small-molecule ligands and thus enables early-stage drug discovery. DEL technology is well-established, numerous cases of discovered hit molecules have been published, and the technology is widely employed throughout the pharmaceutical industry. Nonetheless, DEL selection results can be difficult to interpret, as library member enrichment may derive from not only desired products, but also DNA-conjugated byproducts and starting materials. Note that DELs are generally produced using split-and-pool combinatorial chemistry, and DNA-conjugated byproducts and starting materials cannot be removed from the library mixture. Herein, we describe a method for high-throughput parallel resynthesis of DNA-conjugated molecules such that byproducts, starting materials, and desired products are produced in a single pot, using the same chemical reactions and reagents as during library production. The low-complexity mixtures of DNA-conjugate are then assessed for protein binding by affinity selection mass spectrometry and the molecular weights of the binding ligands ascertained. This workflow is demonstrated to be a practical tool to triage and validate potential hits from DEL selection data.

Selections and screenings of DNA-encoded chemical libraries against enzyme and cellular targets.

The use of DNA-encoded libraries (DELs) has increased greatly over the last decade, and today a majority of pharmaceutical companies employ the technology. The technology may be applied to most soluble and purified targets. However, standard DEL technology has limitations; some targets are challenging to purify, and it is not possible to directly screen for cellular or biochemical activity. Numerous creative methods have been reported to overcome these limitations and expand DEL target scope. Reported proof-of-concept experiments include DEL selections of cell surfaces, and inside of living cells. Additional alternatives include the construction and biochemical screening of one-bead-one-compound (OBOC) DELs using picoliter aqueous droplets or microfabricated wells as containers. In these cases, the small-molecule moiety of the library member is liberated from its DNA barcode, and able to interact freely with the desired target. Lastly, patent literature suggests the ability to conduct cellular functional screens using OBOC DELs.

Palladium-mediated Suzuki-Miyaura Cross-Coupling Reaction of Potassium Boc-protected aminomethyltrifluoroborate with DNA-Conjugated aryl bromides for DNA-Encoded chemical library synthesis.

A mild reaction for DNA-compatible, palladium promoted Suzuki-Miyaura cross-coupling reaction of potassium Boc-protected aminomethyltrifluoroborate with DNA-conjugated aryl bromides has been developed efficiently. This novel DNA encoded chemistry reaction proceeded well with a wide range of functional group tolerance, including aryl bromides and heteroaryl bromides. Further, the utility our DNA conjugated aminomethylated arene products is demonstrated by reaction with various types of reagents (including amide formation with carboxylic acids, alkylation with aldehydes, and carbamoylation with amines) as would be desired for the production of a DNA encoded library.

DNA-Compatible Copper-Catalyzed Oxidative Amidation of Aldehydes with Non-Nucleophilic Arylamines.

We report a DNA-compatible protocol for synthesizing amides from DNA-bound aldehydes and non-nucleophilic arylamines including aza-substituted anilines, 2-aminobenzimidazoles, and 3-aminopyrazoles. The reactions were carried out at room temperature and provided reasonable conversions and wide functional group compatibility. The reactions were also successful when employing aryl and aliphatic aldehydes. In addition, qPCR and NGS data suggested no negative impact on DNA integrity after the copper-mediated oxidative amidation reaction.

DNA-Compatible Cyclization Reaction to Access 1,3,4-Oxadiazoles and 1,2,4-Triazoles.

DNA-encoded chemical library (DECL) technology is a commonly employed screening platform in both the pharmaceutical industry and academia. To expand the chemical space of DECLs, new and robust DNA-compatible reactions are sought after. In particular, DNA-compatible cyclization reactions are highly valued, as these reactions tend to be atom economical and thus may provide lead- and drug-like molecules. Herein, we report two new methodologies employing DNA-conjugated thiosemicarbazides as a common precursor, yielding highly substituted 1,3,4-oxadiazoles and 1,2,4-triazoles. These two novel DNA-compatible reactions feature a high conversion efficiency and broad substrate scope under mild conditions that do not observably degrade DNA.

DNA-Encoded Macrocyclic Peptide Libraries Enable the Discovery of a Neutral MDM2-p53 Inhibitor.

Synthetic macrocyclic peptides are an emerging molecular class for both targeting intracellular protein-protein interactions (PPIs) and providing an oral modality for drug targets typically addressed by biologics. Display technologies, such as mRNA and phage display, often yield peptides that are too large and too polar to achieve passive permeability or oral bioavailability without substantial off-platform medicinal chemistry. Herein, we use DNA-encoded cyclic peptide libraries to discover a neutral nonapeptide, UNP-6457, that inhibits MDM2-p53 interaction with an IC50 of 8.9 nM. X-ray structural analysis of the MDM2-UNP-6457 complex revealed mutual binding interactions and identified key ligand modification points which may be tuned to enhance its pharmacokinetic profile. These studies showcase how tailored DEL libraries can directly yield macrocyclic peptides benefiting from low MW, TPSA, and HBD/HBA counts that are capable of potently inhibiting therapeutically relevant protein-protein interactions.

Analysis of DNA-Encoded Library Screening Data: Selection of Molecules for Synthesis.

DNA-encoded library (DEL) screens are used to discover novel chemical matter capable of modulating the activity of pharmaceutically interesting protein targets. DEL selections are accomplished by immobilizing a target protein on a resin and capturing library molecules that bind to the target. The barcodes of the captured library molecules are then amplified and sequenced. This chapter outlines simple methods for visualizing the resulting screening data (using free open-source software), such that enriched molecules can be selected for synthesis and follow-up activity confirmation. Measures of enrichment and the concept of sub-libraries are also illustrated.

Discovery of SARS-CoV-2 main protease covalent inhibitors from a DNA-encoded library selection.

Covalent inhibitors targeting the main protease (Mpro, or 3CLpro) of SARS-CoV-2 have shown promise in preclinical investigations. Herein, we report the discovery of two new series of molecules that irreversibly bind to SARS-CoV-2 Mpro. These acrylamide containing molecules were discovered using our covalent DNA-encoded library (DEL) screening platform. Following selection against SARS-CoV-2 Mpro, off-DNA compounds were synthesized and investigated to determine their inhibitory effects, the nature of their binding, and to generate preliminary structure-activity relationships. LC-MS analysis indicates a 1:1 (covalent) binding stoichiometry between our hit molecules and SARS-CoV-2 Mpro. Fluorescent staining assay for covalent binding in the presence of cell lysate suggests reasonable selectivity for SARS-CoV-2 Mpro. And lastly, inhibition of enzymatic activity was also observed against a panel of 3CLpro enzymes from different coronavirus strains, with IC50 values ranging from inactive to single digit micromolar. Our results indicate that DEL selection is a useful approach for identifying covalent inhibitors of cysteine proteases.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

DNA-Compatible Click Reaction Employing In Situ Generated Azides from Boronic Acids.

An efficient method for the synthesis of DNA-conjugated 1,2,3-triazoles is copper (II) [Cu(II)-ß-cyclodextrin]-mediated Huisgen cycloaddition ("click reaction") of DNA-conjugated alkynes with azides. However, a diverse array of building blocks is required to produce useful DNA encoded libraries, and the commercial availability of azides is limited. The method described herein generates azides in situ from aryl borates and TMSN3 , which then further react with DNA-conjugated terminal alkynes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Conjugation of PEG linker to DNA headpiece Basic Protocol 2: DNA conjugated terminal alkyne preparation Basic Protocol 3: DNA compatible one-pot click reaction Basic Protocol 4: LCMS monitoring.

Discovery of a Highly Selective BET BD2 Inhibitor from a DNA-Encoded Library Technology Screening Hit.

Second-generation bromodomain and extra terminal (BET) inhibitors, which selectively target one of the two bromodomains in the BET proteins, have begun to emerge in the literature. These inhibitors aim to help determine the roles and functions of each domain and assess whether they can demonstrate an improved safety profile in clinical settings compared to pan-BET inhibitors. Herein, we describe the discovery of a novel BET BD2-selective chemotype using a structure-based drug design from a hit identified by DNA-encoded library technologies, showing a structural differentiation from key previously reported greater than 100-fold BD2-selective chemotypes GSK620, GSK046, and ABBV-744. Following a structure-based hypothesis for the selectivity and optimization of the physicochemical properties of the series, we identified 60 (GSK040), an in vitro ready and in vivo capable BET BD2-inhibitor of unprecedented selectivity (5000-fold) against BET BD1, excellent selectivity against other bromodomains, and good physicochemical properties. This novel chemical probe can be added to the toolbox used in the advancement of epigenetics research.

Exploration of amino alcohol derivatives as novel, potent, and highly selective sphingosine-1-phosphate receptor subtype-1 agonists.

In pursuit of a potent and highly selective sphingosine-1-phosphate receptor agonists with an improved in vivo conversion of the precursor to the active phospho-drug, we have utilized previously reported phenylamide and phenylimidazole scaffolds to identify a selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) within both pharmacophores. SEM and SEO have allowed for over 100 to 500-fold improvement in selectivity for S1P receptor subtype 1 over subtype 3. Utility of SEM and SEO and further SAR study allowed for discovery of a potent and selective preclinical candidate PPI-4955 (21b) with an excellent in vivo potency and dose responsiveness and markedly improved overall in vivo pharmacodynamic properties upon oral administration.

Off-DNA DNA-Encoded Library Affinity Screening.

DNA-encoded library (DEL) technology is emerging as a key element of the small molecule discovery toolbox. Conventional DEL screens (i.e., on-DNA screening) interrogate large combinatorial libraries via affinity selection of DNA-tagged library members that are ligands of a purified and immobilized protein target. In these selections, the DNA tags can materially and undesirably influence target binding and, therefore, the experiment outcome. Here, we use a solid-phase DEL and droplet-based microfluidic screening to separate the DEL member from its DNA tag (i.e., off-DNA screening), for subsequent in-droplet laser-induced fluorescence polarization (FP) detection of target binding, obviating DNA tag interference. Using the receptor tyrosine kinase (RTK) discoidin domain receptor 1 (DDR1) as a proof-of-concept target in a droplet-scale competition-binding assay, we screened a 67 100-member solid-phase DEL of drug-like small molecules for competitive ligands of DDR1 and identified several known RTK inhibitor pharmacophores, including azaindole- and quinazolinone-containing monomers. Off-DNA DEL affinity screening with FP detection is potentially amenable to a wide array of target classes, including nucleic acid binding proteins, proteins that are difficult to overexpress and purify, or targets with no known activity assay.

Synthesis of 1,2-Amino Alcohols by Photoredox-Mediated Decarboxylative Coupling of α-Amino Acids and DNA-Conjugated Carbonyls.

We report a DNA-compatible photoredox decarboxylative coupling of α-amino acids with carbonyl compounds to access DNA-encoded sp3-rich 1,2-amino alcohols. The reaction proceeds efficiently for a wide range of DNA-conjugated aldehydes and ketones and provides the desired 1,2-amino alcohols with conversions generally >50%. Additional utility of the developed protocol is demonstrated by one-pot cyclization of DNA-conjugated 1,2-amino alcohols into oxazolidiones and morpholinones. Lastly, qPCR and sequencing data analysis indicates no significant DNA damage upon photoredox decarboxylative coupling.

Copper-Mediated DNA-Compatible One-Pot Click Reactions of Alkynes with Aryl Borates and TMS-N3.

We report a DNA-compatible copper-mediated efficient synthesis of 1,2,3-triazoles via a one-pot reaction of aryl borates with TMS-N3 followed by a click cycloaddition reaction. Employing the binuclear macrocyclic nanocatalyst Cu(II)-ß-cyclodextrin, the reactions were performed under mild conditions with high conversions and wide functional group tolerance. We also demonstrate the reaction application toward a one-pot DNA-compatible intramolecular macrocyclization. Our optimized reaction protocol results in no significant DNA damage as judged by qPCR analysis and Sanger sequencing data.

Solution-Phase DNA-Compatible Pictet-Spengler Reaction Aided by Machine Learning Building Block Filtering.

The application of machine learning toward DNA encoded library (DEL) technology is lacking despite obvious synergy between these two advancing technologies. Herein, a machine learning algorithm has been developed that predicts the conversion rate for the DNA-compatible reaction of a building block with a model DNA-conjugate. We exemplify the value of this technique with a challenging reaction, the Pictet-Spengler, where acidic conditions are normally required to achieve the desired cyclization between tryptophan and aldehydes to provide tryptolines. This is the first demonstration of using a machine learning algorithm to cull potential building blocks prior to their purchase and testing for DNA-encoded library synthesis. Importantly, this allows for a challenging reaction, with an otherwise very low building block pass rate in the test reaction, to still be used in DEL synthesis. Furthermore, because our protocol is solution phase it is directly applicable to standard plate-based DEL synthesis.

Synthesis and evaluation of arylalkoxy- and biarylalkoxy-phenylamide and phenylimidazoles as potent and selective sphingosine-1-phosphate receptor subtype-1 agonists.

In pursuit of potent and selective sphingosine-1-phosphate receptor agonists, we have utilized previously reported phenylamide and phenylimidazole scaffolds to explore extensive side-chain modifications to generate new molecular entities. A number of designed molecules demonstrate good selectivity and excellent in vitro and in vivo potency in both mouse and rat models. Oral administration of the lead molecule 11c (PPI-4667) demonstrated potent and dose-responsive lymphopenia.

High-Throughput Solid-Phase Building Block Synthesis for DNA-Encoded Libraries.

Herein we provide a generalizable method for the cost-effective synthesis of thousands of building blocks (BBs) employing DNA-incompatible chemistries. The ability to produce large numbers of crude products via solid-phase synthesis has existed for decades; however, our work demonstrates a practical use of such crude reaction mixtures and employs DNA-conjugation to simultaneously encode, purify, and rapidly analyze the desired products. This workflow generated sp3-rich BBs that could be encoded by DNA in a high-throughput manner.

Development of DNA-Compatible Van Leusen Three-Component Imidazole Synthesis.

DNA-encoded libraries (DELs) have generated recent interest due to their ability to provide new small molecule ligands for pharmaceutically important proteins. The chemical diversity of DELs determines their ability to provide potent, novel, and drug-like chemical matter, and DEL chemical diversity is limited by the scope of DNA-compatible chemical reactions. Herein, the one-pot three-component Van Leusen chemistry is applied to DEL synthesis, providing the first reported DNA-compatible method to generate novel highly functionalized imidazoles.