Identification and Evaluation of Reversible Covalent Binders to Cys55 of Bfl-1 from a DNA-Encoded Chemical Library Screen.

Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.

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.

Discovery of a First-in-Class Small-Molecule Ligand for WDR91 Using DNA-Encoded Chemical Library Selection Followed by Machine Learning.

WD40 repeat-containing protein 91 (WDR91) regulates early-to-late endosome conversion and plays vital roles in endosome fusion, recycling, and transport. WDR91 was recently identified as a potential host factor for viral infection. We employed DNA-encoded chemical library (DEL) selection against the WDR domain of WDR91, followed by machine learning to predict ligands from the synthetically accessible Enamine REAL database. Screening of predicted compounds identified a WDR91 selective compound 1, with a KD of 6 ± 2 µM by surface plasmon resonance. The co-crystal structure confirmed the binding of 1 to the WDR91 side pocket, in proximity to cysteine 487, which led to the discovery of covalent analogues 18 and 19. The covalent adduct formation for 18 and 19 was confirmed by intact mass liquid chromatography-mass spectrometry. The discovery of 1, 18, and 19, accompanying structure-activity relationship, and the co-crystal structures provide valuable insights for designing potent and selective chemical tools against WDR91 to evaluate its therapeutic potential.

Discovery of Nanomolar DCAF1 Small Molecule Ligands.

DCAF1 is a substrate receptor of two distinct E3 ligases (CRL4DCAF1 and EDVP), plays a critical physiological role in protein degradation, and is considered a drug target for various cancers. Antagonists of DCAF1 could be used toward the development of therapeutics for cancers and viral treatments. We used the WDR domain of DCAF1 to screen a 114-billion-compound DNA encoded library (DEL) and identified candidate compounds using similarity search and machine learning. This led to the discovery of a compound (Z1391232269) with an SPR KD of 11 µM. Structure-guided hit optimization led to the discovery of OICR-8268 (26e) with an SPR KD of 38 nM and cellular target engagement with EC50 of 10 µM as measured by cellular thermal shift assay (CETSA). OICR-8268 is an excellent tool compound to enable the development of next-generation DCAF1 ligands toward cancer therapeutics, further investigation of DCAF1 functions in cells, and the development of DCAF1-based PROTACs.

Novel irreversible covalent BTK inhibitors discovered using DNA-encoded chemistry.

Libraries of DNA-Encoded small molecules created using combinatorial chemistry and synthetic oligonucleotides are being applied to drug discovery projects across the pharmaceutical industry. The majority of reported projects describe the discovery of reversible, i.e. non-covalent, target modulators. We synthesized multiple DNA-encoded chemical libraries terminated in electrophiles and then used them to discover covalent irreversible inhibitors and report the successful discovery of acrylamide- and epoxide-terminated Bruton's Tyrosine Kinase (BTK) inhibitors. We also demonstrate their selectivity, potency and covalent cysteine engagement using a range of techniques including X-ray crystallography, thermal transition shift assay, reporter displacement assay and intact protein complex mass spectrometry. The epoxide BTK inhibitors described here are the first ever reported to utilize this electrophile for this target.

Generating Selective Leads for Mer Kinase Inhibitors-Example of a Comprehensive Lead-Generation Strategy.

Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.

Machine Learning on DNA-Encoded Libraries: A New Paradigm for Hit Finding.

DNA-encoded small molecule libraries (DELs) have enabled discovery of novel inhibitors for many distinct protein targets of therapeutic value. We demonstrate a new approach applying machine learning to DEL selection data by identifying active molecules from large libraries of commercial and easily synthesizable compounds. We train models using only DEL selection data and apply automated or automatable filters to the predictions. We perform a large prospective study (∼2000 compounds) across three diverse protein targets: sEH (a hydrolase), ERα (a nuclear receptor), and c-KIT (a kinase). The approach is effective, with an overall hit rate of ∼30% at 30 µM and discovery of potent compounds (IC50 < 10 nM) for every target. The system makes useful predictions even for molecules dissimilar to the original DEL, and the compounds identified are diverse, predominantly drug-like, and different from known ligands. This work demonstrates a powerful new approach to hit-finding.

Continuous directed evolution of DNA-binding proteins to improve TALEN specificity.

Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate transcription activator-like effectors nucleases (TALENs) with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Determining the specificities of TALENs, Cas9, and other genome-editing enzymes.

The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases, transcription activator-like effectornucleases, and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction, and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this chapter, we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~10(12)-membered) libraries of potential nuclease substrates.

Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling.

Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.

Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.

Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ∼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.

Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity.

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.