Quantitation of DNA-Encoded Libraries by qPCR.

DNA-encoded library (DEL) yields can be easily measured throughout the selection process using the quantitative polymerase chain reaction (qPCR) (Sannino A, Gabriele E, Bigatti M, Mulatto S, Piazzi J, Scheuermann J, Neri D, Donckele EJ, Samain F, Chembiochem Eur J Chem Biol 20:955-962, 2019). Samples taken throughout the selection process are diluted prior to amplification and compared to standards of known DNA concentration. Here, I describe a general protocol using a double-stranded DNA binding dye for reaction monitoring. This allows the selection process to be assessed at each step prior to preparation for sequencing. The same method has additional applications in the practice of DEL technology.

The Impact of Variable Selection Coverage on Detection of Ligands from a DNA-Encoded Library Screen.

DNA-encoded library (DEL) technology has become a prominent screening platform in drug discovery owing to the capacity to screen billions or trillions of compounds in a single experiment. Although numerous successes with DEL technology have been reported, we are unaware of a rigorous examination of the many different variables that can influence a screen's success. Herein, we explore the impact of variable sample sequencing depth on the detection of tool compounds with known affinities toward a given target while simultaneously probing the effect of initial compound input. Our sequencing data confirm reports that high-affinity compounds can be discovered directly from a DEL screen, but we demonstrate that a mismatch between selection output and sequencing quantity can obscure useful ligands. Our results highlight the importance of selection coverage in grasping the entire picture of a DEL screen where the signal of a weak or underrepresented ligand may be suppressed by the inherent noise of a selection. These potential missed ligands may be critical to the success or failure of a drug discovery program.

Quantitative Comparison of Enrichment from DNA-Encoded Chemical Library Selections.

DNA-encoded chemical libraries (DELs) provide a high-throughput and cost-effective route for screening billions of unique molecules for binding affinity for diverse protein targets. Identifying candidate compounds from these libraries involves affinity selection, DNA sequencing, and measuring enrichment in a sample pool of DNA barcodes. Successful detection of potent binders is affected by many factors, including selection parameters, chemical yields, library amplification, sequencing depth, sequencing errors, library sizes, and the chosen enrichment metric. To date, there has not been a clear consensus about how enrichment from DEL selections should be measured or reported. We propose a normalized  z-score enrichment metric using a binomial distribution model that satisfies important criteria that are relevant for analysis of DEL selection data. The introduced metric is robust with respect to library diversity and sampling and allows for quantitative comparisons of enrichment of n-synthons from parallel DEL selections. These features enable a comparative enrichment analysis strategy that can provide valuable information about hit compounds in early stage drug discovery.

Author Correction: Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

This corrects the article DOI: 10.1038/ncomms16081.

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

Discovery, SAR, and X-ray Binding Mode Study of BCATm Inhibitors from a Novel DNA-Encoded Library.

As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-N-methyl-2'-(methylsulfonamido)-[1,1'-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki-Miyaura cross-coupling showed BCATm inhibitory activity with IC50 = 2.0 µM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors.

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.

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.

Favorable interactions between enfuvirtide and 1-beta-D-2,6-diaminopurine dioxolane in vitro.

We evaluated the in vitro anti-human immunodeficiency virus type 1 (HIV-1) interactions between 1-beta-D-2,6-diaminopurine dioxolane (DAPD) and enfuvirtide (T-20) against clinical isolates sensitive and resistant to reverse transcriptase and protease inhibitors. Interactions between T-20 and DAPD were synergistic to nearly additive, with combination index values ranging from 0.53 to 1.06 at 95% inhibitory concentrations. These studies suggest that a combination of T-20 and DAPD might be useful in the treatment of antiretroviral drug-experienced patients.