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.

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 IC 50 = 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.

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.

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SCH-C (SCH 351125) is a small-molecule antagonist of the human immunodeficiency virus type 1(HIV-1) coreceptor CCR5. It has in vitro activity against R5 viruses with 50% inhibitory concentrations ranging from 1.0 to 30.9 nM. We have studied anti-HIV-1 interactions of SCH-C with other antiretroviral agents in vitro. Synergistic interactions were seen with nucleoside reverse transcriptase inhibitors (zidovudine and lamivudine), nonnucleoside reverse transcriptase inhibitors (efavirenz), and protease inhibitors (indinavir) at all inhibitory concentrations evaluated. We have also studied antiviral interactions between the HIV-1 fusion inhibitor T-20 and SCH-C against a panel of R5 HIV-1 isolates. We found synergistic interactions against all the viruses tested, some of which harbored resistance mutations to reverse transcriptase and protease inhibitors. Anti-HIV-1 synergy was also observed between SCH-C and another R5 virus inhibitor, aminooxypentane-RANTES. These findings suggest that SCH-C may be a useful anti-HIV drug in combination regimens and that a combination of chemokine coreceptor/fusion inhibitors may be useful in the treatment of multidrug-resistant viruses.

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.

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Attachment and entry of HIV-1 into CD4 cells involve a series of events in which different viral envelope proteins interact with specific cell receptors, culminating in fusion of viral and cell membranes. AMD-3100 is a small molecule inhibitor of HIV-1 attachment to the CXCR4 chemokine receptor, and T-20 is a synthetic peptide corresponding to a region of HIV-1 gp41 that blocks fusion to cell membranes. To evaluate the interaction between agents acting at two different steps of the entry process, we conducted in vitro studies of the combination of T-20 and AMD-3100 against an X4 HIV-1 isolate. Single drugs or multiply diluted fixed ratio combinations of drugs were added to peripheral blood mononuclear cells infected with a clinical isolate, 14aPre. Drug interactions were evaluated using the median-effect principle and the combination index technique. The 50% inhibitory concentration (IC50) for T-20 was 0.10 microg/ml and for AMD-3100 was 0.19 microg/ml. Synergy was observed between T-20 and AMD-3100 and this increased with higher inhibitory concentrations, with combination indices ranging from 0.62 at IC50 to 0.02 at IC95. Whether these synergistic interactions translate into clinical benefit will need to be addressed in the context of clinical trials.