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.

Integrated, Continuous Emulsion Creamer.

Automated and reproducible sample handling is a key requirement for high-throughput compound screening and currently demands heavy reliance on expensive robotics in screening centers. Integrated droplet microfluidic screening processors are poised to replace robotic automation by miniaturizing biochemical reactions to the droplet scale. These processors must generate, incubate, and sort droplets for continuous droplet screening, passively handling millions of droplets with complete uniformity, especially during the key step of sample incubation. Here, we disclose an integrated microfluidic emulsion creamer that packs ("creams") assay droplets by draining away excess oil through microfabricated drain channels. The drained oil coflows with creamed emulsion and then reintroduces the oil to disperse the droplets at the circuit terminus for analysis. Creamed emulsion assay incubation time dispersion was 1.7%, 3-fold less than other reported incubators. The integrated, continuous emulsion creamer (ICEcreamer) was used to miniaturize and optimize measurements of various enzymatic activities (phosphodiesterase, kinase, bacterial translation) under multiple- and single-turnover conditions. Combining the ICEcreamer with current integrated microfluidic DNA-encoded library bead processors eliminates potentially cumbersome instrumentation engineering challenges and is compatible with assays of diverse target class activities commonly investigated in drug discovery.

Optimization and Evaluation of Antiparasitic Benzamidobenzoic Acids as Inhibitors of Kinetoplastid Hexokinase†1.

Kinetoplastid-based infections are neglected diseases that represent a significant human health issue. Chemotherapeutic options are limited due to toxicity, parasite susceptibility, and poor patient compliance. In response, we studied a molecular-target-directed approach involving intervention of hexokinase activity-a pivotal enzyme in parasite metabolism. A benzamidobenzoic acid hit with modest biochemical inhibition of Trypanosoma brucei hexokinase 1 (TbHK1, IC50 =9.1 µm), low mammalian cytotoxicity (IMR90 cells, EC50 >25 µm), and no appreciable activity on whole bloodstream-form (BSF) parasites was optimized to afford a probe with improved TbHK1 potency and, significantly, efficacy against whole BSF parasites (TbHK1, IC50 =0.28 µm; BSF, ED50 =1.9 µm). Compounds in this series also inhibited the hexokinase enzyme from Leishmania major (LmHK1), albeit with less potency than toward TbHK1, suggesting that inhibition of the glycolytic pathway may be a promising opportunity to target multiple disease-causing trypanosomatid protozoa.

Evaluation of substituted ebselen derivatives as potential trypanocidal agents.

Human African trypanosomiasis is a disease of sub-Saharan Africa, where millions are at risk for the illness. The disease, commonly referred to as African sleeping sickness, is caused by an infection by the eukaryotic pathogen, Trypanosoma brucei. Previously, a target-based high throughput screen revealed ebselen (EbSe), and its sulfur analog, EbS, to be potent in vitro inhibitors of the T. brucei hexokinase 1 (TbHK1). These molecules also exhibited potent trypanocidal activity in vivo. In this manuscript, we synthesized a series of sixteen EbSe and EbS derivatives bearing electron-withdrawing carboxylic acid and methyl ester functional groups, and evaluated the influence of these substituents on the biological efficacy of the parent scaffold. With the exception of one methyl ester derivative, these modifications ablated or blunted the potent TbHK1 inhibition of the parent scaffold. Nonetheless, a few of the methyl ester derivatives still exhibited trypanocidal effects with single-digit micromolar or high nanomolar EC50 values.

Characterization of an African trypanosome mutant refractory to lectin-induced death.

Incubation of African trypanosomes with the lectin concanavalin A (conA) leads to alteration in cellular DNA content, DNA degradation, and surface membrane blebbing. Here, we report the generation and characterization of a conA-refractory Trypanosoma brucei line. These insect stage parasites were resistant to conA killing, with a mediun lethal dose at least 50-fold greater than the parental line. Fluorescence-based experiments revealed that the resistant cells bound less lectin when compared to the parental line. Western blotting and mass spectrometry confirmed that the resistant line lacked an N-glycan required for conA binding on the cellular receptors, EP procyclin proteins. The failure to N-glycosylate the EP procyclins was not the consequence of altered N-glycan precursor biosynthesis, as another glycosylated protein (Fla1p) was normally modified. These findings support the likelihood that resistance to conA was a consequence of failure to bind the lectin trigger.