Mega-High-Throughput Screening Platform for the Discovery of Biologically Relevant Sequence-Defined Non-Natural Polymers.

Combinatorial methods enable the synthesis of chemical libraries on scales of millions to billions of compounds, but the ability to efficiently screen and sequence such large libraries has remained a major bottleneck for molecular discovery. We developed a novel technology for screening and sequencing libraries of synthetic molecules of up to a billion compounds in size. This platform utilizes the fiber-optic array scanning technology (FAST) to screen bead-based libraries of synthetic compounds at a rate of 5 million compounds per minute (∼83 000 Hz). This ultra-high-throughput screening platform has been used to screen libraries of synthetic "self-readable" non-natural polymers that can be sequenced at the femtomole scale by chemical fragmentation and high-resolution mass spectrometry. The versatility and throughput of the platform were demonstrated by screening two libraries of non-natural polyamide polymers with sizes of 1.77M and 1B compounds against the protein targets K-Ras, asialoglycoprotein receptor 1 (ASGPR), IL-6, IL-6 receptor (IL-6R), and TNFα. Hits with low nanomolar binding affinities were found against all targets, including competitive inhibitors of K-Ras binding to Raf and functionally active uptake ligands for ASGPR facilitating intracellular delivery of a nonglycan ligand.

Discovery and optimization of benzotriazine di-N-oxides targeting replicating and nonreplicating Mycobacterium tuberculosis.

Compounds bactericidal against both replicating and nonreplicating Mtb may shorten the length of TB treatment regimens by eliminating infections more rapidly. Screening of a panel of antimicrobial and anticancer drug classes that are bioreduced into cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs) are potently bactericidal against replicating and nonreplicating Mtb. Medicinal chemistry optimization, guided by semiempirical molecular orbital calculations, identified a new lead compound (20q) from this series with an MIC of 0.31 µg/mL against H37Rv and a cytotoxicity (CC(50)) against Vero cells of 25 µg/mL. 20q also had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that BTOs have the potential to be developed into a new class of antitubercular drugs.

Mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities.

Synthetic derivatives of the natural product antibiotic novobiocin were synthesized in order to improve their physiochemical properties. A Mannich reaction was used to introduce new side chains at a solvent-exposed position of the molecule, and a diverse panel of functional groups was evaluated at this position. Novobiocin and the new derivatives were tested for their binding to gyrase B and their antibacterial activities against Staphylococcus aureus, Mycobacterium tuberculosis, Francisella tularensis and Escherichia coli. While the new derivatives still bound the gyrase B protein potently (0.07-1.8 µM, IC(50)), they had significantly less antibacterial activity. Two compounds were identified with increased antibacterial activity against M. tuberculosis, with a minimum inhibitory concentration of 2.5 µg/ml.