Acoustic Mist Ionization-Mass Spectrometry: A Comparison to Conventional High-Throughput Screening and Compound Profiling Platforms.

Drug discovery usually begins with a high-throughput screen (HTS) of thousands to millions of molecules to identify starting points for medicinal chemistry. Conventional HTS platforms require expensive reagents and typically have complex assay formats. HTS platforms based on radioactivity are expensive, both in terms of reagent cost and disposal. Furthermore, nonspecific interferences common to these technologies result in an extensive attrition of hits during validation experiments. Mass spectrometry (MS) is a highly selective, label-free technology that can quantify multiple analytes in a single experiment. However, most commercial MS platforms typically involve a separation or cleanup prior to analysis and are too slow for large-scale screening campaigns. Recently, an MS platform (AMI-MS) was introduced that uses acoustically generated droplets to deliver analyte molecules directly from microtiter plates into the mass spectrometer at subsecond per well sampling rates. Here, we demonstrate the application of AMI-MS by developing an HTS-compatible assay that measures the inhibition of histone acetyltransferase activity. Real-time kinetic measurements from a single well were used to determine enzyme Km and Vmax values. We compare the AMI-MS readout with conventional platforms in single-shot screening and multipoint profiling modes. The AMI-MS assay identified 86% of hits previously identified, with a pIC50 ≥ 5.0, in a scintillation proximity assay (SPA) HTS at a lower hit rate and with a significantly reduced cost per well compared to the SPA-based readout. Furthermore, pIC50s, as measured by AMI-MS, showed a good correlation with values generated by RapidFire-MS. AMI-MS has the potential to provide significant improvements to high-throughput bioassays.

Thiolactomycin-based ß-ketoacyl-AcpM synthase A (KasA) inhibitors: fragment-based inhibitor discovery using transient one-dimensional nuclear overhauser effect NMR spectroscopy.

Thiolactomycin (TLM) is a natural product inhibitor of KasA, the ß-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy.

Substrate recognition by ß-ketoacyl-ACP synthases.

ß-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a ß-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.

Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin.

Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.

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.

Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens.

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2.

The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.

Crystal structures of Mycobacterium tuberculosis KasA show mode of action within cell wall biosynthesis and its inhibition by thiolactomycin.

Mycobacteria have a unique cell wall consisting of mycolic acids, very-long-chain lipids that provide protection and allow the bacteria to persist within human macrophages. Inhibition of cell wall biosynthesis is fatal for the organism and a starting point for the discovery and development of novel antibiotics. We determined the crystal structures of KasA, a key enzyme involved in the biosynthesis of long-chain fatty acids, in its apo-form and bound to the natural product inhibitor thiolactomycin. Detailed insights into the interaction of the inhibitor with KasA and the identification of a polyethylene glycol molecule that mimics a fatty acid substrate of approximately 40 carbon atoms length, represent the first atomic view of a mycobacterial enzyme involved in the synthesis of long-chain fatty acids and provide a robust platform for the development of novel thiolactomycin analogs with high affinity for KasA.

Substrate recognition by the human fatty-acid synthase.

The human fatty-acid synthase (HFAS) is a potential target for anti-tumor drug discovery. As a prelude to the design of compounds that target the enoyl reductase (ER) component of HFAS, the recognition of NADPH and exogenous substrates by the ER active site has been investigated. Previous studies demonstrate that modification of Lys-1699 by pyridoxal 5'-phosphate results in a specific decrease in ER activity. For the overall HFAS reaction, the K1699A and K1699Q mutations reduced kcat and kcat/KNADPH by 8- and 600-fold, respectively (where KNADPH indicates the Km value for NADPH). Thus, Lys-1699 contributes 4 kcal/mol to stabilization of the rate-limiting transition state following NADPH binding, while also stabilizing the most stable ground state after NADPH binding by 3 kcal/mol. A similar effect of the mutations on the ER partial reaction was observed, in agreement with the proposal that Lys-1699 is located in the ER NADPH-binding site. Most unexpectedly, however, both kcat and kcat/KNADPH for the beta-ketoacyl reductase (BKR) reaction were also impacted by the Lys-1699 mutations, raising the possibility that the ER and BKR activities share a single active site. However, based on previous data indicating that the two reductase activities utilize distinct cofactor binding sites, mutagenesis of Lys-1699 is hypothesized to modulate BKR activity via allosteric effects between the ER and BKR NADPH sites.