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A major focus of our pediatric cancer research is the discovery of chemical probes to further our understanding of the biology of leukemia harboring fusion proteins arising from chromosomal rearrangements, and to develop novel specifically targeted therapies. The NUP98-NSD1 fusion protein occurs in a highly aggressive subtype of acute myeloid leukemia after rearrangement of the genes NUP98 and NSD1. The methyltransferase activity of NSD1 is retained in the fusion, and it gives rise to abnormally high levels of methylation at lysine 36 on histone 3, enforcing oncogene activation. Therefore, inhibition of the methyltransferase activity of NUP98-NSD1 may be considered a viable therapeutic strategy. Here, we report the development and validation of a highly sensitive and robust luminescence-based assay for NSD1 and other methyltransferases that use S-adenosylmethionine (SAM) as a methyl donor. The assay quantifies S-adenosylhomocysteine (SAH), which is produced during methyl transfer from SAM. SAH is converted enzymatically to adenosine monophosphate (AMP); in the process, adenosine triphosphate (ATP) is consumed and the amount of ATP remaining is measured using a luminescent assay kit. The assay was validated by pilot high-throughput screening (HTS), dose-response confirmation of hits, and elimination of artifacts through counterscreening against SAH detection in the absence of NSD1. The known methyltransferase inhibitor suramin was identified, and profiled for selectivity against the histone methyltransferases EZH2, SETD7, and PRMT1. HTS using the luminescent NSD1 assay described here has the potential to deliver selective NSD1 inhibitors that may serve as leads in the development of targeted therapies for NUP98-NSD1-driven leukemias.

RAD51 is a key protein of homologous recombination that plays a critical role in the repair of DNA double-strand breaks (DSB) and interstrand cross links (ICL). To better understand the cellular function(s) of human RAD51, we propose to develop specific RAD51 inhibitors. RAD51 inhibitors may also help to increase the potency of anticancer drugs that act by inducing DSBs or ICLs, e.g., cisplatin or ionizing radiation. In vitro, RAD51 promotes DNA strand exchange between homologous ss- and dsDNA. Here, we developed a DNA strand exchange assay based on fluorescence resonance energy transfer and used this assay to identify RAD51 inhibitors by high throughput screening of the NIH Small Molecule Repository (>200,000 compounds). Seventeen RAD51 inhibitors were identified and analyzed for selectivity using additional non-fluorescent DNA-based assays. As a result, we identified a compound (B02) that specifically inhibited human RAD51 (IC50 = 27.4 μM), but not its E. coli homologue RecA (IC50 > 250 μM). Two other compounds (A03 and A10) were identified that inhibited both RAD51 and RecA, but not the structurally unrelated RAD54 protein. The structure-activity relationship (SAR) analysis allowed us to identify the structural components of B02 that are critical for RAD51 inhibition. The described approach can be used for identification of specific inhibitors of other human proteins that play an important role in DNA repair, e.g., RAD54 or Bloom’s syndrome helicase.

Recently, we identified a thiocarbazate that exhibits potent inhibitory activity against human cathepsin L. Since this structure represents a novel chemotype with potential for activity against the entire cysteine protease family, we designed, synthesized, and assayed a series of analogs to probe the mechanism of action, as well as the structural requirements for cathepsin L activity. Molecular docking studies using coordinates of a papain-inhibitor complex as a model for cathepsin L provided useful insights.

A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.15) with an IC(50) of 56 nM, was developed after a 57,821-compound screen of the National Institutes of Health Molecular Libraries Small Molecule Repository. After a 4-h preincubation with cathepsin L, this compound became even more potent, demonstrating an IC(50) of 1.0 nM. The thiocarbazate was determined to be a slow-binding and slowly reversible competitive inhibitor. Through a transient kinetic analysis for single-step reversibility, inhibition rate constants were k(on) = 24,000 M(-1)s(-1) and k(off) = 2.2 x 10(-5) s(-1) (K(i) = 0.89 nM). Molecular docking studies were undertaken using the experimentally derived X-ray crystal structure of papain/CLIK-148 (1cvz. pdb). These studies revealed critical hydrogen bonding patterns of the thiocarbazate with key active site residues in papain. The thiocarbazate displayed 7- to 151-fold greater selectivity toward cathepsin L than papain and cathepsins B, K, V, and S with no activity against cathepsin G. The inhibitor demonstrated a lack of toxicity in human aortic endothelial cells and zebrafish. In addition, the thiocarbazate inhibited in vitro propagation of malaria parasite Plasmodium falciparum with an IC(50) of 15.4 microM and inhibited Leishmania major with an IC(50) of 12.5 microM.

A miniaturized fluorescent assay is described that monitors the conversion of NADPH to NADP+. The same assay format may also be used to measure NADH to NAD+ conversion. Examples of assay development and validation results are presented to illustrate the use of this protocol to screen an enzyme that consumes NADPH as a cofactor during conversion of substrate to a reduced product. Enzymatic assays are carried out low volume 384-well plates, in which the turnover of NADPH is monitored by the decrease in fluorescent emission at 460 nm between an initial fluorescence measurement and a second reading after 90 minutes. A follow-up assay is used to rule out false positive artifacts arising from compounds that fluoresce at 460 nm.

Background: Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use. Methodology/Principal Findings: We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells. Conclusions/Significance: We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.

High-throughput screening of 201,368 compounds revealed that 1-(3-(5-ethyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one (SID 7975595) inhibited RmlC a TB cell wall biosynthetic enzyme. SID 7975595 acts as a competitive inhibitor of the enzyme's substrate and inhibits RmlC as a fast-on rate, fully reversible inhibitor. An analog of SID 7975595 had a K(i) of 62nM. Computer modeling showed that the binding of the tethered two-ringed system into the active site occurred at the thymidine binding region for one ring system and the sugar region for the other ring system.

Eukaryotic initiation factor 2 (eIF2) B is a guanine nucleotide exchange factor that plays a central role in translation initiation and its control, especially in response to diverse cellular stresses. In addition, inherited mutations in human eIF2B subunits cause a fatal brain disorder commonly called childhood ataxia with central nervous system hypomyelination or leukoencephalopathy with vanishing white matter. In yeast, inhibiting activity of eIF2B up-regulates expression of the transcriptional activator general control nondepressible (GCN) 4. We report here evaluation of high-throughput screening (HTS) using a yeast-based reporter gene assay, in which strains containing either wild-type or a mutant eIF2B were screened in parallel to identify compounds modifying eIF2B-dependent responses. The goals of the HTS were twofold: first, to discover compounds that restore normal function to mutant eIF2B, which may have therapeutic utility for the fatal human disease; and second, to identify compounds that activate a GCN4 response, which might be useful experimental tools. The HTS assay measured cell growth by absorbance, and activation of gene expression via a beta-galactosidase reporter gene fusion. Because mutant eIF2B activates GCN4 in the absence of stress inducers, the mutant strain was screened for reduction in GCN4 activation. HTS revealed apparent mutant-selective inhibitors but did not reliably predict selectivity as these hits affected both wild-type and mutant strains equally on dose-response confirmation. Wild-type strain results from the HTS identified two GCN4 response activators, both of which were confirmed to be wild-type selective in dose-response testing, suggesting that these compounds may activate GCN4 by a mechanism that down-regulates eIF2B activity.

MLL-rearranged leukemia is an aggressive form of cancer characterized by specific chromosomal rearrangements of the mixed lineage leukemia (MLL) gene. In normal cells, MLL, a histone methyltransferase, methylates the lysine 4 residue on histone 3 (H3K4) and thereby regulates the expression of genes which control hematopoietic development. In leukemic cells, chromosomal rearrangements fuse the N-terminus of MLL with one of more than 50 partner proteins, giving rise to oncogenic fusion proteins that lack H3K4 methyltransferase activity. These fusion proteins inappropriately recruit DOT1L, which methylates H3K79, producing an abnormal methylation pattern and activation of genes normally suppressed during differentiation. Elevated expression of HOXA9 in particular leads to a block in normal hematopoetic differentiation and the onset of leukemia. A solid body of evidence has shown DOT1L to be crucial for the development and maintenance of MLL-rearranged leukemias; consequently, it is a promising target for therapeutic intervention. We have focused on developing an assay suitable for high-throughput screening (HTS) to detect compounds that inhibit DOT1L enzymatic activity. DOT1L catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to H3K79 and generates S-adenosylhomocysteine (SAH) as a byproduct. The enzyme displays a very low rate of methylation of histone octamers or nucleosomes and no activity towards individual histones or histone-derived peptides. Thus DOT1L HTS requires a highly sensitive assay that is able to accommodate protein-protein and protein-DNA complexes as substrates. An assay using radiolabeled SAM has sufficient sensitivity, but is relatively laborious and not well-suited for HTS. We tested several previously reported non-radioactive assay formats, but none enabled robust detection of DOT1L activity. Therefore, we developed a coupled enzyme assay in which SAH is converted to homocysteine and adenosine using SAH hydrolase. In a second enzyme-coupled step, adenosine kinase generates AMP and ADP from the SAH-derived adenosine and added ATP. Finally, ADP is detected using the luciferase-based Promega ADP-Glo assay. The luminescent signal provides a highly sensitive measure of ADP at a concentration as low as 10 nM. The readout is directly proportional to SAH production; thus DOT1L inhibitors are identified by a reduction in this signal. We report development of the assay, including careful optimization of the activity of adenosine kinase to enhance sensitivity, and assay validation demonstrating DOT1L inhibition by the non-selective SAM-competitive inhibitor sinefungin, and pilot HTS of a 2,000-compound library. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3878. doi:1538-7445.AM2012-3878