David Shum

Superoxide dismutase 1 (SOD1) is a target for a small molecule identified in a screen for inhibitors of the growth of lung adenocarcinoma cell lines

We previously described four small molecules that reduced the growth of lung adenocarcinoma cell lines with either epidermal growth factor receptor (EGFR) or KRAS mutations in a high-throughout chemical screen. By combining affinity proteomics and gene expression analysis, we now propose superoxide dismutase 1 (SOD1) as the most likely target of one of these small molecules, referred to as lung cancer screen 1 (LCS-1). siRNAs against SOD1 slowed the growth of LCS-1 sensitive cell lines; conversely, expression of a SOD1 cDNA increased proliferation of H358 cells and reduced sensitivity of these cells to LCS-1. In addition, SOD1 enzymatic activity was inhibited in vitro by LCS-1 and two closely related analogs. These results suggest that SOD1 is an LCS-1–binding protein that may act in concert with mutant proteins, such as EGFR and KRAS, to promote cell growth, providing a therapeutic target for compounds like LCS-1.

Synthesis of Antiproliferative Cephalotaxus Esters and Their Evaluation against Several Human Hematopoietic and Solid Tumor Cell Lines: Uncovering Differential Susceptibilities to Multidrug Resistance

Deoxyharringtonine (2), homoharringtonine (3), homodeoxyharringtonine (4), and anhydroharringtonine (5) are reported to be among the most potent members of the antileukemia alkaloids isolated from the Cephalotaxus genus. Convergent syntheses of these four natural products are described, each involving novel synthetic methods and strategies. These syntheses enabled evaluation of several advanced natural and non-natural compounds against an array of human hematopoietic and solid tumor cells. Potent cytotoxicity was observed in several cell lines previously not challenged with these alkaloids. Variations in the structure of the ester chain within this family of alkaloids confer differing activity profiles against vincristine-resistant HL-60/RV+, signalling new avenues for molecular design of these natural products to combat multi-drug resistance.

A high density assay format for the detection of novel cytotoxic agents in large chemical libraries.

In response to the need for inexpensive high throughput assays for anti-cancer drug screening, a 1536-well microtiter plate based assay utilizing the Alamar Blue fluorescent dye as a measure of cellular growth was validated in 10 μL assay volume. Its robustness was assessed in a screen against a library of 2000 known bioactives; with an overall Z′ value of 0.89 for assay robustness, several known cytotoxic agents were identified including and not limited to anthracyclines, cardiac glycosides, gamboges, and quinones. To further test the sensitivity of the assay, IC50 determinations were performed in both 384-well and 1536-well formats and the obtained results show a very good correlation between the two density formats. These findings demonstrate that this newly developed assay is simple to set up, robust, highly sensitive and inexpensive. It could potentially provide a rapid way to screen established and primary tumor cell lines against large chemical libraries.

High-Throughput Identification of Inhibitors of Human Mitochondrial Peptide Deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid—based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)—based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent. (Journal of Biomolecular Screening 2007:521-535)

Inhibition of dengue virus replication by a class of small-molecule compounds that antagonize dopamine receptor d4 and downstream mitogen-activated protein kinase signaling.

ABSTRACT Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds. IMPORTANCE The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.

Small-Molecule Inhibitors of SETD8 with Cellular Activity

SETD8/SET8/Pr-SET7/KMT5A is the sole protein lysine methyltransferase (PKMT) known to monomethylate lysine 20 of histone H4 in vivo. SETD8’s methyltransferase activity has been implicated in many essential cellular processes including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. Developing SETD8 inhibitors with cellular activity is a key step toward elucidating the diverse roles of SETD8 via convenient pharmacological perturbation. From the hits of a prior high throughput screen (HTS), SPS8I1–3 (NSC663284, BVT948, and ryuvidine) were validated as potent SETD8 inhibitors. These compounds contain different structural motifs and inhibit SETD8 via distinct modes. More importantly, these compounds show cellular activity by suppressing the H4K20me1 mark of SETD8 and recapitulate characteristic S/G2/M-phase cell cycle defects as observed for RNAi-mediated SETD8 knockdown. The commonality of SPS8I1–3 against SETD8, together with their distinct structures and mechanisms for SETD8 inhibition, argues for the collective application of these compounds as SETD8 inhibitors.

An Image-Based Biosensor Assay Strategy to Screen for Modulators of the microRNA 21 Biogenesis Pathway

microRNAs (miRNAs) are evolutionary conserved, small endogenous non-coding, RNA molecules. Although their mode of action has been extensively studied, little is known about their biogenesis. As their altered expression has been implicated in many diseases, small molecules that would modulate their expression are sought after. They are generated through the concerted action of several complexes which promote their transcription, maturation, export, trafficking, and loading of mature miRNA into silencing complexes. An increasing number of studies have suggested that each of these steps serves as a regulatory junction in the process, and therefore provides an intervention point. For this purpose, we have developed a simple image-based assay strategy to screen for such modulators. Here, we describe its successful implementation which combines the use of a microRNA 21 (miR-21) synthetic mimic together with an EGFP based reporter cell line, where its expression is under the control of miR-21, to monitor EGFP expression in a format suitable for HTS. The strategy was further validated using a small panel of known gene modulators of the miRNA pathway. A screen was performed in duplicate against a library of 6,912 compounds and identified 48 initial positives exhibiting enhanced EGFP fluorescence intensity. 42 compounds were found to be inherently fluorescent in the green channel leaving the remaining 6 as potential inhibitors and with a positive rate of 0.09%. Taken together, this validated strategy offers the opportunity to discover novel and specific inhibitors of the pathway through the screening of diverse chemical libraries.

Synthesis, antileukemic and antiplatelet activities of 2,3-diaryl-6,7-dihydro-5H-1,4-diazepines

The synthesis, antileukemic and antiplatelet activity evaluation of 2,3-diaryl-6,7-dihydro-5H-1,4-diazepines are described. In general, it was found that compound 17o showed moderate antileukemic activity against MOLT3 human leukemic cancer cell lines. An arachidonic acid induced platelet aggregation effect on washed rat platelets was studied. Compound 17i was found to be the most potent. The antiplatelet properties may be mediated by interference with the arachidonic acid pathway.

Flaviviruses are sensitive to inhibition of thymidine synthesis pathways

ABSTRACT Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.

Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase.

Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their structure-activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.