Caspase inhibition is a promising approach for treating multiple diseases. Using a reconstituted assay and high-throughput screening, we identified a group of non-peptide caspase inhibitors. These inhibitors share common chemical scaffolds, suggesting same mechanism of action. They can inhibit apoptosis in various cell types induced by multiple stimuli; they can also inhibit caspase-1-mediated interleukin generation in macrophages, indicating potential anti-inflammatory application. While these compounds inhibit all the tested caspases, kinetic analysis indicates they do not compete for the catalytic sites of the enzymes. The co-crystal structure of one of these compounds with caspase-7 reveals that it binds to the dimerization interface of the caspase, another common structural element shared by all active caspases. Consistently, biochemical analysis demonstrates that the compound abates caspase-8 dimerization. Based on these kinetic, biochemical, and structural analyses, we suggest that these compounds are allosteric caspase inhibitors that function through binding to the dimerization interface of caspases.
Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z′ values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.
protein methyl transferases; drug discovery; inhibit or; SET7/9; SET8; SETD2; EuHMTase1; SPA technology; red shifted imaging beads
We report the design, synthesis and structure-activity relationship (SAR) of a series of novel pyrido[2,3-d]pyrimidin-7-one compounds as potent Abl kinase inhibitors. We evaluate their specificity profile against a panel of human recombinant kinases, as well as their biological profile toward a panel of well characterized cancer cell lines. Our study reveals that substitutions in the -3 and -4 positions of the phenylamino moiety lead to improved potency and improved selectivity both in target-based and cell based assays. Altogether, our results provide an insight into the SAR of pyrido[2,3-d]pyrimidin-7-ones for the development of drug candidates with improved potency and selectivity for the targeted treatment of CML.
Pyridopyrimidines; CML; Abl kinase; inhibitor
RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener™ ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high-throughput screening.
triphosphatase; drug discovery; high-throughput screening; fluorescence polarization
Intraarterial delivery of chemotherapeutic agents offers a new and exciting opportunity for the treatment of advanced intraocular retinoblastoma. It allows local delivery of relatively high doses of chemo agents while bypassing general blood circulation. For this reason we sought to revisit some of the FDA approved drugs for the treatment of retinoblastoma.
High throughput screening (HTS) of 2,640 approved drugs and bioactive compounds resulted in the identification of cytotoxic agents with potent activity toward both the Y79 and RB355 human retinoblastoma cell lines. Subsequent profiling of the drug candidates was performed in a panel of ocular cancer cell lines. Induction of apoptosis in Y79 cells was assessed by immunofluorescence detection of activated Caspase-3. Therapeutic effect was evaluated in a xenograft model of retinoblastoma.
We have identified several FDA approved drugs with potent cytotoxic activity toward retinoblastoma cell lines in vitro. Among them were several cardiac glycosides, a class of cardenolides historically associated with the prevention and treatment of congestive heart failure. Caspase-3 activation studies provided an insight into the mechanism of action of cardenolides in retinoblastoma cells. When tested in a xenograft model of retinoblastoma, the cardenolide ouabain induced complete tumor regression in the treated mice.
We have identified cardenolides as a new class of antitumor agents for the treatment of retinoblastoma. We propose that members of this class of cardiotonic drugs could be repositioned for retinoblastoma if administered locally via direct intraarterial infusion.
Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates; due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell permeable modulators for development. For this purpose, we have used our recently optimized EGFR biosensor (EGFRB) assay to screen for modulators of EGFR activity. Here, we report on its validation under HTS conditions displaying a S/N ratio of 21 and a Z’ value of 0.56; attributes of a robust cell based assay. We performed a pilot screen against a library of 6,912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95 %, respectively. Follow up dose response studies revealed that 12 out of the 13 known EGFR inhibitors in the library confirmed as hits. ZM-306416, a VEGFR antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic.
EGFR; domain-based biosensor; high content analysis; live cell imaging
Caspases are central to the execution of programmed cell death and their activation constitutes the biochemical hallmark of apoptosis. In this article, we report the successful adaptation of a high content assay method utilizing the DEVD-NucView488™ fluorogenic substrate, and for the first time, we show caspase activation in live cells induced either by drugs or siRNA. The fluorogenic substrate was found to be non-toxic over an exposure period of several days; during which we demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the anti-apoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to Doxorubicin, Etoposide or cell death siRNA. Our method was further validated against two well characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to Erlotinib; where we show a differential time dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, our results demonstrate the feasibility of using this newly adapted and validated high content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells.
High content assay; RNAi HT screening; Chemical HT screening; caspase; apoptosis; cancer; live cells
For many cancers, the lack of potency and the toxicity of current drugs limits the dose achievable in patients and the efficacy of treatment. Among them, retinoblastoma is a rare cancer of the eye for which better chemotherapeutic options are needed. Combination therapy is a compelling approach to enhance the efficacy of current treatment, however clinical trials to test rationally designed combinations of approved drugs are slow and expensive, and limited by our lack of in-depth knowledge of drug specificity. Since many patients already turn to nutraceuticals in hopes of improving their condition, we hypothesized that certain approved drugs could potentially synergize with widely consumed supplements. Following this hypothesis, we devised an alternative screening strategy aimed at taking advantage of a bait compound such as a nutraceutical with potential therapeutic benefits but low potency, by screening chemical libraries for approved drugs that synergize with this companion effector. As a proof of concept, we sought to identify approved drugs with synergetic therapeutic effects toward retinoblastoma cells in combination with the antioxidant resveratrol, popular as a supplement. We systematically tested FDA-approved drugs and known bioactives seeking to identify such pairs, which led to uncovering only a few additive combinations; but to our surprise, we identified a class of anticancer drugs widely used in the clinic whose therapeutic effect is antagonized with resveratrol. Our observations could explain in part why some patients do not respond well to treatment. Our results validate this alternative approach, and we expect that our companion effector strategy could significantly impact both drug discovery and the nutraceutical industry.
Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein–labeled SRC homology 2 domain–based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.
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 struture 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.
Human peptide deformylase; Benzofuran-4,5-diones; Structure activity relationships; Fluorescence polarization; Antiproliferative agents
Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFRα) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z′ value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFRα inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRα inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries.
Prostate specific antigen (PSA) molecules secreted by cancerous and normal prostate cells differ in their N-linked glycan composition, while the peptide backbone appears to be conserved. Antibodies selectively recognizing such differentially glycosylated PSA structures could form a basis for a new diagnostic assay for prostate cancer. Twenty-amino acid PSA fragments carrying di-, tri-, and tetrabranched complex-type glycans were prepared by total synthesis and conjugated to maleimide-modified keyhole limpet hemocyanin (KLH) carrier protein through backbone Cys residues. These glycopeptide/KLH conjugates were then used for antibody generation.
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.
alkaloids; antitumor agents; multidrug resistance; total synthesis
Current limitations to applications of monoclonal antibody (mAb) targeted isotope generators in radioimmunotherapy include the low mAb labeling yields and the non-specific radiation of normal tissues by non-targeted radioimmunoconjugates (RIC). Radiotoxicity occurs in normal organs that metabolize radiolabeled proteins and peptides, primarily liver and kidneys, or in radiosensitive organs with prolonged exposure to the isotope from the blood, such as the bone marrow. Actinium-225 nanogenerators also have the problem of released alpha emitting daughters. We developed two new bifunctional chelating agents (BCA) in order to address these issues. Thiol-maleimide conjugation chemistry was employed to increase the efficiency of the mAb radiolabelings by up to 8 fold. In addition, one bifunctional chelating agent incorporated a cleavable linker to alter the catabolism of the alpha particle emitting mAb conjugate. This linker was designed to be sensitive to cathepsins to allow release and clearance of the chelated radiometal after internalization of the radioimmunoconjugate into the cell. We compared the properties of the cleavable conjugate (mAb-DOTA-G3FC) to non-cleavable constructs (mAb-DOTA-NCS and mAb-DOTA-SH). The cleavable RIC was able to release 80% of its radioactive payload when incubated with purified cathepsin B. The catabolism of the constructs mAb-DOTA-G3FC and mAb-DOTA-NCS was investigated in vitro and in vivo. RIC integrity was retained at 85% over a period of 136 hours in mouse serum in vivo. Both conjugates were degraded over time inside HL-60 cells after internalization and in mouse liver in vivo. While we found that the rates of degradation of the two RICs in those conditions were similar, the amounts of the radiolabeled product residues were different. The cleavable mAb-DOTA-G3FC conjugate yielded a larger proportion of fragments below 6kDa in size in mouse liver in vivo after 12 hours than the DOTA-NCS conjugate. Biodistribution studies in mice showed that the mAb-DOTA-G3FC construct yielded a higher liver dose and prolonged liver retention of radioactivity compared to the mAb-DOTA-NCS conjugate. The accumulation in the liver seemed to be in part caused by the maleimide functionalization of the antibody, since the non-cleavable mAb-DOTA-SH maleimide-functionalized control conjugate displayed the same biodistribution pattern. These results provide an insight into the catabolism of RICs, by demonstrating that the release of the radioisotope from a RIC is not a sufficient condition to allow the radioactive moiety to clear from the body. The excretion mechanisms of radiolabeled fragments seem to constitute a major limiting step in the chain of events leading to their clearance.
Although proteases represent an estimated 5% to 10% of potential drug targets, inhibitors for metalloproteases (MPs) account for only a small proportion of all approved drugs, failures of which have typically been associated with lack of selectivity. In this study, the authors describe a novel and universal binding assay based on an actinonin derivative and show its binding activities for several MPs and its lack of activity toward all the non-MPs tested. This newly developed assay would allow for the rapid screening for inhibitors of a given MP and for the selectivity profiling of the resulting hits. The assay has successfully enabled for the first time simultaneous profiling of 8 well-known inhibitors against a panel of selected MPs. Previously published activities for these inhibitors were confirmed, and the authors have also discovered new molecular targets for some of them. The authors conclude that their profiling platform provides a generic assay solution for the identification of novel metalloprotease inhibitors as well as their selectivity profiling using a simple and homogeneous assay.
metalloprotease; inhibitor; profiling; fluorescence polarization; HTS
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.
human peptide deformylase; high-throughput screening; fluorescence polarization; antiproliferative agents