Stimulators of translation identified during a small molecule screening campaign

In screening a library of natural and synthetic products for eukaryotic translation modulators, we identified two natural products, isohymenialdisine and hymenialdisine, that exhibit stimulatory effects on translation. The characterization of these compounds led to the insight that mRNA used to program the translation extracts during high-throughput assay setup was leading to phosphorylation of eIF2α, a potent negative regulatory event that is mediated by one of four kinases. We identified double-stranded RNA-dependent protein kinase (PKR) as the eIF2α kinase that was being activated by exogenously added mRNA template. Characterization of the mode of action of isohymenialdisine revealed that it directly acts on PKR by inhibiting autophosphorylation, perturbs the PKR–eIF2α phosphorylation axis, and can be modeled into the PKR ATP binding site. Our results identify a source of “false positives” for high-throughput screen campaigns using translation extracts, raising a cautionary note for this type of screen.     

Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries.

In an attempt to clone protein tyrosine kinases, antiphosphotyrosine antibodies were used to screen lambda gt11 cDNA expression libraries. By this method, a 2.5-kilobase cDNA encoding a novel tyrosine kinase was isolated from a mouse liver cDNA library. This new gene is most closely related to the receptor tyrosine kinases ret, fms, and kit.     

Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.     

Ribosomal protein S18 identified as a cofilin-binding protein by using phage display library

We previously reported that an actin-binding protein, cofilin, is involved in superoxide production, phagocytosis, and chemotaxis in activated phagocytes through cytoskeletal reorganization. To elucidate the functions of cofilin in greater detail we tried to identify cofilin-binding proteins by using a phage-displayed cDNA library constructed from human brain mRNAs. Several phage clones capable of binding to cofilin were obtained, and the phage with the strongest binding affinity contained the C-terminal half of ribosomal protein S18. To confirm the interaction between the S18 protein and cofilin, we investigated whether cofilin would bind to His-tagged S18 protein immobilized in Ni-NTA-agarose gel. Cofilin and the S18 protein co-eluted with a low pH (4.5) buffer, suggesting that the proteins interact with each other. Preincubation of cofilin with actin abrogated the binding to protein S18, indicating that cofilin interacts with S18 protein at the actin-binding site, and cofilin co-immunoprecipitated with FLAG-tagged S18 protein expressed in COS-7 cells. These results suggest that some cofilin molecules bind the ribosomal S18 protein under physiological conditions.     

Targeted protein knockdown using small molecule degraders

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Diversity & tractability revisited in collaborative small molecule phenotypic screening library design

Identification of purposeful chemical matter on a broad range of drug targets is of high importance to the pharmaceutical industry. However, disease-relevant but more complex hit-finding plans require flexibility regarding the subset of the compounds that we screen. Herein we describe a strategy to design high-quality small molecule screening subsets of two different sizes to cope with a rapidly changing early discovery portfolio. The approach taken balances chemical tractability, chemical diversity and biological target coverage. Furthermore, using surveys, we actively involved chemists within our company in the selection process of the diversity decks to ensure current medicinal chemistry principles were incorporated. The chemist surveys revealed that not all published PAINS substructure alerts are considered productive by the medicinal chemistry community and in agreement with previously published results from other institutions, QED scores tracked quite well with chemists’ notions of chemical attractiveness.     

Solid-phase assays for small molecule screening using sol-gel entrapped proteins.

With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput screening of small molecule libraries and mixtures.     

Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.     

Novel phenotypes identified by plasma biochemical screening in the mouse

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8-12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.     

Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening

Salmonella enterica polymyxin B (PM) resistance is modulated mainly by substitutions of the acyl chains and the phosphate groups on the lipid A moiety of lipopolysaccharide. These modifications are mediated by genes under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems. In this study, a deletion in the gene encoding the alternative σ⁵⁴ factor, rpoN , was shown to increase PM resistance without affecting protamine sensitivity. The results presented here showed that the increased polymyxin resistance observed in the Δ rpoN mutant occurs through a PmrA/PhoP-independent pathway. Downregulation of one or more genes belonging to the RpoN regulon may provide an additional mechanism of defence against membrane-permeabilizing antimicrobial peptides that helps the pathogen to survive in different environments.     

The endogenous siRNA pathway in Drosophila impacts stress resistance and lifespan by regulating metabolic homeostasis

Small non-coding RNAs regulate gene expression in a sequence-specific manner. In Drosophila, Dicer-2 (Dcr-2) functions in the biogenesis of endogenous small interfering RNAs (endo-siRNAs). We identified 21 distinct proteins that exhibited a ≥ 1.5-fold change as a consequence of loss of dcr-2 function. Most of these were metabolic genes implicated in stress resistance and aging. dcr-2 Mutants had reduced lifespan and were hypersensitive to oxidative, endoplasmic reticulum, starvation, and cold stresses. Furthermore, loss of dcr-2 function led to abnormal lipid and carbohydrate metabolism. Our results suggest roles for the endo-siRNA pathway in metabolic regulation and defense against stress and aging in Drosophila.     

PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of Imd signaling (PIMS), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections.     

Novel VEGFR-2 kinase inhibitors identified by the back-to-front approach

We report a novel VEGFR-2 inhibitor, developed by the back-to-front approach. Docking experiments indicated that the 3-chloromethylphenylurea motif of the lead compound occupied the back pocket of VEGFR-2 kinase. An attempt was made to enhance the binding affinity of 1 by expanding the structure to access the front pocket using a triazole linker. A library of 1,4-(disubstituted)-1H-1,2,3-triazoles were screened in silico, and one compound (VH02) was identified with an IC₅₀ against VEGFR-2 of 0.56 μM. VH02 showed antiangiogenic effects, inhibiting tube formation in HUVEC cells (EA.hy926) at 0.3 μM, 13 times lower than its cytotoxic dose. These enzymatic and cellular activities suggest that VH02 has potential as a lead for further optimization.     

Discovery of novel pyruvate dehydrogenase kinases inhibitors by screening of an in-house small molecule library for anti-lung cancer therapeutics

Pyruvate dehydrogenase kinases (PDKs) are widely over-expressed in various human solid cancers, making them attractive therapeutic targets for cancer treatment. Herein, we report the identification of structurally novel PDKs inhibitors by screening of an in-house small molecule library. Biochemical assay indicated that the identified compounds 1–4 inhibited PDK1 activity with EC₅₀ values of 0.50, 1.99, 4.64, and 0.42?µM, respectively. The ITC analysis suggested that the identified compounds 1–4 were pan-isoform PDK inhibitors, which bound to and inhibited the four PDK isoforms. Moreover, 1–4 dose-dependently reduced pyruvate dehydrogenase complex phosphorylation in NCI-H1975 cell. Molecular docking suggested that the most potent compound 4 docked well in the ATP binding pocket of the four PDK isoforms, forming direct hydrogen bond interactions with the conserved amino acids Thr and Asp in ATP binding pocket of PDKs. The cell viability assay demonstrated that 4 potently blocked NCI-H1975 cell proliferation (IC₅₀?=?3.32?µM), but had little effect on human normal lung cell MRC-5 even with the tested concentration up to 40?µM. All the data demonstrated that 4 was a promising lead for the development of structurally novel PDKs inhibitor for the cancer treatment.     

Identification of potent Yes1 kinase inhibitors using a library screening approach

Yes1 kinase has been implicated as a potential therapeutic target in a number of cancers including melanomas, breast cancers, and rhabdomyosarcomas. Described here is the development of a robust and miniaturized biochemical assay for Yes1 kinase that was applied in a high throughput screen (HTS) of kinase-focused small molecule libraries. The HTS provided 144 (17% hit rate) small molecule compounds with IC₅₀ values in the sub-micromolar range. Three of the most potent Yes1 inhibitors were then examined in a cell-based assay for inhibition of cell survival in rhabdomyosarcoma cell lines. Homology models of Yes1 were generated in active and inactive conformations, and docking of inhibitors supports binding to the active conformation (DFG-in) of Yes1. This is the first report of a large high throughput enzymatic activity screen for identification of Yes1 kinase inhibitors, thereby elucidating the polypharmacology of a variety of small molecules and clinical candidates.     

Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence we examined the role of TOR pathway genes in regulating lifespan by using Drosophila. We show that inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2 (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. We find that overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction.     

Novel candidate substrates of AMP-activated protein kinase identified in red blood cell lysates

AMPK is a metabolic stress-sensing kinase with important functions for red blood cell (RBC) survival. By using a proteomic approach, we identified putative AMPK targets in hemoglobin-depleted lysates of RBC, including metabolic enzymes, cytoskeletal proteins and enzymes involved in the oxidative stress response. These data tie in with the phenotypic observations of AMPKα1-deficient RBC and provide reference for future studies.