Screening For Small Molecule Modulators Research Articles

High-throughput sensitive screening of small molecule modulators of microexon alternative splicing using dual Nano and Firefly luciferase reporters

Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease.

High-Throughput Methods for the Discovery of Small Molecule Modulators of Pancreatic Beta-Cell Function and Regeneration.

The progression of type II diabetes (T2D) is characterized by a complex and highly variable loss of beta-cell mass, resulting in impaired insulin secretion. Many T2D drug discovery efforts aimed at discovering molecules that can protect or restore beta-cell mass and function have been developed using limited beta-cell lines and primary rodent/human pancreatic islets. Various high-throughput screening methods have been used in the context of drug discovery, including luciferase-based reporter assays, glucose-stimulated insulin secretion, and high-content screening. In this context, a cornerstone of small molecule discovery has been the use of immortalized rodent beta-cell lines. Although insightful, this usage has led to a more comprehensive understanding of rodent beta-cell proliferation pathways rather than their human counterparts. Advantages gained in enhanced physiological relevance are offered by three-dimensional (3D) primary islets and pseudoislets in contrast to monolayer cultures, but these approaches have been limited to use in low-throughput experiments. Emerging methods, such as high-throughput 3D islet imaging coupled with machine learning, aim to increase the feasibility of integrating 3D microtissue structures into high-throughput screening. This review explores the current methods used in high-throughput screening for small molecule modulators of beta-cell mass and function, a potentially pivotal strategy for diabetes drug discovery.

Expression and biochemical analyses of proteins involved in the transport of carotenoids and retinoids.

Animals acquire carotenoids from the diet and convert them to retinoids. These lipids must be distributed in the body to support retinoid signaling in peripheral tissues and photoreceptor function in the eyes. However, the hydrophobicity of carotenoids and retinoids limit their diffusion in the aqueous environment of the body. Therefore, membrane proteins and cellular binding proteins transport these lipids between extra- and intracellular compartments and facilitate their metabolism. Mutations in genes encoding these transport proteins are associated with a wide spectrum of blinding disorders. Here, we describe approaches used by our laboratories that have proven successful in expressing these proteins and examining their biochemical properties in the test tube and in cell-based assays. These assays can be utilized for screening of small molecule modulators of their activities to correct pathologies associated with retinoid metabolism.

In silico screening of small molecule modulators and their binding studies against human sirtuin-6 protein

Sirtuin-6 (SIRT6), class III family of deacetylase regulates several biological functions, including transcriptional repression, telomere maintenance, and DNA repair. It is unique among sirtuin family members with diverse enzymatic functions: mono-ADP-ribosylase, deacetylase and defatty-acylase. The studies so far implicated SIRT6 role in lifespan extension, tumor suppression, and is considered as an attractive drug target for aging-related disease. In this study, we have carried out in silico screening for human SIRT6 modulators using NCI Diversity Set III library, molecular dynamic (MD) simulations to analyze the protein-ligand interaction, and validated their binding-affinity () using MicroScale Thermophoresis. This study yielded two novel compounds, ((3Z)-3-((4-(dimethylamino)phenyl)methylidene)-5-(5,6,7,8-tetrahydronaphthalen-2-yl)furan-2-one and 5-phenyl-2-(5-phenyl-2,3-dihydro-1,3-benzoxazol-2-yl)-2,3-dihydro-1,3-benzoxazole showing high-affinity interaction for SIRT6. The structural analysis from MD simulation suggests both compounds might act as substrate-analogs or mimic the nicotinamide binding. On considering the uniqueness of SIRT6 substrate binding acyl channel among sirtuin family member, binding of both compounds to the above site suggesting their specificity for SIRT6 isoform. Therefore, it may form the basis for the development of potential modulators for human SIRT6. Communicated by Ramaswamy H. Sarma

Single-particle cryo-EM at atomic resolution.

The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

A direct fluorometric activity assay for lipid kinases and phosphatases

Lipid kinases and phosphatases play key roles in cell signaling and regulation, are implicated in many human diseases, and are thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here, we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Because of its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided detailed mechanistic information about the product inhibition and substrate acyl-chain selectivity of PI3K and enabled rapid evaluation of small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.

Droplet sample introduction to microchip gel and zone electrophoresis for rapid analysis of protein-protein complexes and enzymatic reactions.

Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6s with 1200V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device. Graphical abstract.

Modulation of Autophagy by a Small Molecule Inverse Agonist of ERRα Is Neuroprotective.

Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA), new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR) independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson’s disease (PD), XCT 790 exerted neuroprotective effects in the dopaminergic neurons of nigra by inducing autophagy to clear toxic protein aggregates and, in addition, ameliorated motor co-ordination deficits. Using a chemical biology approach, we unrevealed the role of ERRα in regulating autophagy and can be therapeutic target for neurodegeneration.

Surface marker profiling of SH-SY5Y cells enables small molecule screens identifying BMP4 as a modulator of neuroblastoma differentiation

Neuroblastoma is the most common extra-cranial solid tumor in children. Its broad spectrum of clinical outcomes reflects the underlying inherent cellular heterogeneity. As current treatments often do not lead to tumor eradication, there is a need to better define therapy-resistant neuroblastoma and to identify new modulatory molecules. To this end, we performed the first comprehensive flow cytometric characterization of surface molecule expression in neuroblastoma cell lines. Exploiting an established clustering algorithm (SPADE) for unbiased visualization of cellular subsets, we conducted a multiwell screen for small molecule modulators of neuroblastoma phenotype. In addition to SH-SY5Y cells, the SH-EP, BE(2)-M17 and Kelly lines were included in follow-up analysis as in vitro models of neuroblastoma. A combinatorial detection of glycoprotein epitopes (CD15, CD24, CD44, CD57, TrkA) and the chemokine receptor CXCR4 (CD184) enabled the quantitative identification of SPADE-defined clusters differentially responding to small molecules. Exposure to bone morphogenetic protein (BMP)-4 was found to enhance a TrkAhigh/CD15−/CD184− neuroblastoma cellular subset, accompanied by a reduction in doublecortin-positive neuroblasts and of NMYC protein expression in SH-SY5Y cells. Beyond yielding novel marker candidates for studying neuroblastoma pathology, our approach may provide tools for improved pharmacological screens towards developing novel avenues of neuroblastoma diagnosis and treatment.

From Protein Folding to Cognition: The Serendipitous Path of Discovery

A screen for small molecule modulators of the unfolded protein response yielded ISRIB, a drug‐like compound that with high efficacy renders cells insensitive to translational inhibition by phosphorylation of eIF2. ISRIB proved to be a cognitive enhancer in rodents, significantly improving long‐term memory of wild‐type animals in behavioral assays.

CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability.

Recent data shows that fibroblast growth factor 14 (FGF14) binds to and controls the function of the voltage-gated sodium (Nav) channel with phenotypic outcomes on neuronal excitability. Mutations in the FGF14 gene in humans have been associated with brain disorders that are partially recapitulated in Fgf14(-/-) mice. Thus, signaling pathways that modulate the FGF14:Nav channel interaction may be important therapeutic targets. Bioluminescence-based screening of small molecule modulators of the FGF14:Nav1.6 complex identified 4,5,6,7 -: tetrabromobenzotriazole (TBB), a potent casein kinase 2 (CK2) inhibitor, as a strong suppressor of FGF14:Nav1.6 interaction. Inhibition of CK2 through TBB reduces the interaction of FGF14 with Nav1.6 and Nav1.2 channels. Mass spectrometry confirmed direct phosphorylation of FGF14 by CK2 at S228 and S230, and mutation to alanine at these sites modified FGF14 modulation of Nav1.6-mediated currents. In 1 d in vitro hippocampal neurons, TBB induced a reduction in FGF14 expression, a decrease in transient Na(+) current amplitude, and a hyperpolarizing shift in the voltage dependence of Nav channel steady-state inactivation. In mature neurons, TBB reduces the axodendritic polarity of FGF14. In cornu ammonis area 1 hippocampal slices from wild-type mice, TBB impairs neuronal excitability by increasing action potential threshold and lowering firing frequency. Importantly, these changes in excitability are recapitulated in Fgf14(-/-) mice, and deletion of Fgf14 occludes TBB-dependent phenotypes observed in wild-type mice. These results suggest that a CK2-FGF14 axis may regulate Nav channels and neuronal excitability.-Hsu, W.-C. J., Scala, F., Nenov, M. N., Wildburger, N. C., Elferink, H., Singh, A. K., Chesson, C. B., Buzhdygan, T., Sohail, M., Shavkunov, A. S., Panova, N. I., Nilsson, C. L., Rudra, J. S., Lichti, C. F., Laezza, F. CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability.

High-throughput screening for small molecule modulators of motor protein Kinesin.

The kinesin superfamily of motor proteins are involved in the active transport of a large number of cargos such as organelles, proteins, and RNAs from the neuronal cell body to distal neuronal processes. Previously, we have shown that kinesin-mediated axonal transport of proteins and RNAs are important for long-term memory storage. Identification of small molecules that can activate or inhibit kinesins is of specific interest due to the significance of kinesin-mediated functions in neuronal health and plasticity. Here, we describe a high-throughput screening assay designed to specifically identify compounds that inhibit or activate adenosine triphosphatase activity of the kinesin 5B of humans. The luminescence-based assay that we developed is highly reproducible and robust. Using this approach, we screened a pharmacologically characterized compound collection and have identified small molecules with either activator or inhibitor-like activity. To further characterize screening hits, we also developed an orthogonal assay based on absorbance and a counter screen assay based on luminescence. Development of such assays is important to help identify small molecules that can be used in potential drug development efforts targeted at modulating the function of kinesin.

Identification of inhibitors of inositol 5-phosphatases through multiple screening strategies.

Phosphoinositides are low abundance membrane phospholipids that have key roles in signaling, membrane trafficking, and cytoskeletal dynamics in all cells. Until recently, strategies for robust and quantitative development of pharmacological tools for manipulating phosphoinositide levels have focused selectively on PI(3,4,5)P3 due to the importance of this lipid in growth factor signaling and cell proliferation. However, drugs that affect levels of other phosphoinositides have potential therapeutic applications and will be powerful research tools. Here, we describe methodology for the high-throughput screening of small molecule modulators of the inositol 5-phosphatases, which dephosphorylate PI(4,5)P2 (the precursor for PI(3,4,5)P3) and PI(3,4,5)P3). We developed three complementary in vitro activity assays, tested hit compounds on a panel of 5-phosphatases, and monitored efficacy toward various substrates. Two prominent chemical scaffolds were identified with high nanomolar/low micromolar activity, with one class showing inhibitory activity toward all 5-phosphatases tested and the other selective activity toward OCRL and INPP5B, which are closely related to each other. One highly soluble OCRL/INPP5B-specific inhibitor shows a direct interaction with the catalytic domain of INPP5B. The efficacy of this compound in living cells was validated through its property to enhance actin nucleation at the cell cortex, a PI(4,5)P2 dependent process, and to inhibit PI(4,5)P2 dephosphorylation by OCRL (both overexpressed and endogenous enzyme). The assays and screening strategies described here are applicable to other phosphoinositide-metabolizing enzymes, at least several of which have major clinical relevance. Most importantly, this study identifies the first OCRL/INPP5B specific inhibitor and provides a platform for the design of more potent inhibitors of this family of enzymes.

Abstract 2471: Chemical optimization of direct and selective molecular activators of pro-apoptotic BAX for cancer therapy.

Abstract BAX is a pro-apoptotic BCL-2 family member that lies dormant in the cytosol until triggered by cellular stress to translocate to the mitochondria and form toxic oligomeric pores. The majority of cancer cells retain wild-type BAX and instead neutralize the death pathway by overexpressing anti-apoptotic BCL-2 family proteins. Thus, direct activation of BAX represents a pharmacologic opportunity to lower the apoptotic threshold in the setting of chemoresistance. Using a Stabilized Alpha-Helix of BCL-2 domain (SAHB) modeled after the BIM BH3 helix, we previously identified the “trigger site” for direct BAX activation at the confluence of alpha-helices 1 and 6. In silico screening for small molecule modulators of BAX led to our identification of BAX activator molecule 7 (BAM7), which selectively engages the trigger site and promotes BAX-mediated cell death (Gavathiotis et al, Nat Chem Biol, 2012). Here, we applied BAM7 to a panel of acute lymphoblastic leukemia cells and observe dose-responsive killing in the micromolar range. To improve the potency of anti-ALL activity, we undertook a systematic medicinal chemistry-based iteration of BAM7’s molecular features. A series of analogs demonstrate improved BAX binding activity based on a FITC-BIM SAHB/BAX competitive fluorescence polarization assay. The most potent binders were advanced to testing in a cellular model of anti-apoptotic protein-dependent chemoresistance. Whereas select BAM7 derivatives exhibited a potency similar to or greater than the BCL-2/BCL-XL inhibitor ABT-737 in a BCL-XL-dependent ALL cell line, the compounds significantly outperformed ABT-737 in the isogenic MCL-1-dependent ALL cells. Preliminary studies revealed favorable pharmacokinetic profiles for lead BAM7 derivatives, including oral bioavailability and central nervous system penetration. These early encouraging structure-activity relationship and pharmacokinetic data on chemically-optimized BAM7 analogs suggest that direct and selective activation of BAX may be a viable strategy for therapeutic induction of apoptosis in the context of cancer chemoresistance. Citation Format: Joseph A. Bellairs, Denis Reyna, Divakaramenon Sethumadhavan, Madhavi Neelagiri, Alex Broadhead, Christopher Baccei, Brian Koss, Joseph T. Opferman, Evripidis Gavathiotis, Loren D. Walensky. Chemical optimization of direct and selective molecular activators of pro-apoptotic BAX for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2013-2471

Label-Free Detection of G Protein–SNARE Interactions and Screening for Small Molecule Modulators

G(i/o)-coupled presynaptic GPCRs are major targets in neuropsychiatric diseases. For example, presynaptic auto- or heteroreceptors include the D(2) dopamine receptor, H(3) histamine receptor, 5HT(1) serotonin receptors, M(4) acetylcholine receptors, GABA(B) receptors, Class II and III metabotropic glutamate receptors, opioid receptors, as well as many other receptors. These GPCRs exert their influence by decreasing exocytosis of synaptic vesicles. One mechanism by which they act is through direct interaction of the Gβγ subunit with members of the SNARE complex downstream of voltage-dependent calcium channels, and specifically with the C-terminus of SNAP25 and the H3 domain of syntaxin1A(1-3). Small molecule inhibitors of the Gβγ-SNARE interaction would allow the study of the relative importance of this mechanism in more detail. We have utilized novel, label-free technology to detect this protein-protein interaction and screen for several small molecule compounds that perturb the interaction, demonstrating the viability of this approach. Interestingly, the screen also produced enhancers of the Gβγ-SNARE interaction.

Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1

Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC(50) values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

A Homogeneous Method to Measure Nucleotide Exchange by α-Subunits of Heterotrimeric G-Proteins Using Fluorescence Polarization

The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [³⁵S]guanosine 5'-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg(2+) concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.

Epolones: novel sesquiterpene-tropolones from fungus OS-F69284 that induce erythropoietin in human cells.

In the course of our screening for small molecule modulators of erythropoietin gene expression, two novel sesquiterpene tropolones and pycnidone were isolated from a culture of OS-F69284 (ATCC 74390). Their structures were elucidated by extensive 1H and 13C NMR spectroscopic studies and chemical reactions. These compounds induced erythropoietin gene expression 5-fold at a concentration of 1-1.6 microM.