A perfusion host-microbe bioreactor (HMB) system that captures dynamic interactions of secreted metabolites between epithelial cells cocultured with a human gut anaerobe.

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.

A Novel Rotavirus Reverse Genetics Platform Supports Flexible Insertion of Exogenous Genes and Enables Rapid Development of a High-Throughput Neutralization Assay.

Despite the success of rotavirus vaccines, rotaviruses remain one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality, especially in low- and middle-income countries. The reverse genetics system enables the manipulation of the rotavirus genome and opens the possibility of using rotavirus as an expression vector for heterologous proteins, such as vaccine antigens and therapeutic payloads. Here, we demonstrate that three positions in rotavirus genome-the C terminus of NSP1, NSP3 and NSP5-can tolerate the insertion of reporter genes. By using rotavirus expressing GFP, we develop a high-throughput neutralization assay and reveal the pre-existing immunity against rotavirus in humans and other animal species. Our work shows the plasticity of the rotavirus genome and establishes a high-throughput assay for interrogating humoral immune responses, benefiting the design of next-generation rotavirus vaccines and the development of rotavirus-based expression platforms.

A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays.

Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (µVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DµVS), that integrates a µVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DµVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DµVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.

Development of a fully automated platform for agar-based measurement of viable bacterial growth.

Dynamic in vitro antibacterial studies provide valuable insight on effective dosing strategies prior to translating to in vivo models. Frequent sampling is required to monitor the pharmacodynamics (PD) of these studies, leading to significant work when quantifying the bacterial load of the samples. Spreading a bacterial suspension on agar to allow colony counting is a proven process for measuring very low levels of growth, but commercial automation equipment to handle agar plating and colony counting at scale is not readily available. We describe a process to greatly decrease the hands-on time required for PD assays by utilizing general-purpose liquid handling robots to plate bacteria and a custom-made plate imager to automate colony counting. The platform developed handles the biological assay from beginning to end as well as sample tracking at each step of the process. The process relies heavily on custom automation scheduling software to enable dynamic process decisions and coordinate data flow throughout. Using the described platform, we can efficiently quantify >100 PD samples per day while maintaining the necessary dynamic range of the assay. Alleviating the main bottleneck in the dynamic antibacterial studies has allowed us to accelerate the rate of experiments to provide antibacterial dosing data within shorter timelines.

In Vitro Pharmacokinetic/Pharmacodynamic Modeling of HIV Latency Reversal by Novel HDAC Inhibitors Using an Automated Platform.

Antiretroviral therapy is able to effectively control but not eradicate HIV infection, which can persist, leading to the need for lifelong therapy. The existence of latently HIV-infected cells is a major barrier to the eradication of chronic HIV infection. Histone deacetylase inhibitors (HDACis), small molecules licensed for oncology indications, have shown the ability to produce HIV transcripts in vitro and in vivo. The pharmacologic parameters that drive optimal HIV latency reversal in vivo are unknown and could be influenced by such factors as the HDACi binding kinetics, concentration of compound, and duration of exposure. This study evaluates how these parameters affect HIV latency reversal for a series of novel HDACis that differ in their enzymatic on and off rates. Varying cellular exposure, using automated washout methods of HDACi in a Jurkat cell model of HIV latency, led to the investigation of the relationship between pharmacokinetic (PK) properties, target engagement (TE), and pharmacodynamic (PD) responses. Using an automated robotic platform enabled miniaturization of a suspension cell-based washout assay that required multiple manipulations over the 48 h duration of the assay. Quantification of histone acetylation (TE) revealed that HDACis showed early peaks and differences in the durability of response between different investigated HDACis. By expanding the sample times, the shift between TE and PD, as measured by green fluorescent protein, could be fully characterized. The comprehensive data set generated by automating the assays described here was used to establish a PK/PD model for HDACi-induced HIV latency reversal.

High-Throughput Agonist Shift Assay Development for the Analysis of M1-Positive Allosteric Modulators.

Agonist shift assays feature cross-titrations of allosteric modulators and orthosteric ligands. Information generated in agonist shift assays can include a modulator's effect on the orthosteric agonist's potency (alpha) and efficacy (beta), as well as direct agonist activity of the allosteric ligand (tauB) and the intrinsic binding affinity of the modulator to the unoccupied receptor (KB). Because of the heavy resource demand and complex data handling, these allosteric parameters are determined infrequently during the course of a drug discovery program and on a relatively small subset of compounds. Automation of agonist shift assays enables this data-rich analysis to evaluate a larger number of compounds, offering the potential to differentiate compound classes earlier and prospectively prioritize based on desired molecular pharmacology. A high-throughput calcium-imaging agonist shift assay was pursued to determine the allosteric parameters of over 1000 positive allosteric modulator (PAM) molecules for the human muscarinic acetylcholine receptor 1 (M1). Control compounds were run repeatedly to demonstrate internal consistency. Comparisons between potency measurements and the allosteric parameter results demonstrate that these different types of measurements do not necessarily correlate, highlighting the importance of fully characterizing and understanding the allosteric properties of leads.

Iterative Focused Screening with Biological Fingerprints Identifies Selective Asc-1 Inhibitors Distinct from Traditional High Throughput Screening.

N-methyl-d-aspartate receptors (NMDARs) mediate glutamatergic signaling that is critical to cognitive processes in the central nervous system, and NMDAR hypofunction is thought to contribute to cognitive impairment observed in both schizophrenia and Alzheimer's disease. One approach to enhance the function of NMDAR is to increase the concentration of an NMDAR coagonist, such as glycine or d-serine, in the synaptic cleft. Inhibition of alanine-serine-cysteine transporter-1 (Asc-1), the primary transporter of d-serine, is attractive because the transporter is localized to neurons in brain regions critical to cognitive function, including the hippocampus and cortical layers III and IV, and is colocalized with d-serine and NMDARs. To identify novel Asc-1 inhibitors, two different screening approaches were performed with whole-cell amino acid uptake in heterologous cells stably expressing human Asc-1: (1) a high-throughput screen (HTS) of 3 M compounds measuring 35S l-cysteine uptake into cells attached to scintillation proximity assay beads in a 1536 well format and (2) an iterative focused screen (IFS) of a 45 000 compound diversity set using a 3H d-serine uptake assay with a liquid scintillation plate reader in a 384 well format. Critically important for both screening approaches was the implementation of counter screens to remove nonspecific inhibitors of radioactive amino acid uptake. Furthermore, a 15 000 compound expansion step incorporating both on- and off-target data into chemical and biological fingerprint-based models for selection of additional hits enabled the identification of novel Asc-1-selective chemical matter from the IFS that was not identified in the full-collection HTS.

Dynamic Compound-Dependent Acoustic Transfer to Investigate Inhibitor Reversibility.

Automated mechanism of action studies are introducing the need for tailored compound delivery, which can be challenging for standard compound management procedures. Jump dilution assays investigating inhibitor reversibility require compound delivery at specific volumes to assay specific concentrations of 10 × IC50 for each inhibitor. Creating custom-made source plates with unique compound concentrations to dispense a uniform single volume can be prohibitively slow. A broadly applicable tool that enables on-the fly dispensing of variable amounts of stock concentrations was developed using the Acoustic Transfer System (ATS). The Dynamic Transfer Modification Program (DTMP) is an integrated LabVIEW program used to automate customized volume transfers from each well based on compound identity within a given source plate. A jump dilution investigating the time-dependent inhibition of the enzyme dipeptidyl peptidase-4 (DPP4) with multiple inhibitors is described here to demonstrate the delivery of specific volumes of various compounds in a high-throughput manner. The ability to automate this process allows for the characterization of inhibitor reversibility earlier in the drug discovery process, resulting in better informed lead candidate selection.

Development of a Platform to Enable Fully Automated Cross-Titration Experiments.

In the triage of hits from a high-throughput screening campaign or during the optimization of a lead compound, it is relatively routine to test compounds at multiple concentrations to determine potency and maximal effect. Additional follow-up experiments, such as agonist shift, can be quite valuable in ascertaining compound mechanism of action (MOA). However, these experiments require cross-titration of a test compound with the activating ligand of the receptor requiring 100-200 data points, severely limiting the number tested in MOA assays in a screening triage. We describe a process to enhance the throughput of such cross-titration experiments through the integration of Hewlett Packard's D300 digital dispenser onto one of our robotics platforms to enable on-the-fly cross-titration of compounds in a 1536-well plate format. The process handles all the compound management and data tracking, as well as the biological assay. The process relies heavily on in-house-built software and hardware, and uses our proprietary control software for the platform. Using this system, we were able to automate the cross-titration of compounds for both positive and negative allosteric modulators of two different G protein-coupled receptors (GPCRs) using two distinct assay detection formats, IP1 and Ca2+ detection, on nearly 100 compounds for each target.

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5.

The NaV1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding NaV1.7, resulting in either loss-of-function (e.g., congenital analgesia) or gain-of-function (e.g., paroxysmal extreme pain disorder) pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of NaV1.7. A full-deck high-throughput screen was developed for both NaV1.7 and cardiac NaV1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve, which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in NaV1.7 relative to 1.5, the AUC measurement and a subsequent refinement of this measurement were able to differentiate compounds with NaV1.7 pharmacological selectivity over NaV1.5 as confirmed in electrophysiology.

Multiplexed random peptide library and phospho-specific antibodies facilitate human polo-like kinase 1 inhibitor screen.

One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.

Identification of small-molecule modulators of mouse SVZ progenitor cell proliferation and differentiation through high-throughput screening.

Adult mouse subventricular zone (SVZ) neural stem/progenitor cells are multipotent self-renewing cells that retain the capacity to generate the major cell types of the central nervous system in vitro and in vivo. The relative ease of expanding SVZ cells in culture as neurospheres makes them an ideal model for carrying out large-scale screening to identify compounds that regulate neural progenitor cell proliferation and differentiation. The authors have developed an adenosine triphosphate-based cell proliferation assay using adult SVZ cells to identify small molecules that activate or inhibit progenitor cell proliferation. This assay was miniaturized to a 1536-well format for high-throughput screening (HTS) of >1 million small-molecule compounds, and 325 and 581 compounds were confirmed as potential inducers of SVZ cell proliferation and differentiation, respectively. A number of these compounds were identified as having a selective proliferative and differentiation effect on SVZ cells versus mouse Neuro2a neuroblastoma cells. These compounds can potentially be useful pharmacological tools to modulate resident stem cells and neurogenesis in the adult brain. This study represents a novel application of primary somatic stem cells in the HTS of a large-scale compound library.

A 1,536-well [(35)S]GTPgammaS scintillation proximity binding assay for ultra-high-throughput screening of an orphan galphai-coupled GPCR.

Members of the superfamily of seven transmembrane receptors, known as G protein-coupled receptors (GPCRs), are important targets for many therapeutic areas in drug discovery. A homogeneous guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) scintillation proximity assay (SPA) binding assay targeting a Galphai-coupled GPCR recombinantly expressed in membranes of Chinese hamster ovary (CHO) cells was developed and miniaturized into 1,536-well plate format. The primary ultra-high-throughput screen of the entire compound collection was accomplished on the Kalypsys (San Diego, CA) robotic platform at a concentration of 8 muM using the 1,536-well [(35)S]GTPgammaS SPA binding functional assay. The signal-to-noise ratio of the primary screen was approximately 2.1-fold, and the plate coefficient of variation for the compound field was approximately 11%. The hit rate from the primary screen for receptor agonists at >35% activity was approximately 0.3%. Primary hits were cherry-picked, confirmed in triplicate, counterscreened against untransfected CHO cell membranes, and further analyzed in a cyclic AMP functional assay, resulting in 34 leads for optimization.

A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production.

Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.

Miniaturization and HTS of a FRET-based membrane potential assay for K(ir) channel inhibitors.

The K(ir) family of potassium-selective ion channels is characterized by their inward (anomalous) rectifying current-voltage relationship. K(ir) channels are widely expressed in mammalian cells and through their role in regulation of the cell membrane potential have been implicated in diverse physiological functions. To enable the identification of novel K(ir) channel inhibitors, a fluorescence resonance energy transfer (FRET)-based membrane potential assay was developed using a Chinese hamster ovary cell line stably expressing a human K(ir) channel. The FRET-based assay incorporates the use of two dyes {N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidylethanolamine (CC2-DMPE) and bis(1,3-diethylthiobarbiturate)trimethine oxonol [DiSBAC(2)(3)]} to track changes in membrane potential, thus enabling all of the advantages of ratiometric readout: reduced inaccuracies arising from well-to-well variation in cell number, dye loading, signal intensities, and plate inconsistencies. The assay was miniaturized to a 1,536-well microtiter plate format and read on a fluorometric imaging plate reader (FLIPR(Tetra), Molecular Devices, Sunnyvale, CA). The assay was automated and utilized to perform a primary high-throughput screening campaign to identify novel inhibitors of the K(ir) channel.

Miniaturization and automation of an ubiquitin ligase cascade enzyme-linked immunosorbent assay in 1,536-well format.

Enzyme-linked immunosorbent assays (ELISAs) are a long established and widely used assay format for drug discovery and diagnostics. They offer many advantages over homogeneous assay formats, including high sensitivity and separation (wash) steps that remove detection-interfering compounds. Many high-throughput screening assays are now performed in miniaturized formats (1,536- and 3,456-well plates) for higher throughput and lower reagent consumption. With miniaturization, separation steps in assays such as ELISA can become difficult to implement. Here we report on the implementation of the Kalypsys, Inc. (San Diego, CA) 1,536-well plate washer to enable the successful miniaturization and full automation of an ELISA that monitors ubiquitin ligase activity. The 1,536-well plate ELISA was robust and used for the high-throughput screening of a large screening collection (>1 million compounds).

Miniaturization of absorbance assays using the fluorescent properties of white microplates.

Miniaturization of high-throughput screening (HTS) assays has several obvious advantages, including increased throughput and lower cost by reduction in reagent consumption. Although absorbance assays are widely used in research laboratories, their application for HTS in a low-volume format has been met with mixed success because they are difficult to miniaturize. Challenges for the miniaturization of absorbance assays include low signal due to short path lengths and meniscus distortions in small well sizes. Here we describe a method to miniaturize absorbance assays to standard, white, low-volume 384-well and 1536-well microplates using a fluorometric plate reader for detection. The premise of this absorbance assay is based on the fluorescent properties of white microplates and the ability of a colored product to quench the fluorescence signal from the plate by absorbing either the excitation light or the emission light. This method was applied to the detection of inorganic phosphate using Quinaldine red and Malachite green dyes and to the monitoring of alkaline phosphatase hydrolysis of p-nitrophenyl phosphate. These assays can be carried out in low volumes, give robust screening statistics, and can be accomplished with a simple, inexpensive fluorometric plate reader.

Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader.

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.

Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system.

Antagonists to the human metabotropic glutamate receptor subtype 5a(mGluR(5a)) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and Parkinson's disease. To discover novel antagonists to the mGluR(5a), a functional assay measuring agonist-induced intracellular calcium release was developed. The assay was used for the high-throughput screening of a large collection of compounds in single wells using a fully automated robotic platform. Primary high-throughput screening hits were subjected to a combination of data analysis and counterscreening assays to identify several compounds with both efficacy and selectivity for the metabotropic glutamate receptor target.