Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.

Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture.

Novel high-throughput screen against Candida albicans identifies antifungal potentiators and agents effective against biofilms.

OBJECTIVES: Microbial adhesion and biofilms have important implications for human health and disease. Candida albicans is an opportunistic pathogen which forms drug-resistant biofilms that contribute to the recalcitrance of disease. We have developed a high-throughput screen for potentiators of clotrimazole, a common therapy for Candida infections, including vaginitis and thrush. The screen was performed against C. albicans biofilms grown in microtitre plates in order to target the most resilient forms of the pathogen. METHODS: Biofilm growth, in individual wells of 384-well plates, was measured using the metabolic indicator alamarBlue® and found to be very consistent and reproducible. This assay was used to test the effect of more than 120 000 small molecule compounds from the NIH Molecular Libraries Small Molecule Repository, and compounds that enhanced the activity of clotrimazole or acted on the biofilms alone were identified as hits. RESULTS: Nineteen compounds (0.016% hit rate) were identified and found to cause more than 30% metabolic inhibition of biofilms compared with clotrimazole alone, which had a modest effect on biofilm viability at the concentration tested. Hits were confirmed for activity against biofilms with dose-response measurements. Several compounds had increased activity in combination with clotrimazole, including a 1,3-benzothiazole scaffold that exhibited a >100-fold improvement against biofilms of three separate C. albicans isolates. Cytotoxicity experiments using human fibroblasts confirmed the presence of lead molecules with favourable antifungal activity relative to cytotoxicity. CONCLUSIONS: We have validated a novel approach to identify antifungal potentiators and completed a high-throughput screen to identify small molecules with activity against C. albicans biofilms. These small molecules may specifically target the biofilm and make currently available antifungals more effective.

Identification of novel inhibitors of dietary lipid absorption using zebrafish.

Pharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lipid absorption using the zebrafish system. A pilot screen of an unbiased chemical library identified novel compounds that inhibited processing of fluorescent lipid analogues in live zebrafish larvae. Secondary assays identified those compounds suitable for testing in mammals and provided insight into mechanism of action, which for several compounds could be distinguished from ezetimibe, a drug used to inhibit cholesterol absorption in humans that broadly inhibited lipid absorption in zebrafish larvae. These findings support the utility of zebrafish screening assays to identify novel compounds that target complex physiological processes.

Discovery of potent small-molecule inhibitors of multidrug-resistant Plasmodium falciparum using a novel miniaturized high-throughput luciferase-based assay.

Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by Plasmodium falciparum. Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites, which necessitates the discovery of new drugs. To identify new antimalarials, we developed an automated 384-well plate screening assay using P. falciparum parasites that stably express cytoplasmic firefly luciferase. After initial optimization, we tested two different types of compound libraries: known bioactive collections (Library of Pharmacologically Active Compounds [LOPAC] and the library from the National Institute of Neurological Disorders and Stroke [NINDS]) and a library of uncharacterized compounds (ChemBridge). A total of 12,320 compounds were screened at 5.5 microM. Selecting only compounds that reduced parasite growth by 85% resulted in 33 hits from the combined bioactive collection and 130 hits from the ChemBridge library. Fifteen novel drug-like compounds from the bioactive collection were found to be active against P. falciparum. Twelve new chemical scaffolds were found from the ChemBridge hits, the most potent of which was a series based on the 1,4-naphthoquinone scaffold, which is structurally similar to the FDA-approved antimalarial atovaquone. However, in contrast to atovaquone, which acts to inhibit the bc(1) complex and block the electron transport chain in parasite mitochondria, we have determined that our new 1,4-napthoquinones act in a novel, non-bc(1)-dependent mechanism and remain potent against atovaquone- and chloroquine-resistant parasites. Ultimately, this study may provide new probes to understand the molecular details of the malaria life cycle and to identify new antimalarials.

Discovery of novel inhibitors targeting enoyl-acyl carrier protein reductase in Plasmodium falciparum by structure-based virtual screening.

There is a dire need for novel therapeutics to treat the virulent malarial parasite, Plasmodium falciparum. Recently, the X-ray crystal structure of enoyl-acyl carrier protein reductase (ENR) in complex with triclosan has been determined and provides an opportunity for the rational design of novel inhibitors targeting the active site of ENR. Here, we report the discovery of several compounds by virtual screening and their experimental validation as high potency PfENR inhibitors.

Synthesis and biological activity of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 2: 2'-substituted triclosan derivatives.

2'-Substituted analogs of triclosan have been synthesized to target inhibition of the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit good potency (EC50<500 nM) against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and modest (IC50=1-20 microM) potency against purified PfENR enzyme. Compared to triclosan, this survey of 2'-substituted derivatives has afforded gains in excess of 20- and 30-fold versus the 3D7 and Dd2 strains of parasite, respectively.

Synthesis, biological activity, and X-ray crystal structural analysis of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 1: 4'-substituted triclosan derivatives.

A structure-based approach has been taken to develop 4'-substituted analogs of triclosan that target the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit nanomolar potency against purified PfENR enzyme and modest (2-10microM) potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite. X-ray crystal structures of nitro 29, aniline 30, methylamide 37, and urea 46 demonstrate the presence of hydrogen-bonding interactions with residues in the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular parasites.