Structural determination of triclosan derivatives as inhibitors of Plasmodium falciparum enoyl reductase (PfENR)

Malaria is a disease that causes more than 1 million deaths per year world wide and more than 400 million clinical cases. Due to the acquired resistance of Plasmodium falciparum to the drugs used to control the infection, searching for new anti-malaria drugs is necessary in modern days. Recent studies have shown that the parasite synthesizes fatty acids using a fatty acid synthase type II (FAS-II) instead of a type-I fatty acid synthase (FAS-I) that is present in other eukaryotes. Plasmodium falciparum enoyl reductase (PfENR) is responsible for the last step of fatty acid biosynthesis in the parasite. This enzyme is located within the apicoplast, a plastid-like organelle that is responsible for several important metabolic pathways, including fatty acid biosynthesis. It is known that triclosan is an inhibitor of ENR in bacteria and we and others have shown that it is also effective against ENR in apicomplexan organisms such as P. falciparum. However triclosan cannot be used to treat malaria in humans because it has metabolic liability (glucoronidation) which limits its inhibitory potency. We have used X-ray crystallography and a Structural Activity Relationship (SAR) strategy to design and cocrystallize a tertiary complex of PfENR with NAD+ and triclosan derivatives to improve their properties as drugs to treat malaria. More than five hundred triclosan derivatives were synthesized, and their in vitro and in vivo inhibitory activity evaluated. Furthermore, structural studies were made of their affinity to interact with residues in PfENR active sites, as well as with the cofactor NAD+. A total of six PfENR-NAD+-triclosan analog/complexes structures were determined. Analogs which had replacements of chloride groups at position 5 of ring A and 4' of ring B were determined, allowing the structural analysis of the binding of these triclosan analogs to PfENR. In addition, the urea derivatives (modification at position 1) as well as phenylsulphonamides (modification at position 2') have shown to be more potent inhibitors than triclosan in the in vivo assay. The analysis of the inhibitory properties and the structure of these analogs bound to PfENR will provide novel compounds in the search for new anti-malarial drugs

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

THREE-DIMENSIONAL MICROFLUIDIC CELL CULTURE OF STEM CELL-DERIVED NEURONAL MODELS OF PARKINSON'S DISEASE

Cell culture models in 3D have become an essential tool for the implementation of cellular models of neurodegenerative diseases. Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons from the substantia nigra. The study of PD at the cellular level requires a cellular model that recapitulates the complexity of those neurons affected in PD. Induced Pluripotent Stem Cells (iPSC) technology is an efficient method for the derivation of dopaminergic neurons from human neuroepithelial stem cells (hNESC), hence proving to be a suitable tool to develop cellular models of PD. To obtain DA neurons from hNESC in a 3D culture, a protocol based on the use of small molecules and growth factors was implemented in a microfluidic device (OrganoPlate). This non PDMS device is based on the use of phaseguide (capillary pressure barriers that guide the liquid air interface) technology and the hydrogel matrigel as an extra cellular matrix surrogate. To characterize the morphological features and the electrophysiological activity of wild type hNESCs differentiated neuronal population, with those differentiated neurons carrying the LRRK2 mutation G2019S, a calcium imaging assay based on the use of a calcium sensitive dye (Fluo-4) and image analysis methods, were implemented. Additionally, several aspects of fluid flow dynamics, rheological properties of matrigel and its use as surrogate extracellular matrix were investigated. Final characterization of the differentiated neuronal population was done using an immunostaining assay and microscopy techniques. The yields of differentiated dopaminergic neurons in the 2 lane OrganoPlate were in the range of 13% to 27%. Morphological (length of processes) and electrophysiological (firing patterns) characteristics of wild type differentiated neurons and those carrying the LRRK2 mutation G2019S, were determined applying an image analysis pipeline. Velocity profiles and shear stress of fluorescent beads in matrigel flowing in culture lanes of the 2 lane OrganoPlate, were estimated using particle image velocimetry techniques. In this thesis, we integrate two new technologies to establish a new in vitro 3D cell based model to study several aspects of PD at the cellular level, aiming to establish a microfluidic cell culture experimental platform to study PD, using a systems biology approach

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

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

Abstract There is a dire need for novel therapeutics to treat the dangerous 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

Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture

Analytical BioScience