Small molecule screening in the zebrafish

The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish.     

Small molecule screen for compounds that affect vascular development in the zebrafish retina

Blood vessel formation in the vertebrate eye is a precisely regulated process. In the human retina, both an excess and a deficiency of blood vessels may lead to a loss of vision. To gain insight into the molecular basis of vessel formation in the vertebrate retina and to develop pharmacological means of manipulating this process in a living organism, we further characterized the embryonic zebrafish eye vasculature, and performed a small molecule screen for compounds that affect blood vessel morphogenesis. The screening of approximately 2000 compounds revealed four small molecules that at specific concentrations affect retinal vessel morphology but do not produce obvious changes in trunk vessels, or in the neuronal architecture of the retina. Of these, two induce a pronounced widening of vessel diameter without a substantial loss of vessel number, one compound produces a loss of retinal blood vessels accompanied by a mild increase of their diameter, and finally one other generates a severe loss of retinal vessels. This work demonstrates the utility of zebrafish as a screening tool for small molecules that affect eye vasculature and presents several compounds of potential therapeutic importance.     

Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice

Tight junctions are well-developed between adjacent endothelial cells of blood vessels in the central nervous system, and play a central role in establishing the blood-brain barrier (BBB). Claudin-5 is a major cell adhesion molecule of tight junctions in brain endothelial cells. To examine its possible involvement in the BBB, claudin-5-deficient mice were generated. In the brains of these mice, the development and morphology of blood vessels were not altered, showing no bleeding or edema. However, tracer experiments and magnetic resonance imaging revealed that in these mice, the BBB against small molecules (<800 D), but not larger molecules, was selectively affected. This unexpected finding (i.e., the size-selective loosening of the BBB) not only provides new insight into the basic molecular physiology of BBB but also opens a new way to deliver potential drugs across the BBB into the central nervous system.     

A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development

Background

Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB) and blood-retinal (BRB) barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP) in blood plasma, as an endogenous tracer.

Results

The temporal establishment of the BBB and BRB was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. We also examined the expression of claudin-5, a component of tight junctions during the first 4 days of development. We observed that the BBB of zebrafish starts to develop by 3 dpf, with expression of claudin-5 in the central arteries preceding it at 2 dpf. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB.

Conclusion

Zebrafish embryos develop BBB and BRB function simultaneously by 3 dpf, which is regulated by tight junction proteins. The Tg(l-fabp:DBP-EGFP) zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model.     

Protection of the blood-brain barrier during acute and chronic hypertension

A new concept about sympathetic nerves has emerged recently: not only is sympathetic tone important in short-term regulation of vascular resistance, but chronic effects of nerves on vessels have important effects. This concept is supported by studies of mechanisms by which sympathetic nerves protect the blood-brain barrier (BBB). The BBB is susceptible to disruption during acute and chronic hypertension. Acute, severe hypertension produces passive dilatation of cerebral vessels with disruption of the BBB. Sympathetic stimulation attenuates the increase in cerebral blood flow during acute hypertension and thereby protects the BBB. During chronic hypertension, we have observed disruption of the barrier, which may contribute to hypertensive encephalopathy. Sympathetic nerves protect against disruption of the BBB during chronic hypertension. This protective effect is apparently related to a trophic effect of nerves in promotion of cerebral vascular hypertrophy during chronic hypertension. Thus, this is the first evidence that, in the same vascular bed, sympathetic nerves have two different protective effects. Protection of the BBB is accomplished acutely by sympathetic neural effects on vascular resistance and chronically by promotion of vascular hypertrophy.     

Identification of the F1F0 mitochondrial ATPase as a target for modulating skin pigmentation by screening a tagged triazine library in zebrafish

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells.     

Selective expression of eGFP in mouse perivascular astrocytes by modification of the Mlc1 gene using T2A‐based ribosome skipping

Perivascular astrocyte end feet closely juxtapose cerebral blood vessels to regulate important developmental and physiological processes including endothelial cell proliferation and sprouting as well as the formation of the blood‐brain barrier (BBB). The mechanisms underlying these events remain largely unknown due to a lack of experimental models for identifying perivascular astrocytes and distinguishing these cell types from other astroglial populations. Megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) is a transmembrane protein that is expressed in perivascular astrocyte end feet where it controls BBB development and homeostasis. On the basis of this knowledge, we used T2A peptide‐skipping strategies to engineer a knock‐in mouse model in which the endogenous Mlc1 gene drives expression of enhanced green fluorescent protein (eGFP), without impacting expression of Mlc1 protein. Analysis of fetal, neonatal and adult Mlc1‐eGFP knock‐in mice revealed a dynamic spatiotemporal expression pattern of eGFP in glial cells, including nestin‐expressing neuroepithelial cells during development and glial fibrillary acidic protein (GFAP)‐expressing perivascular astrocytes in the postnatal brain. EGFP was not expressed in neurons, microglia, oligodendroglia, or cerebral vascular cells. Analysis of angiogenesis in the neonatal retina also revealed enriched Mlc1‐driven eGFP expression in perivascular astrocytes that contact sprouting blood vessels and regulate blood‐retinal barrier permeability. A cortical injury model revealed that Mlc1‐eGFP expression is progressively induced in reactive astrocytes that form a glial scar. Hence, Mlc1‐eGFP knock‐in mice are a new and powerful tool to identify perivascular astrocytes in the brain and retina and characterize how these cell types regulate cerebral blood vessel functions in health and disease.     

HIF prolyl hydroxylase inhibition prior to transient focal cerebral ischaemia is neuroprotective in mice

This study investigated the effects of 2‐(1‐chloro‐4‐hydroxyisoquinoline‐3‐carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia‐inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8‐ to 12‐week‐old C57/B6 mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) either immediately or 24 h after receiving IOX3. Mice receiving IOX3 at 20 mg/kg 24 h prior to the MCAO had better neuroscores and smaller blood–brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60 mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up‐regulated HIF‐1α, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up‐regulated HIF‐1α and delocalized ZO‐1. Pre‐treating IOX3 on RBE4 cells 24 h before oxygen–glucose deprivation had a protective effect on endothelial barrier preservation with ZO‐1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia.

     

LSR/angulin-1 is a tricellular tight junction protein involved in blood–brain barrier formation

The blood–brain barrier (BBB) is a term used to describe the unique properties of central nervous system (CNS) blood vessels. One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs). Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues. We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis. We further demonstrate that the BBB does not seal during embryogenesis in Lsr knockout mice with a leakage to small molecules. Finally, in mouse models in which BBB was disrupted, including an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and a middle cerebral artery occlusion (MCAO) model of stroke, LSR was down-regulated, linking loss of LSR and pathological BBB leakage.     

斑马鱼模型在药物筛选中的应用

斑马鱼具有子代数量多、体外受精、胚胎透明、可以做大规模遗传突变筛选等生物学特性, 因此成为一种良好的脊椎动物模式生物。随着研究的深入, 斑马鱼不仅应用于遗传学和发育生物学研究, 而且拓展和延伸到疾病模型和药物筛选领域。作为一种整体动物模型, 斑马鱼能够全面地检测评估化合物的活性和副作用, 实现高内涵筛选。近年来, 科学家们不断地发展出新的斑马鱼疾病模型和新的筛选技术, 并找到了一批活性化合物。这些化合物大多数在哺乳动物模型中也有相似的效果, 其中前列腺素E2(dmPGE2)和来氟米特(Leflunomide)已经进入临床实验, 分别用来促进脐带血细胞移植后的增殖和治疗黑素瘤。这些成果显示了斑马鱼模型很适合用于药物筛选。文章概括介绍了斑马鱼模型的特点和近年来在疾病模型和药物筛选方面的进展, 希望能够帮助人们了解斑马鱼在新药研发中的应用, 并开展基于斑马鱼模型的药物筛选。     

Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain

Neuronal survival, electrical signaling and synaptic activity require a well-balanced micro-environment in the central nervous system. This is achieved by the blood–brain barrier (BBB), an endothelial barrier situated in the brain capillaries, that controls near-to-all passage in and out of the brain. The endothelial barrier function is highly dependent on signaling interactions with surrounding glial, neuronal and vascular cells, together forming the neuro-glio-vascular unit. Within this functional unit, connexin (Cx) channels are of utmost importance for intercellular communication between the different cellular compartments. Connexins are best known as the building blocks of gap junction (GJ) channels that enable direct cell–cell transfer of metabolic, biochemical and electric signals. In addition, beyond their role in direct intercellular communication, Cxs also form unapposed, non-junctional hemichannels in the plasma membrane that allow the passage of several paracrine messengers, complementing direct GJ communication. Within the NGVU, Cxs are expressed in vascular endothelial cells, including those that form the BBB, and are eminent in astrocytes, especially at their endfoot processes that wrap around cerebral vessels. However, despite the density of Cx channels at this so-called gliovascular interface, it remains unclear as to how Cx-based signaling between astrocytes and BBB endothelial cells may converge control over BBB permeability in health and disease. In this review we describe available evidence that supports a role for astroglial as well as endothelial Cxs in the regulation of BBB permeability during development as well as in disease states.     

Optimisation of Embryonic and Larval ECG Measurement in Zebrafish for Quantifying the Effect of QT Prolonging Drugs

Effective chemical compound toxicity screening is of paramount importance for safe cardiac drug development. Using mammals in preliminary screening for detection of cardiac dysfunction by electrocardiography (ECG) is costly and requires a large number of animals. Alternatively, zebrafish embryos can be used as the ECG waveform is similar to mammals, a minimal amount of chemical is necessary for drug testing, while embryos are abundant, inexpensive and represent replacement in animal research with reduced bioethical concerns. We demonstrate here the utility of pre-feeding stage zebrafish larvae in detection of cardiac dysfunction by electrocardiography. We have optimised an ECG recording system by addressing key parameters such as the form of immobilization, recording temperature, electrode positioning and developmental age. Furthermore, analysis of 3 days post fertilization (dpf) zebrafish embryos treated with known QT prolonging drugs such as terfenadine, verapamil and haloperidol led to reproducible detection of QT prolongation as previously shown for adult zebrafish. In addition, calculation of Z-factor scores revealed that the assay was sensitive and specific enough to detect large drug-induced changes in QTc intervals. Thus, the ECG recording system is a useful drug-screening tool to detect alteration to cardiac cycle components and secondary effects such as heart block and arrhythmias in zebrafish larvae before free feeding stage, and thus provides a suitable replacement for mammalian experimentation.     

The blood-brain barrier: an alternative hypothesis

Brain blood vessels, unlike most vessels elsewhere in the body, exhibit a blood-brain barrier (BBB) to certain substances, e.g. trypan blue. Under some circumstances this barrier is no longer effective and the permeability of the vessels increases. Although capillarization is much less in the brain than in many other organs, e.g. heart muscle, total cerebral blood flow per minute is enormous. Consequently, to accommodate a large blood volume with a limited capillary bed, the velocity of blood through brain vessels must be extremely fast. The hypothesis presented in this paper is that this rapid flow results in a low or negative pressure on the endothelium, and plasma and trypan blue are prevented from passing through the wall. The tight junctions of cerebral endothelial cells may be able to withstand only a limited amount of pressure on their luminal surface. If the velocity of blood in brain capillaries decreases, pressure on the endothelium should increase, and brain vessels, like blood vessels elsewhere in the body, become permeable to vital dyes. Other conditions also increase capillary permeability, e.g. acute arterial hypertension or venous congestion. Although brain vessels can adapt to a moderate, gradual change in systemic pressure, when a significant rise in cerebral arterial pressure is abrupt, the compensatory changes in the postcapillary venous bed may be inadequate and consequently intracapillary pressure and vascular permeability are increased. Venous congestion increases intracapillary pressure by restricting capillary outflow as well as by reducing velocity through capillary beds. Under such conditions increased capillary permeability may be indicated by cerebral edema, and even, on occasion, by petechial hemorrhages. In short, if the flow is fast and unimpeded the BBB will be effective; if the velocity decreases, or intracapillary pressure increases for whatever reason, the permeability of the brain endothelium will be abnormally increased.     

The zebrafish as a tool to identify novel therapies for human cardiovascular disease

Over the past decade, the zebrafish has become an increasingly popular animal model for the study of human cardiovascular disease. Because zebrafish embryos are transparent and their genetic manipulation is straightforward, the zebrafish has been used to recapitulate a number of cardiovascular disease processes ranging from congenital heart defects to arrhythmia to cardiomyopathy. The use of fluorescent reporters has been essential to identify two discrete phases of cardiomyocyte differentiation necessary for normal cardiac development in the zebrafish. These phases are analogous to the differentiation of the two progenitor heart cell populations in mammals, termed the first and second heart fields. The small size of zebrafish embryos has enabled high-throughput chemical screening to identify small-molecule suppressors of fundamental pathways in vasculogenesis, such as the BMP axis, as well as of common vascular defects, such as aortic coarctation. The optical clarity of zebrafish has facilitated studies of valvulogenesis as well as detailed electrophysiological mapping to characterize the early cardiac conduction system. One unique aspect of zebrafish larvae is their ability to oxygenate through diffusion alone, permitting the study of mutations that cause severe cardiomyopathy phenotypes such as silent heart and pickwick^m171, which mimic titin mutations observed in human dilated cardiomyopathy. Above all, the regenerative capacity of zebrafish presents a particularly exciting opportunity to discover new therapies for cardiac injury, including scar formation following myocardial infarction. This Review will summarize the current state of the field and describe future directions to advance our understanding of human cardiovascular disease.KEY WORDS: Cardiovascular, Drug discovery, Zebrafish     

Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases

Previous studies have provided evidence that, in the early hours of ischemic stroke, a luminal membrane blood-brain barrier (BBB) Na-K-Cl cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. Inhibition of BBB NKCC activity by intravenous bumetanide significantly reduces edema and infarct in the rat permanent middle cerebral artery occlusion model of ischemic stroke. We demonstrated previously that the BBB cotransporter is stimulated by hypoxia, aglycemia, and AVP, factors present during cerebral ischemia. However, the underlying mechanisms have not been known. Ischemic conditions have been shown to activate p38 and JNK MAP kinases (MAPKs) in brain, and the p38 and JNK inhibitors SB-239063 and SP-600125, respectively, have been found to reduce brain damage following middle cerebral artery occlusion and subarachnoid hemorrhage, respectively. The present study was conducted to determine whether one or both of these MAPKs participates in ischemic factor stimulation of BBB NKCC activity. Cultured cerebral microvascular endothelial cell NKCC activity was evaluated as bumetanide-sensitive (86)Rb influx. Activities of p38 and JNK were assessed by Western blot and immunofluorescence methods using antibodies that detect total vs. phosphorylated (activated) p38 or JNK. We report that p38 and JNK are present in cultured cerebral microvascular endothelial cells and in BBB endothelial cells in situ and that hypoxia (7% O(2) and 2% O(2)), aglycemia, AVP, and O(2)-glucose deprivation (5- to 120-min exposures) all rapidly activate p38 and JNK in the cells. We also provide evidence that SB-239063 and SP-600125 reduce or abolish ischemic factor stimulation of BBB NKCC activity. These findings support the hypothesis that ischemic factor stimulation of the BBB NKCC involves activation of p38 and JNK MAPKs.     

Development of the zebrafish lymphatic system requires VEGFC signaling

Lymphangiogenesis results in the formation of a vascular network distinct from arteries and veins that serves to drain interstitial fluid from surrounding tissues and plays a pivotal role in the immune defense of vertebrates as well as in the progression of cancer and other diseases . In mammals, lymph vessels are lined by endothelial cells possibly sprouting from embryonic veins, and their development appears to be critically dependent on the function of PROX1 and VEGFC signaling . The existence of a lymphatic system in teleosts has been a matter of debate for decades. Here we show on the morphological, molecular, and functional levels that zebrafish embryos develop a lymphatic vasculature that serves to retrieve components of the interstitium to the lymph system. We demonstrate the existence of vessels that are molecularly and functionally distinct from blood vessels and show that the development of these vessels depends on Vegfc and VEGFR-3/Flt4 signaling. These findings imply that the molecular components controlling lymphangiogenesis in zebrafish and mammals are conserved and that the zebrafish lymphatic system develops early enough to allow in vivo observations, lineage tracing, and genetic as well as pharmacological screens.     

A Method to Predict Blood-Brain Barrier Permeability of Drug-Like Compounds Using Molecular Dynamics Simulations

The blood-brain barrier (BBB) is formed by specialized tight junctions between endothelial cells that line brain capillaries to create a highly selective barrier between the brain and the rest of the body. A major problem to overcome in drug design is the ability of the compound in question to cross the BBB. Neuroactive drugs are required to cross the BBB to function. Conversely, drugs that target other parts of the body ideally should not cross the BBB to avoid possible psychotropic side effects. Thus, the task of predicting the BBB permeability of new compounds is of great importance. Two gold-standard experimental measures of BBB permeability are logBB (the concentration of drug in the brain divided by concentration in the blood) and logPS (permeability surface-area product). Both methods are time-consuming and expensive, and although logPS is considered the more informative measure, it is lower throughput and more resource intensive. With continual increases in computer power and improvements in molecular simulations, in silico methods may provide viable alternatives. Computational predictions of these two parameters for a sample of 12 small molecule compounds were performed. The potential of mean force for each compound through a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer is determined by molecular dynamics simulations. This system setup is often used as a simple BBB mimetic. Additionally, one-dimensional position-dependent diffusion coefficients are calculated from the molecular dynamics trajectories. The diffusion coefficient is combined with the free energy landscape to calculate the effective permeability (P_eff) for each sample compound. The relative values of these permeabilities are compared to experimentally determined logBB and logPS values. Our computational predictions correlate remarkably well with both logBB (R² = 0.94) and logPS (R² = 0.90). Thus, we have demonstrated that this approach may have the potential to provide reliable, quantitatively predictive BBB permeability, using a relatively quick, inexpensive method.     

A Method to Predict Blood-Brain Barrier Permeability of Drug-Like Compounds Using Molecular Dynamics Simulations

The blood-brain barrier (BBB) is formed by specialized tight junctions between endothelial cells that line brain capillaries to create a highly selective barrier between the brain and the rest of the body. A major problem to overcome in drug design is the ability of the compound in question to cross the BBB. Neuroactive drugs are required to cross the BBB to function. Conversely, drugs that target other parts of the body ideally should not cross the BBB to avoid possible psychotropic side effects. Thus, the task of predicting the BBB permeability of new compounds is of great importance. Two gold-standard experimental measures of BBB permeability are logBB (the concentration of drug in the brain divided by concentration in the blood) and logPS (permeability surface-area product). Both methods are time-consuming and expensive, and although logPS is considered the more informative measure, it is lower throughput and more resource intensive. With continual increases in computer power and improvements in molecular simulations, in silico methods may provide viable alternatives. Computational predictions of these two parameters for a sample of 12 small molecule compounds were performed. The potential of mean force for each compound through a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer is determined by molecular dynamics simulations. This system setup is often used as a simple BBB mimetic. Additionally, one-dimensional position-dependent diffusion coefficients are calculated from the molecular dynamics trajectories. The diffusion coefficient is combined with the free energy landscape to calculate the effective permeability (P_eff) for each sample compound. The relative values of these permeabilities are compared to experimentally determined logBB and logPS values. Our computational predictions correlate remarkably well with both logBB (R² = 0.94) and logPS (R² = 0.90). Thus, we have demonstrated that this approach may have the potential to provide reliable, quantitatively predictive BBB permeability, using a relatively quick, inexpensive method.     

Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification

The blood–brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma‐targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high‐throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d‐ amino acids, this approach screens only protease‐resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state‐of‐the‐art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix‐and‐split technique to generate a library based on the following: Ac‐d‐ Arg‐XXXXX‐NH₂, where X were: d‐ Ala (a), d‐ Arg (r), d‐ Ile (i), d‐ Glu (e), d‐ Ser (s), d‐ Trp (w) or d‐ Pro (p). The assays used comprised the in vitro cell‐based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two‐step mass spectrometry approach combining LTQ‐Orbitrap and Q‐trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease‐resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.