Multi-input Drug-Controlled Switches of Mammalian Gene Expression Based on Engineered Nuclear Hormone Receptors.

Protein-based switches that respond to different inputs to regulate cellular outputs, such as gene expression, are central to synthetic biology. For increased controllability, multi-input switches that integrate several cooperating and competing signals for the regulation of a shared output are of particular interest. The nuclear hormone receptor (NHR) superfamily offers promising starting points for engineering multi-input-controlled responses to clinically approved drugs. Starting from the VgEcR/RXR pair, we demonstrate that novel (multi)drug regulation can be achieved by exchange of the ecdysone receptor (EcR) ligand binding domain (LBD) for other human NHR-derived LBDs. For responses activated to saturation by an agonist for the first LBD, we show that outputs can be boosted by an agonist targeting the second LBD. In combination with an antagonist, output levels are tunable by up to three simultaneously present small-molecule drugs. Such high-level control validates NHRs as a versatile, engineerable platform for programming multidrug-controlled responses.

Thiazole derivatives as dual inhibitors of deoxyribonuclease I and 5-lipoxygenase: A promising scaffold for the development of neuroprotective drugs.

A library of 43 thiazole derivatives, including 31 previously and 12 newly synthesized in the present study, was evaluated in vitro for their inhibitory properties against bovine pancreatic DNase I. Nine compounds (including three newly synthesized) inhibited the enzyme showing improved inhibitory properties compared to that of the reference crystal violet (IC50 = 346.39 µM). Two compounds (5 and 29) stood out as the most potent DNase I inhibitors, with IC50 values below 100 µM. The 5-LO inhibitory properties of the investigated derivatives were also analyzed due to the importance of this enzyme in the development of neurodegenerative diseases. Compounds (12 and 29) proved to be the most prominent new 5-LO inhibitors, with IC50 values of 60 nM and 56 nM, respectively, in cell-free assay. Four compounds, including one previously (41) and three newly (12, 29 and 30) synthesized, have the ability to inhibit DNase I with IC50 values below 200 µM and 5-LO with IC50 values below 150 nM in cell-free assay. Molecular docking and molecular dynamics simulations were used to clarify DNase I and 5-LO inhibitory properties of the most potent representatives at the molecular level. The newly synthesized compound 29 (4-((4-(3-bromo-4-morpholinophenyl)thiazol-2-yl)amino)phenol) represents the most promising dual DNase I and 5-LO inhibitor, as it inhibited 5-LO in the nanomolar and DNase I in the double-digit micromolar concentration ranges. The results obtained in the present study, together with our recently published results for 4-(4-chlorophenyl)thiazol-2-amines, represent a good basis for the development of new neuroprotective therapeutics based on dual inhibition of DNase I and 5-LO.

Multi-input drug-controlled switches of mammalian gene expression based on engineered nuclear hormone receptors.

Protein-based switches that respond to different inputs to regulate cellular outputs, such as gene expression, are central to synthetic biology. For increased controllability, multi-input switches that integrate several cooperating and competing signals for the regulation of a shared output are of particular interest. The nuclear hormone receptor (NHR) superfamily offers promising starting points for engineering multi-input-controlled responses to clinically approved drugs. Starting from the VgEcR/RXR pair, we demonstrate that novel (multi-)drug regulation can be achieved by exchange of the ecdysone receptor (EcR) ligand binding domain (LBD) for other human NHR-derived LBDs. For responses activated to saturation by an agonist for the first LBD, we show that outputs can be boosted by an agonist targeting the second LBD. In combination with an antagonist, output levels are tunable by up to three simultaneously present small-molecule drugs. Such high-level control validates NHRs as a versatile, engineerable platform for programming multi-drug-controlled responses.

Advances in the Computational Design of Small-Molecule-Controlled Protein-Based Circuits for Synthetic Biology.

Synthetic biology approaches living systems with an engineering perspective and promises to deliver solutions to global challenges in healthcare and sustainability. A critical component is the design of biomolecular circuits with programmable input-output behaviors. Such circuits typically rely on a sensor module that recognizes molecular inputs, which is coupled to a functional output via protein-level circuits or regulating the expression of a target gene. While gene expression outputs can be customized relatively easily by exchanging the target genes, sensing new inputs is a major limitation. There is a limited repertoire of sensors found in nature, and there are often difficulties with interfacing them with engineered circuits. Computational protein design could be a key enabling technology to address these challenges, as it allows for the engineering of modular and tunable sensors that can be tailored to the circuit's application. In this article, we review recent computational approaches to design protein-based sensors for small-molecule inputs with particular focus on those based on the widely used Rosetta software suite. Furthermore, we review mechanisms that have been harnessed to couple ligand inputs to functional outputs. Based on recent literature, we illustrate how the combination of protein design and synthetic biology enables new sensors for diverse applications ranging from biomedicine to metabolic engineering. We conclude with a perspective on how strategies to address frontiers in protein design and cellular circuit design may enable the next generation of sense-response networks, which may increasingly be assembled from de novo components to display diverse and engineerable input-output behaviors.

Ex-vivo evaluation of miniaturized probes for endoscopic optical coherence tomography in urothelial cancer diagnostics.

Background: The gold standard for detecting bladder cancer is white light cystoscopy (WLC) and resection of suspicious lesions. In this study, we evaluate two miniaturized Optical Coherence Tomography (OCT) probes for endoscopic use, regarding their applicability in diagnosing urothelial cancer. Materials and methods: In total, 33 patients who underwent a radical cystectomy were included. Preoperative oncological staging and determining the indication for the surgical intervention were done following the latest European Association of Urology (EAU) guidelines. Samples were taken from bladder tissue after bladder removal and prepared for OCT measurement. Additionally, porcine bladder samples were used as reference tissue. We took measurements using two miniaturized probes: a bimodal probe and a single modality OCT probe. A non-miniaturized standard OCT scanner was used as a reference. Results: Histopathological examination revealed urothelial cancer in all but three patients. Measurements on porcine tissue revealed a clear distinction between the urothelial layers for all probes. Furthermore, we detected improved image quality thanks to the stretching of the tissue. We took 271 measurements in human samples. While the urothelial layers were well delineated in healthy tissue, all the probes revealed a loss of these structures in cancerous regions. While the single-modality probe delivered an image quality equaling the reference images, it was possible to detect cancerous areas with the bimodal probe. Conclusion: We demonstrate that endoscopic probes for OCT imaging are technologically feasible and deliver acceptable image quality. A distinction between healthy and abnormal tissue is possible. We propose combining different endoscopic imaging modalities as a promising approach for urothelial cancer diagnostics.

Increasing MinD's Membrane Affinity Yields Standing Wave Oscillations and Functional Gradients on Flat Membranes.

The formation of large-scale patterns through molecular self-organization is a basic principle of life. Accordingly, the engineering of protein patterns and gradients is of prime relevance for synthetic biology. As a paradigm for such pattern formation, the bacterial MinDE protein system is based on self-organization of the ATPase MinD and ATPase-activating protein MinE on lipid membranes. Min patterns can be tightly regulated by tuning physical or biochemical parameters. Among the biochemically engineerable modules, MinD's membrane targeting sequence, despite being a key regulating element, has received little attention. Here we attempt to engineer patterns by modulating the membrane affinity of MinD. Unlike the traveling waves or stationary patterns commonly observed in vitro on flat supported membranes, standing-wave oscillations emerge upon elongating MinD's membrane targeting sequence via rationally guided mutagenesis. These patterns are capable of forming gradients and thereby spatially target co-reconstituted downstream proteins, highlighting their functional potential in designing new life-like systems.

Non-Equilibrium Large-Scale Membrane Transformations Driven by MinDE Biochemical Reaction Cycles.

The MinDE proteins from E. coli have received great attention as a paradigmatic biological pattern-forming system. Recently, it has surfaced that these proteins do not only generate oscillating concentration gradients driven by ATP hydrolysis, but that they can reversibly deform giant vesicles. In order to explore the potential of Min proteins to actually perform mechanical work, we introduce a new model membrane system, flat vesicle stacks on top of a supported lipid bilayer. MinDE oscillations can repeatedly deform these flat vesicles into tubules and promote progressive membrane spreading through membrane adhesion. Dependent on membrane and buffer compositions, Min oscillations further induce robust bud formation. Altogether, we demonstrate that under specific conditions, MinDE self-organization can result in work performed on biomimetic systems and achieve a straightforward mechanochemical coupling between the MinDE biochemical reaction cycle and membrane transformation.

Design, Synthesis, and Structure-Activity Relationship Studies of Dual Inhibitors of Soluble Epoxide Hydrolase and 5-Lipoxygenase.

Inhibition of multiple enzymes of the arachidonic acid cascade leads to synergistic anti-inflammatory effects. Merging of 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH) pharmacophores led to the discovery of a dual 5-LOX/sEH inhibitor, which was subsequently optimized in terms of potency toward both targets and metabolic stability. The optimized lead structure displayed cellular activity in human polymorphonuclear leukocytes, oral bioavailability, and target engagement in vivo and demonstrated profound anti-inflammatory and anti-fibrotic efficiency in a kidney injury model caused by unilateral ureteral obstruction in mice. These results pave the way for investigating the therapeutic potential of dual 5-LOX/sEH inhibitors in other inflammation- and fibrosis-related disease models.

Morpho-molecular ex vivo detection and grading of non-muscle-invasive bladder cancer using forward imaging probe based multimodal optical coherence tomography and Raman spectroscopy.

Non-muscle-invasive bladder cancer affects millions of people worldwide, resulting in significant discomfort to the patient and potential death. Today, cystoscopy is the gold standard for bladder cancer assessment, using white light endoscopy to detect tumor suspected lesion areas, followed by resection of these areas and subsequent histopathological evaluation. Not only does the pathological examination take days, but due to the invasive nature, the performed biopsy can result in significant harm to the patient. Nowadays, optical modalities, such as optical coherence tomography (OCT) and Raman spectroscopy (RS), have proven to detect cancer in real time and can provide more detailed clinical information of a lesion, e.g. its penetration depth (stage) and the differentiation of the cells (grade). In this paper, we present an ex vivo study performed with a combined piezoelectric tube-based OCT-probe and fiber optic RS-probe imaging system that allows large field-of-view imaging of bladder biopsies, using both modalities and co-registered visualization, detection and grading of cancerous bladder lesions. In the present study, 119 examined biopsies were characterized, showing that fiber-optic based OCT provides a sensitivity of 78% and a specificity of 69% for the detection of non-muscle-invasive bladder cancer, while RS, on the other hand, provides a sensitivity of 81% and a specificity of 61% for the grading of low- and high-grade tissues. Moreover, the study shows that a piezoelectric tube-based OCT probe can have significant endurance, suitable for future long-lasting in vivo applications. These results also indicate that combined OCT and RS fiber probe-based characterization offers an exciting possibility for label-free and morpho-chemical optical biopsies for bladder cancer diagnostics.

Comparison of optical coherence tomography angiography and narrow-band imaging using a bimodal endoscope.

We present coregistered images of tissue vasculature that allow a direct comparison between the performance of narrow-band imaging (NBI) and optical coherence tomography angiography (OCTA). Images were generated with a bimodal endomicroscope having a size of 15 × 2.4 × 3.3 3 ( l , w , h ) that combines two imaging channels. The white light imaging channel was used to perform NBI, the current gold standard for endoscopic visualization of vessels. The second channel allowed the simultaneous acquisition of optical coherence tomography (OCT) and OCTA images, enabling a three-dimensional (3-D) visualization of morphological as well as functional tissue information. In order to obtain 3-D OCT images scanning of the light-transmitting fiber was implemented by a small piezoelectric tube. A field of view of ∼1.1 mm was achieved for both modalities. Under the assumption that OCTA can address current limitations of NBI, their performance was studied and compared during in vivo experiments. The preliminary results show the potential of OCT regarding an improved visualization and localization of vessel beds, which can be beneficial for diagnosis of pathological conditions.

Synthetic cell division via membrane-transforming molecular assemblies.

Reproduction, i.e. the ability to produce new individuals from a parent organism, is a hallmark of living matter. Even the simplest forms of reproduction require cell division: attempts to create a designer cell therefore should include a synthetic cell division machinery. In this review, we will illustrate how nature solves this task, describing membrane remodelling processes in general and focusing on bacterial cell division in particular. We discuss recent progress made in their in vitro reconstitution, identify open challenges, and suggest how purely synthetic building blocks could provide an additional and attractive route to creating artificial cell division machineries.

Endoscopic optical coherence tomography angiography using a forward imaging piezo scanner probe.

A forward imaging endoscope for optical coherence tomography angiography (OCTA) featuring a piezoelectric fiber scanner is presented. Imaging is performed with an optical coherence tomography (OCT) system incorporating an akinetic light source with a center wavelength of 1300 nm, bandwidth of 90 nm and A-line rate of 173 kHz. The endoscope operates in contact mode to avoid motion artifacts, in particular, beneficial for OCTA measurements, and achieves a transversal resolution of 12 µm in air at a rigid probe size of 4 mm in diameter and 11.3 mm in length. A spiral scan pattern is generated at a scanning frequency of 360 Hz to sample a maximum field of view of 1.3 mm. OCT images of a human finger as well as visualization of microvasculature of the human palm are presented both in two and three dimensions. The combination of morphological tissue contrast with qualitative dynamic blood flow information within this endoscopic imaging approach potentially enables improved early diagnostic capabilities of internal organs for diseases such as bladder cancer.

Stationary Patterns in a Two-Protein Reaction-Diffusion System.

Patterns formed by reaction-diffusion mechanisms are crucial for the development or sustenance of most organisms in nature. Patterns include dynamic waves, but are more often found as static distributions, such as animal skin patterns. Yet, a simplistic biological model system to reproduce and quantitatively investigate static reaction-diffusion patterns has been missing so far. Here, we demonstrate that the Escherichia coli Min system, known for its oscillatory behavior between the cell poles, is under certain conditions capable of transitioning to quasi-stationary protein distributions on membranes closely resembling Turing patterns. We systematically titrated both proteins, MinD and MinE, and found that removing all purification tags and linkers from the N-terminus of MinE was critical for static patterns to occur. At small bulk heights, dynamic patterns dominate, such as in rod-shaped microcompartments. We see implications of this work for studying pattern formation in general, but also for creating artificial gradients as downstream cues in synthetic biology applications.

MinE conformational switching confers robustness on self-organized Min protein patterns.

Protein patterning is vital for many fundamental cellular processes. This raises two intriguing questions: Can such intrinsically complex processes be reduced to certain core principles and, if so, what roles do the molecular details play in individual systems? A prototypical example for protein patterning is the bacterial Min system, in which self-organized pole-to-pole oscillations of MinCDE proteins guide the cell division machinery to midcell. These oscillations are based on cycling of the ATPase MinD and its activating protein MinE between the membrane and the cytoplasm. Recent biochemical evidence suggests that MinE undergoes a reversible, MinD-dependent conformational switch from a latent to a reactive state. However, the functional relevance of this switch for the Min network and pattern formation remains unclear. By combining mathematical modeling and in vitro reconstitution of mutant proteins, we dissect the two aspects of MinE's switch, persistent membrane binding and a change in MinE's affinity for MinD. Our study shows that the MinD-dependent change in MinE's binding affinity for MinD is essential for patterns to emerge over a broad and physiological range of protein concentrations. Mechanistically, our results suggest that conformational switching of an ATPase-activating protein can lead to the spatial separation of its distinct functional states and thereby confer robustness on an intracellular protein network with vital roles in bacterial cell division.

Reverse and forward engineering of protein pattern formation.

Living systems employ protein pattern formation to regulate important life processes in space and time. Although pattern-forming protein networks have been identified in various prokaryotes and eukaryotes, their systematic experimental characterization is challenging owing to the complex environment of living cells. In turn, cell-free systems are ideally suited for this goal, as they offer defined molecular environments that can be precisely controlled and manipulated. Towards revealing the molecular basis of protein pattern formation, we outline two complementary approaches: the biochemical reverse engineering of reconstituted networks and the de novo design, or forward engineering, of artificial self-organizing systems. We first illustrate the reverse engineering approach by the example of the Escherichia coli Min system, a model system for protein self-organization based on the reversible and energy-dependent interaction of the ATPase MinD and its activating protein MinE with a lipid membrane. By reconstituting MinE mutants impaired in ATPase stimulation, we demonstrate how large-scale Min protein patterns are modulated by MinE activity and concentration. We then provide a perspective on the de novo design of self-organizing protein networks. Tightly integrated reverse and forward engineering approaches will be key to understanding and engineering the intriguing phenomenon of protein pattern formation.This article is part of the theme issue 'Self-organization in cell biology'.

Optical Control of a Biological Reaction-Diffusion System.

Patterns formed by reaction and diffusion are the foundation for many phenomena in biology. However, the experimental study of reaction-diffusion (R-D) systems has so far been dominated by chemical oscillators, for which many tools are available. In this work, we developed a photoswitch for the Min system of Escherichia coli, a versatile biological in vitro R-D system consisting of the antagonistic proteins MinD and MinE. A MinE-derived peptide of 19 amino acids was covalently modified with a photoisomerizable crosslinker based on azobenzene to externally control peptide-mediated depletion of MinD from the membrane. In addition to providing an on-off switch for pattern formation, we achieve frequency-locked resonance with a precise 2D spatial memory, thus allowing new insights into Min protein action on the membrane. Taken together, we provide a tool to study phenomena in pattern formation using biological agents.

Large-scale modulation of reconstituted Min protein patterns and gradients by defined mutations in MinE's membrane targeting sequence.

The E. coli MinDE oscillator is a paradigm for protein self-organization and gradient formation. Previously, we reconstituted Min protein wave patterns on flat membranes as well as gradient-forming pole-to-pole oscillations in cell-shaped PDMS microcompartments. These oscillations appeared to require direct membrane interaction of the ATPase activating protein MinE. However, it remained unclear how exactly Min protein dynamics are regulated by MinE membrane binding. Here, we dissect the role of MinE's membrane targeting sequence (MTS) by reconstituting various MinE mutants in 2D and 3D geometries. We demonstrate that the MTS defines the lower limit of the concentration-dependent wavelength of Min protein patterns while restraining MinE's ability to stimulate MinD's ATPase activity. Strikingly, a markedly reduced length scale-obtainable even by single mutations-is associated with a rich variety of multistable dynamic modes in cell-shaped compartments. This dramatic remodeling in response to biochemical changes reveals a remarkable trade-off between robustness and versatility of the Min oscillator.

Defined chromosome structure in the genome-reduced bacterium Mycoplasma pneumoniae.

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.

Characterization of the molecular mechanism of 5-lipoxygenase inhibition by 2-aminothiazoles.

5-Lipoxygenase (5-LO, EC1.13.11.34) has been implicated in the pathogenesis of inflammatory and immune diseases. Recently, aminothiazole comprising inhibitors have been discovered for this valuable target. Yet, the molecular mode of action of this class of substances is only poorly understood. Here, we present the detailed molecular mechanism of action of the compound class and the in vitro pharmacological profile of two lead compounds ST-1853 and ST-1906. Mechanistic studies with recombinant proteins as well as intact cell assays enabled us to define this class as a novel type of 5-LO inhibitors with unique characteristics. The parent compounds herein presented a certain reactivity concerning oxidation and thiol binding: Unsubstituted aminophenols bound covalently to C159 and C418 of human 5-LO. Yet, dimethyl substitution of the aminophenol prevented this reactivity and slowed down phase II metabolism. Both ST-1853 and ST-1906 confirmed their lead likeness by retaining their high potency in physiologically relevant 5-LO activity assays, high metabolic stability, high specificity and non-cytotoxicity.

Michael acceptor containing drugs are a novel class of 5-lipoxygenase inhibitor targeting the surface cysteines C416 and C418.

Recently, we published that nitro-fatty acids (NFA) are potent electrophilic molecules which inhibit 5-lipoxygenase (5-LO) by interacting catalytically with cysteine residues next to a substrate entry channel. The electrophilicity is derived from an intramolecular Michael acceptor moiety consisting of an electron-withdrawing group in close proximity to a double bond. The potential of the Michael acceptor moiety to interact with functionally relevant cysteines of proteins potentially renders them effective and sustained enzyme activity modulators. We screened a large library of naturally derived and synthetic electrophilic compounds to investigate whether other types of Michael acceptor containing drugs suppress 5-LO enzyme activity. The activity was measured by assessing the effect on the 5-LO product formation of intact human polymorphonuclear leukocytes. We demonstrated that a number of structurally different compounds were suppressive in the activity assays and showed that Michael acceptors of the quinone and nitro-alkene group produced the strongest inhibition of 5-LO product formation. Reactivity with the catalytically relevant cysteines 416 and 418 was confirmed using mutated recombinant 5-LO and mass spectrometric analysis (MALDI-MS). In the present study, we show for the first time that a number of well-recognized naturally occurring or synthetic anti-inflammatory compounds carrying a Michael acceptor, such as thymoquinone (TQ), the paracetamol metabolite NAPQI, the 5-LO inhibitor AA-861, and bardoxolone methyl (also known as RTA 402 or CDDO-methyl ester) are direct covalent 5-LO enzyme inhibitors that target the catalytically relevant cysteines 416 and 418.