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Fluorescent azobenzene-confined coiled-coil mesofibers

Punia, Kamia; Britton, Dustin; Hüll, Katharina; Yin, Liming; Wang, Yifei; Renfrew, P Douglas; Gilchrist, M Lane; Bonneau, Richard; Trauner, Dirk; Montclare, Jin K
Fluorescent protein biomaterials have important applications such as bioimaging in pharmacological studies. Self-assembly of proteins, especially into fibrils, is known to produce fluorescence in the blue band. Capable of self-assembly into nanofibers, we have shown we can modulate its aggregation into mesofibers by encapsulation of a small hydrophobic molecule. Conversely, azobenzenes are hydrophobic small molecules that are virtually non-fluorescent in solution due to their highly efficient photoisomerization. However, they demonstrate fluorogenic properties upon confinement in nanoscale assemblies by reducing the non-radiative photoisomerization. Here, we report the fluorescence of a hybrid protein-small molecule system in which azobenzene is confined in our protein assembly leading to fiber thickening and increased fluorescence. We show our engineered protein Q encapsulates AzoCholine, bearing a photoswitchable azobenzene moiety, in the hydrophobic pore to produce fluorescent mesofibers. This study further investigates the photocontrol of protein conformation as well as fluorescence of an azobenze-containing biomaterial.
PMID: 36538008
ISSN: 1744-6848
CID: 5409292

Medium-Chain Lipid Conjugation Facilitates Cell-Permeability and Bioactivity

Morstein, Johannes; Capecchi, Alice; Hinnah, Konstantin; Park, ByungUk; Petit-Jacques, Jerome; Van Lehn, Reid C; Reymond, Jean-Louis; Trauner, Dirk
The majority of bioactive molecules act on membrane proteins or intracellular targets and therefore needs to partition into or cross biological membranes. Natural products often exhibit lipid modifications to facilitate critical molecule-membrane interactions, and in many cases their bioactivity is markedly reduced upon removal of a lipid group. However, despite its importance in nature, lipid-conjugation of small molecules is not commonly used in chemical biology and medicinal chemistry, and the effect of such conjugation has not been systematically studied. To understand the composition of lipids found in natural products, we carried out a chemoinformatic characterization of the "natural product lipidome". According to this analysis, lipidated natural products predominantly contain saturated medium-chain lipids (MCLs), which are significantly shorter than the long-chain lipids (LCLs) found in membranes and lipidated proteins. To study the usefulness of such modifications in probe design, we systematically explored the effect of lipid conjugation on five different small molecule chemotypes and find that permeability, cellular retention, subcellular localization, and bioactivity can be significantly modulated depending on the type of lipid tail used. We demonstrate that MCL conjugation can render molecules cell-permeable and modulate their bioactivity. With all explored chemotypes, MCL-conjugates consistently exhibited superior uptake or bioactivity compared to LCL-conjugates and either comparable or superior uptake or bioactivity to short-chain lipid (SCL)-conjugates. Together, our findings suggest that conjugation of small molecules with MCLs could be a powerful strategy for the design of probes and drugs.
PMID: 36178375
ISSN: 1520-5126
CID: 5334602

A Photohormone for Light-Dependent Control of PPARα in Live Cells

Willems, Sabine; Morstein, Johannes; Hinnah, Konstantin; Trauner, Dirk; Merk, Daniel
Photopharmacology enables the optical control of several biochemical processes using small-molecule photoswitches that exhibit different bioactivities in their cis- and trans-conformations. Such tool compounds allow for high spatiotemporal control of biological signaling, and the approach also holds promise for the development of drug molecules that can be locally activated to reduce target-mediated adverse effects. Herein, we present the expansion of the photopharmacological arsenal to two new members of the peroxisome proliferator-activated receptor (PPAR) family, PPARα and PPARδ. We have developed a set of highly potent PPARα and PPARδ targeting photohormones derived from the weak pan-PPAR agonist GL479 that can be deactivated by light. The photohormone 6 selectively activated PPARα in its trans-conformation with high selectivity over the related PPAR subtypes and was used in live cells to switch PPARα activity on and off in a light- and time-dependent fashion.
PMID: 34213899
ISSN: 1520-4804
CID: 4950342

Optical control of targeted protein degradation

Reynders, Martin; Trauner, Dirk
Molecular glues and proteolysis targeting chimeras (PROTACs) have emerged as small-molecule tools that selectively induce the degradation of a chosen protein and have shown therapeutic promise. Recently, several approaches employing light as an additional stimulus to control induced protein degradation have been reported. Here, we analyze the principles guiding the design of such systems, provide a survey of the literature published to date, and discuss opportunities for further development. Light-responsive degraders enable the precise temporal and spatial control of protein levels, making them useful research tools but also potential candidates for human precision medicine.
PMID: 34115971
ISSN: 2451-9448
CID: 4964802

Perfect and Defective13C-Furan-Derived Nanothreads from Modest-Pressure Synthesis Analyzed by13C NMR

Matsuura, B S; Huss, S; Zheng, Z; Yuan, S; Wang, T; Chen, B; Badding, J V; Trauner, D; Elacqua, E; van, Duin A C T; Crespi, V H; Schmidt-Rohr, K
The molecular structure of nanothreads produced by the slow compression of13C4-furan was studied by advanced solid-state NMR. Spectral editing showed that >95% of carbon atoms were bonded to one hydrogen (C-H) and that there were 2-4% CH2, 0.6% CO, and <0.3% CH3groups. Alkenes accounted for 18% of the CH moieties, while trapped, unreacted furan made up 7%. Two-dimensional (2D)13C-13C and1H-13C NMR identified 12% of all carbon in asymmetric O-CHCH-CH-CH- and 24% in symmetric O-CH-CHCH-CH- rings. While the former represented defects or chain ends, some of the latter appeared to form repeating thread segments. Around 10% of carbon atoms were found in highly ordered, fully saturated nanothread segments. Unusually slow13C spin-exchange with sites outside the perfect thread segments documented a length of at least 14 bonds; the small width of the perfect-thread signals also implied a fairly long, regular structure. Carbons in the perfect threads underwent relatively slow spin-lattice relaxation, indicating slow spin exchange with other threads and smaller amplitude motions. Through partial inversion recovery, the signals of the perfect threads were observed and analyzed selectively. Previously consideredsyn-threads with four different C-H bond orientations were ruled out by centerband-only detection of exchange NMR, which was, on the contrary, consistent withanti-threads. The observed13C chemical shifts were matched well by quantum-chemical calculations foranti-threads but not for more complex structures likesyn/anti-threads. These observations represent the first direct determination of the atomic-level structure of fully saturated nanothreads.
Copyright
EMBASE:2013594262
ISSN: 0002-7863
CID: 4978682

Selective Photoswitchable Allosteric Agonist of a G Protein-Coupled Receptor

Donthamsetti, Prashant; Konrad, David B; Hetzler, Belinda; Fu, Zhu; Trauner, Dirk; Isacoff, Ehud Y
G protein-coupled receptors (GPCRs) are the most common targets of drug discovery. However, the similarity between related GPCRs combined with the complex spatiotemporal dynamics of receptor activation in vivo has hindered drug development. Photopharmacology offers the possibility of using light to control the location and timing of drug action by incorporating a photoisomerizable azobenzene into a GPCR ligand, enabling rapid and reversible switching between an inactive and active configuration. Recent advances in this area include (i) photoagonists and photoantagonists that directly control receptor activity but are nonselective because they bind conserved sites, and (ii) photoallosteric modulators that bind selectively to nonconserved sites but indirectly control receptor activity by modulating the response to endogenous ligand. In this study, we designed a photoswitchable allosteric agonist that targets a nonconserved allosteric site for selectivity and activates the receptor on its own to provide direct control. This work culminated in the development of aBINA, a photoswitchable allosteric agonist that selectively activates the Gi/o-coupled metabotropic glutamate receptor 2 (mGluR2). aBINA is the first example of a new class of precision drugs for GPCRs and other clinically important signaling proteins.
PMCID:8227462
PMID: 34115935
ISSN: 1520-5126
CID: 4950202

How Photoswitchable Lipids Affect the Order and Dynamics of Lipid Bilayers and Embedded Proteins

Doroudgar, Mahmoudreza; Morstein, Johannes; Becker-Baldus, Johanna; Trauner, Dirk; Glaubitz, Clemens
Altering the properties of phospholipid membranes by light is an attractive option for the noninvasive manipulation of membrane proteins and cellular functions. Lipids with an azobenzene group within their acyl chains such as AzoPC are suitable tools for manipulating lipid order and dynamics through a light-induced trans-to-cis isomerization. However, the action of these photoswitchable lipids at the atomic level is still poorly understood. Here, liposomes containing AzoPC, POPE, and POPG have been characterized by solid-state NMR through chemical shift and dipolar CH order parameter measurements. Upon UV-light illumination, an efficient trans-to-cis conversion can be achieved resulting in a localized reduction of the CH order parameter within the bulk lipid acyl chains. This effect is even more pronounced in liposomes containing the integral membrane protein E. coli diacylglycerol kinase. The protein responds to the light-induced trans-to-cis isomerization by a site-specific increase in the molecular dynamics as observed by altered cross peak intensities in NCA spectra. This study represents a proof-of-concept demonstration for the use of photoswitchable lipids to modulate membrane properties by light for inducing dynamic changes within an embedded membrane protein.
PMID: 34133158
ISSN: 1520-5126
CID: 4950242

Perfect and Defective 13C-Furan-Derived Nanothreads from Modest-Pressure Synthesis Analyzed by 13C NMR

Matsuura, Bryan S; Huss, Steven; Zheng, Zhaoxi; Yuan, Shichen; Wang, Tao; Chen, Bo; Badding, John V; Trauner, Dirk; Elacqua, Elizabeth; van Duin, Adri C T; Crespi, Vincent H; Schmidt-Rohr, Klaus
The molecular structure of nanothreads produced by the slow compression of 13C4-furan was studied by advanced solid-state NMR. Spectral editing showed that >95% of carbon atoms were bonded to one hydrogen (C-H) and that there were 2-4% CH2, 0.6% C═O, and <0.3% CH3 groups. Alkenes accounted for 18% of the CH moieties, while trapped, unreacted furan made up 7%. Two-dimensional (2D) 13C-13C and 1H-13C NMR identified 12% of all carbon in asymmetric O-CH═CH-CH-CH- and 24% in symmetric O-CH-CH═CH-CH- rings. While the former represented defects or chain ends, some of the latter appeared to form repeating thread segments. Around 10% of carbon atoms were found in highly ordered, fully saturated nanothread segments. Unusually slow 13C spin-exchange with sites outside the perfect thread segments documented a length of at least 14 bonds; the small width of the perfect-thread signals also implied a fairly long, regular structure. Carbons in the perfect threads underwent relatively slow spin-lattice relaxation, indicating slow spin exchange with other threads and smaller amplitude motions. Through partial inversion recovery, the signals of the perfect threads were observed and analyzed selectively. Previously considered syn-threads with four different C-H bond orientations were ruled out by centerband-only detection of exchange NMR, which was, on the contrary, consistent with anti-threads. The observed 13C chemical shifts were matched well by quantum-chemical calculations for anti-threads but not for more complex structures like syn/anti-threads. These observations represent the first direct determination of the atomic-level structure of fully saturated nanothreads.
PMID: 34130458
ISSN: 1520-5126
CID: 4950232

Optimized Photoactivatable Lipid Nanoparticles Enable Red Light Triggered Drug Release

Chander, Nisha; Morstein, Johannes; Bolten, Jan S; Shemet, Andrej; Cullis, Pieter R; Trauner, Dirk; Witzigmann, Dominik
Encapsulation of small molecule drugs in long-circulating lipid nanoparticles (LNPs) can reduce toxic side effects and enhance accumulation at tumor sites. A fundamental problem, however, is the slow release of encapsulated drugs from these liposomal systems at the disease site resulting in limited therapeutic benefit. Methods to trigger release at specific sites are highly warranted. Here, it is demonstrated that incorporation of ultraviolet (UV-A) or red-light photoswitchable-phosphatidylcholine analogs (AzoPC and redAzoPC) in conventional LNPs generates photoactivatable LNPs (paLNPs) having comparable structural integrity, drug loading capacity, and size distribution to the parent DSPC-cholesterol liposomes. It is shown that 65-70% drug release (doxorubicin) can be induced from these systems by irradiation with pulsed light based on trans-to-cis azobenzene isomerization. In vitro it is confirmed that paLNPs are non-toxic in the dark but convey cytotoxicity upon irradiation in a human cancer cell line. In vivo studies in zebrafish embryos demonstrate prolonged blood circulation and extravasation of paLNPs comparable to clinically approved formulations, with enhanced drug release following irradiation with pulsed light. Conclusively, paLNPs closely mimic the properties of clinically approved LNPs with the added benefit of light-induced drug release making them promising candidates for clinical development.
PMID: 33880882
ISSN: 1613-6829
CID: 4858792

Short Photoswitchable Ceramides Enable Optical Control of Apoptosis

Morstein, Johannes; Kol, Matthijs; Novak, Alexander J E; Feng, Suihan; Khayyo, Shadi; Hinnah, Konstantin; Li-Purcell, Nasi; Pan, Grace; Williams, Benjamin M; Riezman, Howard; Atilla-Gokcumen, G Ekin; Holthuis, Joost C M; Trauner, Dirk
We report short ceramide analogs that can be activated with light and further functionalized using azide-alkyne click chemistry. These molecules, termed scaCers, exhibit increased cell permeability compared to their long-chain analogs as demonstrated using mass spectrometry and imaging. Notably, scaCers enable optical control of apoptosis, which is not observed with long-chain variants. Additionally, they function as photoswitchable substrates for sphingomyelin synthase 2 (SMS2), exhibiting inverted light-dependence compared to their extended analogs.
PMID: 33586946
ISSN: 1554-8937
CID: 4808022