Understanding the Interactions of Ubiquitin-Specific Protease 7 with Its Substrates through Molecular Dynamics Simulations: Insights into the Role of Its C-Terminal Domains in Substrate Recognition.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme that plays a critical role in regulating various cellular processes by cleaving ubiquitin molecules from target proteins. The C-terminal loop (CTL) motif is a specific region at the C-terminal end of the USP7 enzyme. Recent experiments suggest that the CTL motif plays a role in USP7's catalytic activity by contributing to the enzyme's structural stability, substrate recognition, and catalytic efficiency. The objective of this work is to elucidate these roles through the utilization of computational methods for molecular simulations. For this, we conducted extensive molecular dynamics (MD) simulations to investigate the conformational dynamics and protein-protein interactions within the USP7 enzyme-substrate complex with the substrate consisting of the ubiquitin tagged with the fluorescent label rhodamine 110-gly (Ub-Rho). Our results demonstrate that the CTL motif plays a crucial role in stabilizing the Ubl domains' conformation and augmenting the stability of active conformations within the enzyme-substrate complex. Conversely, the absence of the CTL motif results in increased flexibility and variability in Ubl domains' motion, leading to a reduced percentage of active conformations. Furthermore, our analysis of protein-protein interactions highlights the significance of the CTL motif in anchoring the Ubl45 domains to the catalytic domain (CD), thereby facilitating stable interactions with the substrate. Overall, our findings provide valuable insights into the conformational dynamics and protein-protein interactions inherent in the USP7 enzyme-substrate complex. These insights shed light on some mechanistic details of USP7 concerning the substrate's recognition before its catalytic action.

Super-resolution imaging of mitochondrial cristae using a more hydrophobic far-red Si-rhodamine probe.

Mitochondrial probe SiRPFA was synthesized by attaching a long perfluoroalkyl chain on Si-rhodamine cationic dye. High lipophilicity endowed SiRPFA with mitochondrial membrane potential independent properties. Under stimulated emission depletion microscopy, SiRPFA clearly revealed changes in mitochondrial cristae morphology during autophagy induced by starvation or apoptosis.

Mitochondria-Targeting 1,5-Diazacyclooctane-Spacered Triterpene Rhodamine Conjugates Exhibit Cytotoxicity at Sub-Nanomolar Concentration against Breast Cancer Cells.

1,5-Diazacyclooctane was prepared by a simple synthetic sequence and coupled to pentacyclic triterpenoic acids oleanolic acid, ursolic acid, betulinic acid, platanic acid, and asiatic acid; these amides were activated with oxalyl chloride and reacted with rhodamine B or rhodamine 101 to yield conjugates. The conjugates were screened in SRB assays with various human breast cancer cell lines (MDA-MB-231, HS578T, MCF-7, and T47D) and found to exert cytotoxic activity even at a low concentration. Therefore, for an asiatic acid rhodamine 101 conjugate (28), an IC50 = 0.60 nM was determined and found to induce apoptosis in MDA-MB-231 and HS578T cells. Extra experiments showed the compound to act as a mitocan and to induce inhibition of proliferation or growth arrest in MDA-MB-231 cells at lower doses followed by an induction of apoptosis at higher doses. Furthermore, differential responses to proliferation inhibition and apoptosis induction may explain differential sensitivity of mammary cell lines to compound 28.

Combinatorial drug-loaded quality by design adapted transliposome gel formulation for dermal delivery: In vitro and dermatokinetic study.

BACKGROUND: Ursolic acid is a powerful drug that possesses many therapeutic properties, such as hepatoprotection, immunomodulation, anti-inflammatory, antidiabetic, antibacterial, antiviral, antiulcer, and anticancer activity. Centella asiatica (L.) Urban (Umbelliferae) contains a triterpene called asiatic acid, which has been used effectively in traditional Chinese and Indian medicine system for centuries. Anticancer, anti-inflammatory, and neuroprotective properties are only some of the many pharmacological actions previously attributed to asiatic acid . AIM: The present work developed an optimized combinatorial drug-loaded nano-formulation by Quality by design approach. MATERIALS AND METHODS: The optimize transliposome for accentuated dermal delivery of dual drug. The optimization of drug-loaded transliposome was done using the "Box-Behnken design." The optimized formulation was characterized for vesicles size, entrapment efficiency (%), and in vitro drug release. Additionally, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), and dermatokinetic study were performed for further evaluation of drug-loaded optimized transliposome formulation. RESULTS: The optimized combinatorial drug-loaded transliposome formulation showed a particle size of 86.36 ± 2.54 nm, polydispersity index (PDI) 0.230 ± 0.008, and an entrapment efficiency of 87.43 ± 2.66% which depicted good entrapment efficiency. In vitro drug release of ursolic acid and asiatic acid transliposomes was found to be 85.12 ± 2.54% and 80.23 ± 3.23%, respectively, as compared to optimized ursolic acid and asiatic acid transliposome gel drug release that was 67.18 ± 2.85% and 60.28 ± 4.12%, respectively. The skin permeation study of ursolic and asiatic acid conventional formulation was only 32.48 ± 2.42%, compared with optimized combinatorial drug-loaded transliposome gel (79.83 ± 4.52%) at 12 h. After applying combinatorial drug-loaded transliposome gel, rhodamine was able to more easily cross rat skin, as observed by confocal laser scanning microscopy, in comparison with when the rhodamine control solution was used. DISCUSSION: The UA_AA-TL gel formulation absorbed more ursolic acid and asiatic acid than the UA_AA-CF gel formulation, as per dermatokinetic study. Even after being incorporated into transliposome vesicles, the antioxidant effects of ursolic and asiatic acid were still detectable. In most cases, transliposomes vesicular systems generate depots in the skin's deeper layers and gradually release the medicine over time, allowing for fewer applications. CONCLUSION: In overall our studies, it may be concluded that developed dual drug-loaded transliposomal formulation has great potential for effective topical drug delivery for skin cancer.

Tuning the Cellular Uptake and Retention of Rhodamine Dyes by Molecular Engineering for High-Contrast Imaging of Cancer Cells.

Probes allowing high-contrast discrimination of cancer cells and effective retention are powerful tools for the early diagnosis and treatment of cancer. However, conventional small-molecule probes often show limited performance in both aspects. Herein, we report an ingenious molecular engineering strategy for tuning the cellular uptake and retention of rhodamine dyes. Introduction of polar aminoethyl leads to the increased brightness and reduced cellular uptake of dyes, and this change can be reversed by amino acetylation. Moreover, these modifications allow cancer cells to take up more dyes than normal cells (16-fold) through active transport. Specifically, we further improve the signal contrast (56-fold) between cancer and normal cells by constructing activatable probes and confirm that the released fluorophore can remain in cancer cells with extended time, enabling long-term and specific tumor imaging.

P-glycoprotein, FK-binding Protein-12, and the Intracellular Tacrolimus Concentration in T-lymphocytes and Monocytes of Kidney Transplant Recipients.

BACKGROUND: . Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C 0 ). The intra-immune cellular concentration is potentially a better target than C 0 . However, little is known regarding intracellular tacrolimus concentration in T-lymphocytes and monocytes. We investigated the tacrolimus concentrations in both cell types and their relation with the expression and activity of FK-binding protein (FKBP)-12 and P-glycoprotein (P-gp). METHODS: . T-lymphocytes and monocytes were isolated from kidney transplant recipients followed by intracellular tacrolimus concentration measurement. FKBP-12 and P-gp were quantified with Western blot, flow cytometry, and the Rhodamine-123 assay. Interleukin-2 and interferon-γ in T-lymphocytes were measured to quantify the effect of tacrolimus. RESULTS: . Tacrolimus concentration in T-lymphocytes was lower than in monocytes (15.3 [8.5-33.4] versus 131.0 [73.5-225.1] pg/million cells; P < 0.001). The activity of P-gp (measured by Rhodamine-123 assay) was higher in T-lymphocytes than in monocytes. Flow cytometry demonstrated a higher expression of P-gp (normalized mean fluorescence intensity 1.5 [1.2-1.7] versus 1.2 [1.1-1.4]; P = 0.012) and a lower expression of FKBP-12 (normalized mean fluorescence intensity 1.3 [1.2-1.7] versus 1.5 [1.4-2.0]; P = 0.011) in T-lymphocytes than monocytes. Western blot confirmed these observations. The addition of verapamil, a P-gp inhibitor, resulted in a 2-fold higher intra-T-cell tacrolimus concentration. This was accompanied by a significantly fewer cytokine-producing cells. CONCLUSIONS: . T-lymphocytes have a higher activity of P-gp and lower concentration of the FKBP-12 compared with monocytes. This explains the relatively lower tacrolimus concentration in T-lymphocytes. The addition of verapamil prevents loss of intracellular tacrolimus during the cell isolation process and is required to ensure adequate intracellular concentration measurement.

[Study on transport of small molecule rhodamine B within different layers of cartilage].

The small molecule nutrients and cell growth factors required for the normal metabolism of chondrocyte mainly transport into the cartilage through free diffusion. However, the specific mass transfer law in the cartilage remains to be studied. In this study, using small molecule rhodamine B as tracer, the mass transfer models of cartilage were built under different pathways including surface pathway, lateral pathway and composite pathway. Sections of cartilage at different mass transfer times were observed by using laser confocal microscopy and the transport law of small molecules within different layers of cartilage was studied. The results showed that rhodamine B diffused into the whole cartilage layer through surface pathway within 2 h. The fluorescence intensity in the whole cartilage layer increased with the increase of mass transfer time. Compared to mass transfer of 2 h, the mean fluorescence intensity in the superficial, middle, and deep layers of cartilage increased by 1.83, 1.95, and 3.64 times, respectively, after 24 h of mass transfer. Under lateral path condition, rhodamine B was transported along the cartilage width, and the molecular transport distance increased with increasing mass transfer time. It is noted that rhodamine B could be transported to 2 mm away from cartilage side after 24 h of mass transfer. The effect of mass transfer under the composite path was better than those under the surface path and the lateral path, and especially the mass transfer in the deep layer of cartilage was improved. This study may provide a reference for the treatment and repair of cartilage injury.

Structural and functional investigation of ABC transporter STE6-2p from Pichia pastoris reveals unexpected interaction with sterol molecules.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast Pichia pastoris, a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated the Pichia ABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter from P. pastoris in the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function.

Eco-toxicological effect of a commercial dye Rhodamine B on freshwater microalgae Chlorella vulgaris.

In this study, the acute toxicity effects of a fluorescent xanthene dye, Rhodamine B (RhB), widely used in textile, paper, and leather industries was investigated on a freshwater microalgae Chlorella vulgaris. The acute toxicity of RhB on C. vulgaris was determined by examining the growth, cell morphology, pigment production, protein content, and the activities of oxidative stress enzymes. Based on the results of the toxicity study of 24-96 h, the median inhibitory concentration (IC50) values ranged from 69.94 to 31.29 mg L-1. The growth of C. vulgaris was conspicuously inhibited by RhB exposure, and the cell surfaces appeared to be seriously shrunk in SEM analysis. The growth of C. vulgaris was hindered after exposure to graded concentrations (10-50 mg L-1) of RhB. A significant reduction in growth rate, pigment synthesis (chlorophyll a, chlorophyll b, and carotenoid), and protein content was recorded in a dose-dependent manner. After 96 h exposure of C. vulgaris to 50 mg L-1 RhB, chlorophyll a, chlorophyll b, carotenoids, and protein contents were reduced by 71.59, 74.90, 65.84, and 74.20%, respectively. The activities of the antioxidant enzymes peroxidase (POD), and catalase (CAT) also increased markedly in the presence of RhB. A notable effect was observed on oxidative enzymes catalase and peroxidase, indicating that oxidative stress may be the primary factor in the inhibition of growth and pigment synthesis. Consequently, the experimental acute toxicity data were compared to the QSAR prediction made by the ECOSAR programme. Results showed that the experimental acute toxicity values were 67.74-fold lower than the ECOSAR predicted values. The study provides convincing evidence for the metabolic disruption in the ubiquitous microalgae C. vulgaris due to the RhB dye toxicity.

Magnetic alignment of rhodamine/magnetite dual-labeled microtubules probed with inverted fluorescence microscopy.

Molecular machines, as exemplified by the kinesin and microtubule system, are responsible for molecular transport in cells. The monitoring of the cellular machinery has attracted much attention in recent years, requiring sophisticated techniques such as optical tweezers, and dark field hyperspectral and fluorescence microscopies. It also demands suitable procedures for immobilization and labeling with functional agents such as dyes, plasmonic nanoparticles and quantum dots. In this work, microtubules were co-polymerized by incubating a tubulin mix consisting of 7 biotinylated tubulin to 3 rhodamine tubulin. Rhodamine provided the fluorescent tag, while biotin was the anchoring group for receiving streptavidin containing species. To control the microtubule alignment and consequently, the molecular gliding directions, functionalized iron oxide nanoparticles were employed in the presence of an external magnet field. Such iron oxide nanoparticles, (MagNPs) were previously coated with silica and (3-aminopro-pyl)triethoxysilane (APTS) and then modified with streptavidin (SA) for linking to the biotin-functionalized microtubules. In this way, the binding has been successfully performed, and the magnetic alignment probed by Inverted Fluorescence Microscopy. The proposed strategy has proved promising, as tested with one of the most important biological structures of the cellular machinery.

Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles.

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter) for optically monitoring membrane potential changes in the ER of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO is voltage-sensitive and that the LUnAR RhoVR can be targeted to an intact ER in living cells. Using the LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use the LUnAR RhoVR to directly visualize functional coupling between the plasma-ER membranes in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that the LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology.

Rapid visualization of mammary gland tumor lesions of dogs using the enzyme-activated fluorogenic probe; γ-glutamyl hydroxymethyl rhodamine green.

Since gamma-glutamyl transpeptidase (GGT) is highly and locally expressed in human breast cancer, a GGT-enzymatically activatable fluorescent probe, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), has been developed to detect the human breast cancer area with high performance. In this study, GGT expression and the efficacy of gGlu-HMRG on visualization were investigated in canine mammary gland tumors (MGT). Seventeen non-fixed fresh-frozen MGT specimens and each peritumoral control tissue were utilized. The GGT mRNA levels were highly observed in the tumor specimens compared with the control. GGT immunostaining was mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues. These signals were strongly positive in several cases while they were mild to not observed in other cases. When gGlu-HMRG solution was dropped to the non-fixed tissue pieces of MGT or control tissues, the fluorescence intensities (FIs) were measured using Maestro in-vivo imaging device. FIs in MGT tissues were significantly higher than each control tissue 20 min after treatment. Based on Youden index method, the maximum sensitivity and specificity of FI was 82.4% and 82.4%. These findings suggest that GGT is highly expressed in several MGTs in dogs and gGlu-HMRG could visualize at least a part of MGT tissues in dogs. Nevertheless, it should be needed to assess the false-negative areas more carefully in canine than human cases.

Identification of deubiquitinase inhibitors via high-throughput screening using a fluorogenic ubiquitin-rhodamine assay.

Identification of selective deubiquitinase (DUB) inhibitors is critical for probe development to further understand and explore DUB biological function. Here, we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs. In screening the compound library against multiple DUBs in parallel, we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery. For complete details on the use and execution of this protocol, please refer to Varca et al. (2021).

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5.

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker's yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.

Magneto- and opto-stimuli responsive nanofibers as a controlled drug delivery system.

The drawbacks of conventional drug administration include repeated administration, non-specific biodistribution in the body's systems, the long-term unsustainability of drug molecules, and high global cytotoxicity, posing a challenge for the efficient treatment of chronic diseases that require varying drug dosages over time for optimal therapeutic efficacy. Most controlled-release methods encapsulate drug molecules in biodegradable materials that dissolve over time to release the drug, making it difficult to deliver drugs on a schedule. To address these limitations, we developed a magneto-, opto-stimuli responsive drug delivery system based on functionalized electrospun nanofibers loaded with superparamagnetic iron oxide nanoparticles (SPIONs). We exploited the Néel relaxation effect of SPIONs, where heat generated from vibrating SPIONs under exogenously applied magnetic fields or laser illumination induced structural changes of the thermo-sensitive nanofibers that encapsulate the particles. We showed that this structural change of nanofibers is the governing factor in controlling the release of dye molecules, used as a model drug and co-encapsulated within the nanofibers. We also showed that the degree of nanofiber structural change depends on SPION loading and duration of stimulation, demonstrating the tunability of the drug release profile. Overall, we demonstrated the potential of SPION-embedded thermoplastic nanofibers as an attractive platform for on-demand drug delivery.

Soluble microneedle patch with photothermal and NO-release properties for painless and precise treatment of ischemic perforator flaps.

Skin necrosis is the most serious complication of flap plastic surgery, which means the failure of the operation. Systemic administration rarely benefits the local area and can lead to side effects, while topical administration has poor permeability due to the skin barrier function. Currently, few of these common medical interventions can totally respond to the blood supply of the skin after surgery. Herein, a soluble microneedle (MN) patch made of hyaluronic acid was used to target the ischemic area in a painless and precise manner for transdermal drug delivery. Based on the important role of nitric oxide (NO) in angiogenesis, the thermosensitive NO donor (BNN6) and gold nanorods (GNRs) acting as photothermal agents were introduced into the microneedles (MNs). The hyperthermia induced by GNRs under near infrared (NIR, 808 nm) irradiation could enhance the penetration of drugs and facilitate NO release from BNN6. A series of corresponding experiments proved that the system played a significant promotion role in vascular regeneration, providing a painless, precise and NO-assisted treatment method for the ischemic perforator flaps.

Journey of Poly(ethylene Glycol) in Living Cells.

As the gold standard for stealth polymer materials, poly(ethylene glycol) (PEG) has been widely used in drug delivery with excellent properties such as low toxicity, reduced immunogenicity, good water solubility, and so forth. However, lack of understanding for the fate of PEG and PEGylated delivery systems at the cellular level has limited the application of PEGylated molecules in diagnosis and therapy. Here, we chose linear PEG 5k as a representative model and focused on the internalization behavior and mechanism, intracellular trafficking, sub-cellular localization, and cellular exocytosis of PEG and PEGylated molecules in living cells. Our investigation showed that PEG could be internalized into cells in 1 h. The internalized PEG was localized to lysosome, cytosol, endoplasmic reticulum (ER) and mitochondria. Importantly, the fate of PEG in cells could be regulated by conjugating different small molecules. PEGylated rhodamine B (PEG-RB) as the positively charged macromolecule was internalized into cells by micropinocytosis and then transported in lysosomes, ER, and mitochondria via vesicles sequentially. In contrast, PEGylated pyropheophorbide-a (PEG-PPa) as the negatively charged macromolecule was internalized into cells and transported to lysosomes ultimately. PEGylation slowed down the exocytosis process of RB and PPa and significantly prolonged their residence time inside the cells. These findings improve the understanding of how PEG and PEGylated molecules interact with the biological system at cellular and sub-cellular levels, which is of significance to rational PEGylation design for drug delivery.

Rhodamine 6G-Ligand Influencing G-Quadruplex Stability and Topology.

The involvement of G-quadruplex (G4) structures in nucleic acids in various molecular processes in cells such as replication, gene-pausing, the expression of crucial cancer-related genes and DNA damage repair is well known. The compounds targeting G4 usually bind directly to the G4 structure, but some ligands can also facilitate the G4 folding of unfolded G-rich sequences and stabilize them even without the presence of monovalent ions such as sodium or potassium. Interestingly, some G4-ligand complexes can show a clear induced CD signal, a feature which is indirect proof of the ligand interaction. Based on the dichroic spectral profile it is not only possible to confirm the presence of a G4 structure but also to determine its topology. In this study we examine the potential of the commercially available Rhodamine 6G (RhG) as a G4 ligand. RhG tends to convert antiparallel G4 structures to parallel forms in a manner similar to that of Thiazole Orange. Our results confirm the very high selectivity of this ligand to the G4 structure. Moreover, the parallel topology of G4 can be verified unambiguously based on the specific induced CD profile of the G4-RhG complex. This feature has been verified on more than 50 different DNA sequences forming various non-canonical structural motifs.

Photoacoustic-fluorescence microendoscopy in vivo.

A miniature endoscope capable of imaging multiple tissue contrasts in high resolution is highly attractive, because it can provide complementary and detailed tissue information of internal organs. Here we present a photoacoustic (PA)-fluorescence (FL) endoscope for optical-resolution PA microscopy (PAM) and FL microscopy (FLM). The endoscope with a diameter of 2.8 mm achieves high lateral resolutions of 5.5 and 6.3 µm for PAM and FLM modes, respectively. In vivo imaging of zebrafish larvae and a mouse ear is conducted, and high-quality images are obtained. Additionally, in vivo endoscopic imaging of a rat rectum is demonstrated, showing the endoscopic imaging capability of our endoscope. By providing dual contrasts with high resolution, the endoscope may open up new opportunities for clinical endoscopic imaging applications.

Macrocyclic FKBP51 Ligands Define a Transient Binding Mode with Enhanced Selectivity.

Subtype selectivity represents a challenge in many drug discovery campaigns. A typical example is the FK506 binding protein 51 (FKBP51), which has emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs. FKBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During a macrocyclization pilot study, we observed that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with a new binding mode featuring a transient conformation, which is disfavored for the small FKBPs. Using a conformation-sensitive assay we show that this binding mode occurs in solution and is characteristic for this new class of compounds. The discovered macrocycles are non-immunosuppressive, engage FKBP51 in cells, and block the cellular effect of FKBP51 on IKKα. Our findings provide a new chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.