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The toxicity of tacrolimus metabolites and their potential pharmacodynamic (PD) interactions with tacrolimus might respectively explain the surprising combination of higher toxicity and lower efficacy of tacrolimus despite normal blood concentrations, described in extensive metabolizers. To evaluate such interactions, we produced tacrolimus metabolites in vitro and characterized them by high resolution mass spectrometry (HRMS, for all) and nuclear magnetic resonance (NMR, for the most abundant, M-I). We quantified tacrolimus metabolites and checked their structure in patient whole blood and peripheral blood mononuclear cells (PBMC). We explored the interactions of M-I with tacrolimus in silico, in vitro and ex vivo. In vitro metabolization produced isoforms of tacrolimus and of its metabolites M-I and M-III, whose HRMS fragmentation suggested an open-ring structure. M-I and M-III open-ring isomers were also observed in patient blood. By contrast, NMR could not detect these open-ring forms. Transplant patients expressing CYP3A5 exhibited higher M-I/TAC ratios in blood and PBMC than non-expressers. Molecular Dynamics simulations showed that: all possible tacrolimus metabolites and isomers bind FKPB12; and the hypothetical open-ring structures induce looser binding between FKBP12 and calcineurins, leading to lower CN inhibition. In vitro, tacrolimus bound FKPB12 with more affinity than purified M-I, and the pool of tacrolimus metabolites and purified M-I had only weak inhibitory activity on IL2 secretion and not at all on NFAT nuclear translocation. M-I showed no competitive effect with tacrolimus on either test. Finally, M-I or the metabolite pool did not significantly interact with tacrolimus MLR suppression, thus eliminating a pharmacodynamic interaction., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Melanie Campana reports administrative support and equipment, drugs, or supplies were provided by MedinCell SA. Rudy Mevizou reports administrative support and equipment, drugs, or supplies were provided by MedinCell SA. Pierre Marquet reports a relationship with MedinCell SA that includes: consulting or advisory, equity or stocks, and funding grants. Melanie Campana reports a relationship with MedinCell SA that includes: employment and equity or stocks. Alfonso Lopez-Noriega reports a relationship with MedinCell SA that includes: employment and equity or stocks. Pierre Marquet reports a relationship with Sandoz France that includes: consulting or advisory and funding grants. Pierre Marquet reports a relationship with Chiesi SA France that includes: consulting or advisory and funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chimeric antigen receptor (CAR) T cells from allogeneic donors promise "off-the-shelf" availability by overcoming challenges associated with autologous cell manufacturing. However, recipient immunologic rejection of allogeneic CAR-T cells may decrease their in vivo lifespan and limit treatment efficacy. Here, we demonstrate that the immunosuppressants rapamycin and tacrolimus effectively mitigate allorejection of HLA-mismatched CAR-T cells in immunocompetent humanized mice, extending their in vivo persistence to that of syngeneic humanized mouse-derived CAR-T cells. In turn, genetic knockout (KO) of FKBP prolyl isomerase 1A (FKBP1A), which encodes a protein targeted by both drugs, was necessary to confer CD19-specific CAR-T cells (19CAR) robust functional resistance to these immunosuppressants. FKBP1A KO 19CAR-T cells maintained potent in vitro functional profiles and controlled in vivo tumor progression similarly to untreated 19CAR-T cells. Moreover, immunosuppressant treatment averted in vivo allorejection permitting FKBP1A KO 19CAR-T cell-driven B cell aplasia. Thus, we demonstrate that genome engineering enables immunosuppressant treatment to improve the therapeutic potential of universal donor-derived CAR-T cells., Competing Interests: Declaration of interests C.R.M., A.C.M., P.B.B., A.J.C., F.M.M., M.L.W., J.J.R., L.J.C., C.K., F.L., and B.Y. were employees of Beam Therapeutics when the work was conducted and are shareholders in the company. Beam Therapeutics has filed patent applications based on this work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FK506-binding protein 12 (FKBP12)-rapamycin-binding (FRB) domain of human target of rapamycin kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FRB domain fused to the target proteins with the human FKBP fused to S-phase kinase-associated protein 1 or Cullin 1, subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein dynamin-related protein 5B and E2 transcription factor, a regulator of the G1/S transition, within 2 to 3 h after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Subcellular protein localization regulates protein function and can be corrupted in cancers 1 and neurodegenerative diseases 2,3 . The rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules that harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could enforce targeted protein relocalization and rewire the interactome. Here we identify a collection of shuttle proteins with potent ligands amenable to incorporation into targeted relocalization-activating molecules (TRAMs), and use these to relocalize endogenous proteins. Using a custom imaging analysis pipeline, we show that protein steady-state localization can be modulated through molecular coupling to shuttle proteins containing sufficiently strong localization sequences and expressed in the necessary abundance. We analyse the TRAM-induced relocalization of different proteins and then use nuclear hormone receptors as shuttles to redistribute disease-driving mutant proteins such as SMARCB1 Q318X , TDP43 ΔNLS and FUS R495X . TRAM-mediated relocalization of FUS R495X to the nucleus from the cytoplasm correlated with a reduction in the number of stress granules in a model of cellular stress. With methionyl aminopeptidase 2 and poly(ADP-ribose) polymerase 1 as endogenous cytoplasmic and nuclear shuttles, respectively, we demonstrate relocalization of endogenous PRMT9, SOS1 and FKBP12. Small-molecule-mediated redistribution of nicotinamide nucleotide adenylyltransferase 1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the genetic WldS gain-of-function phenotype in mice resistant to certain types of neurodegeneration 4 . The concept of targeted protein relocalization could therefore inspire approaches for treating disease through interactome rewiring., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The ryanodine receptor type 1 (RyR1) is a Ca2+ release channel that regulates skeletal muscle contraction by controlling Ca2+ release from the sarcoplasmic reticulum (SR). Posttranslational modifications (PTMs) of RyR1, such as phosphorylation, S-nitrosylation, and carbonylation are known to increase RyR1 open probability (Po), contributing to SR Ca2+ leak and skeletal muscle dysfunction. PTMs on RyR1 have been linked to muscle dysfunction in diseases like breast cancer, rheumatoid arthritis, Duchenne muscle dystrophy, and aging. While reactive oxygen species (ROS) and oxidative stress induce PTMs, the impact of stable oxidative modifications like 3-nitrotyrosine (3-NT) and malondialdehyde adducts (MDA) on RyR1 gating remains unclear. Mass spectrometry and single-channel recordings were used to study how 3-NT and MDA modify RyR1 and affect Po. Both modifications increased Po in a dose-dependent manner, with mass spectrometry identifying 30 modified residues out of 5035 amino acids per RyR1 monomer. Key modifications were found in domains critical for protein interaction and channel activation, including Y808/3NT in SPRY1, Y1081/3NT and H1254/MDA in SPRY2&3, and Q2107/MDA and Y2128/3NT in JSol, near the binding site of FKBP12. Though these modifications did not directly overlap with FKBP12 binding residues, they promoted FKBP12 dissociation from RyR1. These findings provide detailed insights into how stable oxidative PTMs on RyR1 residues alter channel gating, advancing our understanding of RyR1-mediated Ca2+ release in conditions associated with oxidative stress and muscle weakness., (© 2024 Steinz et al.)

Targeted protein degradation (TPD) relies on small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation by the proteasome. Only a few of the approximately 600 human E3 ligases are currently amenable to this strategy. This limits the actionable target space and clinical opportunities and thus establishes the necessity to expand to additional ligases. Here we identify and characterize SP3N, a specific degrader of the prolyl isomerase FKBP12. SP3N features a minimal design, where a known FKBP12 ligand is appended with a flexible alkylamine tail that conveys degradation properties. We found that SP3N is a precursor and that the alkylamine is metabolized to an active aldehyde species that recruits the SCF FBXO22 ligase for FKBP12 degradation. Target engagement occurs via covalent adduction of Cys326 in the FBXO22 C-terminal domain, which is critical for ternary complex formation, ubiquitylation and degradation. This mechanism is conserved for two recently reported alkylamine-based degraders of NSD2 and XIAP, thus establishing alkylamine tethering and covalent hijacking of FBXO22 as a generalizable TPD strategy., (© 2024. The Author(s).)

Glioma stem cells (GSCs) exhibit diverse molecular subtypes with the mesenchymal (MES) population representing the most malignant variant. The oncogenic potential of Salmonella pathogenicity island 1 (SPI1), an oncogenic transcription factor, has been established across various human malignancies. In this study, we explored the association between the SPI1 pathway and the MES GSC phenotype. Through comprehensive analysis of the Cancer Genome Atlas and Chinese Glioma Genome Atlas glioma databases, along with patient-derived GSC cultures, we analyzed SPI1 expression. Using genetic knockdown and overexpression techniques, we assessed the functional impact of SPI1 on GSC MES marker expression, invasion, proliferation, self-renewal, and sensitivity to radiation in vitro, as well as its influence on tumor formation in vivo. Additionally, we investigated the downstream signaling cascades activated by SPI1. Our findings revealed a positive correlation between elevated SPI1 expression and the MES phenotype, which in turn, correlated with poor survival. SPI1 enhanced GSC MES differentiation, self-renewal, and radioresistance in vitro, promoting tumorigenicity in vivo. Mechanistically, SPI1 augmented the transcriptional activity of both TGF-β1 and FKBP12 while activating the non-canonical PI3K/Akt pathway. Notably, inhibition of TGF-β1/PI3K/Akt signaling partially attenuated SPI1-induced GSC MES differentiation and its associated malignant phenotype. Collectively, our results underscore SPI1's role in activating TGF-β1/PI3K/Akt signaling through transcriptional upregulation of FKBP12, thereby supporting the aggressive MES phenotype of GSCs. Therefore, SPI1 emerges as a potential therapeutic target in glioma treatment., (© 2023 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

FKBP12 is the archetype of the FK506 binding domains that define the family of FKBP proteins which participate in the regulation of various distinct physiological signaling processes. As the drugs FK506 and rapamycin inhibit many of these FKBP proteins, there is need to develop therapeutics which exhibit selectivity within this family. The long β 4 -β 5 loop of the FKBP domain is known to regulate transcriptional activity for the steroid hormone receptors and appears to participate in regulating calcium channel activity for the cardiac and skeletal muscle ryanodine receptors. The β 4 -β 5 loop of FKBP12 has been shown to undergo extensive conformational dynamics, and here we report hydrogen exchange measurements for a series of mutational variants in that loop which indicate deviations from a two-state kinetics for those dynamics. In addition to a previously characterized local transition near the tip of this loop, evidence is presented for a second site of conformational dynamics in the stem of this loop. These mutation-dependent hydrogen exchange effects extend beyond the β 4 -β 5 loop, primarily by disrupting the hydrogen bond between the Gly 58 amide and the Tyr 80 carbonyl oxygen which links the two halves of the structural rim that surrounds the active site cleft. Mutationally-induced opening of the cleft between Gly 58 and Tyr 80 not only modulates the global stability of the protein, it promotes a conformational transition in the distant β 2 -β 3a hairpin that modulates the binding affinity for a FKBP51-selective inhibitor previously designed to exploit a localized conformational transition at the homologous site., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Wadsworth Center, New York State Department of Health. Published by Elsevier B.V. All rights reserved.)

Esophageal carcinoma is amongst the prevalent malignancies worldwide, characterized by unclear molecular classifications and varying clinical outcomes. The PI3K/AKT/mTOR signaling, one of the frequently perturbed dysregulated pathways in human malignancies, has instigated the development of various inhibitory agents targeting this pathway, but many ESCC patients exhibit intrinsic or adaptive resistance to these inhibitors. Here, we aim to explore the reasons for the insensitivity of ESCC patients to mTOR inhibitors. We assessed the sensitivity to rapamycin in various ESCC cell lines by determining their respective IC50 values and found that cells with a low level of HMGA1 were more tolerant to rapamycin. Subsequent experiments have supported this finding. Through a transcriptome sequencing, we identified a crucial downstream effector of HMGA1, FKBP12, and found that FKBP12 was necessary for HMGA1-induced cell sensitivity to rapamycin. HMGA1 interacted with ETS1, and facilitated the transcription of FKBP12. Finally, we validated this regulatory axis in in vivo experiments, where HMGA1 deficiency in transplanted tumors rendered them resistance to rapamycin. Therefore, we speculate that mTOR inhibitor therapy for individuals exhibiting a reduced level of HMGA1 or FKBP12 may not work. Conversely, individuals exhibiting an elevated level of HMGA1 or FKBP12 are more suitable candidates for mTOR inhibitor treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using T andem A ffinity P urification for identification of D rug- B inding P roteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.

The FK506-binding protein 51 (FKBP51) is a promising target in a variety of disorders including depression, chronic pain, and obesity. Previous FKBP51-targeting strategies were restricted to occupation of the FK506-binding site, which does not affect core functions of FKBP51. Here, we report the discovery of the first FKBP51 proteolysis targeting chimera (PROTAC) that enables degradation of FKBP51 abolishing its scaffolding function. Initial synthesis of 220 FKBP-focused PROTACs yielded a plethora of active PROTACs for FKBP12, six for FKBP51, and none for FKBP52. Structural analysis of a binary FKBP12:PROTAC complex revealed the molecular basis for negative cooperativity. Linker-based optimization of first generation FKBP51 PROTACs led to the PROTAC SelDeg51 with improved cellular activity, selectivity, and high cooperativity. The structure of the ternary FKBP51:SelDeg51:VCB complex revealed how SelDeg51 establishes cooperativity by dimerizing FKBP51 and the von Hippel-Lindau protein (VHL) in a glue-like fashion. SelDeg51 efficiently depletes FKBP51 and reactivates glucocorticoid receptor (GR)-signalling, highlighting the enhanced efficacy of full protein degradation compared to classical FKBP51 binding., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

For a potential application of FK506 in the treatment of acute kidney failure only the FKBP12 binding capability of the compound is required, while the immunosuppressive activity via calcineurin binding is considered as a likely risk to the patients. The methoxy groups at C13 and C15 are thought to have significant influence on the immunosuppressive activity of the molecule. Consequently, FK506 analogs with different functionalities at C13 and C15 were generated by targeted CRISPR editing of the AT domains in module 7 and 8 of the biosynthetic assembly line in Streptomyces tsukubaensis. In addition, the corresponding FK520 (C21 ethyl derivative of FK506) analogs could be obtained by media adjustments. The compounds were tested for their bioactivity in regards to FKBP12 binding, BMP potentiation and calcineurin sparing. 15-desmethoxy FK506 was superior to the other tested analogs as it did not inhibit calcineurin but retained high potency towards FKBP12 binding and BMP potentiation., (© 2023 Wiley-VCH GmbH.)

Conditional depletion of proteins is a potential strategy to elucidate protein function, especially in complex cellular processes like meiosis. Several methods are available to effectively deplete a protein in a conditional manner. Conditional loss of a protein function can be achieved by depleting it from its region of action by degrading it. A conditional loss of protein function can also be achieved by sequestering it to a functionally unavailable compartment inside the cell. This chapter describes anchor away, a conditional depletion tool that can deplete a protein both temporally and spatially by translocation. It utilizes the affinity of FRB to bind FKBP12 in the presence of rapamycin for a quick and efficient translocation of the protein to a designated location. Anchor away is a reliable tool for the study of meiotic proteins, as only small quantities of rapamycin are required to efficiently and rapidly translocate the protein of interest without compromising meiotic progression., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Skeletal muscle atrophy is a hallmark of cachexia, a wasting condition typical of chronic pathologies, that still represents an unmet medical need. Bone morphogenetic protein (BMP)-Smad1/5/8 signaling alterations are emerging drivers of muscle catabolism, hence, characterizing these perturbations is pivotal to develop therapeutic approaches. We identified two promoters of "BMP resistance" in cancer cachexia, specifically the BMP scavenger erythroferrone (ERFE) and the intracellular inhibitor FKBP12. ERFE is upregulated in cachectic cancer patients' muscle biopsies and in murine cachexia models, where its expression is driven by STAT3. Moreover, the knock down of Erfe or Fkbp12 reduces muscle wasting in cachectic mice. To bypass the BMP resistance mediated by ERFE and release the brake on the signaling, we targeted FKBP12 with low-dose FK506. FK506 restores BMP-Smad1/5/8 signaling, rescuing myotube atrophy by inducing protein synthesis. In cachectic tumor-bearing mice, FK506 prevents muscle and body weight loss and protects from neuromuscular junction alteration, suggesting therapeutic potential for targeting the ERFE-FKBP12 axis., Competing Interests: Declaration of interests E.M., V.R., L.S., and P.E.P. declare to be inventors of the patent PCTIB2022050175 (WO2022/149113)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The thermodynamic and biophysical implications of the introduction of a co-solvent during protein-ligand binding remain elusive. Using ternary complexes of 12-kDa FK506 binding protein (FKBP12), FKBP-rapamycin binding (FRB) domain of the mammalian/mechanistic target of rapamycin (mTOR) kinase, and rapamycin analogs (rapalogs) in glycerol-water mixtures, the influence of solvent composition on ligand binding dynamics was explored. The pharmaceutical potential of rapalogs and the utility of glycerol as a co-solvent in drug delivery applications were critical in deciding the system to be studied. Consolidation of existing studies on rapamycin modification was first performed to strategically design a new rapalog called T1. The results from 100-ns dual-boost Gaussian accelerated molecular dynamics simulations showed that protein stability was induced in the presence of glycerol. Reweighting of the trajectories revealed that the glycerol-rich solvent system lowers the energy barrier in the conformational space of the protein while also preserving native contacts between the ligand and the residues in the binding site. Calculated binding free energies using MM/GBSA also showed that electrostatic energy and polar contribution of solvation energy are heavily influenced by the changes in solvation. Glycerol molecules are preferentially excluded through electrostatic interactions from the solvation shell which induce complex stability as seen in existing experiments. Hence, using glycerol as a co-solvent in rapamycin delivery has a significant role in maintaining stability. In addition, compound T1 is a potential mTORC1-selective inhibitor with strong affinity for the FKBP12-FRB complex. This study aims to provide insights on the design of new rapalogs, and the applicability of glycerol as co-solvent for FKBP12-rapalog-FRB complexes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The expression of the iron regulatory hormone hepcidin in hepatocytes is regulated by the BMP-SMAD pathway through the type I receptors ALK2 and ALK3, the type II receptors ACVR2A and BMPR2, and the ligands BMP2 and BMP6. We previously identified the immunophilin FKBP12 as a new hepcidin inhibitor that acts by blocking ALK2. Both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug Tacrolimus (TAC) displace FKBP12 from ALK2 and activate the signaling. However, the molecular mechanism whereby FKBP12 regulates BMP-SMAD pathway activity and thus hepcidin expression remains unclear. Here, we show that FKBP12 acts by modulating BMP receptor interactions and ligand responsiveness. We first demonstrate that in primary murine hepatocytes TAC regulates hepcidin expression exclusively via FKBP12. Downregulation of the BMP receptors reveals that ALK2, to a lesser extent ALK3, and ACVR2A are required for hepcidin upregulation in response to both BMP6 and TAC. Mechanistically, TAC and BMP6 increase ALK2 homo-oligomerization and ALK2-ALK3 hetero-oligomerization and the interaction between ALK2 and the type II receptors. By acting on the same receptors, TAC and BMP6 cooperate in BMP pathway activation and hepcidin expression both in vitro and in vivo. Interestingly, the activation state of ALK3 modulates its interaction with FKBP12, which may explain the cell-specific activity of FKBP12. Overall, our results identify the mechanism whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression in hepatocytes, and suggest that FKBP12-ALK2 interaction is a potential pharmacologic target in disorders caused by defective BMP-SMAD signaling and characterized by low hepcidin and high BMP6 expression., (© 2023 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Background : Solute Carrier Family 3 Member 2 (SLC3A2) is a member of the solute carrier family that plays pivotal roles in regulation of intracellular calcium levels and transports L-type amino acids. However, there are insufficient scientific researches on the prognostic and immunological roles of SLC3A2 in breast cancer (BC) and whether everolimus regulates novel SLC3A2 related molecular mechanism in the immuno-oncology context of the tumor microenvironment (TME), therefore, we see a necessity to conduct the current in silico and biological experimental study. Methods : Using diverse online databases, we investigated the role of SLC3A2 in therapy response, clinicopathological characteristics, tumor immune infiltration, genetic alteration, methylation and single cell sequencing in BC. WB, Co-IP, cell proliferation assay, Edu staining, ROS and GSH assay and in vivo tumor xenograft assays were performed to verify FKBP1A/SLC3A2 axis in everolimus inducing ferroptosis of breast cancer. Co-cultures and IL-9 ELISA were performed to demonstrate the T lymphocyte function. Results : We demonstrated that SLC3A2 was aberrantly expressed among various BC cohorts. Our results also suggested that SLC3A2 expression was associated with chemotherapeutic outcome in BC patients. Our results further indicated that SLC3A2 was associated with tumor infiltration of cytotoxic T cell but not other immune cells among BC TME. The alterations in SLC3A2 gene had a significant correlation to relapse free survival and contributed a significant impact on BC tumor mutational burden. Finally, SLC3A2 was illustrated to be expressed in diverse BC cellular populations at single cell level, and negatively linked to angiogenesis, inflammation and quiescence, but positively correlated with other functional phenotypes. Noteworthily, everolimus (a targeted therapy drug for BC) related protein, FK506-binding protein 1A (FKBP1A) was found to bind with SLC3A2, and negatively regulated SLC3A2 expression during the processes of everolimus inducing ferroptosis of BC cells and promoting anti-proliferation of Th9 lymphocytes. Conclusions : Altogether, our study strongly implies that SLC3A2 is an immuno-oncogenic factor and FKBP1A/SLC3A2 axis would provide insights for a novel immunotherapy approach for the treatment of BC in the context of TME., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest., Competing Interests: Declaration of interests PSL valorisation has submitted a patent related to the J-DNA (PCT FR2018/053533) with D.K., T.S., and C.G. among the inventors., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cardiac ryanodine receptors (RyR2) release the Ca 2+ from intracellular stores that is essential for cardiac myocyte contraction. The ion channel opening is tightly regulated by intracellular factors, including the FK506 binding proteins, FKBP12 and FKBP12.6. The impact of these proteins on RyR2 activity and cardiac contraction is debated, with often apparently contradictory experimental results, particularly for FKBP12. The isoform that regulates RyR2 has generally been considered to be FKBP12.6, despite the fact that FKBP12 is the major isoform associated with RyR2 in some species and is bound in similar proportions to FKBP12.6 in others, including sheep and humans. Here, we show time- and concentration-dependent effects of adding FKBP12 to RyR2 channels that were partly depleted of FKBP12/12.6 during isolation. The added FKBP12 displaced most remaining endogenous FKBP12/12.6. The results suggest that FKBP12 activates RyR2 with high affinity and inhibits RyR2 with lower affinity, consistent with a model of negative cooperativity in FKBP12 binding to each of the four subunits in the RyR tetramer. The easy dissociation of some FKBP12/12.6 could dynamically alter RyR2 activity in response to changes in in vivo regulatory factors, indicating a significant role for FKBP12/12.6 in Ca 2+ signalling and cardiac function in healthy and diseased hearts. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

We examine whether calcineurin or protein phosphatase 2B (PP2B) regulates the basolateral inwardly rectifying potassium channel Kir4.1/Kir5.1 in the distal convoluted tubule (DCT). Application of tacrolimus (FK506) or cyclosporine A (CsA) increased whole-cell Kir4.1/Kir5.1-mediated K+ currents and hyperpolarized the DCT membrane. Moreover, FK506-induced stimulation of Kir4.1/Kir5.1 was absent in kidney tubule-specific 12 kDa FK506-binding protein-knockout mice (Ks-FKBP-12-KO). In contrast, CsA stimulated Kir4.1/Kir5.1 of the DCT in Ks-FKBP-12-KO mice, suggesting that FK506-induced stimulation of Kir4.1/Kir5.1 was due to inhibiting PP2B. Single-channel patch-clamp experiments demonstrated that FK506 or CsA stimulated the basolateral Kir4.1/Kir5.1 activity of the DCT, defined by NPo (a product of channel number and open probability). However, this effect was absent in the DCT treated with Src family protein tyrosine kinase (SFK) inhibitor or hydroxyl peroxide. Fluorescence imaging demonstrated that CsA treatment increased membrane staining intensity of Kir4.1 in the DCT of Kcnj10fl/fl mice. Moreover, CsA treatment had no obvious effect on phosphorylated NaCl cotransporter (pNCC) expression in Ks-Kir4.1-KO mice. Immunoblotting showed acute FK506 treatment increased pNCC expression in Kcnj10fl/fl mice, but this effect was attenuated in Ks-Kir4.1-KO mice. In vivo measurement of thiazide-induced renal Na+ excretion demonstrated that FK506 enhanced thiazide-induced natriuresis. This effect was absent in Ks-FKBP-12-KO mice and blunted in Ks-Kir4.1-KO mice. We conclude that inhibition of PP2B stimulates Kir4.1/Kir5.1 of the DCT and NCC and that PP2B inhibition-induced stimulation of NCC is partially achieved by stimulation of the basolateral Kir4.1/Kir5.1.

Alzheimer's disease and related tauopathies are characterized by the pathogenic misfolding and aggregation of the microtubule-associated protein tau. Understanding how endogenous chaperones modulate tau misfolding could guide future therapies. Here, we show that the immunophilin FKBP12, the 12-kDa FK506-binding protein (also known as FKBP prolyl isomerase 1A), regulates the neuronal resilience by chaperoning a specific structure in monomeric tau. Using a combination of mouse and cell experiments, in vitro aggregation experiments, nuclear magnetic resonance-based structural analysis of monomeric tau, site-specific phosphorylation and mutation, as well as structure-based analysis using the neural network-based structure prediction program AlphaFold, we define the molecular factors that govern the binding of FKBP12 to tau and its influence on tau-induced neurotoxicity. We further demonstrate that tyrosine phosphorylation of tau blocks the binding of FKBP12 to two highly specific structural motifs in tau. Our data together with previous results demonstrating FKBP12/tau colocalization in neurons and neurofibrillary tangles support a critical role of FKBP12 in regulating tau pathology.

Light is a particularly important environmental cue that regulates a variety of diverse plant developmental processes, such as photomorphogenesis. Blue light promotes photomorphogenesis mainly through the activation of the photoreceptor cryptochrome 1 (CRY1). However, the mechanism underlying the CRY1-mediated regulation of growth is not fully understood. Here, we found that blue light induced N 6 -methyladenosine (m 6 A) RNA modification during photomorphogenesis partially via CRY1. Cryptochrome 1 mediates blue light-induced expression of FKBP12-interacting protein 37 (FIP37), which is a component of m 6 A writer. Moreover, we showed that CRY1 physically interacted with FIP37 in vitro and in vivo, and mediated blue light activation of FIP37 binding to RNA. Furthermore, CRY1 and FIP37 modulated m 6 A on photomorphogenesis-related genes PIF3, PIF4, and PIF5, thereby accelerating the decay of their transcripts. Genetically, FIP37 repressed hypocotyl elongation under blue light, and fip37 mutation could partially rescue the short-hypocotyl phenotype of CRY1-overexpressing plants. Together, our results provide a new insight into CRY1 signal in modulating m 6 A methylation and stability of PIFs, and establish an essential molecular link between m 6 A modification and determination of photomorphogenesis in plants., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

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., Competing Interests: D.A.H. has received grant support (paid to the Erasmus MC) from Astellas Pharma and Chiesi Pharma and consulting fees from Astellas Pharma, Chiesi Pharma, MedinCell, Novartis Pharma and Sangamo Therapeutics. The other authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The immunophilin FKBP12 binds to TGF-β family type I receptors, including the BMP type I receptor ALK2. FKBP12 keeps the type I receptor in an inactive state and controls signaling activity. Removal of FKBP12 with drugs such as the FKBP-ligand FK506 enhances BMP activity in various cell types. In multiple myeloma cells, activation of SMAD1/5/8 leads to apoptosis. We hypothesized that removing FKBP12 from ALK2 in myeloma cells would potentiate BMP-induced ALK2-SMAD1/5/8 activity and in consequence cell death., Methods: Multiple myeloma cell lines were treated with FK506, or other FKBP-binding compounds, combined with different BMPs before analyzing SMAD1/5/8 activity and cell viability. SMAD1/5/8 activity was also investigated using a reporter cell line, INA-6 BRE-luc. To characterize the functional signaling receptor complex, we genetically manipulated receptor expression by siRNA, shRNA and CRISPR/Cas9 technology., Results: FK506 potentiated BMP-induced SMAD1/5/8 activation and apoptosis in multiple myeloma cell lines. By using FKBP-binding compounds with different affinity profiles, and siRNA targeting FKBP12, we show that the FK506 effect is mediated by binding to FKBP12. Ligands that typically signal via ALK3 in myeloma cells, BMP2, BMP4, and BMP10, did not induce apoptosis in cells lacking ALK3. Notably, BMP10 competed with BMP6 and BMP9 and antagonized their activity via ALK2. However, upon addition of FK506, we saw a surprising shift in specificity, as the ALK3 ligands gained the ability to signal via ALK2 and induce apoptosis. This indicates that the receptor complex can switch from an inactive non-signaling complex (NSC) to an active one by adding FK506. This gain of activity was also seen in other cell types, indicating that the observed effects have broader relevance. BMP2, BMP4 and BMP10 depended on BMPR2 as type II receptor to signal, which contrasts with BMP6 and BMP9, that activate ALK2 more potently when BMPR2 is knocked down., Conclusions: In summary, our data suggest that FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells, partly by switching an NSC into an active signaling complex. FKBP12 targeting compounds devoid of immunosuppressing activity could have potential in novel treatment strategies aiming at reducing multiple myeloma tumor load. Video Abstract., (© 2023. The Author(s).)

Proteolysis-targeting chimeras (PROTACs) bring a protein of interest (POI) into spatial proximity of an E3 ubiquitin ligase, promoting POI ubiquitylation and proteasomal degradation. PROTACs rely on endogenous cellular machinery to mediate POI degradation, therefore the subcellular location of the POI and access to the E3 ligase being recruited potentially impacts PROTAC efficacy. To interrogate whether the subcellular context of the POI influences PROTAC-mediated degradation, we expressed either Halo or FKBP12 F36V (dTAG) constructs consisting of varying localization signals and tested the efficacy of their degradation by von Hippel-Lindau (VHL)- or cereblon (CRBN)-recruiting PROTACs targeting either Halo or dTAG. POIs were localized to the nucleus, cytoplasm, outer mitochondrial membrane, endoplasmic reticulum, Golgi, peroxisome or lysosome. Differentially localized Halo or FKBP12 F36V proteins displayed varying levels of degradation using the same respective PROTACs, suggesting therefore that the subcellular context of the POI can influence the efficacy of PROTAC-mediated POI degradation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mammalian target of rapamycin (mTOR) is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism, and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in Saccharomyces cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a rapamycin-induced developmental delay in Drosophila .

On-target-off-tissue drug engagement is an important source of adverse effects that constrains the therapeutic window of drug candidates 1,2 . In diseases of the central nervous system, drugs with brain-restricted pharmacology are highly desirable. Here we report a strategy to achieve inhibition of mammalian target of rapamycin (mTOR) while sparing mTOR activity elsewhere through the use of the brain-permeable mTOR inhibitor RapaLink-1 and the brain-impermeable FKBP12 ligand RapaBlock. We show that this drug combination mitigates the systemic effects of mTOR inhibitors but retains the efficacy of RapaLink-1 in glioblastoma xenografts. We further present a general method to design cell-permeable, FKBP12-dependent kinase inhibitors from known drug scaffolds. These inhibitors are sensitive to deactivation by RapaBlock, enabling the brain-restricted inhibition of their respective kinase targets., (© 2022. The Author(s).)

Calcineurin is an essential virulence factor that is conserved across human fungal pathogens, including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals, and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a nonconserved region of FKBP12 that can be exploited for fungus-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C-22 position, which was less immunosuppressive yet maintained antifungal activity. We now report high-resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C-22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05, demonstrates broad-spectrum antifungal activity in vitro and is nonimmunosuppressive in vivo . In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside the C-22 and C-21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. IMPORTANCE Due to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin-bound inhibitor complexes has enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound with increased fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and maintained therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.

Intracellular peptides (InPeps) generated by proteasomes were previously suggested as putative natural regulators of protein-protein interactions (PPI). Here, the main aim was to investigate the intracellular effects of intracellular peptide VFDVELL (VFD7) and related peptides on PPI. The internalization of the peptides was achieved using a C-terminus covalently bound cell-penetrating peptide (cpp; YGRKKRRQRRR). The possible inhibition of PPI was investigated using a NanoBiT ® luciferase structural complementation reporter system, with a pair of plasmids vectors each encoding, simultaneously, either FK506-binding protein (FKBP) or FKBP-binding domain (FRB) of mechanistic target of rapamycin complex 1 (mTORC1). The interaction of FKBP-FRB within cells occurs under rapamycin induction. Results shown that rapamycin-induced interaction between FKBP-FRB within human embryonic kidney 293 (HEK293) cells was inhibited by VFD7-cpp (10-500 nM) and FDVELLYGRKKRRQRRR (VFD6-cpp; 1-500 nM); additional VFD7-cpp derivatives were either less or not effective in inhibiting FKBP-FRB interaction induced by rapamycin. Molecular dynamics simulations suggested that selected peptides, such as VFD7-cpp, VFD6-cpp, VFAVELLYGRKKKRRQRRR (VFA7-cpp), and VFEVELLYGRKKKRRQRRR (VFA7-cpp), bind to FKBP and to FRB protein surfaces. However, only VFD7-cpp and VFD6-cpp induced changes on FKBP structure, which could help with understanding their mechanism of PPI inhibition. InPeps extracted from HEK293 cells were found mainly associated with macromolecular components (i.e., proteins and/or nucleic acids), contributing to understanding InPeps' intracellular proteolytic stability and mechanism of action-inhibiting PPI within cells. In a model of cell death induced by hypoxia-reoxygenation, VFD6-cpp (1 µM) increased the viability of mouse embryonic fibroblasts cells (MEF) expressing mTORC1-regulated autophagy-related gene 5 (Atg5), but not in autophagy-deficient MEF cells lacking the expression of Atg5. These data suggest that VFD6-cpp could have therapeutic applications reducing undesired side effects of rapamycin long-term treatments. In summary, the present report provides further evidence that InPeps have biological significance and could be valuable tools for the rational design of therapeutic molecules targeting intracellular PPI.

Anchor away is a sequestering method designed to acutely and timely abrogate the function of a protein of interest by anchoring to a cell compartment different from its target. This method induces the binding of the target protein to the anchor by either the addition of rapamycin to Drosophila food or cell media. Rapamycin mediates the formation of a ternary complex between the anchor, which is tagged with the FK506-binding protein (FKBP12), and the target protein fused with the FKB12 rapamycin-binding (FRB) domain of mammalian target of rapamycin (mTOR). The rapamycin-bound target protein stays sequestered away from its compartment, where it cannot perform its biological function., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein-protein interactions are central to most cellular processes and their dysregulation has been related to the development of various diseases. Proximity-based labeling methods are used to identify the endogenous interaction partners of specific proteins of interest (POIs). The POI is fused to promiscuous enzymes, which generate reactive species in vivo and label proteins in close vicinity. APEX-based proximity labeling techniques utilize an engineered ascorbate peroxidase, which in the presence of H 2 O 2 oxidizes biotin-phenol to short lived biotin-phenoxyl radicals that biotinylate nearby proteins. The biotinylated proteins are enriched by biotin affinity capture and identified by mass spectrometry. We devised an advanced method, RAPIDS, in which the peroxidase is physically separated from the POI and only a rapamycin-induced dimerization using the FRB-FKBP12 system brings the two proteins together. RAPIDS improves the specificity of APEX-based interactome analysis by strictly eliminating false positives. In this chapter, we describe this method in detail, with VAPB as a protein of interest and versions of APEX2 with different subcellular localizations. VAPB localizing to different cellular compartments, the endoplasmic reticulum and the inner nuclear membrane, yielded distinct sets of proximity partners as identified by RAPIDS., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides , and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C 15 , C 16 , C 18 , C 36 , and C 37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C 15 to C 18 and C 36 to C 37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

Bone morphogenetic protein (BMP) signaling contributes to acute kidney injury (AKI), a common disease without adequate treatment options. In this issue of Cell Chemical Biology, Larraufie, Gao and colleagues (Larraufie et al., 2021) show that FKBP12 inhibition can be a target for treatment of AKI via activation of BMP signaling., Competing Interests: Declaration of interests F.H. is an inventor and owner on patents covering FKBP ligands (US9914732B2, EP2877470B1, EP 3 097 103 B1, US 9 371 322 B2), including ligands for FKBP12. A follow-up patent application on FKBP ligands has been filed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The hypothesis of rescuing FKBP12/RyR1 interaction and intracellular calcium homeostasis through molecular "reshaping" of FKBP12 was investigated. To this end, novel 4-arylthioalkyl-1-carboxyalkyl-1,2,3-triazoles were designed and synthesized, and their efficacy was tested in human myotubes. A library of 17 compounds (10a-n) designed to dock the FKBP12/RyR1 hot-spot interface contact residues, was readily prepared from free α-amino acids and arylthioalkynes using CuAAC "click" protocols amenable to one-pot transformations in high overall yields and total configurational integrity. To model nitro-oxidative stress, human myotubes were treated with the peroxynitrite donor SIN1, and evidence was found that some triazoles 10 were able to normalize calcium levels, as well as FKBP12/RyR1 interaction. For example, compound 10 b at 150 nM rescued 46% of FKBP12/RyR1 interaction and up to 70% of resting cytosolic calcium levels in human myotubes under nitro-oxidative stress. All compounds 10 analyzed showed target engagement to FKBP12 and low levels of cytotoxicity in vitro. Compounds 10b, 10c, 10h, and 10iR were identified as potential therapeutic candidates to protect myotubes in muscle disorders with underlying nitro-oxidative stress, FKBP12/RyR1 dysfunction and calcium dysregulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Computer aided drug-design methods proved to be powerful tools for the identification of new therapeutic agents. We employed a structure-based workflow to identify new inhibitors targeting mTOR kinase at rapamycin binding site. By combining molecular dynamics (MD) simulation and pharmacophore modelling, a simplified structure-based pharmacophore hypothesis was built starting from the FKBP12-rapamycin-FRB ternary complex retrieved from RCSB Protein Data Bank (PDB code 1FAP). Then, the obtained model was used as filter to screen the ZINC biogenic compounds library, containing molecules derived from natural sources or natural-inspired compounds. The resulting hits were clustered according to their similarity; moreover, compounds showing the highest pharmacophore fit-score were chosen from each cluster. The selected molecules were subjected to docking studies to clarify their putative binding mode. The binding free energy of the obtained complexes was calculated by MM/GBSA method and the hits characterized by the lowest ΔG bind values were identified as potential mTOR inhibitors. Furthermore, the stability of the resulting complexes was studied by means of MD simulation which revealed that the selected compounds were able to form a stable ternary complex with FKBP12 and FRB domain, thus underlining their potential ability to inhibit mTOR with a rapamycin-like mechanism.

Post-transcriptional regulation of ATP-binding cassette (ABC) proteins has been so far shown to encompass protein phosphorylation, maturation, and ubiquitination. Yet, recent accumulating evidence implicates FK506-binding proteins (FKBPs), a type of peptidylprolyl cis-trans isomerase (PPIase) proteins, in ABC transporter regulation. In this perspective article, we summarize current knowledge on ABC transporter regulation by FKBPs, which seems to be conserved over kingdoms and ABC subfamilies. We uncover striking functional similarities but also differences between regulatory FKBP-ABC modules in plants and mammals. We dissect a PPIase- and HSP90-dependent and independent impact of FKBPs on ABC biogenesis and transport activity. We propose and discuss a putative new mode of transient ABC transporter regulation by cis-trans isomerization of X-prolyl bonds., (© 2020 Federation of European Biochemical Societies.)

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAG V -1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12 F36V -tagged proteins. dTAG V -1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12., Competing Interests: Competing interest statement: U.K.S., S.-J.L., M.E.S., B.R.B., K.R., M.Z., K.H.P., D.T.S., J.A.V.B., D.W.U., A.T.R., A.S.M., S.M., Z.W., M.S., K.K., J.P.M., E.P., D.C.G., R.M.P., S.A.T., and G.L.V. were employees of Warp Drive Bio, which has since become a wholly owned subsidiary of Revolution Medicines, Inc. G.L.V., R.D.K., and B.R.B. are minority shareholders in Revolution Medicines, Inc. R.D.K. is an employee of Lyell Immunopharma. U.K.S. is an employee of LifeMine Therapeutics. S.-J.L. is an employee of Beam Therapeutics. M.E.S. is an employee of C4 Therapeutics. B.R.B. is an employee of Inzen Therapeutics. K.R., D.T.S., K.K., J.P.M., and D.C.G. are employees of Ginko Bioworks. M.Z. is an employee of Kronos Bio. K.H.P. is an employee of A*STAR. A.S.M. is an employee of Agios Pharmaceuticals. S.M. is an employee of Morphic Therapeutic. Z.W. is an employee of Blueprint Medicines. M.S. is an employee of Bristol-Myers Squibb. E.P. is an employee of Amgen. S.A.T. is an employee of Kymera Therapeutics. G.L.V. is an employee of FOG Pharmaceuticals, Inc. and LifeMine Therapeutics., (Copyright © 2020 the Author(s). Published by PNAS.)

A series of fluorescent thiazole-pyrazoline derivatives was synthesized and their structures were characterized by 1 H NMR, 13 C NMR, and HRMS. Biological evaluation demonstrated that these compounds could effectively inhibit the growth of human non-small cell lung cancer (NSCLC) A549 cells in a dose- and time-dependent manner in vitro and inhibit tumor growth in vivo. The structure-activity relationship (SAR) of the compounds was analyzed. Further mechanism research revealed they could induce autophagy and cell cycle arrest while had no influence on cell necrosis. Compound 5e inhibited the activity of mTOR via FKBP12, which could be reversed by 3BDO, an mTOR activator and autophagy inhibitor. Compound 5e inhibited growth, promoted autophagy of A549 cells in vivo. Moreover, compound 5e showed good selectivity with no influence on normal vascular endothelial cell growth and the normal chick embryo chorioallantoic membrane (CAM) capillary formation. Therefore, our research provides potential lead compounds for the development of new anticancer drugs against human lung cancer.

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80's loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40's loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40's and 50's loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80's loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo., Competing Interests: Declaration of competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background/aims: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells., Methods: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern., Results: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15 P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172 P-Cdk4/Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors increased the protein expression of FKBP12 and FKBP51 that appeared to play pro-survival role. Interestingly, the administration of immunosuppressants yields a specific pattern of miRNAs. Tacrolimus and mTOR inhibitors decreased miR-92a-1-5p, miR-197-3p, miR-483-3p and miR-720, and increased miR-22-3p, miR-376a-3p, miR-663b, miR-886-5p, miR-1300 and miR-1303 expressions in HepG2 cells., Conclusion: The more potent pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with an increased activation of PERK and p53 signaling, and p21 protein expression. FKBP12 and FKBP51 appeared to be the most relevant partners of Tacrolimus and mTOR inhibitors exerting a pro-survival effect in HepG2 cells. The observed effects of immunosuppressants were related to a specific miRNA signature in liver cancer cells., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12-rapamycin binding domain (cpFRB). By permuting the topology of the four helices within FRB, we have created cpFRB-FKBP pairs that respond to ligand with varying activation kinetics and dynamics. The cpRAPID system enables chemical-controllable subcellular redistribution of proteins, as well as inducible transcriptional activation when coupled with the CRISPR activation (CRISPRa) technology to induce a GFP reporter and endogenous gene expression. We have further demonstrated the use of cpRAPID to generate chemically switchable split nanobody (designated Chessbody) for ligand-gated antigen recognition in living cells. Collectively, the circular permutation approach offers a powerful means for diversifying the chemogenetics toolbox to benefit the burgeoning synthetic biology field., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The inducible activation system is valuable for investigating spatiotemporal roles of molecules. A chemically inducible activation system for Fas (CD95/APO-1), which works efficiently to induce apoptosis and leads non-apoptotic pathways, has not yet been developed. Here, we engineered a rapamycin-induced dimerization system of Fas consisting of FKBP and FRB proteins. Treatment of rapamycin specifically induces cellular apoptosis. In neurons and cells with high c-FLIP expression, rapamycin-induced Fas activation triggered the activation of the non-apoptotic pathway components instead of cell death. Intracranial delivery of the system could be utilized to induce apoptosis of tumor cells upon rapamycin treatment. Our results demonstrate a novel inducible Fas activation system which operates with high efficiency and temporal precision in vitro and in vivo promising a potential therapeutic strategy., Competing Interests: Declaration of competing interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

PROTACs (PROteolysis TArgeting Chimeras) are bifunctional molecules that target proteins for ubiquitylation by an E3 ligase complex and subsequent degradation by the proteasome. They have emerged as powerful tools to control the levels of specific cellular proteins. We now introduce photoswitchable PROTACs that can be activated with the spatiotemporal precision that light provides. These trifunctional molecules, which we named PHOTACs (PHOtochemically TArgeting Chimeras), consist of a ligand for an E3 ligase, a photoswitch, and a ligand for a protein of interest. We demonstrate this concept by using PHOTACs that target either BET family proteins (BRD2,3,4) or FKBP12. Our lead compounds display little or no activity in the dark but can be reversibly activated with different wavelengths of light. Our modular approach provides a method for the optical control of protein levels with photopharmacology and could lead to new types of precision therapeutics that avoid undesired systemic toxicity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Augmenting live cells with new signal transduction capabilities is a key objective in genetic engineering and synthetic biology. We showed earlier that two-component signaling pathways could function in mammalian cells, albeit while losing their ligand sensitivity. Here, we show how to transduce small-molecule ligands in a dose-dependent fashion into gene expression in mammalian cells using two-component signaling machinery. First, we engineer mutually complementing truncated mutants of a histidine kinase unable to dimerize and phosphorylate the response regulator. Next, we fuse these mutants to protein domains capable of ligand-induced dimerization, which restores the phosphoryl transfer in a ligand-dependent manner. Cytoplasmic ligands are transduced by facilitating mutant dimerization in the cytoplasm, while extracellular ligands trigger dimerization at the inner side of a plasma membrane. These findings point to the potential of two-component regulatory systems as enabling tools for orthogonal signaling pathways in mammalian cells.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs used to prevent graft rejection after organ transplant. Common side effects include renal magnesium wasting and hypomagnesemia, which may contribute to new-onset diabetes mellitus, and hypercalciuria, which may contribute to post-transplant osteoporosis. Previous work suggested that CNIs reduce the abundance of key divalent cation transport proteins, expressed along the distal convoluted tubule, causing renal magnesium and calcium wasting. It has not been clear, however, whether these effects are specific for the distal convoluted tubule, and whether these represent off-target toxic drug effects, or result from inhibition of calcineurin. The CNI tacrolimus can inhibit calcineurin only when it binds with the immunophilin, FKBP12; we previously generated mice in which FKBP12 could be deleted along the nephron, to test whether calcineurin inhibition is involved, these mice are normal at baseline. Here, we confirmed that tacrolimus-treated control mice developed hypomagnesemia and urinary calcium wasting, with decreased protein and mRNA abundance of key magnesium and calcium transport proteins (NCX-1 and Calbindin-D 28k ). However, qPCR also showed decreased mRNA expression of NCX-1 and Calbindin-D 28k , and TRPM6. In contrast, KS-FKBP12 -/- mice treated with tacrolimus were completely protected from these effects. These results indicate that tacrolimus affects calcium and magnesium transport along the distal convoluted tubule and strongly suggests that inhibition of the phosphatase, calcineurin, is directly involved., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.