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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

Activation of PPAR alpha by fenofibrate inhibits apoptosis in vascular adventitial fibroblasts partly through SIRT1-mediated deacetylation of FoxO1

Recent studies demonstrated that the ligand-activated transcription factor peroxisome proliferator-activated receptorα (PPARα) acts in association with histone deacetylase sirtuin 1 (SIRT1) in the regulation of metabolism and inflammation involved in cardiovascular diseases. PPARα activation also participates in the modulation of cell apoptosis. Our previous study found that SIRT1 inhibits the apoptosis of vascular adventitial fibroblasts (VAFs). However, whether the role of PPARα in apoptosis of VAFs is mediated by SIRT1 remains unknown. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on cell apoptosis and SIRT1 expression and related mechanisms in ApoE{sup −/−} mice and VAFs in vitro. We found that fenofibrate inhibited cell apoptosis in vascular adventitia and up-regulated SIRT1 expression in aorta of ApoE{sup −/−} mice. Moreover, SIRT1 activator resveratrol (RSV) further enhanced these effects of fenofibrate. In vitro study showed that activation of PPARα by fenofibrate inhibited TNF-α-induced cell apoptosis and cell cycle arrest in VAFs. Meanwhile, fenofibrate up-regulated SIRT1 expression and inhibited SIRT1 translocation from nucleus to cytoplasm in VAFs stimulated with TNF-α. Moreover, the effects of fenofibrate on cell apoptosis and SIRT1 expression in VAFs were reversed by PPARα antagonist GW6471. Importantly, treatment of VAFs with SIRT1 siRNA or pcDNA3.1(+)-SIRT1 showed that the inhibitory effect of fenofibrate on cell apoptosis in VAFs through SIRT1. On the other hand, knockdown of FoxO1 decreased cell apoptosis of VAFs compared with fenofibrate group. Overexpression of FoxO1 increased cell apoptosis of VAFs compared with fenofibrate group. Further study found that fenofibrate decreased the expression of acetylated-FoxO1 in TNF-α-stimulated VAFs, which was abolished by SIRT1 knockdown. Taken together, these findings indicate that activation of PPARα by fenofibrate inhibits cell apoptosis in VAFs partly through the SIRT1-mediated deacetylation of FoxO1. - Highlights: • Fenofibrate inhibits apoptosis and increases SIRT1 expression in vivo and in vitro. • Fenofibrate inhibits cell apoptosis in VAFs through SIRT1. • Fenofibrate inhibits apoptosis through SIRT1-mediated deacetylation of FoxO1. • Fenofibrate inhibits apoptosis and increases SIRT1 expression via PPARα activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts

Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing, immune responses and invasive tumors all require the orchestrated movement of cells to specific locations. Histone demethylase proteins alter transcription by regulating the chromatin state at specific gene loci. FBXL10 is a conserved and ubiquitously expressed member of the JmjC domain-containing histone demethylase family and is implicated in the demethylation of H3K4me3 and H3K36me2 and thereby removing active chromatin marks. However, the physiological role of FBXL10 in vivo remains largely unknown. Therefore, we established an inducible gain of function model to analyze the role of Fbxl10 and compared wild-type with Fbxl10 overexpressing mouse embryonic fibroblasts (MEFs). Our study shows that overexpression of Fbxl10 in MEFs doesn’t influence the proliferation capability but leads to an enhanced migration capacity in comparison to wild-type MEFs. Transcriptome and ChIP-seq experiments demonstrated that Fbxl10 binds to genes involved in migration like Areg, Mdk, Lmnb1, Thbs1, Mgp and Cxcl12. Taken together, our results strongly suggest that Fbxl10 plays a critical role in migration by binding to the promoter region of migration-associated genes and thereby might influences cell behaviour to a possibly more aggressive phenotype. - Highlights: • Migration capability of MEFs is enhanced after Fbxl10 upregulation. • Overexpression of Fbxl10 induced migration-associated genes. • Fbxl10 binds directly to migration-associated genes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Glucocorticoids indirectly decrease colon cancer cell proliferation and invasion via effects on cancer-associated fibroblasts

Highlights: • Indirect effects of glucocorticoid treatment on cancer cells via CAFs are described. • Glucocorticoids decrease expression of MMP-2 by CAFs. • Glucocorticoids neutralize growth-promoting and pro-invasive effects of CAFs. • In vivo, glucocorticoid treatment inhibits tumor progression. Cancer-associated fibroblasts (CAFs) support cancer growth, invasion, and metastasis. Glucocorticoids (GCs), drugs often administered together with chemotherapy, are steroidal ligands of the glucocorticoid receptor (GR), a transcription factor which upon activation regulates expression of multiple genes involved in suppression of inflammation. We have previously shown that in dexamethasone (Dex)-treated CAFs derived from colon cancer, production and secretion of several factors related to cancer progression, such as tenascin C (TNC) and hepatocyte growth factor (HGF), were strongly suppressed. In this study we show that GCs can neutralize the cancer cell-promoting properties of CAFs. Conditioned medium from solvent-treated CAFs (CM{sup CTRL}) stimulates proliferation, motility and stretched morphotype of GR-deficient HCT8/E11 colon cancer cells. Yet, HCT8/E11 proliferation and stretched morphotype are impaired upon treatment with conditioned medium from Dex-treated CAFs (CM{sup DEX}), but HCT8/E11 cell migration is slightly increased under these conditions. Moreover, expression and potential activity of MMP-2 is also reduced in CM{sup DEX} compared with CM{sup CTRL}. These combined in vitro results concur with the results from in vivo chick chorioallantoic membrane assays, where the co-cultures of CAFs with colon cancer cells displayed impaired tumor formation and cancer cell invasion due to Dex administration. Combined, GC treatment influences cancer cell behavior indirectly through effects on CAFs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

LncRNA HOTTIP from synovial fibroblast-derived exosomes: A novel molecular target for rheumatoid arthritis through the miR-1908–5p/STAT3 axis

Rheumatoid arthritis (RA) is a chronic inflammation mediated by autoimmune responses. HOTTIP, a long noncoding RNA (lncRNA), participates in cell proliferation and invasion. However, the correlation between HOTTIP and RA remains unclear. Therefore, this study aimed to clarify how HOTTIP works in RA and to investigate its role in the development of RA. Flow cytometry was used to analyze cell cycle progression. Binding between HOTTIP, signal transducer and activator of transcription 3 (STAT3) and miR-1908–5p was demonstrated by dual-luciferase assays. Quantitative real-time polymerase chain reaction (qRT–PCR) was used to measure the expression of T cell differentiation-related proteins. We found that HOTTIP was upregulated in rheumatoid arthritis synovial fibroblasts (RASFs). HOTTIP directly bound to miR-1908–5p and negatively modulated miR-1908–5p expression while positively regulating STAT3. The effects of HOTTIP overexpression on regulating the balance of the Th17/Treg cell ratio were partly reversed by miR-1908–5p overexpression. In addition, in vivo experiments demonstrated that overexpression of HOTTIP aggravated inflammation in RA mice, which was demonstrated by hematoxylin and eosin (HE) staining and the increased expression levels of CD4{sup +} interleukin (IL)-17{sup +}, forkhead Box P3 (FOXP3) and retinoid-related orphan receptor gamma-t (RORγt). In summary, our study suggests that HOTTIP plays a damaging role in RA by promoting inflammation, which may be related to the regulation of miR-1908–5p expression and the STAT3 signaling pathway. These results suggest that the regulation of HOTTIP may be a promising therapeutic strategy for RA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Mimicking the Bioactivity of Fibroblast Growth Factor-2 Using Supramolecular Nanoribbons

Fibroblast growth factor (FGF-2) is a multifunctional growth factor that has pleiotropic effects in different tissues and organs. In particular, FGF-2 has a special role in angiogenesis, an important process in development, wound healing, cell survival, and differentiation. Therefore, incorporating biological agents like FGF-2 within therapeutic biomaterials is a potential strategy to create angiogenic bioactivity for the repair of damaged tissue caused by trauma or complications that arise from age and/or disease. However, the use of growth factors as therapeutic agents can be costly and does not always bring about efficient tissue repair due to rapid clearance from the targeted site. An alternative would be a stable supramolecular nanostructure with the capacity to activate the FGF-2 receptor that can also assemble into a scaffold deliverable to tissue. We report here on peptide amphiphiles that incorporate a peptide known to activate the FGF-2 receptor and peptide domains that drive its self-assembly into supramolecular nanoribbons. These FGF2-PA nanoribbons displayed the ability to increase the proliferation and migration of the human umbilical vein endothelial cells (HUVECs) in vitro to the same extent as the native FGF-2 protein at certain concentrations. We confirmed that this activity was specific to the FGFR1 signaling pathway by tracking the phosphorylation of downstream signaling effectors such ERK1/2 and pH3. These results indicated the specificity of FGF2-PA nanoribbons in activating the FGF-2 signaling pathway and its potential application as a supramolecular scaffold that can be used in vivo as an alternative to the encapsulation and delivery of the native FGF-2 protein.

36 MATERIALS SCIENCE↗
DOE OSTI2012

Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

16O/18O labeling↗
DOE OSTI2013

Comparison of the biological effectiveness of 45 MeV C-ions and {gamma}-rays in inducing early and late effects in normal human primary fibroblasts

Investigation of the mechanisms underlying the biological effects induced by densely ionizing radiation has relevant implications in both radiation protection and therapy. In particular, the possible advantages of hadrontherapy with respect to conventional radiotherapy in terms of high conformal tumor treatment and sparing of healthy tissues are well known. Further improvements are limited by lack of radiobiological knowledge, particularly about the specific cellular response to the damage induced by particles of potential interest for tumor treatment. This study compares early and late effects induced in AG01522 normal human primary fibroblasts by {gamma}-rays and C-ions having E {approx} 45 MeV/u at the cell entrance, corresponding to LET (in water) {approx} 49 keV/{mu}m. Different end points have been investigated, namely: cell killing and lethal mutation, evaluated as early and delayed reproductive cell death, respectively; chromosome damage, as measured by micronuclei induction (MN); DNA damage, in terms of DSB induction and repair, as measured by the H2AX phosphorylation/dephosphorylation kinetics. Linear dose-response relationships were found for cell killing and induction of lethal mutations, with RBEs of about 1.3 and 1.6 respectively, indicating that the presence of genomic instability is greater in the progeny of C-ions irradiated cells. H2AX phosphorylation/dephosphorylation kinetics have shown a maximum foci number at 30 min after irradiation, higher for {gamma}-rays than for C-ions. However, in the first 12 h the fraction of residual {gamma}-H2AX foci was higher for C-ions irradiated cells, indicating a lower removal rate, possibly related to multiple/more complex damage along the particle track, with respect to the sparse lesions produced by {gamma}-rays. MN induction, observed after 72 h from irradiation, was also greater for C-ions. Overall, these data indicate a more severe DNA damage induced by 45 MeV/u C-ions with respect to {gamma}-rays, likely responsible of an increased cellular misrepair, leading to the greater observed levels of chromosome damage and, eventually, of genomic instability. They give strong support to the idea that higher damage severity at molecular level, determined by the typical deposition pattern of densely ionizing radiation, is the earliest relevant factor for the more severe late effects at cellular level.

61 RADIATION PROTECTION AND DOSIMETRY↗
DOE OSTI2015

Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2018

Agonistic β -Klotho antibody mimics fibroblast growth factor 21 (FGF21) functions

Fibroblast growth factor 21 (FGF21), an endocrine hormone in the FGF family, plays a critical role in regulating metabolic homeostasis and has emerged as a therapeutic target for metabolic diseases, including Type 2 diabetes mellitus. FGF21 functions through a receptor complex that consists of an FGF receptor (FGFR) and a co-receptor β-Klotho. Here, we identify and biochemically and structurally characterize 39F7, a high-affinity agonistic monoclonal antibody (mAb) against β-Klotho that mimics FGF21 function. The co-crystal structure of β-Klotho KL1 domain in complex with 39F7 Fab revealed that the recognition of 39F7 is centered on Trp-295 of β-Klotho in a FGF21 noncompetitive manner. KL1 adopts a (β/α) 8 TIM barrel fold which resembles that of β-glycosylceramidase, but lacks molecular features for enzymatic activity, suggesting that KL1 functions as a scaffold protein instead. In vitro characterization demonstrated that, although 39F7 does not compete with FGF21, it is specific for β-Klotho/FGFR1c activation. Furthermore, the agonistic activity of 39F7 required the full IgG molecule to be bivalent, suggesting that 39F7 functions by promoting receptor/co-receptor dimerization. Supported by negative stain EM analysis of full-length β-Klotho, we propose a molecular model wherein the agonistic antibody 39F7 acts in a β-Klotho– and FGFR1c-dependent manner, mimicking FGF21 activity. More importantly, 39F7 offers promising therapeutic potential in the axis of FGF21 signaling as an antibody therapy alternative to FGF21 analogs for treatment of metabolic diseases.

agonist↗
DOE OSTI2019

Probing Mechanical Characteristics of Living Fibroblasts via Atomic Force Microscopy

Atomic force microscopy (AFM) measurements have revealed hardening of intact fibroblasts after treatment with colchicine. The reliability of AFM measurements was confirmed by the identification of cells the lateral response of which to indentation did not disturb the normal force contribution. AFM data on the mechanical characteristics of such cells admit an unambiguous interpretation.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Imaging of Fibroblast Activation Protein Alpha Expression in a Preclinical Mouse Model of Glioma Using Positron Emission Tomography

Glioblastoma multiforme (GBM) is the most aggressive glioma of the primary central nervous system. Due to the lack of effective treatment options, the prognosis for patients remains bleak. Fibroblast activation protein alpha (FAP), a 170 kDa type II transmembrane serine protease was observed to be expressed on glioma cells and within the glioma tumor microenvironment. To understand the utility of targeting FAP in this tumor type, the immuno-PET radiopharmaceutical [ 89 Zr]Zr-Df-Bz-F19 mAb was prepared and Lindmo analysis was used for its in vitro evaluation using the U87MG cell line, which expresses FAP endogenously. Lindmo analysis revealed an association constant (K a ) of 10 -8 M -1 and an immunoreactivity of 52%. Biodistribution studies in U87MG tumor-bearing mice revealed increasing radiotracer retention in tumors over time, leading to average tumor-to-muscle ratios of 3.1, 7.3, 7.2, and 8.3 at 2, 24, 48 and 72 h, respectively. Small animal PET corroborated the biodistribution studies; tumor-to-muscle ratios at 2, 24, 48, and 72 h were 2.0, 5.0, 6.1 and 7.8, respectively. Autoradiography demonstrated accumulated activity throughout the interior of FAP + tumors, while sequential tumor sections stained positively for FAP expression. Conversely, FAP - tissues retained minimal radioactivity and were negative for FAP expression by immunohistochemistry. These results demonstrate FAP as a promising biomarker that may be exploited to diagnose and potentially treat GBM and other neuroepithelial cancers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2013

Low dose radiation hypersensitivity and clustered DNA damages in human fibroblasts exposed to low dose and dose rate protons or 137CS y-rays

Effective radioprotection for human space travelers hinges upon understanding the individual properties of charged particles. A significant fraction of particle radiation astronauts will encounter in space exploratory missions will come from high energy protons in galactic cosmic radiation (GCR) and/or possible exposures to lower energy proton flux from solar particle events (SPEs). These potential exposures present major concerns for NASA and others, in planning and executing long term space exploratory missions. We recently reported cell survival and transformation (acquisition of anchorage-independent growth in soft agar) frequencies in apparently normal NFF-28 primary human fibroblasts exposed to 0-30 cGy of 50MeV, 100MeV (SPE-like), or 1000 MeV (GCR-like) monoenergetic protons. These were modeled after 1989 SPE energies at an SPE-like low dose-rate (LDR) of 1.65 cGy/min or high dose rate (HDR) of 33.3 cGy/min delivered at the NASA Space Radiation Laboratory (NSRL) at BNL.

61 RADIATION PROTECTION AND DOSIMETRY↗
DOE OSTI2016

Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells. - Highlights: • Hsc70 siRNA treatment suppressed the expression of Hsc70 but induced the expression of Hsp70 in NRK-49F cells. • Hsc70 siRNA treatment suppressed the activation of Smad2/3 in the cells treated with TGF-β. • Hsc70 interacted with Smad2/3 on stimulation with TGF-β in the cells. • Hsp70 did not influence the TGF-β-induced activation of Smad2/3 in the cells overexpressing Hsp70.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Synchrotron based infrared imaging and spectroscopy via focal plane array on live fibroblasts in D2O enriched medium

We tested the viability of using synchrotron based infrared imaging to study biochemical processes inside living cells. As a model system, we studied fibroblast cells exposed to a medium highly enriched with D2O. We could show that the experimental technique allows us to reproduce at the cellular level measurements that are normally performed on purified biological molecules. We can obtain information about lipid conformation and distribution, kinetics of hydrogen/deuterium exchange, and the formation of concentration gradients of H and O isotopes in water that are associated with cell metabolism. The implementation of the full field technique in a sequential imaging format gives a description of cellular biochemistry and biophysics that contains both spatial and temporal information.

Cell Lipids↗
DOE OSTI2024

Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. In conclusion, the transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2016

Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. In this work, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous “second generation” form of FGF-1 for therapeutic application.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Folding of Fibroblast Growth Factor 1 Is Critical for Its Nonclassical Release

Fibroblast growth factor 1 (FGF1), a ubiquitously expressed pro-angiogenic protein that is involved in tissue repair, carcinogenesis, and maintenance of vasculature stability, is released from the cells via a stress-dependent nonclassical secretory pathway. FGF1 secretion is a result of transmembrane translocation of this protein. It correlates with the ability of FGF1 to permeabilize membranes composed of acidic phospholipids. Like several other nonclassically exported proteins, FGF1 exhibits β-barrel folding. In order to assess the role of folding of FGF1 in its secretion, we applied targeted mutagenesis in combination with a complex of biophysical methods and molecular dynamics studies, followed by artificial membrane permeabilization and stress-induced release experiments. It has been demonstrated that a mutation of proline 135 located in the C-terminus of FGF1 results in (i) partial unfolding of FGF1, (ii) a decrease in FGF1’s ability to permeabilize bilayers composed of phosphatidylserine, and (iii) drastic inhibition of stress-induced FGF1 export. Thus, folding of FGF1 is critical for its nonclassical secretion.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Identification of Small-Molecule Inhibitors of Fibroblast Growth Factor 23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, the FGF receptor (FGFR), and α-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, burosumab. Small-molecule drugs that disrupt protein/protein interactions necessary for the ternary complex formation offer an alternative to disrupting FGF23 signaling. In this study, the FGF23:α-Klotho interface was targeted to identify small-molecule protein/protein interaction inhibitors since it was computationally predicted to have a large fraction of hot spots and two druggable residues on α-Klotho. We further identified Tyr433 on the KL1 domain of α-Klotho as a promising hot spot and α-Klotho as an appropriate drug-binding target at this interface. Subsequently, we performed in silico docking of ~5.5 million compounds from the ZINC database to the interface region of α-Klotho from the ternary crystal structure. Following docking, 24 and 20 compounds were in the final list based on the lowest binding free energies to α-Klotho and the largest number of contacts with Tyr433, respectively. Herein, five compounds were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduced activities significantly. Both these compounds were predicted to have favorable binding affinities to α-Klotho but not have a large number of contacts with the hot spot Tyr433. ZINC12409120 was found experimentally to disrupt FGF23:α-Klotho interaction to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 μM. Molecular dynamics (MD) simulations of the ZINC12409120:α-Klotho complex starting from in silico docking poses reveal that the ligand exhibits contacts with residues on the KL1 domain, the KL1–KL2 linker, and the KL2 domain of α-Klotho simultaneously, thereby possibly disrupting the regular function of α-Klotho and impeding FGF23:α-Klotho interaction. ZINC12409120 is a candidate for lead optimization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗