Biology subjects

Smith, J. C.

Publications and source records attributed to Smith, J. C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response

The FAM83 (Family with sequence similarity 83) family is highly conserved in vertebrates, but little is known of the functions of these proteins beyond their association with oncogenesis. Of the family, FAM83F is of particular interest because it is the only membrane-targeted FAM83 protein. When over-expressed, FAM83F activates the canonical Wnt signalling pathway and binds to and stabilizes p53; it therefore interacts with two pathways often dysregulated in disease. Insights into gene function can often be gained by studying the roles they play during development, and here we report the generation of fam83f knock-out (KO) zebrafish, which we have used to study the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the hatching gland of developing zebrafish embryos, and that fam83f KO embryos hatch earlier than their wild-type (WT) counterparts, despite developing at a comparable rate. We also demonstrate that fam83f KO embryos are more sensitive to ionizing radiation than WT embryos--an unexpected finding, bearing in mind the previously-reported ability of FAM83F to stabilize p53. Transcriptomic analysis shows that loss of fam83f leads to downregulation of phosphatidylinositol-3-phosphate (PI(3)P) binding proteins and impairment of cellular degradation pathways, particularly autophagy, a crucial component of the DNA damage response. Finally, we show that Fam83f protein is itself targeted to the lysosome when over-expressed in HEK293T cells, and that this localization is dependent upon a C terminal signal sequence. The zebrafish lines we have generated suggest that Fam83f plays an important role in autophagic/lysosomal processes, resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo.

developmental biology↗

bioRxiv2023

TCR-H: Machine Learning Prediction of T-cell Receptor Epitope Binding on Unseen Datasets

AI/ML approaches to predicting T-cell receptor (TCR) epitope specificity achieve high performance metrics on test datasets which include sequences that are also part of the training set but fail to generalize to test sets consisting of epitopes and TCRs that are absent from the training set, i.e., unseen. We present TCR-H, a supervised classification Support Vector Machines model using physicochemical features trained on the largest dataset available to date using only experimentally validated non-binders as negative datapoints. TCR-H exhibits an area under the curve of the receiver-operator characteristic (AUC of ROC) of 0.87 for epitope hard splitting (i.e., on test sets with all epitopes unseen), 0.92 for TCR hard splitting and 0.89 for strict splitting in which neither the epitopes nor the TCRs in the test set are seen in the training data. TCR-H may thus represent a significant step towards general applicability of epitope:TCR specificity prediction.

immunology↗

bioRxiv2023

Tailoring Tfh Profiles Enhances Antibody Persistence to a Clade C HIV-1 Vaccine in Rhesus Macaques

CD4 T follicular helper cells (Tfh) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Insights into Tfh subsets that promote antibody persistence and functional capacity can critically inform vaccine design. Based on the Tfh profiles evoked by the live attenuated measles virus vaccine, renowned for its ability to establish durable humoral immunity, we investigated the potential of a Tfh1/17 recall response during the boost phase to enhance persistence of HIV-1 Envelope (Env) antibodies in rhesus macaques. Using a DNA-prime encoding gp160 antigen and Tfh polarizing cytokines (interferon protein-10 (IP-10) and interleukin-6 (IL-6)), followed by a gp140 protein boost formulated in a cationic liposome-based adjuvant (CAF01), we successfully generated germinal center (GC) Tfh1/17 cells. In contrast, a similar DNA-prime (including IP-10) followed by gp140 formulated with monophosphoryl lipid A (MPLA)+QS-21 adjuvant predominantly induced GC Tfh1 cells. While the generation of GC Tfh1/17 cells with CAF01 and GC Tfh1 cells with MPLA+QS-21 induced comparable peak Env antibodies, the latter group demonstrated significantly greater antibody concentrations at week 8 after final immunization which persisted up to 30 weeks (gp140 IgG ng/ml-MPLA; 5500; CAF01, 2155; p <0.05). Notably, interferon {gamma}+ Env-specific Tfh responses were consistently higher with gp140 in MPLA+QS-21 and positively correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC Tfh1 induction promote persistent Env antibodies, important for protective immunity against HIV.

immunology↗

bioRxiv2023

MCAK Inhibitors Induce Aneuploidy in Triple Negative Breast Cancer Models

Standard of care for triple negative breast cancer (TNBC) involves the use of microtubule poisons like paclitaxel, which are proposed to work by inducing lethal levels of aneuploidy in tumor cells. While these drugs are initially effective in treating cancer, dose-limiting peripheral neuropathies are common. Unfortunately, patients often relapse with drug resistant tumors. Identifying agents against targets that limit aneuploidy may be a valuable approach for therapeutic development. One potential target is the microtubule depolymerizing kinesin, MCAK, which limits aneuploidy by regulating microtubule dynamics during mitosis. Using publicly available datasets, we found that MCAK is upregulated in triple negative breast cancer and is associated with poorer prognoses. Knockdown of MCAK in tumor-derived cell lines caused a two- to five-fold reduction in the IC50 for paclitaxel, without affecting normal cells. Using FRET and image-based assays, we screened compounds from the ChemBridge 50k library and discovered three putative MCAK inhibitors. These compounds reproduced the aneuploidy-inducing phenotype of MCAK loss, reduced clonogenic survival of TNBC cells regardless of taxane-resistance, and the most potent of the three, C4, sensitized TNBC cells to paclitaxel. Collectively, our work shows promise that MCAK may serve as both a biomarker of prognosis and as a therapeutic target. Simple SummaryTriple negative breast cancer (TNBC) is the most lethal breast cancer subtype with few treatment options available. Standard of care for TNBC involves the use of taxanes, which are initially effective, but dose limiting toxicities are common, and patients often relapse with resistant tumors. Specific drugs that produce taxane-like effects may be able to improve patient quality of life and prognosis. In this study we identify three novel inhibitors of the Kinesin-13 MCAK. MCAK inhibition induces aneuploidy; similar to cells treated with taxanes. We demonstrate that MCAK is upregulated in TNBC and is associated with poorer prognoses. These MCAK inhibitors reduce the clonogenic survival of TNBC cells, and the most potent of the three inhibitors, C4, sensitizes TNBC cells to taxanes, similar to the effects of MCAK knockdown. This work will expand the field of precision medicine to include aneuploidy-inducing drugs that have the potential to improve patient outcomes.

cancer biology↗

bioRxiv2023

Pose prediction accuracy in ligand docking to RNA

Structure-based virtual high-throughput screening is used in early-stage drug discovery. Over the years, docking protocols and scoring functions for protein-ligand complexes have evolved to improve accuracy in the computation of binding strengths and poses. In the last decade, RNA has also emerged as a target class for new small molecule drugs. However, most ligand docking programs have been validated and tested for proteins and not RNA. Here, we test the docking power (pose prediction accuracy) of three state-of-the-art docking protocols on [~]173 RNA-small molecule crystal structures. The programs are AutoDock4 (AD4) and AutoDock Vina (Vina), which were designed for protein targets, and rDock, which was designed for both protein and nucleic acid targets. AD4 performed relatively poorly. For RNA targets for which a crystal structure of a bound ligand is available, and the goal is to identify new molecules for the same pocket, rDock performs slightly better than Vina. However, in the more common type of early-stage drug discovery setting, in which no structure of a ligand:target complex is known, rDock performed similar to Vina, with a low success rate of [~]27 %. Vina was found to bias for ligands with certain physicochemical properties whereas rDock performs similarly for all ligand properties. Thus, for projects where no ligand:protein structure already exists, Vina and rDock are both applicable. However, the relatively poor performance of all methods relative to protein target docking illustrates a need for further methods refinement.

biophysics↗

bioRxiv2023

Oncogene-like addiction to aneuploidy in human cancers

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses TP53 signaling, and we show that TP53 mutations are mutually-exclusive with 1q aneuploidy in human cancers. Thus, specific aneuploidies play essential roles in tumorigenesis, raising the possibility that targeting these "aneuploidy addictions" could represent a novel approach for cancer treatment.

cancer biology↗

bioRxiv2022

A drug's most potent target is not necessarily the source of its anti-cancer activity

The small-molecule drug ralimetinib was developed as an inhibitor of the kinase p38, and it has advanced to phase 2 clinical trials in oncology. Here, we apply a multi-modal approach to demonstrate that ralimetinibs anti-cancer activity occurs due to its ability to inhibit EGFR, rather than p38. We find that cancer cell lines driven by EGFR mutations exhibit the greatest sensitivity to ralimetinib treatment, and ralimetinib phenocopies established EGFR inhibitors in pharmacogenomic profiling experiments. We further demonstrate that ralimetinib inhibits EGFR kinase activity in vitro and in cellulo, albeit at >30-fold higher concentrations than it inhibits p38. Finally, while deletion of the gene encoding p38 has no effect on ralimetinib sensitivity, expression of the EGFR-T790M gatekeeper mutation confers resistance to ralimetinib treatment. These findings suggest that future clinical trials involving ralimetinib could incorporate EGFR mutation status as a biomarker to identify sensitive patients. Moreover, our results demonstrate that a compounds anti-cancer effects should not necessarily be attributed to the protein that it inhibits most strongly, and instead, comprehensive cellular and genetic profiling is required to understand a drugs mechanism-of-action.

cancer biology↗

bioRxiv2022

Differential Boric Acid and Water Transport in Type I and Type II Pores of Arabidopsis Nodulin 26-Intrinsic Protein

Nodulin-26 intrinsic proteins (NIPs) are plant-specific multifunctional aquaporin-like channels that are phylogenetically and structurally segregated into three subfamilies: NIP I, II, and III. Each subfamily has a characteristic selectivity filter sequence (the "aromatic-arginine" region, or ar/R) that controls substrate transport specificity based on steric constraints, hydrophobicity, and the spatial orientation of hydrogen bonding moieties. All three NIP subfamilies transport metalloid hydroxides, both beneficial as well as toxic, but with different selectivities. Here we investigated the B, As, and water selectivity of representative Arabidopsis thaliana NIP I and II proteins as well as their ar/R mutants in transport assays as well as through B complementation analysis in the B sensitive nip5;1 mutant background. All NIP proteins, and their ar/R mutants, showed equal permeability to arsenite, but showed differences in boric acid and aquaporin activities that was linked to the amino acid at the helix 2 (H2) position of the ar/R filter (Ala for NIP II and Trp for NIP I). The presence of an alanine at this position in NIP II proteins enhances boric acid permeability and drastically reduces the aquaporin/water permeability of the channel. A NIP II structural model generated from the AlphaFold2 resource and evaluated by MD simulation shows that the alanine results in a wider ar/R pore that accommodates the trigonal boric acid molecule and may allow gating of the pore in a manner that affects water permeability. In contrast, NIP I proteins adopt a more classical aquaporin/glyceroporin arrangement in the ar/R that allows metalloid permeability, although with greater selectivity, as well as permeation by water.

biochemistry↗

bioRxiv2022

Loss of retinogeniculate synaptic function in the DBA/2J mouse model of glaucoma.

BackgroundRetinal ganglion cell (RGC) axons comprise the optic nerve and carry information to the dorsolateral geniculate nucleus (dLGN) that is relayed to the cortex for conscious vision. Glaucoma is a blinding neurodegenerative disease that commonly results from intraocular pressure (IOP)-associated injury leading to RGC axonal pathology, disruption of RGC outputs to the brain, and eventual apoptotic loss of RGC somata. The consequences of elevated IOP and glaucomatous pathology on RGC signaling to the dLGN are largely unknown and likely to be important contributors to visual system dysfunction in glaucoma. Thus, the goal of this study was to determine how glaucoma affects RGC outputs to the dLGN. MethodsWe used a combination of anatomical and physiological approaches to study the structure and function of retinogeniculate synapses in male and female DBA/2J mice at multiple ages before and after IOP elevation. These included measures of anterograde axonal transport, immunofluorescence staining of RGC axon terminals, patch-clamp recording retinogeniculate (RG) synapses in living brain slices, Sholl analysis of thalamocortical relay neuron dendrites, measurements of RGC somatic density, and treatment with a topical ophthalmic alpha-2 adrenergic agonist (brimonidine). ResultsDBA/2J mice showed progressive loss of anterograde optic tract transport to the dLGN and vGlut2 labeling of RGC axon terminals. Patch-clamp measurements of RG synaptic function showed that the strength of synaptic transmission was lower in 9 and 12-month DBA/2J mice and that this was the result of loss of individual RGC axon contributions. TC neuron dendrites showed a reduction in complexity at 12 months, suggestive of a delayed reorganization following reduced synaptic input. There was no detectable change in RGC soma density in 11-12m DBA/2J retinas indicating that observed effects occurred prior to RGC somatic loss. Finally, treatment with brimonidine eye drops prevented the loss of vGlut2-labeled RGC terminals in the dLGN. ConclusionsThese findings identify glaucoma- and IOP-associated functional deficits in an important subcortical RGC projection target. This sheds light on the processes linking IOP to vision loss and will be critical for informing future diagnostic approaches and vision-restoration therapies.

neuroscience↗

bioRxiv2022

Microstructures of Ant Chemosensory Sensilla Support a Dual Function in Detecting Both Volatile and Contact-Mediated Cues

Ants and other eusocial insects emit and receive chemical signals to communicate important information within the colony. In ants, nestmate recognition, task allocation, and reproductive distribution of labor are largely mediated through the detection of cuticular hydrocarbons (CHCs) that cover the exoskeleton. With their large size and limited volatility, these CHCs are believed to be primarily detected through direct contact with the antennae during behavioral interactions. Here we use scanning electron microscopy to investigate the unique morphological features of CHC-sensitive basiconic sensilla of two ant species, the black carpenter ant Camponotus pennsylvanicus and the Indian jumping ant Harpegnathos saltator. These basiconic sensilla possess an abundance of small pores typical of most insect olfactory sensilla, but also have a large concave depression at the terminal end. Basiconic sensilla are enriched at the distal segments of the antennae in both species, further supporting their proposed role in contact chemosensation. A survey of these sensilla across other ant subfamilies shows varied microstructures at their tips, but each possess surface textures that would also increase sensory surface area. These unique ant chemosensory sensilla represent yet another example of how specialized structures have evolved to serve the functional requirements of eusocial communication.

zoology↗

bioRxiv2022

Seasonal changes in membrane structure and excitability in central neurons of goldfish (Carassius auratus) under constant environmental conditions

Seasonal modifications in the structure of cellular membranes occur as an adaptive measure to withstand exposure to prolonged environmental change. Little is known about whether such changes may occur independently of external cues, such as photoperiod or temperature, or how they may impact the central nervous system (CNS). We compared membrane properties of central neurons isolated from the retina of goldfish (Carassius auratus), an organism well-adapted to extreme environmental change, during the summer and winter months. Goldfish were maintained in a facility under constant environmental conditions throughout the year. Analysis of whole-retina phospholipid composition using mass spectrometry-based lipidomics revealed a two-fold increase in phosphatidylethanolamine species during the winter, suggesting an increase in cell membrane fluidity. Atomic force microscopy was used to produce localized, nanoscale-force deformation of neuronal membranes. Measurement of Youngs modulus indicated increased membrane stiffness (or decreased elasticity) in neurons isolated during the winter. Voltage-clamp electrophysiology was used to assess physiological changes in neurons between seasons. Winter neurons displayed a hyperpolarized reversal potential (Vrev) and a significantly lower input resistance (Rin) compared to summer neurons. This was indicative of a decrease in membrane excitability during the winter. Subsequent measurement of intracellular Ca2+ activity using Fura-2 microspectrofluorometry confirmed a reduction in action potential activity, including duration and action potential profile, in neurons isolated during the winter. These studies demonstrate chemical and biophysical changes that occur in central neurons of goldfish throughout the year without exposure to seasonal cues, and suggest a novel mechanism of seasonal regulation of CNS activity. SUMMARY STATEMENTCentral neurons isolated from the retina of goldfish held under constant environmental conditions undergo seasonal changes in membrane structure and excitability.

neuroscience↗

bioRxiv2022

Osteocalcin binds to the GPRC6A Venus fly trap allosteric site to positively modulate GPRC6A signaling

GPRC6A is a member of the Family C G-protein coupled receptors that is activated by cations, L-amino acids, the osteocalcin (Ocn) peptide, and testosterone. GPRC6A functions as a master regulator of energy metabolism and sex hormone production. Based on homology to the related receptors mGluR5 and CaSR, GPRC6As multiple ligand specificity is likely based on an orthosteric ligand binding site in the bilobed Venus fly trap (VFT) domain together with two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7TM domain. Here, we show that Ocn acts as a PAM for GPRC6A by binding to a site in the VFT that is distinct from the orthosteric site for calcium and L-amino acids. In agreement with this finding, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the predicted Ocn binding site, K352E and H355P, prevent Ocn activation of GPRC6A. These observations provide a structural framework for understanding the ability of multiple distinct classes of compounds to activate GPRC6A and set the stage to develop novel small molecules to activate and inhibit this receptor.

molecular biology↗

bioRxiv2021

Salmonella enterica serovar Typhimurium from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type

Salmonella enterica serovar Typhimurium is typically considered a host generalist, however certain strains are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium strains from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most strains originating from domestic animal sources, and genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the U.S. are likely host-adapted, and the representative genomic dataset examined in this study can improve source prediction and facilitate outbreak investigation. IMPORTANCEWithin-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the U.S. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.

microbiology↗

bioRxiv2021

Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator

Previously our computational modeling studies (Phillips et al., 2019) proposed that neuronal persistent sodium current (INaP) and calcium-activated non-selective cation current (ICAN) are key biophysical factors that, respectively, generate inspiratory rhythm and burst pattern in the mammalian preBotzinger complex (preBotC) respiratory oscillator. Here, we experimentally tested and confirmed three predictions of the model from new simulations concerning the roles of INaP and ICAN: (1) INaP and ICAN blockade have opposite effects on the relationship between network excitability and preBotC rhythmic activity; (2) INaP is essential for preBotC rhythmogenesis; (3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. These predictions were confirmed via optogenetic manipulations of preBotC network excitability during graded INaP or ICAN blockade by pharmacological manipulations in neonatal mouse slices in vitro. Our results support and advance the hypothesis that INaP and ICAN mechanistically underlie rhythm and inspiratory burst pattern generation, respectively, in the isolated preBotC.

neuroscience↗

bioRxiv2021

Genome-wide identification and analysis of prognostic features in human cancers

Clinical decisions in cancer rely on precisely assessing patient risk. To improve our ability to accurately identify the most aggressive malignancies, we constructed genome-wide survival models using gene expression, copy number, methylation, and mutation data from 10,884 patients with known clinical outcomes. We identified more than 100,000 significant prognostic biomarkers and demonstrate that these genomic features can predict patient outcomes in clinically-ambiguous situations. While adverse biomarkers are commonly believed to represent cancer driver genes and promising therapeutic targets, we show that cancer features associated with shorter survival times are not enriched for either oncogenes or for successful drug targets. Instead, the strongest adverse biomarkers represent widely-expressed housekeeping genes with roles in cell cycle progression, and, correspondingly, nearly all therapies directed against these features have failed in clinical trials. In total, our analysis establishes a rich resource for prognostic biomarker analysis and clarifies the use of patient survival data in preclinical cancer research and therapeutic development.

cancer biology↗

bioRxiv2020

Saracatinib is an efficacious clinical candidate for fibrodysplasia ossificans progressiva

Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues due to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2/ACVR1. From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2 compared with other receptors of the BMP/TGF{beta} signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D transgenic mouse line, which provides a model of heterotopic ossification, as well as an inducible ACVR1R206H knock-in, which serves as a genetically and physiologically faithful model of FOP. In both models, saracatinib was well tolerated and potently inhibited the development of heterotopic ossification even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in the treatment of FOP, offering an accelerated path to clinical proof of efficacy studies and potentially significant benefits to individuals with this devastating condition.

molecular biology↗

bioRxiv2020

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an -ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N{delta} (HD) and N{epsilon} (HE) protonation of His41 and His164, respectively, the -ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

biophysics↗

bioRxiv2020

Small molecule FGF23 inhibitors increase serum phosphate and improve skeletal abnormalities in Hyp mice

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that binds to binary FGFR/-Klotho receptor complexes in the kidney tubules to inhibit phosphate reabsorption and 1,25(OH)2D production. Excess FGF23 causes X-linked hypophosphatemia (XLH) and tumor induced osteomalacia (TIO). Recently, Burosumab, an FGF23 blocking antibody, was approved for treating these hypophosphatemic disorders. A small molecule inhibitor that specifically binds to FGF23 to prevent activation of FGFR/-Klotho complexes has potential advantages over a systemically administered FGF23 blocking antibody. We previously identified the small molecule ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a therapeutic lead FGF23 antagonist. Additional structure-activity studies developed a series of ZINC13407541 analogues with enhanced drug-like properties. In this study, we tested in a pre-clinical Hyp mouse homologue of XLH a direct connect analogue (8n) [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] that exhibited the greatest stability in microsomal assays, and 13a [(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] that exhibited increased in vitro potency. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and significantly increased serum phosphate and 1,25(OH)2D concentrations in Hyp mice. Long-term parenteral treatment with 8n or 13a also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in Hyp mice. The more potent 13a compound showed greater therapeutic efficacy in Hyp mice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat FGF23-mediated disorders.

pharmacology and toxicology↗